Assay Platform for Clinically Relevant Metallo-β-lactamases...-蚂蚁淘在线

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RETURN TO ISSUEPREVArticleNEXTAssay Platform for Clinically Relevant Metallo-β-lactamasesSander S. van Berkel†,Jürgen Brem†,Anna M. Rydzik†,Ramya Salimraj‡,Ricky Cain⊥,Anil Verma§∥,Raymond J. Owens§∥,Colin W. G. Fishwick⊥,James Spencer‡,andChristopher J. Schofield*†View Author Information† Chemistry Research Laboratory, University of Oxford, 12 Mansfield Road, Oxford OX1 3TA, United Kingdom‡ School of Cellular and Molecular Medicine, University of Bristol, Medical Sciences Building, Bristol BS8 1TD, United Kingdom§ Oxford Protein Production Facility UK, The Research Complex at Harwell, Rutherford Appleton Laboratory, Harwell Science and Innovation Campus, Oxfordshire OX11 0FA, United Kingdom∥ Division of Structural Biology, University of Oxford, Henry Wellcome Building for Genomic Medicine, Oxford OX3 7BN, United Kingdom⊥ School of Chemistry, University of Leeds, Leeds LS2 9JT, United Kingdom*Phone: +44 (0) 1865 275625. E-mail: [email protected]Cite this: J. Med. Chem. 2013, 56, 17, 6945–6953Publication Date (Web):July 30, 2013Publication History Received24 May 2013Published online16 August 2013Published inissue 12 September 2013https://doi.org/10.1021/jm400769bCopyright © 2013 American Chemical SocietyRIGHTS & PERMISSIONSACS AuthorChoicewith CC-BYlicenseArticle Views4316Altmetric-Citations73LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated.Get e-AlertsAbstractHigh Resolution ImageDownload MS PowerPoint SlideMetallo-β-lactamases (MBLs) are a growing threat to the use of almost all clinically used β-lactam antibiotics. The identification of broad-spectrum MBL inhibitors is hampered by the lack of a suitable screening platform, consisting of appropriate substrates and a set of clinically relevant MBLs. We report procedures for the preparation of a set of clinically relevant metallo-β-lactamases (i.e., NDM-1 (New Delhi MBL), IMP-1 (Imipenemase), SPM-1 (São Paulo MBL), and VIM-2 (Verona integron-encoded MBL)) and the identification of suitable fluorogenic substrates (umbelliferone-derived cephalosporins). The fluorogenic substrates were compared to chromogenic substrates (CENTA, nitrocefin, and imipenem), showing improved sensitivity and kinetic parameters. The efficiency of the fluorogenic substrates was exemplified by inhibitor screening, identifying 4-chloroisoquinolinols as potential pan MBL inhibitors.IntroductionARTICLE SECTIONSJump ToSelection pressure caused by frequent use of β-lactam antibiotics, including penicillins, cephalosporins, carbapenems, and monobactams, has resulted in the emergence and dissemination of highly efficient resistance mechanisms in clinically relevant bacterial pathogens. This rapid adaptation of bacterial strains to commonly used antibiotics is an increasing global threat to public health care.(1, 2) An important mechanism of resistance to β-lactam antibiotics, including clinically challenging Gram-negative organisms, involves β-lactamase catalysis. β-Lactamases can be classified into those employing a nucleophilic serinyl residue at their active site (serine-β-lactamases, SBLs, classes A, C, and D) and those employing one or two zinc ions to promote hydrolysis (metallo-β-lactamases, MBLs, class B).(3) The combination of a penicillin antibiotic and an SBL inhibitor has substantially extended the utility of the former, as, for example, in the case of amoxicillin and clavulanic acid (Augmentin).(4) In addition to clavulanic acid, two other class A (penicillinase) β-lactamase inhibitors are used, tazobactam and sulbactam; all three inhibitors are themselves β-lactams, though in contrast to penicillins they react with SBLs to form relatively stable acyl-enzyme complexes.(5) These SBL inhibitors, however, appear to have no effect on MBLs.(6) β-Lactam antibiotics have been developed that possess resistance to SBLs, or as in the case of carbapenems, they have been developed to be inhibitors or relatively poor substrates.(7) However, since the pioneering introduction of the SBL-targeting inhibitors, progress in the development of clinically useful, β-lactamase-specific inhibitors has been limited. In the case of SBLs, at least one compound, Avibactam, is currently in clinical trials as a combined class A and class C SBL inhibitor.(8)MBLs, which, unlike the SBLs, are not structurally or mechanistically related to the penicillin-binding proteins (PBPs), were first identified approximately 50 years ago.(9) However, as a result of their apparently restricted distribution to the chromosomes of less pathogenic species, they were long not considered to represent a significant threat to the clinical effectiveness of β-lactams. The emergence of horizontally acquired MBLs, where horizontal gene transfer permits genetic material to be transferred between individual bacteria of the same species or between different species, has led to their extensive dissemination across geographical and species boundaries and now threatens the effectiveness of almost all β-lactams, including carbapenems (i.e., \"last resort” antibiotics);(10) the monobactam aztreonam is a current exception. MBLs identified on transferrable plasmids (e.g., the NDM-, IMP-, VIM-, GIM-, and SPM-type enzymes) are considered a prevalent and immediate clinical problem.(11) Various structurally diverse types of MBL inhibitors have been described, including carboxylic/succinic acids,(12) triazoles/tetrazoles,(13) thiols,(14) trifluoromethyl ketones,(15) and others.(16) To date, no clinically useful MBL inhibitor has been reported. This, in part, may reflect the technical and scientific challenges in the development of an MBL−β-lactam-based combination therapy. There is also extensive structural diversity in the MBL family, particularly in the proposed mobile loop regions around the active site.(17) For an MBL inhibitor to be clinically useful, it is possible that more than one MBL will need to be inhibited. In an effort to help enable work that will lead to the development of useful MBL inhibitors, we here describe an assay platform for clinically relevant MBLs, including protein production procedures and assay conditions using both chromogenic and fluorogenic substrates.Results and DiscussionARTICLE SECTIONSJump ToSeveral screening methods for the in vitro and in vivo detection of β-lactamases have been reported.(18) Representative substrates currently in use for β-lactamases include chromogenic cephalosporin-based substrates, such as CENTA,(19) PADAC,(20) and nitrocefin,(21) cephalosporin-based fluorogenic substrates,(22) bioluminescent probes,(23) and fluorescence resonance energy transfer (FRET)-based substrates.(24) These substrates have mainly been applied in research centered around SBLs and only in one case on MBLs (L1 and CcrA).(22c) The large-scale application of these type of compounds in automated high-throughput screening (HTS) for MBL inhibitors is, however, hampered by their lengthy and often difficult synthesis and the high costs associated with these substrates. More importantly, these substrates suffer from poor substrate recognition by some MBLs as a result of the high diversity of this enzyme family whose members vary significantly in sequence, structure, and substrate specificity, thus making it hard to use a single substrate for broad MBL activity screening. The development of new assays for broad-range MBL activity screening, based on hydrolysis of chromogenic or fluorogenic β-lactams, would, however, significantly facilitate inhibitor identification. Not unimportantly, large-scale inhibitor screening against different MBLs needs an inexpensive substrate, thus requiring a simple, scalable, and cost-efficient synthetic route. To develop assay procedures suitable for a range of MBLs, we targeted four clinically relevant enzymes as well as the MBL from Bacillus cereus (BcII), which has been extensively used as a model enzyme. The clinically relevant enzymes chosen were NDM-1 (New Delhi MBL), IMP-1 (Imipenemase), SPM-1 (São Paulo MBL), and VIM-2 (Verona integron-encoded MBL). To identify MBL substrates useful for efficient inhibitor screening, we prepared and tested substrates on the above-mentioned panel of MBLs. These substrates included three chromogenic substrates, i.e., imipenem, nitrocefin, and CENTA, and five coumarin-linked substrates, i.e., FC1–FC5 (Figure 1). Fluorescent substrates FC1 and FC2 release 7-mercapto-4-methylcoumarin upon hydrolysis, resulting in a decrease of fluorescence. Conversely, fluorogenic substrates FC3–FC5 liberate 7-hydroxycoumarin upon hydrolysis of the lactam ring, producing an increased fluorescence signal (Figure 2).Figure 1Figure 1. Substrates for metallo-β-lactamase activity measurements.High Resolution ImageDownload MS PowerPoint SlideFigure 2Figure 2. Hydrolysis of substrates by MBLs, resulting in either an increase or a decrease of fluorescence.High Resolution ImageDownload MS PowerPoint Slide Protein ProductionDetails are provided in the Supporting Information. For the production of recombinant BcII, IMP-1, and NDM-1 MBLs in Escherichia coli, vectors that have been previously described were used, i.e., the pET9a-BcII, pET-26b IMP-1, and pOPINF NDM-1 plasmids.(25) In the case of the MBLs VIM-2 and SPM-1, new pTriEx-based pOPINF vectors were constructed.(26) These vectors encode the mature VIM-2 and SPM-1 sequences (i.e., with the periplasmic export sequences removed) fused to N-terminal hexahistidine tags, cleavable with 3C protease. The VIM-2 and SPM-1 genes were codon optimized for E. coli (Genscript, Piscataway, NJ) and inserted into the pOPINF vector. All the MBLs were produced using in-house-constructed E. coli BL21(DE3) pLyS cells, which were grown in 2TY medium. For the protein purifications standard procedures were used. The purity of the MBL proteins was determined to be 95% by SDS–PAGE analysis (see the Supporting Information, Figure SI_4). Synthesis of SubstratesCENTA was prepared using a modified literature procedure.(27) Cleavage of 5,5′-dithiobis(2-nitrobenzoic acid) by dithiothreitol (DTT) yielded 3-carboxyl-4-nitrothiophenol, which was subsequently used to convert commercially available cephalothin into CENTA by heating (65 °C, 3 h). Use of chromatographically purified 3-carboxyl-4-nitrothiophenol gave CENTA in high purity and satisfactory yield after a single acid/base extraction.(28)Fluorogenic cephalosporin 1 (FC1) was prepared from commercially available p-methoxybenzyl (PMB) ester-protected chlorocephalosporin (CC) via substitution with 7-mercapto-4-methylcoumarin (MMC), followed by acid-mediated PMB deprotection (Scheme 1A). Treatment of CC with MMC resulted in the formation of both the Δ2- and Δ3-alkene isomers of compound 1. Subjecting the Δ2–Δ3 isomer mixture to acid-mediated PMB deprotection conditions led to complete deprotection within 1 h, generating FC1 (Δ2–Δ3 mix). To circumvent the Δ2–Δ3 isomerization issue, oxidation of the thiazine sulfur atom with mCPBA was performed prior to alkylation (Scheme 1A).(29) The Δ3-alkene (S)-sulfoxide 2 was obtained in good yield (72%); subsequent alkylation with MMC in the presence of N,N′-diisopropylethylamine gave 3 as a single isolate isomer in reasonable yield (49%). Acid-mediated PMB deprotection yielded FC2 in good yield (88%). Notably, MMC appears to be nonfluorescent in its free form, whereas the thioether cephalosporin derivatives (i.e., 1 and 3) showed significant fluorescence. While this was not our objective, FC2 has potential as an \"inverse fluorogenic” substrate (for spectroscopic data see the Supporting Information, Figure SI_1).Scheme 1Scheme 1. Synthesis of FC1-5: (A) Thiacoumarin Cephalosporin, (B) Hydroxylcoumarin CephalosporinsaHigh Resolution ImageDownload MS PowerPoint SlideScheme aReagents and conditions: (a) MMC, DiPEA, DMF, rt, 2 h; (b) TFA/anisole (5:1), 0 °C, 30 min; (c) mCPBA (1 equiv), CH2Cl2, 0 °C, 1 h; (d) NaI (10 equiv), acetone, rt, 2 h, (e) 7-HC, K2CO3, MeCN, rt, 4 h; (f) mCPBA (2 equiv), CH2Cl2, 0 °C, 3 h.With the aim of developing a fluorogenic MBL substrate, we focused on 7-hydroxycoumarin (7-HC) cephalosporin derivatives, with the knowledge that umbelliferone shows strong fluorescence in its free form while being nearly nonfluorescent when ether-derivatized.(30) During the course of our investigations, Rao and co-workers reported the synthesis and application of FC3 and FC4 as SBL fluorogenic substrates (specific for BalC over TEM-1).(31) Their reported procedure (with minimal Δ2–Δ3 isomerization) for the synthesis of FC3 and FC4 proved to be efficient and yielded the desired fluorogenic probes (FC3 and FC4) in satisfactory yields (Scheme 1B). Besides substrates FC3 (sulfide) and FC4 ((S)-sulfoxide), we also prepared substrate FC5 (sulfone). Treatment of compound 4 with 2 equiv of mCPBA gave clean conversion to compound 6, which upon PMB deprotection yielded substrate FC5 in good yield. Metallo-β-lactamase AssaysInitially time-dependent changes in absorbance and/or emission spectra upon hydrolysis of the potential MBL substrates were recorded to investigate the optimal wavelengths for analysis (see the Supporting Information, Figures SI_2 and SI_3). For the frequently used chromogenic substrates, i.e., imipenem (carbapenem) and nitrocefin and CENTA (cephalosporins), this resulted in preferred readout absorption wavelengths of 300, 405, and 492 nm, respectively. For fluorogenic substrates FC3–FC5 the absorption and emission spectra of umbelliferon in HEPES assay buffer (pH 7.5) were measured and their corresponding optimal wavelengths applied in the fluorescence-based assay (λex = 380 nm and λem = 460 nm).(32)We then determined kinetic parameters for different MBL–substrate combinations. The data for the chromogenic substrates (Table 1) are generally in good agreement with previously reported data,(27, 39) with the only substantial dissimilarity being the lower KM values obtained by us in the case of CENTA, possibly because of increased purity of the substrate. Direct quantitative comparison of the obtained data with reported values is, however, difficult as a result of variations in protein production conditions, concentration of metal ions added, etc. The improved stability of CENTA compared to imipenem renders it an attractive substrate for inhibitor identification. However, MBL inhibition by the hydrolyzed cephalosporin products (for BcII,(40) CphA,(41) and Sfh-I(42)) or the liberated thiophenol from CENTA (for IMP-1) could complicate interpretation of the results. Examination of the data in Table 1 reveals the high sensitivity of the fluorogenic MBL substrates, which enable the use of enzyme concentrations (generally ≫1 nM) well below those used with chromogenic substrates (generally 1–50 nM).Table 1. Kinetic Data for Chromogenic and Fluorogenic Substrates on MBLs literature dataentryenzymesubstrate[E]a (pM)KM (μM)kcat (s–1)kcat/KM (μM–1·s–1)kcat/KM (μM–1·s–1)ref1NDM-1nitrocefin10008.8 ± 1.125.32.94.1332 CENTA100034.6 ± 6.692.52.7NDbND3 imipenem250111.2 ± 11.7398.53.60.5334 FC35017.6 ± 1.7102.65.8 5 FC4504.0 ± 0.8125.732.0 6 FC5502.4 ± 0.2298124.1 7VIM-2nitrocefin1007.2 ± 0.6225.631.242.7348 CENTA100026.1 ± 3.148.21.8NDND9 imipenem100037.8 ± 11.541.61.13.83410 FC4506.3 ± 0.4125.520.0 11 FC510015.2 ± 1.2291.219.1 12IMP-1nitrocefin5055.7 ± 4.8279450.22.33513 CENTA10017.1 ± 2.0431.925.32.02714 imipenem5042.7 ± 6.7120028.11.23515 FC4115.2 ± 0.712200807 16 FC5516.8 ± 1.98706517 17SPM-1nitrocefin50 × 10316.0 ± 3.7c0.70.040.123618 CENTA10 × 10325.3 ± 6.16.10.2NDND19 imipenem10 × 103330.9 ± 35.537.20.11.03620 FC45002.5 ± 0.28.73.5 21 FC510002.7 ± 0.227.3810.3 22BcIInitrocefin10008.1 ± 1.014.91.80.643723 CENTA1000135.9 ± 16.48.90.060.152724 imipenem1000 10005830.40.133825 FC4100024.4 ± 1.717.10.7 26 FC5100040.4 ± 3.3151.13.7 aThe enzyme concentrations of the purified proteins were determined using a NanoDrop spectrometer; concentrations of diluted solutions used in the assay were calculated from the original concentration.bND = not determined.cThe following apparent Ki value for substrate/product inhibition was determined: 36.6 ± 15.2.In the case of the combination of NDM-1 and FC3–FC5, up to 5–20 times lower enzyme concentration was used as compared to enzyme concentrations required for imipenem or nitrocefin. Further testing of NDM-1 with the three fluorogenic substrates FC3–FC5, with different sulfur oxidation states, disclosed significantly different kcat/KM values (up to 20-fold), implying catalytic efficiency is altered by the oxidation state of the thiazine sulfur atom. We observed that the stability of FC3 in aqueous solution is substantially lower than that of FC4 and FC5. Autohydrolysis studies in buffer at room temperature showed substantial hydrolysis of FC3 after 3 h, while both FC4 and FC5 were found to be stable for at least 8 h. Solutions of FC4 and FC5 could be used for 1–2 days when stored on ice. Moreover, DMSO stock solutions, stored at −20 °C, could be used several months after preparation without any detectable decomposition. Storage of compounds FC4 and FC5 as solids at −20 °C enabled use of these compounds over a prolonged period of time (i.e., months). As a result, FC4 and FC5 were tested on the other MBLs.For nearly all the clinically important MBLs, FC4 and FC5 showed higher kcat/KM values than the chromogenic substrates (with VIM-2 being the only exception). On analysis of the VIM-2 results, we found that FC5 is preferred over FC4 and performs equally well as nitrocefin, having similar kcat values though slightly different KM values (7.2 μM vs 15.2 μM, respectively) and consequently different kcat/KM values (31.2 μM–1·s–1 vs 19.1 μM–1·s–1).The kinetic data of the five substrates for IMP-1 reveal that FC4 outperforms all other substrates mainly as a result of its high kcat value (i.e., 807 μM–1·s–1). As depicted in Figure 2A substrate concentrations up to 100 μM could be applied without perturbing the measurement. Although FC4 has a low KM compared to the other substrates tested on IMP-1, its high sensitivity allows for accurate measurement as indicted by the low interexperimental error (Figure 3A, experiment performed in triplicate).(43) As a result of the high sensitivity of both FC4 and FC5, a 20-fold lower IMP-1 concentration could be used (i.e., picomolar enzyme concentration range) while retaining a KM value in the micromolar range, thus allowing for the detection of weak binding fragments or slow-binding inhibitors.(44)Figure 3Figure 3. (Top) IMP-1 with FC4. (Bottom) SPM-1 with nitrocefin (substrate or product inhibition). Errors are reported as standard errors, n = 3.High Resolution ImageDownload MS PowerPoint SlideIn the case of SPM-1, both FC4 and FC5 outperform all the tested chromogenic substrates. Interestingly, we found that SPM-1 is inhibited by nitrocefin/nitrocefin-derived species at a concentration of 36 μM (see Figure 3B and Table 1). Although β-lactams can act as inhibitors of MBLs, as is apparent for nitrocefin and imipenem at high concentrations (e.g., nitrocefin for SPM-1, this paper, and for CphA and Sfh-I, refs 45 and 42, respectively); the high sensitivity of the here reported fluorogenic substrates (FC4 and FC5) allows for the use of extremely low substrate concentrations. In Silico StudiesIn an attempt to investigate the different substrate binding constants of nitrocefin, imipenem, CENTA, and FC4 for the different MBLs, we performed in silico docking studies. Docking of nitrocefin, imipenem, CENTA, and FC4 in IMP-1, NDM-1, and VIM-2 (PDB IDs 1JJT,(12b)3Q6X,(46) and 1KO3,(47) respectively) was performed using AutoDock 4(48) and SPROUT software.(49) After analysis of the obtained unhydrolyzed–enzyme models (see the Supporting Information), the different docking poses, calculated by Autodock 4 and SPROUT, were scored and compared to the substrate binding constants (KM) (Table SI_1, Supporting Information). Assessment of the SPROUT docking scores generally reveals a better correlation with the kinetic parameters obtained in vitro (specifically KM values in the cases of NDM-1 and VIM-2) compared to the docking scores generated by Autodock 4. However, for all three enzymes, the highest ranking substrate in terms of the SPROUT and Autodock scores is FC4, which was also measured to have the highest binding affinity for all three enzymes. Although in-depth structural analyses are required, these modeling results suggest that the cephalosporins may bind to the active site in more than one orientation, including catalytically nonproductive ones. Overall, these results emphasize the importance of experimentally determining optimal MBL–substrate combinations and the need for more structural analyses on MBL–substrate complexes. Inhibitor ScreeningTo achieve clinical utility, MBL inhibitors may need to act on more than one MBL subfamily. This is potentially challenging because of the generally low sequence similarity between MBLs of different subfamilies (B1, B2, B3). Most clinically relevant MBLs are from the B1 subfamily; however, SPM-1 presents a high similarity with both the B1 and B2 subfamilies(50) and hence was used as a starting point for proof of principle inhibitor screening. To validate our screening method, we screened a series of 4-chloroisoquinolinol derivatives (7a–7g)(28) against SPM-1 using FC4 as a reporter substrate (Table 2). This set of compounds was selected as a result of their previously reported inhibition of metal-binding oxygenases.(51) Screening for residual activity of compounds 7a–7g, at concentrations of 200 μM and 1 mM, revealed that the 4-chloroisoquinolinols bearing a Phe-, Tyr-, or Val-based side chain were poor inhibitors. In contrast, the compounds derivatized with a Trp, Leu, Asp, or Glu residue inhibited SPM-1, with the tryptophan-functionalized chloroisoquinolinols (S)-7d and (R)-7d giving the most promising results (3% and 8% residual activity (RA) at 200 μM, respectively).Table 2. Residual Activities (RAs) on SPM-1 at 1 mM and 200 μMa,bTable aFor the synthesis of compounds 7a–7g see the Supporting Information.Table bThe remaining activity was measured by applying a 10 min preincubation time used in previous MBL work; see refs 18a and 44.Following from the positive screening results obtained with the SPM-1/FC4 pair, IC50 values were determined for (S)-7d and (R)-7d against our MBL panel using FC4 and the conditions reported in Table 1; the FC4 concentrations correspond to the experimentally determined KM values (Table 1). The IC50 values (Table 3) reveal that both (S)-7d and (R)-7d are relatively weak, but broad-spectrum MBL inhibitors with IC50 values ranging from 20 to 130 μM. Since the inhibitor activity of these compounds is in the low micromolar range, they may serve as good starting points for further optimization.Table 3. IC50 Values (μM) for a Panel of Metallo-β-lactamasesaMBL(S)-7d(R)-7dMBL(S)-7d(R)-7dSPM-123.2 ± 1.146.6 ± 1.1VIM-254.7 ± 1.455.3 ± 1.3IMP-175.6 ± 1.574.1 ± 1.6Bc II61.3 ± 1.3132.4 ± 1.3NDM-161.4 ± 1.347.1 ± 1.1 aIC50 determinations were performed in duplicate over a range of 0.25–200 μM.To further validate our assay platform, we measured the IC50 value of the reported inhibitor l-captopril(52) against NDM-1. Measuring at the KM value of FC4 on NDM-1, we obtained an IC50 value of 10.0 ± 1.9 μM corresponding to a Ki of 5.0 μM for l-captopril.(53) Using imipenem as the reported substrate, an IC50 of 13.2 ± 1.5 μM corresponding to a Ki of 6.6 μM were determined. Thus, under the same assay conditions, we found that the fluorogenic substrate (FC4) and the chromogenic substrate (imipenem) produced similar IC50 and Ki values. However, our values are lower than the previously reported IC50 and Ki values for l-captopril on NDM-1 using imipenem as the reported substrate (202 μM(54) and 39 μM,(52b) respectively), possibly reflecting differences in assay conditions, protein constructs, and enzyme production procedures.ConclusionsARTICLE SECTIONSJump ToThe development of broad-screen MBL inhibitors has been hampered by the lack of an appropriate, efficient screening platform for multiple MBLs. The development of such a screening platform has been hindered by the lack of suitable detection substrates. The results of our comparative study, employing five substrates and five MBLs, reveal striking differences in the kinetic parameters for different enzyme–substrate combinations. Fluorogenic substrates FC4 and FC5 are highly sensitive and efficient substrates for the clinically relevant MBLs, with one or both of them having kcat/KM values of 10 μM–1·s–1. The suitability of FC4 for inhibitor screening was demonstrated by testing a set of 4-chloroisoquinolinols as MBL inhibitors. This work yielded two compounds ((S)-7d and (R)-7d) that displayed inhibitory activity against our panel of MBLs, albeit at moderate inhibition concentrations (μM); nonetheless, the results reveal the feasibility of broad-spectrum MBL inhibition.Experimental SectionARTICLE SECTIONSJump ToThe Supporting Information contains a complete general experimental section, including all procedures and equipment used. In general, chemicals were purchased from commonly used suppliers (Aldrich, Acros, Alfa Aesar, and TCI) and were used without further purification. (6R,7R)-4-Methoxybenzyl 3-(chloromethyl)-8-oxo-7-(2-phenylacetamido)-5-thia-1-aza-bicyclo[4.2.0]oct-2-ene-2-carboxylate (PMB-protected Cep-Cl) was obtained from Activate Scientific (Prien, Germany). (6R,7R)-4-Methoxybenzyl 8-Oxo-3-(((2-oxo-2H-chromen-7-yl)oxy)methyl)-7-(2-phenylacetamido)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate (4)To a suspension of Cl-Cep-OPMB ester (680 mg, 1.4 mmol) in acetone (10 mL) was added NaI (2.10 g, 14.0 mmol, 10 equiv). The reaction was stirred at rt for 2 h, after which the solvent was removed in vacuo. The crude mixture was partitioned between H2O (10 mL) and EtOAc (10 mL), and the layers were separated. The H2O layer was extracted with EtOAc (2 × 15 mL), after which the combined organic layers were washed with a 5% NaS2O3 aq solution (2 × 20 mL) and brine (20 mL). The organic layer was dried over Na2SO4, filtered, and concentrated in vacuo. The crude product was dissolved in MeCN (15 mL), and 7-hydroxycoumarin (454 mg, 2.8 mmol, 2 equiv) and K2CO3 (4.2 mmol, 4 equiv) were added. The conversion of the reaction was monitored by TLC analysis (cHex/EtOAc, 1:1), and completion was reached after 4 h. The solvent was evaporated in vacuo, and the resulting crude mixture was partitioned between H2O (10 mL) and EtOAc (10 mL). After separation of the layers, the H2O layer was extracted with EtOAc (2 × 15 mL), after which the combined organic layers were washed with a 5% NaS2O3 aq solution (2 × 20 mL) and brine (20 mL). The organic layer was dried over Na2SO4, filtered, and concentrated in vacuo. The crude product was purified by column chromatography (cHex/EtOAc, 1:1) to yield compound 4 as a light brown solid (392 mg, 46%). Rf = 0.30 (cHex/EtOAc, 1:1). 1H NMR (400 MHz, DMSO-d6): δ = 9.20 (d, J = 8.0 Hz, 1H), 7.98 (d, J = 9.5 Hz, 1H), 7.59 (d, J = 8.5 Hz, 1H), 7.17 - 7.33 (m, 8H), 6.92 (d, J = 1.5 Hz, 2H), 6.87 (dd, J = 8.5, 2.5 Hz, 1H), 6.79 (d, J = 8.5 Hz, 2H), 6.30 (d, J = 9.5 Hz, 1H), 5.44 (dd, J = 7.5, 4.0 Hz, 1H), 5.11–5.19 (m, 2H), 5.01–5.10 (m, app d, 2H), 4.64–4.76 (m, app q, 2H), 3.69 (s, 3H), 3.47–3.57 (m, 2H) ppm. 13C NMR (101 MHz, DMSO-d6): δ = 170.9, 167.0, 163.8, 160.9, 160.3, 159.3, 155.2, 144.3, 135.7, 130.0 (2C), 129.5, 129.1 (2C), 128.2 (2C), 126.9, 126.5, 122.4, 118.8, 113.7 (2C), 112.9, 112.7 (2C), 101.4, 69.8, 67.2, 60.8, 55.0, 52.9, 49.7, 41.6 ppm. LRMS: mass calcd for C33H27N2O8S (M – H) 611.15, mass found (M – H) 611.0. (6R,7R)-4-Methoxybenzyl 8-Oxo-3-(((2-oxo-2H-chromen-7-yl)oxy)methyl)-7-(2-phenylacetamido)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate 5-Oxide (5)To a suspension of compound 4 (122 mg, 0.2 mmol) in dry CH2Cl2 (5 mL) cooled to 0 °C was added mCPBA (45 mg, 0.2 mmol, 1 equiv). The reaction was stirred at 0 °C for 30 min followed by an additional 1 h at rt (a white precipitate was formed). The crude product was dry-loaded onto silica and purified by column chromatography (CH2Cl2/EtOAc, 8:2) to yield compound 5 as a cream white solid (60 mg, 48%). Rf = 0.80 (CH2Cl2/MeOH, 9:1). 1H NMR (400 MHz CDCl3): δ = 7.64 (d, J = 9.5 Hz, 1H), 7.27–7.40 (m, 8H), 6.91 (d, J = 8.5 Hz, 2H), 6.74–6.78 (m, 2H), 6.67 (d, J = 10.0 Hz, 1H), 6.30 (d, J = 9.5 Hz, 1H), 6.10 (dd, J = 10.0, 5.0 Hz, 1H), 5.22–5.35 (m, app mix of d and q, 3H), 4.80 (d, J = 13.5 Hz, 1H), 4.45 (dd, J = 5.0, 1.5 Hz, 1H), 3.99 (d, J = 19.0 Hz, 1H), 3.81 (s, 3H) 3.65 (m, app d, J = 5.5 Hz, 2H), 3.28 (d, J = 19.0 Hz, 1H) ppm. 13C NMR (101 MHz, DMSO-d6): δ = 171.1, 164.5, 160.7 (2C), 160.2, 159.4, 155.2, 144.3, 135.8, 130.4 (2C), 129.5, 129.1 (2C), 128.3 (2C), 126.8, 126.6, 125.0, 119.6, 113.7 (2C), 112.8, 101.6, 67.5, 67.3, 66.5, 58.4, 55.1, 45.4, 41.4 ppm. LRMS: mass calcd for C33H27N2O9S (M – H) 627.14, mass found (M – H) 627.0. (6R,7R)-4-Methoxybenzyl 8-Oxo-3-(((2-oxo-2H-chromen-7-yl)oxy)methyl)-7-(2-phenylacetamido)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate 5,5-Dioxide (6)To a suspension of compound 4 (100 mg, 0.16 mmol) in dry CH2Cl2 (10 mL) cooled to 0 °C was added mCPBA (74 mg, 0.35 mmol, 2 equiv). The reaction was stirred at 0 °C for 30 min (formation of sulfoxide observed), after which the reaction was warmed to rt and stirred overnight. Upon completion of the reaction, the crude product was dry-loaded onto silica and purified by column chromatography (CH2Cl2/MeOH, 9:1) to yield the desired product as a white solid (35 mg, 34%). Rf = 0.85 (CH2Cl2/MeOH, 9:1). 1H NMR (400 MHz, DMSO-d6) (small amount of mCBA present): δ = 8.93 (d, J = 8.5 Hz, 1H), 8.01 (d, J = 9.5 Hz, 1H), 7.87–7.93 (m, 1H), 7.63 (d, J = 8.5 Hz, 1H), 7.18–7.34 (m, 7H), 6.94–6.88 (m, 2H, 2 signals overlapping), 6.81–6.86 (m, app d, 2H), 6.33 (d, J = 9.5 Hz, 1H), 6.01 (dd, J = 8.5, 5.0 Hz, 1H), 5.43 (d, J = 4.5 Hz, 1H), 5.22 (s, 2H), 4.89–4.84 (m, app d, 2H), 4.46 (d, J = 18.5 Hz, 1H) (part of AB system), 4.23 (d, J = 18.5 Hz, 1H) (part of AB system), 3.70 (s, 3H), 3.60 (d, J = 5.0 Hz, 2H) ppm. 13C NMR (101 MHz, DMSO-d6): δ = 170.9, 164.5, 160.6 (2C), 160.2, 159.4, 155.2, 144.3, 135.6, 130.4 (2C), 129.5, 129.2 (2C), 128.2 (2C), 126.6, 126.5, 124.6, 124.0, 113.7 (2C), 112.9, 112.8, 101.6, 67.8, 66.8, 66.2, 58.4, 55.1, 50.9, 41.2 ppm. LRMS: mass calcd for C33H27N2O10S (M – H) 643.14, mass found (M – H) 643.0. (6R,7R)-8-Oxo-3-(((2-oxo-2H-chromen-7-yl)oxy)methyl)-7-(2-phenylacetamido)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic Acid 5-Oxide (FC4)Compound 5 (50 mg, 81.6 μmol) was cooled to 0 °C prior to the addition of TFA/anisole (5:1, 3 mL). The resulting reaction mixture was stirred at 0 °C for 30 min with constant monitoring of the conversion by TLC analysis (CH2Cl2/EtOAC, 8:2). Upon completion of the reaction, cold Et2O (5 mL) was added, which resulted in the formation of a precipitate. The solid material was filtered off, washed with Et2O (2 × 5 mL), and subsequently dried under high vacuum to yield the desired product as an off-white solid (15 mg, 37%). Rf = 0.10 (CH2Cl2/MeOH +AcOH, 9:1). 1H NMR (400 MHz, DMSO-d6): δ = 8.45 (d, J = 8.5 Hz, 1H), 8.00 (d, J = 9.5 Hz, 1H), 7.65 (d, J = 8.5 Hz, 1H), 7.27–7.32 (m, 4H), 7.24 (td, J = 8.5, 4.0 Hz, 1H), 6.93–7.01 (m, 1H), 6.31 (d, J = 9.5 Hz, 1H), 5.81 (dd, J = 8.0, 4.5 Hz, 1H), 5.14 (d, J = 12.5 Hz, 1H), 4.87–4.93 (m, 1H), 3.98 (d, J = 18.0 Hz, 1H), 3.70 (d, J = 14.0 Hz, 1H) (part of AB system), 3.62 (d, J = 18.5 Hz, 1H), 3.54 (d, J = 14.0 Hz, 1H) (part of AB system) ppm. 13C NMR (101 MHz, DMSO-d6): δ = 171.1, 164.2, 162.2, 161.1, 155.3, 144.3, 135.8, 129.6, 129.1 (2C), 128.3 (2C), 126.6, 112.8 (2C), 101.6, 67.5, 66.3, 58.3, 45.3, 41.5 ppm. LRMS: mass calcd for C25H19N2O8S (M – H) 507.09, mass found (M – H) 507.0. Retention time: 7.15 min (purity 99%). (6R,7R)-8-Oxo-3-(((2-oxo-2H-chromen-7-yl)oxy)methyl)-7-(2-phenylacetamido)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic Acid 5,5-Dioxide (FC5)Compound 6 (20 mg, 0.03 mmol) was cooled to 0 °C prior to the addition of TFA/anisole (5:1, 1.2 mL). The reaction was stirred at 0 °C for 30 min followed by 30 min at rt with constant monitoring of the conversion by TLC analysis (CH2Cl2/MeOH, 9:1). Upon completion of the reaction, cold Et2O (5 mL) was added, which resulted in the formation of a precipitate. The solid material was filtered off, washed with Et2O (2 × 3 mL), and subsequently dried under high vacuum to yield the desired product as an off-white solid (10 mg, 63%). Rf = 0.10 (CH2Cl2/MeOH +AcOH, 9:1). 1H NMR (400 MHz, DMSO-d6): δ = 8.92 (d, J = 9.0 Hz, 1H), 8.01 (d, J = 9.5 Hz, 1H), 7.66 (d, J = 8.5 Hz, 1H), 7.19–7.31 (m, 5H) 7.04 (d, J = 2.5 Hz, 1H), 6.99 (dd, J = 8.5, 2.5 Hz, 1H), 6.32 (d, J = 9.5 Hz, 1H), 5.97 (dd, J = 8.5, 4.5 Hz, 1H), 5.42 (d, J = 4.5 Hz, 1H), 4.90–5.00 (m, app q, 2H), 4.41 (d, J = 18.0 Hz, 1H), 4.19 (d, J = 18.0 Hz, 1H), 3.54–3.65 (m, app q, 2H) ppm. 13C NMR (101 MHz, DMSO-d6): δ = 170.9, 164.3, 162.1, 160.8, 160.2, 155.2, 144.3, 135.6, 129.6, 129.2 (2C), 128.2 (2C), 126.5, 125.2, 123.8, 112.9, 112.9, 101.7, 66.8, 66.3, 58.3, 50.8, 41.2 ppm. LRMS: mass calcd for C25H19N2O8S (M – H) 507.09, mass found (M – H) 507.0. Retention time: 8.98 min (purity 95%).Supporting InformationARTICLE SECTIONSJump ToExperimental procedures, characterization of intermediates and target compounds, description of protein production and purification, biological assays, determination of residual activity measurements and IC50 values, and in silico docking data. This material is available free of charge via the Internet at http://pubs.acs.org.jm400769b_si_001.pdf (957.55 kb) Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html. Author InformationARTICLE SECTIONSJump ToCorresponding AuthorChristopher J. Schofield - Chemistry Research Laboratory, University of Oxford, 12 Mansfield Road, Oxford OX13TA, United Kingdom; Email: [email protected]AuthorsSander S. van Berkel - Chemistry Research Laboratory, University of Oxford, 12 Mansfield Road, Oxford OX13TA, United KingdomJürgen Brem - Chemistry Research Laboratory, University of Oxford, 12 Mansfield Road, Oxford OX13TA, United KingdomAnna M. Rydzik - Chemistry Research Laboratory, University of Oxford, 12 Mansfield Road, Oxford OX13TA, United KingdomRamya Salimraj - Schoolof Cellular and MolecularMedicine, University of Bristol, MedicalSciences Building, Bristol BS8 1TD, United KingdomRicky Cain - School of Chemistry, University of Leeds, Leeds LS2 9JT, United KingdomAnil Verma - Oxford Protein Production FacilityUK, The Research Complex at Harwell, Rutherford AppletonLaboratory, Harwell Science and Innovation Campus, OxfordshireOX11 0FA, United Kingdom; Division of Structural Biology, University of Oxford, Henry Wellcome Building for GenomicMedicine, Oxford OX3 7BN, United KingdomRaymond J. Owens - Oxford Protein Production FacilityUK, The Research Complex at Harwell, Rutherford AppletonLaboratory, Harwell Science and Innovation Campus, OxfordshireOX11 0FA, United Kingdom; Division of Structural Biology, University of Oxford, Henry Wellcome Building for GenomicMedicine, Oxford OX3 7BN, United KingdomColin W. G. Fishwick - School of Chemistry, University of Leeds, Leeds LS2 9JT, United KingdomJames Spencer - Schoolof Cellular and MolecularMedicine, University of Bristol, MedicalSciences Building, Bristol BS8 1TD, United KingdomAuthor ContributionsS.S.v.B. and J.B. contributed equally to this work.NotesThe authors declare no competing financial interests.AcknowledgmentARTICLE SECTIONSJump ToWe thank the Medical Research Council (MRC)/Canadian Grant G1100135 and Biotechnology and Biological Sciences Research Council (BBSRC) for support of J.B., R.S., R.C., and A.V. Cancer Research UK (CRUK) is kindly acknowledged for the support of S.S.v.B. The Oxford Protein Production Facility UK (OPPF-UK) is supported by the MRC and BBSRC. Abbreviations UsedMBLmetallo-β-lactamaseSBLserine-β-lactamaseFCfluorogenic cephalosporinDiPEAN,N′-diisopropylethylamineDMFdimethylformamide7-HC7-hydroxycoumarinMeCNacetonitrilemCPBAm-chloroperbenzoic acidMMC7-mercapto-4-methylcoumarinPMBp-methoxybenzylAcOHacetic acidTFAtrifluoroacetic acidReferencesARTICLE SECTIONSJump To This article references 54 other publications. 1Spellberg, B.; Guidos, R.; Gilbert, D.; Bradley, J.; Boucher, H. W.; Scheld, W. M.; Bartlett, J. G.; Edwards, J., Jr. The epidemic of antibiotic-resistant infections: A call to action for the medical community from the Infectious Diseases Society of America Clin. Infect. 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The Infectious Diseases Society of America (IDSA) followed in 2004 with its own report, \"Bad Bugs, No Drugs: As Antibiotic Discovery Stagnates, A Public Health Crisis Brews,\" which proposed incentives to reinvigorate pharmaceutical investment in antibiotic research and development. The IDSA\'s subsequent lobbying efforts led to the introduction of promising legislation in the 109 th US Congress (January 2005-December 2006). Unfortunately, the legislation was not enacted. During the 110 th Congress, the IDSA has continued to work with congressional leaders on promising legislation to address antibiotic-resistant infection. Nevertheless, despite intensive public relations and lobbying efforts, it remains unclear whether sufficiently robust legislation will be enacted. In the meantime, microbes continue to become more resistant, the antibiotic pipeline continues to diminish, and the majority of the public remains unaware of this critical situation. The result of insufficient federal funding; insufficient surveillance, prevention, and control; insufficient research and development activities; misguided regulation of antibiotics in agriculture and, in particular, for food animals; and insufficient overall coordination of US (and international) efforts could mean a literal return to the preantibiotic era for many types of infections. If we are to address the antimicrobial resistance crisis, a concerted, grassroots effort led by the medical community will be required. >> More from SciFinder https://chemport.cas.org/services/resolver?origin=ACS resolution=options coi=1%3ACAS%3A280%3ADC%252BD1c%252FgslGitw%253D%253D md5=52bb108e9c29dc9da3031f7127cba99f2World Health Organization. The evolving threat of antimicrobialresistance: Options for action, (2012. http://whqlibdoc.who.int/publications/2012/9789241503181_eng.pdf (accessed December 2012).Google ScholarThere is no corresponding record for this reference.3Bush, K.; Jacoby, G. A. 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Major groupings generally correlate with the more broadly based mol. classification. The updated system includes group 1 (class C) cephalosporinases; group 2 (classes A and D) broad-spectrum, inhibitor-resistant, and extended-spectrum β-lactamases and serine carbapenemases; and group 3 metallo-β-lactamases. Several new subgroups of each of the major groups are described, based on specific attributes of individual enzymes. A list of attributes is also suggested for the description of a new β-lactamase, including the requisite microbiol. properties, substrate and inhibitor profiles, and mol. sequence data that provide an adequate characterization for a new β-lactam-hydrolyzing enzyme. >> More from SciFinder https://chemport.cas.org/services/resolver?origin=ACS resolution=options coi=1%3ACAS%3A528%3ADC%252BC3cXjtFWqsr4%253D md5=89b376d90ff64b78f81bd43ed5925ea24White, A. R.; Kaye, C.; Poupard, J.; Pypstra, R.; Woodnutt, G.; Wynne, B. J. 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In order to overcome β-lactamase-mediated resistance, β-lactamase inhibitors (clavulanate, sulbactam, and tazobactam) were introduced into clin. practice. These inhibitors greatly enhance the efficacy of their partner β-lactams (amoxicillin, ampicillin, piperacillin, and ticarcillin) in the treatment of serious Enterobacteriaceae and penicillin-resistant staphylococcal infections. However, selective pressure from excess antibiotic use accelerated the emergence of resistance to β-lactam-β-lactamase inhibitor combinations. Furthermore, the prevalence of clin. relevant β-lactamases from other classes that are resistant to inhibition is rapidly increasing. There is an urgent need for effective inhibitors that can restore the activity of β-lactams. Here, we review the catalytic mechanisms of each β-lactamase class. We then discuss approaches for circumventing β-lactamase-mediated resistance, including properties and characteristics of mechanism-based inactivators. We next highlight the mechanisms of action and salient clin. and microbiol. features of β-lactamase inhibitors. We also emphasize their therapeutic applications. We close by focusing on novel compds. and the chem. features of these agents that may contribute to a \"second generation\" of inhibitors. The goal for the next 3 decades will be to design inhibitors that will be effective for more than a single class of β-lactamases. >> More from SciFinder https://chemport.cas.org/services/resolver?origin=ACS resolution=options coi=1%3ACAS%3A528%3ADC%252BC3cXks1Sktbs%253D md5=27cd9fca0d848185d9c61e5a25d2c6016Oelschlaeger, P.; Ai, N.; DuPrez, K. T.; Welsh, W. J.; Toney, J. H. Evolving carbapenemases: Can medicinal chemists advance one step ahead of the coming storm? J. Med. Chem. 2010, 53, 3013– 3027[ACS Full Text ], Google ScholarThere is no corresponding record for this reference.7Papp-Wallace, K. M.; Endimiani, A.; Taracila, M. A.; Bonomo, R. 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Summary: Metallo-β-lactamases are resistance determinants of increasing clin. relevance in Gram-neg. bacteria. Because of their broad range, potent carbapenemase activity and resistance to inhibitors, these enzymes can confer resistance to almost all β-lactams. Since the 1990s, several metallo-β-lactamases encoded by mobile DNA have emerged in important Gram-neg. pathogens (ie, in Enterobacteriaceae, Pseudomonas aeruginosa, and Acinetobacter baumannii). Some of these enzymes (eg, VIM-1 and NDM-1) have been involved in the recent crisis resulting from the international dissemination of carbapenem-resistant Klebsiella pneumoniae and other enterobacteria. Although substantial knowledge about the mol. biol. and genetics of metallo-β-lactamases is available, epidemiol. data are inconsistent and clin. experience is still lacking; therefore, several unsolved or debatable issues remain about the management of infections caused by producers of metallo-β-lactamase. The spread of metallo-β-lactamases presents a major challenge both for treatment of individual patients and for policies of infection control, exposing the substantial unpreparedness of public health structures in facing up to this emergency. >> More from SciFinder https://chemport.cas.org/services/resolver?origin=ACS resolution=options coi=1%3ACAS%3A528%3ADC%252BC3MXlsVGntL4%253D md5=a6097adade71d508091de28f14cc6a0a12(a) Chen, P.; Horton, L. B.; Mikulski, R. L.; Deng, L.; Sundriyal, S.; Palzkill, T.; Song, Y. 2-Substituted 4,5-dihydrothiazole-4-carboxylic acids are novel inhibitors of metallo-β-lactamases Bioorg. Med. Chem. Lett. 2012, 22, 6229– 6232Google ScholarThere is no corresponding record for this reference.(b) Toney, J. H.; Hammond, G. G.; Fitzgerald, P. M. D.; Sharma, N.; Balkovec, J. M.; Rouen, G. P.; Olson, S. H.; Hammond, M. L.; Greenlee, M. L.; Gao, Y. D. Succinic acids as potent inhibitors of plasmid-borne IMP-1 metallo-β-lactamase J. Biol. Chem. 2001, 276, 31913– 31918Google ScholarThere is no corresponding record for this reference.13(a) Toney, J. H.; Fitzgerald, P. M.; Grover-Sharma, N.; Olson, S. H.; May, W. J.; Sundelof, J. G.; Vanderwall, D. E.; Cleary, K. A.; Grant, S. K.; Wu, J. K.; Kozarich, J. W.; Pompliano, D. L.; Hammond, G. G. Antibiotic sensitization using biphenyl tetrazoles as potent inhibitors of Bacteroides fragilis metallo-β-lactamase Chem. Biol. 1998, 5, 185– 196[Crossref], [PubMed], [CAS], Google Scholar13aAntibiotic sensitization using biphenyl tetrazoles as potent inhibitors of Bacteroides fragilis metallo-β-lactamaseToney, Jeffrey H.; Fitzgerald, Paula M. D.; Grover-Sharma, Nandini; Olson, Steven H.; May, Walter J.; Sundelof, Jon G.; Vanderwall, Dana E.; Cleary, Kelly A.; Grant, Stephan K.; Wu, Joseph K.; Kozarich, John W.; Pompliano, David L.; Hammond, Gail G.Chemistry Biology (1998), (4), 185-196CODEN: CBOLE2; ISSN:1074-5521. (Current Biology Ltd.) High level resistance to carbapenem antibiotics in Gram-neg. bacteria such as Bacteroides fragilis is caused, in part, by expression of a wide-spectrum metallo-β-lactamase that hydrolyzes the drug to an inactive form. Co-administration of metallo-β-lactamase inhibitors to resistant bacteria is expected to restore the antibacterial activity of carbapenems. Biphenyl tetrazoles (BPTs) are a structural class of potent competitive inhibitors of metallo-β-lactamase identified through screening and predicted using mol. modeling of the enzyme structure. The X-ray crystal structure of the enzyme bound to the BPT L-159,061 shows that the tetrazole moiety of the inhibitor interacts directly with one of the two zinc atoms in the active site, replacing a metal-bound water mol. Inhibition of metallo-β-lactamase by BPTs in vitro correlates well with antibiotic sensitization of resistant B. fragilis. It is shown here that BPT inhibitors can sensitize a resistant B. fragilis clin. isolate expressing metallo-β-lactamase to the antibiotics imipenem or penicillin G but not to rifampicin. >> More from SciFinder https://chemport.cas.org/services/resolver?origin=ACS resolution=options coi=1%3ACAS%3A528%3ADyaK1cXjtVynu7g%253D md5=32f20851a6d66a200be162350b67eccc(b) Weide, T.; Saldanha, S. A.; Minond, D.; Spicer, T. P.; Fotsing, J. R.; Spaargaren, M.; Frère, J.-M.; Bebrone, C.; Sharpless, K. B.; Hodder, P. S.; Fokin, V. V. NH-1,2,3-Triazole-based inhibitors of the VIM-2 metallo-β-lactamase: Synthesis and structure–activity studies ACS Med. Chem. Lett. 2010, 1, 150– 154[ACS Full Text ], Google ScholarThere is no corresponding record for this reference.14(a) Liénard, B. M. R.; Garau, G.; Horsfall, L.; Karsisiotis, A. I.; Damblon, C.; Lassaux, P.; Papamicael, C.; Roberts, G. C. K.; Galleni, M.; Dideberg, O.; Frère, J.-M.; Schofield, C. J. Structural basis for the broad-spectrum inhibition of metallo-β-lactamases by thiols Org. Biomol. Chem. 2008, 6, 2282– 2294Google ScholarThere is no corresponding record for this reference.(b) Liénard, B. M., R.; Hüting, R.; Lassaux, P.; Galleni, M.; Frère, J.-M.; Schofield, C. J. Dynamic combinatorial mass spectrometry leads to metallo-β-lactamase inhibitors J. Med. Chem. 2008, 51, 684– 688[ACS Full Text ], Google ScholarThere is no corresponding record for this reference.15Walter, M. W.; Felici, A.; Galleni, M.; Paul Soto, R.; Adlington, R. M.; Baldwin, J. E.; Frère, J.-M.; Gololobov, M.; Schofield, C. J. Trifluoromethyl alcohol and ketone inhibitors of metallo-β-lactamases Bioorg. Med. Chem. Lett. 1996, 6, 2455– 2458Google ScholarThere is no corresponding record for this reference.16(a) Fast, W.; Sutton, L. D. Metallo-β-lactamase: Inhibitors and reporter substrates Biochim. Biophys. Acta, Proteins Proteomics 2013, 1834, 1648– 1659[Crossref], [PubMed], [CAS], Google Scholar16aMetallo-β-lactamase: Inhibitors and reporter substratesFast, Walter; Sutton, Larry D.Biochimica et Biophysica Acta, Proteins and Proteomics (2013), 1834 (8), 1648-1659CODEN: BBAPBW; ISSN:1570-9639. (Elsevier B. V.) A review. Metallo-β-lactamases represent an emerging clin. threat due to their ability to render ineffective an entire class of antibiotics. Accordingly, this family of enzymes has been suggested as an attractive target for drug design. Progress toward developing effective inhibitors as well as the development of reporter substrates is reviewed. Inhibitors are classified into six classes and known binding interactions with metallo-β-lactamases are summarized. The development of chromogenic and fluorogenic reporter substrates is also reviewed with respect to current and prospective applications to future inhibitor and diagnostic discovery, mechanistic studies, and biol. imaging. Despite progress in mol. probe development, the sequence and structural diversity within the metallo-β-lactamase family continue to present substantial hurdles for rational ligand design. >> More from SciFinder https://chemport.cas.org/services/resolver?origin=ACS resolution=options coi=1%3ACAS%3A528%3ADC%252BC3sXhtVehu73J md5=234faf23a238484f8b612a3bd3f28b8f(b) Toney, J. H.; Moloughney, J. G. Metallo-β-lactamase inhibitors: Promise for the future? Curr. Opin. Invest. Drugs 2004, 5, 823– 826Google ScholarThere is no corresponding record for this reference.(c) Spencer, J.; Walsh, T. R.; New, A. Approach to the inhibition of metallo-β-lactamases Angew. Chem., Int. Ed. 2006, 45, 1022– 1026Google ScholarThere is no corresponding record for this reference.17(a) Moali, C.; Anne, C.; Lamotte-Brasseur, J.; Groslambert, S.; Devreese, B.; Van Beeumen, J.; Galleni, M.; Frère, J.-M. Analysis of the importance of the metallo-β-lactamase active site loop in substrate binding and catalysis Chem. Biol. 2003, 10, 319– 329[Crossref], [PubMed], [CAS], Google Scholar17aAnalysis of the importance of the metallo-β-lactamase active site loop in substrate binding and catalysisMoali, Catherine; Anne, Christine; Lamotte-Brasseur, Josette; Groslambert, Sylvie; Devreese, Bart; Van Beeumen, Jozef; Galleni, Moreno; Frere, Jean-MarieChemistry Biology (2003), (4), 319-329CODEN: CBOLE2; ISSN:1074-5521. (Cell Press) The role of the mobile loop comprising residues 60-66 in metallo-β-lactamases was studied by site-directed mutagenesis, detn. of kinetic parameters for 6 substrates and 2 inhibitors, pre-steady-state characterization of the interaction with chromogenic nitrocefin, and mol. modeling. The W64A mutation was performed in β-lactamases IMP-1 and BcII (after replacement of the BcII 60-66 peptide by that of IMP-1) and always resulted in increased Ki and Km values and decreased kcat/Km values, an effect reinforced by complete deletion of the loop. The kcat values were, by contrast, much more diversely affected, indicating that the loop did not systematically favor the best relative positioning of substrate and enzyme catalytic groups. The hydrophobic nature of the ligand was also crucial to strong interactions with the loop, since imipenem was almost insensitive to loop modifications. >> More from SciFinder https://chemport.cas.org/services/resolver?origin=ACS resolution=options coi=1%3ACAS%3A528%3ADC%252BD3sXjt1Kntrg%253D md5=b9ddf52e077b003935447c72f82a319a(b) Saradhi Borra, P.; Samuelsen, Ø.; Spencer, J.; Walsh, T. R.; Sjo Lorentzen, M.; Leirose, H.-K. S. Crystal structures of Pseudomonas aeruginosa GIM-1: Active-site plasticity in metallo-β-lactamases Antimicrob. Agents Chemother. 2013, 57, 848– 854Google ScholarThere is no corresponding record for this reference.18(a) Viswanatha, T.; Marrone, L.; Goodfellow, V.; Dmitrienko, G. I. Assays for β-lactamase activity and inhibition Methods Mol. Med. 2008, 142, 239– 260[Crossref], [PubMed], [CAS], Google Scholar18aAssays for β-lactamase activity and inhibitionViswanatha, Thammaiah; Marrone, Laura; Goodfellow, Valerie; Dmitrienko, Gary I.Methods in Molecular Medicine (2008), (New Antibiotic Targets), 239-260CODEN: MMMEFN ISSN:. (Humana Press Inc.) A review. The ability, either innate or acquired, to produce β-lactamases, enzymes capable of hydrolyzing the endocyclic peptide bond in β-lactam antibiotics, would appear to be a primary contributor to the ever-increasing incidences of resistance to this class of antibiotics. To date, four distinct classes, A, B, C, and D, of β-lactamases have been identified. Of these, enzymes in classes A, C, and D utilize a serine residue as a nucleophile in their catalytic mechanism while class B members are Zn2+-dependent for their function. Efforts have been and still continue to be made toward the development of potent inhibitors of these enzymes as a means to ensure the efficacy of β-lactam antibiotics in clin. medicine. This chapter concerns procedures for the evaluation of the catalytic activity of β-lactamases as a means to screen compds. for their inhibitory potency. >> More from SciFinder https://chemport.cas.org/services/resolver?origin=ACS resolution=options coi=1%3ACAS%3A528%3ADC%252BD1cXhvVOhtLY%253D md5=123a5b54e474a5a25cc6d703be9ba3ba(b) Kocaoglu, O.; Calvo, R. A.; Sham, L.-T.; Cozy, L. M.; Lanning, B. R.; Francis, S.; Winkler, M. E.; Kearns, D. B.; Carlson, E. E. Selective penicillin-binding protein imaging probes reveal substructure in bacterial cell division ACS Chem. Biol. 2012, 7, 1746– 1753[ACS Full Text ], [CAS], Google Scholar18bSelective penicillin-binding protein imaging probes reveal substructure in bacterial cell divisionKocaoglu, Ozden; Calvo, Rebecca A.; Sham, Lok-To; Cozy, Loralyn M.; Lanning, Bryan R.; Francis, Samson; Winkler, Malcolm E.; Kearns, Daniel B.; Carlson, Erin E.ACS Chemical Biology (2012), (10), 1746-1753CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society) The peptidoglycan cell wall is a common target for antibiotic therapy, but its structure and assembly are only partially understood. Peptidoglycan synthesis requires a suite of penicillin-binding proteins (PBPs), the individual roles of which are difficult to det. because each enzyme is often dispensable for growth perhaps due to functional redundancy. To address this challenge, the authors sought to generate tools that would enable selective examn. of a subset of PBPs. They designed and synthesized fluorescent and biotin derivs. of the β-lactam-contg. antibiotic cephalosporin C. These probes facilitated specific in vivo labeling of active PBPs in both Bacillus subtilis PY79 and an unencapsulated deriv. of D39 Streptococcus pneumoniae. Microscopy and gel-based anal. indicated that the cephalosporin C-based probes are more selective than BOCILLIN-FL, a com. available penicillin V analog, which labels all PBPs. Dual labeling of live cells performed by satn. of cephalosporin C-susceptible PBPs followed by tagging of the remaining PBP population with BOCILLIN-FL demonstrated that the two sets of PBPs are not co-localized. This suggests that even PBPs that are located at a particular site (e.g., septum) are not all intermixed, but rather that PBP subpopulations are discretely localized. Accordingly, the Ceph C probes represent new tools to explore a subset of PBPs and have the potential to facilitate a deeper understand of the roles of this crit. class of proteins. >> More from SciFinder https://chemport.cas.org/services/resolver?origin=ACS resolution=options coi=1%3ACAS%3A528%3ADC%252BC38Xht1eks7fP md5=5fbfeaaff9a0363111fe4f1689f30f9c(c) Zheng, X.; Sallium, U. W.; Verma, S.; Athar, H.; Evans, C. L.; Hasan, T. Exploiting a bacterial drug-resistance mechanism: A light-activated construct for the destruction of MRSA Angew. Chem., Int. Ed. 2009, 48, 2148– 2151[Crossref], [CAS], Google Scholar18cExploiting a bacterial drug-resistance mechanism: a light-activated construct for the destruction of MRSAZheng, Xiang; Sallum, Ulysses W.; Verma, Sarika; Athar, Humra; Evans, Conor L.; Hasan, TayyabaAngewandte Chemie, International Edition (2009), (12), 2148-2151CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH Co. KGaA) An enzyme-specific mol. construct exploits the overexpression of β-lactamase in several drug-resistant bacteria. Ed Specific photodynamic toxicity was detected towards β-lactam-resistant methicillin-resistant Staphylococcus aureus (MRSA), whereby the usual mechanism for antibiotic resistance (cleavage of the β-lactam ring) releases the phototoxic component from the prodrug. >> More from SciFinder https://chemport.cas.org/services/resolver?origin=ACS resolution=options coi=1%3ACAS%3A528%3ADC%252BD1MXktFegtr4%253D md5=aa3ee1067024a7174bae31b1b240fcde19Jones, R. N.; Wilson, H. W.; Novick, W. J., Jr.; Barry, A. L.; Thornberry, C. In vitro evaluation of CENTA, a new β-lactamase-susceptible chromogenic cephalosporin reagent J. Clin. Microbiol. 1982, 15, 954– 958[PubMed], [CAS], Google Scholar19In vitro evaluation of CENTA, a new beta-lactamase-susceptible chromogenic cephalosporin reagentJones, Ronald N.; Wilson, Harold W.; Novick, William J., Jr.; Barry, Arthur L.; Thornsberry, ClydeJournal of Clinical Microbiology (1982), (5), 954-8CODEN: JCMIDW; ISSN:0095-1137. CENTA is a newly synthesized, β-lactamase-labile, chromogenic cephalosporin reagent which changes color from light yellow (λ max. ∼340 nm) to chrome yellow (λ max. ∼405 nm) concomitant with hydrolysis of the β-lactam ring. This compd. offers promise as a diagnostic reagent comparable to other chromogens (PADAC and nitrocefin) for the early detection of β-lactamase-producing clin. isolates, while retaining some antimicrobial effect against Escherichia coli, Klebsiella, Proteus mirabilis, Staphylococcus aureus, and nonenterococcal Streptococcus. CENTA is relatively unaffected by commonly used microbiol. media and human serum. >> More from SciFinder https://chemport.cas.org/services/resolver?origin=ACS resolution=options coi=1%3ACAS%3A528%3ADyaL38XktVentrs%253D md5=4d2203ff80d47cab37a5e11ba775ce1120Jones, R. N.; Wilson, H. W.; Novick, Jr. In vitro evaluation of pyridine-2-azo-p-dimethylaniline cephalosporin, a new diagnostic chromogenic reagent, and comparison with nitrocefin, cephacetrile, and other β-lactam compounds J. Clin. Microbiol. 1982, 15, 677– 683[PubMed], [CAS], Google Scholar20In vitro evaluation of pyridine-2-azo-p-dimethylaniline cephalosporin, a new diagnostic chromogenic reagent, and comparison with nitrocefin, cephacetrile, and other beta-lactam compoundsJones, Ronald N.; Wilson, Harold W.; Novick, William J., Jr.Journal of Clinical Microbiology (1982), (4), 677-83CODEN: JCMIDW; ISSN:0095-1137. Pyridine-2-azo-p-dimethylanaline cephalosporin (PADAC), a chromogenic reagent which is purple and changes to yellow upon cleavage of its β-lactam ring, was evaluated in comparison with other chromogenic cephalosporins. PADAC exhibited little antimicrobial activity against gram-neg. bacteria, but it did have good activity (min. inhibitory concn., 0.12-0.5 μg/mL) against Staphylococcus aureus, a quality comparable to nitrocefin. Nitrocefin, however, demonstrated an unexpected and uniquely potent activity against Streptococcus faecalis (min. inhibitory concn., ≤0.06-0.12 μg/mL). The relative hydrolysis rate of PADAC when subjected to 6 different β-lactamases was substantially greater than that of cephacetrile but less than that of nitrocefin. The relative hydrolysis rates of PADAC and nitrocefin were comparable with type IIIa β-lactamase and that derived from Bacillus cereus. The inhibition of β-lactamase hydrolysis of the chromogenic cephalosporin substrates by 6 enzyme-stable inhibitors was generally greater with PADAC than with nitrocefin. Unlike nitrocefin, PADAC mixed with 50% human serum or various broth culture media showed no evidence of color change or degrdn. over several hours. The subsequent enzyme hydrolysis rates of such mixts. were the same as in phosphate buffer. β-Lactamase-contg. bacterial suspensions and clin. specimens contg. such bacteria produced pos. visual and spectrophotometric color changes when mixed with PADAC or nitrocefin. Although color changes occurred more slowly with PADAC than with nitrocefin, PADAC was not adversely influenced (nonenzyme-related color change) by the protein content of specimens. PADAC may be a promising alternative for β-lactamase diagnostic testing in the clin. and research microbiol. lab. >> More from SciFinder https://chemport.cas.org/services/resolver?origin=ACS resolution=options coi=1%3ACAS%3A528%3ADyaL38XitFCitLo%253D md5=d0c7f43594b9dc0cec5755406314424821Shannon, K.; Phillips, I. β-Lactamase detection by three simple methods: Intralactam, nitrocefin and acidimetric J. Antimicrob. Chemother. 1980, 6, 617– 621[Crossref], [PubMed], [CAS], Google Scholar21β-Lactamase detection by three simple methods: Intralactam, nitrocefin and acidimetricShannon, Kevin; Phillips, IanJournal of Antimicrobial Chemotherapy (1980), (5), 617-21CODEN: JACHDX; ISSN:0305-7453. Intralactam, an acidimetric paper-strip test for the detection of β-lactamase (I) [9073-60-3], was compared with the nitrocefin test and a tube-acidimetric method. Min. inhibitory concns. (MICs) of appropriate antibiotics were detd. for the organisms studied. All 3 methods detected the I-producing isolates of Neisseria gonorrhoeae and Haemophilus influenzae. Intralactam and nitrocefin detected I in highly carbenicillin (II) [4697-36-3]-resistant isolates of Pseudomonas aeruginosa (MICs 4096 mg/L) as did the tube acidimetric method when cells were subjected to ultrasonic disintegration. Other isolates of P. aeruginosa (II MICs, 32-512 mg/L) were neg. by all methods. The tests also detected benzylpenicillin [61-33-6]-resistant isolates of Staphylococcus aureus. When Enterobacteriaceae were tested, there were many discrepancies among the methods. The nitrocefin test, performed on disintegrated cell suspensions, was the most sensitive method. Intralactam was more sensitive than the tube acidimetric method. None of the methods reliably predicted sensitivity of Enterobacteriaceae to ampicillin [69-53-4] or cephaloridine [50-59-9], as assessed by MICs. >> More from SciFinder https://chemport.cas.org/services/resolver?origin=ACS resolution=options coi=1%3ACAS%3A528%3ADyaL3MXmsFE%253D md5=43b451e6f3f29ed249a97075c7af95c322(a) Gao, W.; Xing, B.; Tsien, R. Y.; Rao, J. Novel fluorogenic substrates for imaging β-lactamase gene expression J. Am. Chem. Soc. 2003, 125, 11146– 11147[ACS Full Text ], [CAS], Google Scholar22aNovel Fluorogenic Substrates for Imaging β-Lactamase Gene ExpressionGao, Wenzhong; Xing, Bengang; Tsien, Roger Y.; Rao, JianghongJournal of the American Chemical Society (2003), (37), 11146-11147CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society) A new class of small nonfluorescent fluorogenic substrates, based on release of a phenolic dye from a vinylogous cephalosporin, becomes brightly fluorescent after β-lactamase hydrolysis with up to 153-fold enhancement in the fluorescence intensity. Less than 500 fM of β-lactamase in cell lysates can be readily detected, and β-lactamase expression in living cells can be imaged with a red fluorescence deriv. These new fluorogenic substrates should find uses in clin. diagnostics and facilitate the applications of β-lactamase as a biosensor. >> More from SciFinder https://chemport.cas.org/services/resolver?origin=ACS resolution=options coi=1%3ACAS%3A528%3ADC%252BD3sXmsVelsrk%253D md5=3ff7d10e2798d1adb0174414da8cb98d(b) Rukavishnikov, A.; Gee, K. R.; Johnson, I.; Corry, S. Fluorogenic cephalosporin substrates for β-lactamase TEM-1 Anal. Biochem. 2011, 419, 9– 16Google ScholarThere is no corresponding record for this reference.(c) Zhang, Y.-L.; Xiao, J.-M.; Feng, J.-L.; Yang, K.-W.; Feng, L.; Zhou, L.-S.; Crowder, M. W. A novel fluorogenic substrate for dinuclear Zn(II)-containing metallo-β-lactamases Bioorg. Med. Chem. Lett. 2013, 23, 1676– 1679Google ScholarThere is no corresponding record for this reference.23Yao, H.; So, M.-K.; Rao, J.; Bioluminogenic, A. Substrate for in vivo imaging of β-lactamase activity Angew. Chem., Int. Ed. 2007, 46, 7031– 7034Google ScholarThere is no corresponding record for this reference.24(a) Watanabe, S.; Mizukami, S.; Hori, Y.; Kikuchi, K. Multicolor protein labeling in living cells using mutant β-lactamase-tag technology Bioconjugate Chem. 2010, 21, 2320– 2326[ACS Full Text ], [CAS], Google Scholar24aMulticolor Protein Labeling in Living Cells Using Mutant β-Lactamase-Tag TechnologyWatanabe, Shuji; Mizukami, Shin; Hori, Yuichiro; Kikuchi, KazuyaBioconjugate Chemistry (2010), (12), 2320-2326CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society) Protein labeling techniques using small mol. probes have become important as practical alternatives to the use of fluorescent proteins (FPs) in live cell imaging. These labeling techniques can be applied to more sophisticated fluorescence imaging studies such as pulse-chase imaging. Previously, the authors reported a novel protein labeling system based on the combination of a mutant β-lactamase (BL-tag) with coumarin-derivatized probes and its application to specific protein labeling on cell membranes. In this paper, the authors demonstrated the broad applicability of the authors\' BL-tag technol. to live cell imaging by the development of a series of fluorescence labeling probes for this technol., and the examn. of the functions of target proteins. These new probes have a fluorescein or rhodamine chromophore, each of which provides enhanced photophys. properties relative to coumarins for the purpose of cellular imaging. These probes were used to specifically label the BL-tag protein and could be used with other small mol. fluorescent probes. Simultaneous labeling using the authors\' new probes with another protein labeling technol. was effective. In addn., it was also confirmed that this technol. has a low interference with respect to the functions of target proteins in comparison to GFP. Highly specific and fast covalent labeling properties of this labeling technol. is expected to provide robust tools for investigating protein functions in living cells, and future applications can be improved by combining the BL-tag technol. with conventional imaging techniques. The combination of probe synthesis and mol. biol. techniques provides the advantages of both techniques and can enable the design of expts. that cannot currently be performed using existing tools. >> More from SciFinder https://chemport.cas.org/services/resolver?origin=ACS resolution=options coi=1%3ACAS%3A528%3ADC%252BC3cXhtlWhsr%252FP md5=3a0506ea01083309ef47f6989a2a25b0(b) Mizukami, S.; Watanabe, S.; Akimoto, Y.; Kikuchi, K. No-wash protein labeling with designed fluorogenic probes and application to real-time pulse-chase analysis J. Am. Chem. Soc. 2012, 134, 1623– 1629[ACS Full Text ], [CAS], Google Scholar24bNo-Wash Protein Labeling with Designed Fluorogenic Probes and Application to Real-Time Pulse-Chase AnalysisMizukami, Shin; Watanabe, Shuji; Akimoto, Yuri; Kikuchi, KazuyaJournal of the American Chemical Society (2012), (3), 1623-1629CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society) Small mol. labeling techniques for cellular proteins under physiol. conditions are very promising for revealing new biol. functions. The authors developed a no-wash fluorogenic labeling system by exploiting fluorescence resonance energy transfer (FRET)-based fluorescein-cephalosporin-azopyridinium probes and a mutant β-lactamase tag. Fast quencher elimination, hydrophilicity, and high resistance against autodegrdn. were achieved by rational refinement of the structure. By applying the probe to real-time pulse-chase anal., the trafficking of epidermal growth factor receptors between cell surface and intracellular region was imaged. In addn., membrane-permeable derivatization of the probe enabled no-wash fluorogenic labeling of intracellular proteins. >> More from SciFinder https://chemport.cas.org/services/resolver?origin=ACS resolution=options coi=1%3ACAS%3A528%3ADC%252BC38XktVGntw%253D%253D md5=da0f68ecab3c42879250946fefd12211(c) Shao, Q.; Xing, B. Enzyme responsive luminescent ruthenium(II) cephalosporin probe for intracellular imaging and photoinactivation of antibiotics resistant bacteria Chem. Commun. 2012, 48, 1739– 1741Google ScholarThere is no corresponding record for this reference.25References for plasmid production:(a) Griffin, D. H.; Richmond, T. K.; Sanchez, C.; Jon Moller, A.; Breece, R. M.; Tierney, D. L.; Bennett, B.; Crowder, M. W. Structural and kinetic studies on metallo-β-lactamase IMP-1 Biochemistry 2011, 50, 9125– 9134(IMP-1)[ACS Full Text ], Google ScholarThere is no corresponding record for this reference.(b) de Seny, D.; Prosperi-Meys, C.; Bebrone, C.; Maria Rossolini, G.; Page, M. I.; Noel, P.; Frère, J.-M.; Galleni, M. Mutational analysis of the two zinc-binding sites of the Bacillus cereus 569/H/9 metallo-β-lactamase Biochem. J. 2002, 363, 687– 696(Bc II)Google ScholarThere is no corresponding record for this reference.(c) Green, V. L.; Verma, A.; Owens, R. J.; Phillipsa, S. E. V.; Carr, S. B. Structure of New Delhi metallo-β-lactamase 1 (NDM-1) Acta Crystallogr. 2011, F67, 1160– 1164(NDM-1)Google ScholarThere is no corresponding record for this reference.26Berrow, N. S.; Alderton, D.; Sainsbury, S.; Nettleship, J.; Assenberg, R.; Rahman, N.; Stuart, D. I.; Owens, R. J. A versatile ligation-independent cloning method suitable for high-throughput expression screening applications Nucleic Acids Res. 2007, 35, e45Google ScholarThere is no corresponding record for this reference.27Bebrone, C.; Moali, C.; Mahy, F.; Rival, S.; Docquier, J.-D.; Maria Rossolini, G.; Fastrez, J.; Pratt, R. F.; Frère, J.-M.; Galleni, M. CENTA as a chromogenic substrate for studying β-lactamases Antimicrob. Agents Chemother. 2001, 45, 1868– 1871Google ScholarThere is no corresponding record for this reference.28For experimental details see the Supporting Information.There is no corresponding record for this reference.29Typically mCPBA oxidation to give the (S)-sulfoxide; see:Kaiser, G. V.; Cooper, R . D. G.; Koehler, R. E.; Murphy, C. F.; Webber, J. A.; Wright, I. G.; van Heyningen, E. M. Transformation of Δ2-cephem to Δ3-cephem by oxidation-reduction at sulfur J. Org. Chem. 1970, 35, 2430– 2433[ACS Full Text ], Google ScholarThere is no corresponding record for this reference.30Goddard, J.-P.; Reymond, J.-L. Enzyme assays for high-throughput screening Curr. Opin. Biotechnol. 2004, 15, 314– 322[Crossref], [PubMed], [CAS], Google Scholar30Enzyme assays for high-throughput screeningGoddard, Jean-Philippe; Reymond, Jean-LouisCurrent Opinion in Biotechnology (2004), (4), 314-322CODEN: CUOBE3; ISSN:0958-1669. (Elsevier Ltd.) A review. Assaying enzyme-catalyzed transformations in high-throughput is crucial to enzyme discovery, enzyme engineering and the drug discovery process. In enzyme assays, catalytic activity is detected using labeled substrates or indirect sensor systems that produce a detectable spectroscopic signal upon reaction. Recent advances in the development of high-throughput enzyme assays have identified new labels and chromophores to detect a wide range of enzymes activities. Enzyme activity profiling and fingerprinting have also been used as tools for identification and classification, while microarray formats have been devised to increase throughput. >> More from SciFinder https://chemport.cas.org/services/resolver?origin=ACS resolution=options coi=1%3ACAS%3A528%3ADC%252BD2cXmsVKms7c%253D md5=5f1248d6031258e0f91a8c4d3f3bee8d31Xie, H.; Mire, J.; Kong, Y.; Chang, M.; Hassounah, H. A.; Thornton, C. N.; Sacchettini, J. C.; Cirillo, J. D.; Rao, J. Rapid point-of-care detection of the tuberculosis pathogen using a BlaC-specific fluorogenic probe Nat. Chem. 2012, 4, 802– 809[Crossref], [PubMed], [CAS], Google Scholar31Rapid point-of-care detection of the tuberculosis pathogen using a BlaC-specific fluorogenic probeXie, Hexin; Mire, Joseph; Kong, Ying; Chang, Mi Hee; Hassounah, Hany A.; Thornton, Chris N.; Sacchettini, James C.; Cirillo, Jeffrey D.; Rao, JianghongNature Chemistry (2012), (10), 802-809CODEN: NCAHBB; ISSN:1755-4330. (Nature Publishing Group) Early diagnosis of tuberculosis can dramatically reduce both its transmission and the assocd. death rate. The extremely slow growth rate of the causative pathogen, Mycobacterium tuberculosis (Mtb), however, makes this challenging at the point of care, particularly in resource-limited settings. Here the authors report the use of BlaC (an enzyme naturally expressed/secreted by tubercle bacilli) as a marker and the design of BlaC-specific fluorogenic substrates as probes for Mtb detection. These probes showed an enhancement by 100-200 times in fluorescence emission on BlaC activation and a 1000-fold selectivity for BlaC over TEM-1 β-lactamase, an important factor in reducing false-pos. diagnoses. Insight into the BlaC specificity was revealed by successful co-crystn. of the probe/enzyme mutant complex. A refined green fluorescent probe (CDG-OMe) enabled the successful detection of live pathogen in less than ten minutes, even in unprocessed human sputum. This system offers the opportunity for the rapid, accurate detection of very low nos. of Mtb for the clin. diagnosis of tuberculosis in sputum and other specimens. >> More from SciFinder https://chemport.cas.org/services/resolver?origin=ACS resolution=options coi=1%3ACAS%3A528%3ADC%252BC38Xht12jtrnP md5=1ad9514e98464cc6b7940f0fda45a3bb32The optimal absorption wavelength for umbelliferone in HEPES buffer was found to be 330 nm with a second absorption band at 380 nm. Fluorogenic substrates FC3–FC5 did not show this second absorption band at 380 nm, allowing the specific excitation of umbelliferone at this wavelength.There is no corresponding record for this reference.33Kim, Y.; Tesar, C.; Mire, J.; Jedrzejczak, R.; Binkowski, A.; Babnigg, G.; Sacchettini, J.; Joachimiak, A. Structure of apo- and monometalated forms of NDM-1—A highly potent carbapenem-hydrolyzing metallo-β-lactamase PLoS One 2011, 6, e24621Google ScholarThere is no corresponding record for this reference.34Docquier, J.-D.; Lamotte-Brasseur, J.; Galleni, M.; Amicosante, G.; Frère, J.-M.; Maria Rossolini, G. On functional and structural heterogeneity of VIM-type metallo-β-lactamases J. Antimicrob. Chemother. 2003, 51, 257– 266[Crossref], [PubMed], [CAS], Google Scholar34On functional and structural heterogeneity of VIM-type metallo-β-lactamasesDocquier, Jean-Denis; Lamotte-Brasseur, Josette; Galleni, Moreno; Amicosante, Gianfranco; Frere, Jean-Marie; Rossolini, Gian MariaJournal of Antimicrobial Chemotherapy (2003), (2), 257-266CODEN: JACHDX; ISSN:0305-7453. (Oxford University Press) The VIM metallo-β-lactamases are emerging resistance determinants, encoded by mobile genetic elements, that have recently been detected in multidrug-resistant nosocomial isolates of Pseudomonas aeruginosa and other Gram-neg. pathogens. In this work a T7-based expression system for overprodn. of the VIM-2 enzyme by Escherichia coli was developed, which yielded ∼80 mg of protein per L of culture. The enzyme was mostly released into the medium, from which it was recovered at 99% purity by an initial ammonium sulfate pptn. followed by two chromatog. steps, with almost 80% efficiency. Detn. of kinetic parameters of VIM-2 under the same exptl. conditions previously used for VIM-1 (the first VIM-type enzyme detected in clin. isolates, which is 93% identical to VIM-2) revealed significant differences in Km values and/or turnover rates with several substrates, including penicillins, cephalosporins and carbapenems. Compared with VIM-1, VIM-2 is more susceptible to inactivation by chelators, indicating that the zinc ions of the latter are probably more loosely bound. These data indicated that at least some of the amino acid differences between the two proteins have functional significance. Mol. modeling of the two enzymes identified some amino acid substitutions, including those at positions 223, 224 and 228 (in the BBL numbering), that could be relevant to the changes in catalytic behavior. >> More from SciFinder https://chemport.cas.org/services/resolver?origin=ACS resolution=options coi=1%3ACAS%3A528%3ADC%252BD3sXovFehtA%253D%253D md5=136c3dfaac6f129c5006d9a9c344733735Laraki, N.; Franceschini, N.; Rossolini, G. M.; Santucci, P.; Meunier, C.; de Pauw, E.; Amicosante, G.; Frère, J.-M.; Galleni, M. Biochemical characterization of the Pseudomonas aeruginosa 101/1477 metallo-β-lactamase IMP-1 produced by Escherichia coli Antimicrob. Agents Chemother. 1999, 43, 902– 906Google ScholarThere is no corresponding record for this reference.36Murphy, T. A.; Simm, A. M.; Toleman, M. A.; Jones, R. N.; Walsh, T. R. Biochemical characterization of the acquired metallo-β-lactamase SPM-1 from Pseudomonas aeruginosa Antimicrob. Agents Chemother. 2003, 47, 582– 587Google ScholarThere is no corresponding record for this reference.37Paul-Soto, R.; Hernadez-Valladares, M.; Fonzé, E.; Goussard, S.; Courvalin, P.; Frère, J.-M. Mono- and binuclear Zn-β-lactamase from Bacteroides fragilis: Catalytic and structural roles of the zinc ions FEBS Lett. 1998, 438, 137– 140Google ScholarThere is no corresponding record for this reference.38Felici, A.; Amicosante, G. Kinetic Analysis of extension of substrate specificity with Xanthomonas maltophilia, Aeromonas hydrophila, and Bacillus cereus metallo-β-lactamases Antimicrob. Agents Chemother. 1995, 39, 192– 199[Crossref], [PubMed], [CAS], Google Scholar38Kinetic analysis of extension of substrate specificity with Xanthomonas maltophilia, Aeromonas hydrophila, and Bacillus cereus metallo-β-lactamasesFelici, Antonio; Amicosante, GianfrancoAntimicrobial Agents and Chemotherapy (1995), (1), 192-9CODEN: AMACCQ; ISSN:0066-4804. (American Society for Microbiology) Twenty β-lactam mols., including penicillins, cephalosporins, penems, carbapenems, and monobactams, were investigated as potential substrates for Xanthomonas maltophilia ULA-511, Aeromonas hydrophila AE036, and Bacillus cereus 5/B/6 metallo-β-lactamases. A detailed anal. of the kinetic parameters examd. confirmed these enzymes to be broad-spectrum β-lactamases with different ranges of catalyst efficiency. Cefoxitin and moxalactam, substrates for the β-lactamases from X. maltophilia ULA-511 and B. cereus 5/B/6, behaved as inactivators of the A. hydrophila AE036 metallo-β-lactamase, which appeared to be unique among the enzymes tested in this study. In addn., we report a new, faster, and reliable purifn. procedure for the B. cereus 5/B/6 metallo-β-lactamase, cloned in Escherichia coli HB101. >> More from SciFinder https://chemport.cas.org/services/resolver?origin=ACS resolution=options coi=1%3ACAS%3A528%3ADyaK2MXislKksbo%253D md5=23b390c06b4e125d01dffcdc36c70c2139Young, D.; Toleman, M. A.; Giske, G. C.; Cho, C. H.; Sundman, K.; Lee, K.; Walsh, T. R. Characterization of a new metallo-β-lactamase gene, blaNDM-1, and a novel erythromycin esterase gene carried on a unique genetic structure in Klebsiella pneumoniae sequence type 14 from India Antimicrob. Agents Chemother. 2009, 53, 5046– 5054Google ScholarThere is no corresponding record for this reference.40Badarau, A.; Llinás, A.; Laws, A. P.; Damblon, C.; Page, M. I. Inhibitors of metallo-β-lactamase generated from β-lactam antibiotics Biochemistry 2005, 44, 8578– 8589[ACS Full Text ], Google ScholarThere is no corresponding record for this reference.41Astrid Zervosen, A.; Hernandez Valladares, M.; Devreese, B.; Prosperi-Meys, C.; Adolph, H.-W.; Sandra Mercuri, P.; Vanhove, M.; Amicosante, G.; van Beeumen, J.; Frère, J.-M.; Galleni, M. Inactivation of Aeromonas hydrophila metallo-β-lactamase by cephamycins and moxalactam Eur. J. Biochem. 2001, 268, 3840– 3850[Crossref], [PubMed], Google ScholarThere is no corresponding record for this reference.42Fonseca, F.; Arthur, C. J.; Bromley, E. H. C.; Samyn, B.; Moerman, P.; Saavedra, M. J.; Correia, A.; Spencer, J. Biochemical characterization of Sfh-I, a subclass B2 metallo-β-lactamase from Serratia fonticola UTAD54 Antimicrob. Agents Chemother. 2011, 55, 5392– 5395Google ScholarThere is no corresponding record for this reference.43Low KM values can result in a low signal readout; in particular, when chromogenic substrates are used, this can decrease the sensitivity of the method (i.e., lead to high interexperimental error during kinetic measurements). Moreover, by using a high-affinity substrate (low KM), it is generally not possible to detect potential slow-binding inhibitors as, for example, in the case of NDM-1/nitrocefin, where the substrate presented a low micromolar range KM, with the enzyme concentration being in the same micromolar range (also see ref 44).There is no corresponding record for this reference.44Siemann, S.; Clarke, A. J.; Viswanatha, T.; Dmitrienko, G. I. Thiols as classical and slow-binding inhibitors of IMP-1 and other binuclear metallo-β-lactamases Biochemistry 2003, 42, 1673– 1683[ACS Full Text ], Google ScholarThere is no corresponding record for this reference.45(a) Badarau, A.; Llinas, A.; Laws, A. P.; Damblon, C.; Page, M. I. Inhibitors of metallo-β-lactamase generated from β-lactam antibiotics Biochemistry 2005, 44, 8578– 8589[ACS Full Text ], Google ScholarThere is no corresponding record for this reference.(b) Murphy, T. A; Catto, L. E.; Halford, S. E.; Hadfield, A. T.; Minor, W.; Walsh, T. R.; Spencer, J. Crystal structure of Pseudomonas aeruginosa SPM-1 provides insights into variable zinc affinity of metallo-β-lactamases J. Mol. Biol. 2006, 357, 890– 903Google ScholarThere is no corresponding record for this reference.46Zhang, H.; Hao, Q. Crystal structure of NDM-1 reveals a common β-lactam hydrolysis mechanism FASEB J. 2011, 25, 2574– 2582[Crossref], [PubMed], [CAS], Google Scholar46Crystal structure of NDM-1 reveals a common β-lactam hydrolysis mechanismZhang, Hong Min; Hao, QuanFASEB Journal (2011), (8), 2574-2582, 10.1096/fj.11-184036CODEN: FAJOEC; ISSN:0892-6638. (Federation of American Societies for Experimental Biology) Metallo-β-lactamases (MBLs) hydrolyze most β-lactam antibiotics, and bacteria contg. this kind of enzyme pose a serious threat to the public health. The newly identified New Delhi MBL (NDM-1) is a new member of this family that shows tight binding to penicillin and cephalosporins. The rapid dissemination of NDM-1 in clin. relevant bacteria has become a global concern. However, no clin. useful inhibitors against MBLs exist, partly due to the lack of knowledge about the catalysis mechanism of this kind of enzyme. Here we report the crystal structure of this novel enzyme in complex with a hydrolyzed ampicillin at its active site at 1.3-Å resoln. Structural comparison with other MBLs revealed a new hydrolysis mechanism applicable to all three subclasses of MBLs, which might help the design of mechanism-based inhibitors. >> More from SciFinder https://chemport.cas.org/services/resolver?origin=ACS resolution=options coi=1%3ACAS%3A528%3ADC%252BC3MXpvFersrk%253D md5=32c83d7babafe70cef7ff785b855e14247Garcia-Saez, I.; Docquier, J. D.; Rossolini, G. M.; Dideberg, O. The three-dimensional structure of VIM-2, a Zn-β-lactamase from Pseudomonas aeruginosa in its reduced and oxidised form J. Mol. Biol. 2008, 375, 604– 611[Crossref], [PubMed], [CAS], Google Scholar47The Three-Dimensional Structure of VIM-2, a Zn-β-Lactamase from Pseudomonas aeruginosa in Its Reduced and Oxidized FormGarcia-Saez, I.; Docquier, J.-D.; Rossolini, G. M.; Dideberg, O.Journal of Molecular Biology (2008), (3), 604-611CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.) The crystal structures of the universally widespread metallo-β-lactamase (MBL) Verona integron-encoded MBL (VIM)-2 from Pseudomonas aeruginosa have been solved in their native form as well as in an unexpected oxidized form. This carbapenem-hydrolyzing enzyme belongs to the so-called B1 subfamily of MBLs and shares the folding of αβ/βα sandwich, consisting of a core of β-sheet surrounded by α-helixes. Surprisingly, it showed a high tendency to be strongly oxidized at the catalytic cysteine located in the Cys site, Cys 221, which, in the oxidized structure, becomes a cysteinesulfonic residue. Its native structure was obtained only in the presence of Tris(2-carboxyethyl)phosphine. This oxidn. might be a consequence of a lower affinity for the second Zn located in the Cys site that would also explain the obsd. susceptibility of VIM-2 to chelating agents. This modification, if present in nature, might play a role in catalytic down-regulation. Comparison between native and oxidized VIM-2 and a predicted model of VIM-1 (which shows one residue different in the Cys site compared with VIM-2) is performed to explain the different activities and antibiotic specificities. >> More from SciFinder https://chemport.cas.org/services/resolver?origin=ACS resolution=options coi=1%3ACAS%3A528%3ADC%252BD2sXhsVOlt73K md5=8dfa1db3d5cb7727be63df010892101f48(a) Goodsell, D. S.; Morris, G. M.; Olson, A. J. Automated docking of flexible ligands: Applications of AutoDock J. Mol. Recognit. 1996, 9, 1– 5[Crossref], [PubMed], [CAS], Google Scholar48aAutomated docking of flexible ligands: application of AutoDockGoodsell, David S.; Morris, Garrett M.; Olson, Arthur J.Journal of Molecular Recognition (1996), (1), 1-5CODEN: JMORE4; ISSN:0952-3499. (Wiley) A review with 23 refs. AutoDock is a suite of C programs used to predict the bound conformations of a small, flexible ligand to a macromol. target of known structure. The technique combines simulated annealing for conformation searching with a rapid grid-based method of energy evaluation. This paper reviews recent applications of the technique and describes the enhancement included in the current release. >> More from SciFinder https://chemport.cas.org/services/resolver?origin=ACS resolution=options coi=1%3ACAS%3A528%3ADyaK28Xis1SqtrY%253D md5=8f8f3964ba0c51da8f8b351e65a40a59(b) Morris, G. M.; Huey, R.; Lindstrom, W.; Sanner, M. F.; Belew, R. K.; Goodsell, D. S.; Olson, A. J. AutoDock4 and AutoDockTools4: Automated docking with selective receptor flexibility J. Comput. Chem. 2009, 30, 2785– 2791[Crossref], [PubMed], [CAS], Google Scholar48bAutoDock and AutoDockTools: Automated docking with selective receptor flexibilityMorris, Garrett M.; Huey, Ruth; Lindstrom, William; Sanner, Michel F.; Belew, Richard K.; Goodsell, David S.; Olson, Arthur J.Journal of Computational Chemistry (2009), (16), 2785-2791CODEN: JCCHDD; ISSN:0192-8651. (John Wiley Sons, Inc.) We describe the testing and release of AutoDock4 and the accompanying graphical user interface AutoDockTools. AutoDock4 incorporates limited flexibility in the receptor. Several tests are reported here, including a redocking expt. with 188 diverse ligand-protein complexes and a cross-docking expt. using flexible sidechains in 87 HIV protease complexes. We also report its utility in anal. of covalently bound ligands, using both a grid-based docking method and a modification of the flexible sidechain technique. © 2009 Wiley Periodicals, Inc. J Comput Chem, 2009. >> More from SciFinder https://chemport.cas.org/services/resolver?origin=ACS resolution=options coi=1%3ACAS%3A528%3ADC%252BD1MXht1GitrnK md5=679ce22fc50e9291c9aa16e7a185584549(a) Gillet, V.; Johnson, A. P.; Mata, P.; Sike, S.; Williams, P. SPROUT: A program for structure generation J. Comput.-Aided Mol. Des. 1993, 7, 127– 153[Crossref], [PubMed], [CAS], Google Scholar49aSPROUT: A program for structure generationGillet, Valerie; Johnson, A. Peter; Mata, Pauline; Sike, Sandor; Williams, PhilipJournal of Computer-Aided Molecular Design (1993), (2), 127-53CODEN: JCADEQ; ISSN:0920-654X. SPROUT is a new computer program for constrained structure generation that is designed to generate mols. for a range of applications in mol. recognition. It uses artificial intelligence techniques to moderate the combinatorial explosion that is inherent in structure generation. The program is presented here for the design of enzyme inhibitors. Structure generation is divided into the following 2 phases: (i) primary structure generation to produce mol. graphs to fit the steric constraints; and (ii) secondary structure generation which is the process of introducing appropriate functionality to the graphs to produce mols. that satisfy the secondary constraints, e.g., electrostatics and hydrophobicity. Primary structure generation has been tested on 2 enzyme receptor sites, i.e., the p-amidinophenylpyruvate binding site of trypsin and the acetyl pepstatin binding site of HIV-1 protease. The program successfully generates structures that resemble known substrates and, more importantly, the predictive power of the program has been demonstrated by its ability to suggest novel structures. >> More from SciFinder https://chemport.cas.org/services/resolver?origin=ACS resolution=options coi=1%3ACAS%3A528%3ADyaK3sXmt1Ghtrk%253D md5=2664279a8e55d30e453810235f57837d(b) Gillet, V. J.; Newell, W.; Mata, P.; Myatt, G.; Sike, S.; Zsoldos, Z.; Johnson, A. P. SPROUT: Recent developments in the de novo design of molecules J. Chem. Inf. Comput. Sci. 1994, 34, 207– 217[ACS Full Text ], [CAS], Google Scholar49bSPROUT: Recent developments in the de novo design of moleculesGillet, Valerie J.; Newell, William; Mata, Paulina; Myatt, Glenn; Sike, Sandor; Zsoldos, Zsolt; Johnson, A. PeterJournal of Chemical Information and Computer Sciences (1994), (1), 207-17CODEN: JCISD8; ISSN:0095-2338. SPROUT is a computer program for constrained structure generation. It is designed to generate mols. for a range of applications in mol. recognition. The program uses a no. of approxns. that enable a wide variety of diverse structures to be generated. Practical use of the program is demonstrated in two examples. The first demonstrates the ability of the program to generate candidate inhibitors for a receptor site of known 3D structure, specifically the GDP binding site of p21. In the 2nd example, structures are generated to fit a pharmacophore hypothesis that models morphine agonists. >> More from SciFinder https://chemport.cas.org/services/resolver?origin=ACS resolution=options coi=1%3ACAS%3A528%3ADyaK2cXns1GqtQ%253D%253D md5=cb5c2f6abe6b8a0657968c46fabf784e50Cadag, E.; Vitalis, E.; Lennox, K. P.; Zhou, C. L. E.; Zemla, A. T. Computational analysis of pathogen-borne metallo β-lactamases reveals discriminating structural features between B1 types BMC Res. Notes 2012, 5, 96[Crossref], [CAS], Google Scholar50Computational analysis of pathogen-borne metallo β-lactamases reveals discriminating structural features between B1 typesCadag, Eithon; Vitalis, Elizabeth; Lennox, Kristin P.; Zhou, Carol L. Ecale; Zemla, Adam T.BMC Research Notes (2012), 96CODEN: BRNMAT; ISSN:1756-0500. (BioMed Central Ltd.) Background: Genes conferring antibiotic resistance to groups of bacterial pathogens are cause for considerable concern, as many once-reliable antibiotics continue to see a redn. in efficacy. The recent discovery of the metallo β-lactamase blaNDM-1 gene, which appears to grant antibiotic resistance to a variety of Enterobacteriaceae via a mobile plasmid, is one example of this distressing trend. The following work describes a computational anal. of pathogen-borne MBLs that focuses on the structural aspects of characterized proteins. Results: Using both sequence and structural analyses, we examine residues and structural features specific to various pathogen-borne MBL types. This anal. identifies a linker region within MBL-like folds that may act as a discriminating structural feature between these proteins and specifically resistance-assocd. acquirable MBLs. Recently released crystal structures of the newly emerged NDM-1 protein were aligned against related MBL structures using a variety of global and local structural alignment methods and the overall fold conformation is examd. for structural conservation. Conservation appears to be present in most areas of the protein, yet is strikingly absent within a linker region, making NDM-1 unique with respect to a linker-based classification scheme. Variability anal. of the NDM-1 crystal structure highlights unique residues in key regions as well as identifying several characteristics shared with other transferable MBLs. Conclusions: A discriminating linker region identified in MBL proteins is highlighted and examd. in the context of NDM-1 and primarily three other MBL types: IMP-1, VIM-2 and ccrA. The presence of an unusual linker region variant and uncommon amino acid compn. at specific structurally important sites may help to explain the unusually broad kinetic profile of NDM-1 and may aid in directing research attention to areas of this protein and possibly other MBLs, that may be targeted for inactivation or attenuation of enzymic activity. >> More from SciFinder https://chemport.cas.org/services/resolver?origin=ACS resolution=options coi=1%3ACAS%3A528%3ADC%252BC38XkvFektb0%253D md5=55c75b24400ec5bab78a49a6f383d8c751(a) Stubbs, C. J.; Loenarz, C.; Mecinović, J.; Kheng Yeoh, K.; Hindley, N.; Liénard, B. M.; Sobott, F.; Schofield, C. J.; Flashman, E. Application of a proteolysis/mass spectrometry method for investigating the effects of inhibitors on hydroxylase structure J. Med. Chem. 2009, 52, 2799– 2805[ACS Full Text ], Google ScholarThere is no corresponding record for this reference.(b) Tian, Y.-M.; Yeoh, K. K.; Lee, M. K.; Eriksson, T.; Kessler, B. M.; Kramer, H. B.; Edelmann, M. J.; Willam, C.; Pugh, C. W.; Schofield, C. J.; Ratcliffe, P. J. Differential sensitivity of hypoxia inducible factor hydroxylation sites to hypoxia and hydroxylase inhibitors J. Biol. Chem. 2011, 286, 13041– 13051Google ScholarThere is no corresponding record for this reference.52(a) Heinz, U.; Bauer, R.; Wommer, S.; Meyer-Klaucke, W.; Papamichaels, C.; Bateson, J.; Adolph, H.-W. Coordination geometries of metal ions in D- or L-captopril-inhibited metallo-β-lactamases J. Biol. Chem. 2003, 278, 20659– 20666Google ScholarThere is no corresponding record for this reference.(b) King, D. T.; Worrall, L. J.; Gruninger, R.; Strynadka, N. C. New Delhi metallo-β-lactamase: Structural insights into β-lactam recognition and inhibition J. Am. Chem. Soc. 2012, 134, 11362– 11365[ACS Full Text ], [CAS], Google Scholar52bNew Delhi metallo-β-lactamase: structural insights into β-lactam recognition and inhibitionKing, Dustin T.; Worrall, Liam J.; Gruninger, Robert; Strynadka, Natalie C. J.Journal of the American Chemical Society (2012), (28), 11362-11365CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society) The β-lactam antibiotics have long been a cornerstone for the treatment of bacterial disease. Recently, a readily transferable antibiotic resistance factor called New Delhi metallo-β-lactamase-1 (NDM-1) was been found to confer enteric bacteria resistance to nearly all β-lactams, including the heralded carbapenems, posing a serious threat to human health. The crystal structure of NDM-1 bound to meropenem showed for the 1st time the mol. details of how carbapenem antibiotics are recognized by di-Zn-contg. metallo-β-lactamases. Addnl., product complex structures of hydrolyzed benzylpenicillin-, methicillin-, and oxacillin-bound NDM-1 were solved to 1.8, 1.2, and 1.2 Å, resp., and represent the highest-resoln. structural data for any metallo-β-lactamase reported to date. Finally, the authors present the crystal structure of NDM-1 bound to the potent competitive inhibitor L-captopril, which revealed a unique binding mechanism. An anal. of the NDM-1 active site in these structures revealed key features important for the informed design of novel inhibitors of NDM-1 and other metallo-β-lactamases. >> More from SciFinder https://chemport.cas.org/services/resolver?origin=ACS resolution=options coi=1%3ACAS%3A528%3ADC%252BC38XoslGgsr8%253D md5=3388110aa1369787d2e91e04829a03b453Conversion of IC50 to Ki was performed according to Cer et al.:Cer, R. Z.; Mudunuri, U.; Stephens, R.; Lebeda, F. J. IC50-to-Ki: A Web-based tool for converting IC50 to Ki values for inhibitors of enzyme activity and ligand binding Nucleic Acids Res. 2009, 37, W441– W445Google ScholarThere is no corresponding record for this reference.54Guo, Y.; Wang, J.; Niu, G.; Shui, W.; Sun, Y.; Zhou, H.; Zhang, Y.; Yang, C.; Lou, Z.; Rao, Z. A structural view of the antibiotic degradation enzyme NDM-1 from a superbug Protein Cell 2011, 2, 384– 394[Crossref], [PubMed], [CAS], Google Scholar54A structural view of the antibiotic degradation enzyme NDM-1 from a superbugGuo, Yu; Wang, Jing; Niu, Guojun; Shui, Wenqing; Sun, Yuna; Zhou, Honggang; Zhang, Yaozhou; Yang, Cheng; Lou, Zhiyong; Rao, ZiheProtein Cell (2011), (5), 384-394CODEN: PCREFB; ISSN:1674-800X. (Higher Education Press) Gram-neg. Enterobacteriaceae with resistance to carbapenem conferred by New Delhi metallo-β-lactamase 1 (NDM-1) are a type of newly discovered antibiotic-resistant bacteria. The rapid pandemic spread of NDM-1 bacteria worldwide (spreading to India, Pakistan, Europe, America, and Chinese Taiwan) in less than 2 mo characterizes these microbes as a potentially major global health problem. The drug resistance of NDM-1 bacteria is largely due to plasmids contg. the blaNDM-1 gene shuttling through bacterial populations. The NDM-1 enzyme encoded by the blaNDM-1 gene hydrolyzes β-lactam antibiotics, allowing the bacteria to escape the action of antibiotics. Although biol. functions and structural features of NDM-1 have been proposed according to results from functional and structural investigation of its homologs, the precise mol. characteristics and mechanism of action of NDM-1 have not been clarified. Here, the authors report the 3-dimensional structure of NDM-1 with 2 catalytic Zn2+ ions in its active site. Biol. and mass spectroscopy results revealed that D-captopril could effectively inhibit NDM-1 by binding to its active site with high binding affinity. The unique features concerning the primary sequence and structural conformation of its active site distinguish NDM-1 from other reported metallo-β-lactamases (MBLs) and implicate its role in wide spectrum drug resistance. The authors also discuss the mol. mechanism of NDM-1 action and its essential role in the pandemic of drug-resistant NDM-1 bacteria. These results will provide helpful information for future drug discovery targeting drug resistance caused by NDM-1 and related metallo-β-lactamases. >> More from SciFinder https://chemport.cas.org/services/resolver?origin=ACS resolution=options coi=1%3ACAS%3A528%3ADC%252BC3MXntlahur0%253D md5=cbc06076c1714c860c353bb2bff35454Cited ByThis article is cited by 73 publications.Alistair J. M. Farley, Yuri Ermolovich, Karina Calvopiña, Patrick Rabe, Tharindi Panduwawala, Jürgen Brem, Fredrik Björkling, Christopher J. Schofield. Structural Basis of Metallo-β-lactamase Inhibition by N-Sulfamoylpyrrole-2-carboxylates. 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Rydzik, Jürgen Brem, Sander S. van Berkel, Inga Pfeffer, Anne Makena, Timothy D. W. Claridge, Christopher J. Schofield. Monitoring Conformational Changes in the NDM-1 Metallo-β-lactamase by F NMR Spectroscopy. Angewandte Chemie 2014, 126 (12) , 3193-3197. https://doi.org/10.1002/ange.201310866Anna M. Rydzik, Jürgen Brem, Sander S. van Berkel, Inga Pfeffer, Anne Makena, Timothy D. W. Claridge, Christopher J. Schofield. Monitoring Conformational Changes in the NDM-1 Metallo-β-lactamase by F NMR Spectroscopy. Angewandte Chemie International Edition 2014, 53 (12) , 3129-3133. https://doi.org/10.1002/anie.201310866Derek A Nichols, Adam R Renslo, Yu Chen. Fragment-based inhibitor discovery against β-lactamase. Future Medicinal Chemistry 2014, 6 , 413-427. https://doi.org/10.4155/fmc.14.10Benito Alcaide, Pedro Almendros. Four-Membered Ring Systems. 2014,,, 85-113. https://doi.org/10.1016/B978-0-08-100017-5.00004-2Peter J. Rayner, Giacomo Gelardi, Peter O\'Brien, Richard A. J. Horan, David C. Blakemore. On the synthesis of α-amino sulfoxides. Org. Biomol. Chem. 2014, 12 (21) , 3499-3512. https://doi.org/10.1039/C4OB00567HAnne Makena, Sander S. van Berkel, Clarisse Lejeune, Raymond J. Owens, Anil Verma, Ramya Salimraj, James Spencer, Jürgen Brem, Christopher J. Schofield. Chromophore-Linked Substrate (CLS405): Probing Metallo-β-Lactamase Activity and Inhibition. ChemMedChem 2013, 8 (12) , 1923-1929. https://doi.org/10.1002/cmdc.201300350FiguresReferencesSupport InfoAbstractHigh Resolution ImageDownload MS PowerPoint SlideFigure 1Figure 1. Substrates for metallo-β-lactamase activity measurements.High Resolution ImageDownload MS PowerPoint SlideFigure 2Figure 2. Hydrolysis of substrates by MBLs, resulting in either an increase or a decrease of fluorescence.High Resolution ImageDownload MS PowerPoint SlideScheme 1Scheme 1. Synthesis of FC1-5: (A) Thiacoumarin Cephalosporin, (B) Hydroxylcoumarin CephalosporinsaHigh Resolution ImageDownload MS PowerPoint SlideScheme aReagents and conditions: (a) MMC, DiPEA, DMF, rt, 2 h; (b) TFA/anisole (5:1), 0 °C, 30 min; (c) mCPBA (1 equiv), CH2Cl2, 0 °C, 1 h; (d) NaI (10 equiv), acetone, rt, 2 h, (e) 7-HC, K2CO3, MeCN, rt, 4 h; (f) mCPBA (2 equiv), CH2Cl2, 0 °C, 3 h.Figure 3Figure 3. (Top) IMP-1 with FC4. (Bottom) SPM-1 with nitrocefin (substrate or product inhibition). Errors are reported as standard errors, n = 3.High Resolution ImageDownload MS PowerPoint SlideReferencesARTICLE SECTIONSJump To This article references 54 other publications. 1Spellberg, B.; Guidos, R.; Gilbert, D.; Bradley, J.; Boucher, H. W.; Scheld, W. M.; Bartlett, J. G.; Edwards, J., Jr. The epidemic of antibiotic-resistant infections: A call to action for the medical community from the Infectious Diseases Society of America Clin. Infect. Dis. 2008, 46, 155– 164[Crossref], [PubMed], [CAS], Google Scholar1The epidemic of antibiotic-resistant infections: a call to action for the medical community from the Infectious Diseases Society of AmericaSpellberg Brad; Guidos Robert; Gilbert David; Bradley John; Boucher Helen W; Scheld W Michael; Bartlett John G; Edwards John JrClinical infectious diseases : an official publication of the Infectious Diseases Society of America (2008), (2), 155-64 ISSN:. The ongoing explosion of antibiotic-resistant infections continues to plague global and US health care. Meanwhile, an equally alarming decline has occurred in the research and development of new antibiotics to deal with the threat. In response to this microbial \"perfect storm,\" in 2001, the federal Interagency Task Force on Antimicrobial Resistance released the \"Action Plan to Combat Antimicrobial Resistance; Part 1: Domestic\" to strengthen the response in the United States. The Infectious Diseases Society of America (IDSA) followed in 2004 with its own report, \"Bad Bugs, No Drugs: As Antibiotic Discovery Stagnates, A Public Health Crisis Brews,\" which proposed incentives to reinvigorate pharmaceutical investment in antibiotic research and development. The IDSA\'s subsequent lobbying efforts led to the introduction of promising legislation in the 109 th US Congress (January 2005-December 2006). Unfortunately, the legislation was not enacted. During the 110 th Congress, the IDSA has continued to work with congressional leaders on promising legislation to address antibiotic-resistant infection. Nevertheless, despite intensive public relations and lobbying efforts, it remains unclear whether sufficiently robust legislation will be enacted. In the meantime, microbes continue to become more resistant, the antibiotic pipeline continues to diminish, and the majority of the public remains unaware of this critical situation. The result of insufficient federal funding; insufficient surveillance, prevention, and control; insufficient research and development activities; misguided regulation of antibiotics in agriculture and, in particular, for food animals; and insufficient overall coordination of US (and international) efforts could mean a literal return to the preantibiotic era for many types of infections. If we are to address the antimicrobial resistance crisis, a concerted, grassroots effort led by the medical community will be required. >> More from SciFinder https://chemport.cas.org/services/resolver?origin=ACS resolution=options coi=1%3ACAS%3A280%3ADC%252BD1c%252FgslGitw%253D%253D md5=52bb108e9c29dc9da3031f7127cba99f2World Health Organization. The evolving threat of antimicrobialresistance: Options for action, (2012. http://whqlibdoc.who.int/publications/2012/9789241503181_eng.pdf (accessed December 2012).Google ScholarThere is no corresponding record for this reference.3Bush, K.; Jacoby, G. A. Updated functional classification of β-lactamases Antimicrob. Agents Chemother. 2010) 54, 969– 976[Crossref], [PubMed], [CAS], Google Scholar3Updated functional classification of β-lactamasesBush, Karen; Jacoby, George A.Antimicrobial Agents and Chemotherapy (2010), (3), 969-976CODEN: AMACCQ; ISSN:0066-4804. (American Society for Microbiology) A review. Two classification schemes for β-lactamases are currently in use. The mol. classification is based on the amino acid sequence and divides β-lactamases into class A, C, and D enzymes which utilize serine for β-lactam hydrolysis and class B metalloenzymes which require Zn2+ ions for substrate hydrolysis. The functional classification scheme updated here is based on the proposal by K. Bush et al. (1995). It takes into account substrate and inhibitor profiles in an attempt to group the enzymes in ways that can be correlated with their phenotype in clin. isolates. Major groupings generally correlate with the more broadly based mol. classification. The updated system includes group 1 (class C) cephalosporinases; group 2 (classes A and D) broad-spectrum, inhibitor-resistant, and extended-spectrum β-lactamases and serine carbapenemases; and group 3 metallo-β-lactamases. Several new subgroups of each of the major groups are described, based on specific attributes of individual enzymes. A list of attributes is also suggested for the description of a new β-lactamase, including the requisite microbiol. properties, substrate and inhibitor profiles, and mol. sequence data that provide an adequate characterization for a new β-lactam-hydrolyzing enzyme. >> More from SciFinder https://chemport.cas.org/services/resolver?origin=ACS resolution=options coi=1%3ACAS%3A528%3ADC%252BC3cXjtFWqsr4%253D md5=89b376d90ff64b78f81bd43ed5925ea24White, A. R.; Kaye, C.; Poupard, J.; Pypstra, R.; Woodnutt, G.; Wynne, B. J. Augmentin (amoxicillin/clavulanate) in the treatment of community-acquired respiratory tract infection: A review of the continuing development of an innovative antimicrobial agent Antimicrob. Chemother. 2004, 53 (Suppl. S1) i3– i20Google ScholarThere is no corresponding record for this reference.5Drawz, S. M.; Bonomo, R. A. Three decades of β-lactamase inhibitors Clin. Microbiol. Rev. 2010, 23, 160– 201[Crossref], [PubMed], [CAS], Google Scholar5Three decades of β-lactamase inhibitorsDrawz, Sarah M.; Bonomo, Robert A.Clinical Microbiology Reviews (2010), (1), 160-201CODEN: CMIREX; ISSN:0893-8512. (American Society for Microbiology) A review. Since the introduction of penicillin, β-lactam antibiotics have been the antimicrobial agents of choice. Unfortunately, the efficacy of these life-saving antibiotics is significantly threatened by bacterial β-lactamases. β-Lactamases are now responsible for resistance to penicillins, extended-spectrum cephalosporins, monobactams, and carbapenems. In order to overcome β-lactamase-mediated resistance, β-lactamase inhibitors (clavulanate, sulbactam, and tazobactam) were introduced into clin. practice. These inhibitors greatly enhance the efficacy of their partner β-lactams (amoxicillin, ampicillin, piperacillin, and ticarcillin) in the treatment of serious Enterobacteriaceae and penicillin-resistant staphylococcal infections. However, selective pressure from excess antibiotic use accelerated the emergence of resistance to β-lactam-β-lactamase inhibitor combinations. Furthermore, the prevalence of clin. relevant β-lactamases from other classes that are resistant to inhibition is rapidly increasing. There is an urgent need for effective inhibitors that can restore the activity of β-lactams. Here, we review the catalytic mechanisms of each β-lactamase class. We then discuss approaches for circumventing β-lactamase-mediated resistance, including properties and characteristics of mechanism-based inactivators. We next highlight the mechanisms of action and salient clin. and microbiol. features of β-lactamase inhibitors. We also emphasize their therapeutic applications. We close by focusing on novel compds. and the chem. features of these agents that may contribute to a \"second generation\" of inhibitors. The goal for the next 3 decades will be to design inhibitors that will be effective for more than a single class of β-lactamases. >> More from SciFinder https://chemport.cas.org/services/resolver?origin=ACS resolution=options coi=1%3ACAS%3A528%3ADC%252BC3cXks1Sktbs%253D md5=27cd9fca0d848185d9c61e5a25d2c6016Oelschlaeger, P.; Ai, N.; DuPrez, K. T.; Welsh, W. J.; Toney, J. H. Evolving carbapenemases: Can medicinal chemists advance one step ahead of the coming storm? J. Med. Chem. 2010, 53, 3013– 3027[ACS Full Text ], Google ScholarThere is no corresponding record for this reference.7Papp-Wallace, K. M.; Endimiani, A.; Taracila, M. A.; Bonomo, R. A Carbapenems: Past, present, and future Antimicrob. Agents Chemother. 2011, 5, 4943– 4960Google ScholarThere is no corresponding record for this reference.8Ehmann, D. E.; Jahić, H.; Ross, P. L.; Gu, R.-F.; Hu, J.; Kern, G.; Walkup, G. K.; Fisher, S. L. Avibactam is a covalent, reversible, non-β-lactam β-lactamase inhibitor Proc. Natl. Acad. Sci. U.S.A. 2012, 109, 11663– 11668[Crossref], [PubMed], [CAS], Google Scholar8Avibactam is a covalent, reversible, non-β-lactam β-lactamase inhibitorEhmann, David E.; Jahic, Haris; Ross, Philip L.; Gu, Rong-Fang; Hu, Jun; Kern, Gunther; Walkup, Grant K.; Fisher, Stewart L.Proceedings of the National Academy of Sciences of the United States of America (2012), (29), 11663-11668, S11663/1-S11663/5CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences) Avibactam is a β-lactamase inhibitor that is in clin. development, combined with P-lactam partners, for the treatment of bacterial infections comprising Gram-neg. organisms. Avibactam is a structural class of inhibitor that does not contain a β-lactam core but maintains the capacity to covalently acylate its β-lactamase targets. Using the TEM-1 enzyme, we characterized avibactam inhibition by measuring the on-rate for acylation and the off-rate for deacylation. The deacylation off-rate was 0.045 min-1, which allowed investigation of the deacylation route from TEM-1. Using NMR and MS, we showed that deacylation proceeds through regeneration of intact avibactam and not hydrolysis. Other than TEM-1, four addnl. clin. relevant β-lactamases were shown to release intact avibactam after being acylated. We showed that avibactam is a covalent, slowly reversible inhibitor, which is a unique mechanism of inhibition among β-lactamase inhibitors. >> More from SciFinder https://chemport.cas.org/services/resolver?origin=ACS resolution=options coi=1%3ACAS%3A528%3ADC%252BC38Xht1Ciu73K md5=4da3c333df0a1eeb6ab84cabd65ab8ec9Ellar, D. J.; Lundgren, D. G. Fine structure of sporulation in Bacillus cereus grown in a chemically defined medium J. Bacteriol. 1966, 92, 1748– 1764[PubMed], [CAS], Google Scholar9Fine structure of sporulation in Bacillus cereus grown in a chemically defined mediumEllar, D. J.; Lundgren, Donald G.Journal of Bacteriology (1966), (6), 1748-64CODEN: JOBAAY; ISSN:0021-9193. A study was made of the fine structure of sporulating cells of B. cereus grown in a chem. defined medium. The developmental stages of sporulation occurred in a fairly synchronous manner and were complete by 14 hrs. This time period was shortened when spore-wall peptide components were added to the medium, but the addn. had no effect on fine structure except to thicken the cell wall. Sporulation could be sepd. into six morphological stages which generally agreed with those for other sporulating bacteria. 40 references. >> More from SciFinder https://chemport.cas.org/services/resolver?origin=ACS resolution=options coi=1%3ACAS%3A528%3ADyaF2sXovFWh md5=e2d1da31b6efbed3d287407c7791dd0f10Walsh, T. R. Emerging carbapenemases: A global perspective Int. J. Antimicrob. Agents 2010, 36 (Suppl. 3) S8– S14Google ScholarThere is no corresponding record for this reference.11Cornaglia, G.; Giamarellou, H.; Maria Rossolini, G. Metallo-β-lactamases: A last frontier for β-lactams? Lancet Infect. Dis. 2011, 11, 381– 393[Crossref], [PubMed], [CAS], Google Scholar11Metallo-β-lactamases: a last frontier for β-lactams?Cornaglia, Giuseppe; Giamarellou, Helen; Rossolini, Gian MariaLancet Infectious Diseases (2011), (5), 381-393CODEN: LIDABP; ISSN:1473-3099. (Elsevier Ltd.) A review. Summary: Metallo-β-lactamases are resistance determinants of increasing clin. relevance in Gram-neg. bacteria. Because of their broad range, potent carbapenemase activity and resistance to inhibitors, these enzymes can confer resistance to almost all β-lactams. Since the 1990s, several metallo-β-lactamases encoded by mobile DNA have emerged in important Gram-neg. pathogens (ie, in Enterobacteriaceae, Pseudomonas aeruginosa, and Acinetobacter baumannii). Some of these enzymes (eg, VIM-1 and NDM-1) have been involved in the recent crisis resulting from the international dissemination of carbapenem-resistant Klebsiella pneumoniae and other enterobacteria. Although substantial knowledge about the mol. biol. and genetics of metallo-β-lactamases is available, epidemiol. data are inconsistent and clin. experience is still lacking; therefore, several unsolved or debatable issues remain about the management of infections caused by producers of metallo-β-lactamase. The spread of metallo-β-lactamases presents a major challenge both for treatment of individual patients and for policies of infection control, exposing the substantial unpreparedness of public health structures in facing up to this emergency. >> More from SciFinder https://chemport.cas.org/services/resolver?origin=ACS resolution=options coi=1%3ACAS%3A528%3ADC%252BC3MXlsVGntL4%253D md5=a6097adade71d508091de28f14cc6a0a12(a) Chen, P.; Horton, L. B.; Mikulski, R. L.; Deng, L.; Sundriyal, S.; Palzkill, T.; Song, Y. 2-Substituted 4,5-dihydrothiazole-4-carboxylic acids are novel inhibitors of metallo-β-lactamases Bioorg. Med. Chem. Lett. 2012, 22, 6229– 6232Google ScholarThere is no corresponding record for this reference.(b) Toney, J. H.; Hammond, G. G.; Fitzgerald, P. M. D.; Sharma, N.; Balkovec, J. M.; Rouen, G. P.; Olson, S. H.; Hammond, M. L.; Greenlee, M. L.; Gao, Y. D. Succinic acids as potent inhibitors of plasmid-borne IMP-1 metallo-β-lactamase J. Biol. Chem. 2001, 276, 31913– 31918Google ScholarThere is no corresponding record for this reference.13(a) Toney, J. H.; Fitzgerald, P. M.; Grover-Sharma, N.; Olson, S. H.; May, W. J.; Sundelof, J. G.; Vanderwall, D. E.; Cleary, K. A.; Grant, S. K.; Wu, J. K.; Kozarich, J. W.; Pompliano, D. L.; Hammond, G. G. Antibiotic sensitization using biphenyl tetrazoles as potent inhibitors of Bacteroides fragilis metallo-β-lactamase Chem. Biol. 1998, 5, 185– 196[Crossref], [PubMed], [CAS], Google Scholar13aAntibiotic sensitization using biphenyl tetrazoles as potent inhibitors of Bacteroides fragilis metallo-β-lactamaseToney, Jeffrey H.; Fitzgerald, Paula M. D.; Grover-Sharma, Nandini; Olson, Steven H.; May, Walter J.; Sundelof, Jon G.; Vanderwall, Dana E.; Cleary, Kelly A.; Grant, Stephan K.; Wu, Joseph K.; Kozarich, John W.; Pompliano, David L.; Hammond, Gail G.Chemistry Biology (1998), (4), 185-196CODEN: CBOLE2; ISSN:1074-5521. (Current Biology Ltd.) High level resistance to carbapenem antibiotics in Gram-neg. bacteria such as Bacteroides fragilis is caused, in part, by expression of a wide-spectrum metallo-β-lactamase that hydrolyzes the drug to an inactive form. Co-administration of metallo-β-lactamase inhibitors to resistant bacteria is expected to restore the antibacterial activity of carbapenems. Biphenyl tetrazoles (BPTs) are a structural class of potent competitive inhibitors of metallo-β-lactamase identified through screening and predicted using mol. modeling of the enzyme structure. The X-ray crystal structure of the enzyme bound to the BPT L-159,061 shows that the tetrazole moiety of the inhibitor interacts directly with one of the two zinc atoms in the active site, replacing a metal-bound water mol. Inhibition of metallo-β-lactamase by BPTs in vitro correlates well with antibiotic sensitization of resistant B. fragilis. It is shown here that BPT inhibitors can sensitize a resistant B. fragilis clin. isolate expressing metallo-β-lactamase to the antibiotics imipenem or penicillin G but not to rifampicin. >> More from SciFinder https://chemport.cas.org/services/resolver?origin=ACS resolution=options coi=1%3ACAS%3A528%3ADyaK1cXjtVynu7g%253D md5=32f20851a6d66a200be162350b67eccc(b) Weide, T.; Saldanha, S. A.; Minond, D.; Spicer, T. P.; Fotsing, J. R.; Spaargaren, M.; Frère, J.-M.; Bebrone, C.; Sharpless, K. B.; Hodder, P. S.; Fokin, V. V. NH-1,2,3-Triazole-based inhibitors of the VIM-2 metallo-β-lactamase: Synthesis and structure–activity studies ACS Med. Chem. Lett. 2010, 1, 150– 154[ACS Full Text ], Google ScholarThere is no corresponding record for this reference.14(a) Liénard, B. M. R.; Garau, G.; Horsfall, L.; Karsisiotis, A. I.; Damblon, C.; Lassaux, P.; Papamicael, C.; Roberts, G. C. K.; Galleni, M.; Dideberg, O.; Frère, J.-M.; Schofield, C. J. Structural basis for the broad-spectrum inhibition of metallo-β-lactamases by thiols Org. Biomol. Chem. 2008, 6, 2282– 2294Google ScholarThere is no corresponding record for this reference.(b) Liénard, B. M., R.; Hüting, R.; Lassaux, P.; Galleni, M.; Frère, J.-M.; Schofield, C. J. Dynamic combinatorial mass spectrometry leads to metallo-β-lactamase inhibitors J. Med. Chem. 2008, 51, 684– 688[ACS Full Text ], Google ScholarThere is no corresponding record for this reference.15Walter, M. W.; Felici, A.; Galleni, M.; Paul Soto, R.; Adlington, R. M.; Baldwin, J. E.; Frère, J.-M.; Gololobov, M.; Schofield, C. J. Trifluoromethyl alcohol and ketone inhibitors of metallo-β-lactamases Bioorg. Med. Chem. Lett. 1996, 6, 2455– 2458Google ScholarThere is no corresponding record for this reference.16(a) Fast, W.; Sutton, L. D. Metallo-β-lactamase: Inhibitors and reporter substrates Biochim. Biophys. Acta, Proteins Proteomics 2013, 1834, 1648– 1659[Crossref], [PubMed], [CAS], Google Scholar16aMetallo-β-lactamase: Inhibitors and reporter substratesFast, Walter; Sutton, Larry D.Biochimica et Biophysica Acta, Proteins and Proteomics (2013), 1834 (8), 1648-1659CODEN: BBAPBW; ISSN:1570-9639. (Elsevier B. V.) A review. Metallo-β-lactamases represent an emerging clin. threat due to their ability to render ineffective an entire class of antibiotics. Accordingly, this family of enzymes has been suggested as an attractive target for drug design. Progress toward developing effective inhibitors as well as the development of reporter substrates is reviewed. Inhibitors are classified into six classes and known binding interactions with metallo-β-lactamases are summarized. The development of chromogenic and fluorogenic reporter substrates is also reviewed with respect to current and prospective applications to future inhibitor and diagnostic discovery, mechanistic studies, and biol. imaging. Despite progress in mol. probe development, the sequence and structural diversity within the metallo-β-lactamase family continue to present substantial hurdles for rational ligand design. >> More from SciFinder https://chemport.cas.org/services/resolver?origin=ACS resolution=options coi=1%3ACAS%3A528%3ADC%252BC3sXhtVehu73J md5=234faf23a238484f8b612a3bd3f28b8f(b) Toney, J. H.; Moloughney, J. G. Metallo-β-lactamase inhibitors: Promise for the future? Curr. Opin. Invest. Drugs 2004, 5, 823– 826Google ScholarThere is no corresponding record for this reference.(c) Spencer, J.; Walsh, T. R.; New, A. Approach to the inhibition of metallo-β-lactamases Angew. Chem., Int. Ed. 2006, 45, 1022– 1026Google ScholarThere is no corresponding record for this reference.17(a) Moali, C.; Anne, C.; Lamotte-Brasseur, J.; Groslambert, S.; Devreese, B.; Van Beeumen, J.; Galleni, M.; Frère, J.-M. Analysis of the importance of the metallo-β-lactamase active site loop in substrate binding and catalysis Chem. Biol. 2003, 10, 319– 329[Crossref], [PubMed], [CAS], Google Scholar17aAnalysis of the importance of the metallo-β-lactamase active site loop in substrate binding and catalysisMoali, Catherine; Anne, Christine; Lamotte-Brasseur, Josette; Groslambert, Sylvie; Devreese, Bart; Van Beeumen, Jozef; Galleni, Moreno; Frere, Jean-MarieChemistry Biology (2003), (4), 319-329CODEN: CBOLE2; ISSN:1074-5521. (Cell Press) The role of the mobile loop comprising residues 60-66 in metallo-β-lactamases was studied by site-directed mutagenesis, detn. of kinetic parameters for 6 substrates and 2 inhibitors, pre-steady-state characterization of the interaction with chromogenic nitrocefin, and mol. modeling. The W64A mutation was performed in β-lactamases IMP-1 and BcII (after replacement of the BcII 60-66 peptide by that of IMP-1) and always resulted in increased Ki and Km values and decreased kcat/Km values, an effect reinforced by complete deletion of the loop. The kcat values were, by contrast, much more diversely affected, indicating that the loop did not systematically favor the best relative positioning of substrate and enzyme catalytic groups. The hydrophobic nature of the ligand was also crucial to strong interactions with the loop, since imipenem was almost insensitive to loop modifications. >> More from SciFinder https://chemport.cas.org/services/resolver?origin=ACS resolution=options coi=1%3ACAS%3A528%3ADC%252BD3sXjt1Kntrg%253D md5=b9ddf52e077b003935447c72f82a319a(b) Saradhi Borra, P.; Samuelsen, Ø.; Spencer, J.; Walsh, T. R.; Sjo Lorentzen, M.; Leirose, H.-K. S. Crystal structures of Pseudomonas aeruginosa GIM-1: Active-site plasticity in metallo-β-lactamases Antimicrob. Agents Chemother. 2013, 57, 848– 854Google ScholarThere is no corresponding record for this reference.18(a) Viswanatha, T.; Marrone, L.; Goodfellow, V.; Dmitrienko, G. I. Assays for β-lactamase activity and inhibition Methods Mol. Med. 2008, 142, 239– 260[Crossref], [PubMed], [CAS], Google Scholar18aAssays for β-lactamase activity and inhibitionViswanatha, Thammaiah; Marrone, Laura; Goodfellow, Valerie; Dmitrienko, Gary I.Methods in Molecular Medicine (2008), (New Antibiotic Targets), 239-260CODEN: MMMEFN ISSN:. (Humana Press Inc.) A review. The ability, either innate or acquired, to produce β-lactamases, enzymes capable of hydrolyzing the endocyclic peptide bond in β-lactam antibiotics, would appear to be a primary contributor to the ever-increasing incidences of resistance to this class of antibiotics. To date, four distinct classes, A, B, C, and D, of β-lactamases have been identified. Of these, enzymes in classes A, C, and D utilize a serine residue as a nucleophile in their catalytic mechanism while class B members are Zn2+-dependent for their function. Efforts have been and still continue to be made toward the development of potent inhibitors of these enzymes as a means to ensure the efficacy of β-lactam antibiotics in clin. medicine. This chapter concerns procedures for the evaluation of the catalytic activity of β-lactamases as a means to screen compds. for their inhibitory potency. >> More from SciFinder https://chemport.cas.org/services/resolver?origin=ACS resolution=options coi=1%3ACAS%3A528%3ADC%252BD1cXhvVOhtLY%253D md5=123a5b54e474a5a25cc6d703be9ba3ba(b) Kocaoglu, O.; Calvo, R. A.; Sham, L.-T.; Cozy, L. M.; Lanning, B. R.; Francis, S.; Winkler, M. E.; Kearns, D. B.; Carlson, E. E. Selective penicillin-binding protein imaging probes reveal substructure in bacterial cell division ACS Chem. Biol. 2012, 7, 1746– 1753[ACS Full Text ], [CAS], Google Scholar18bSelective penicillin-binding protein imaging probes reveal substructure in bacterial cell divisionKocaoglu, Ozden; Calvo, Rebecca A.; Sham, Lok-To; Cozy, Loralyn M.; Lanning, Bryan R.; Francis, Samson; Winkler, Malcolm E.; Kearns, Daniel B.; Carlson, Erin E.ACS Chemical Biology (2012), (10), 1746-1753CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society) The peptidoglycan cell wall is a common target for antibiotic therapy, but its structure and assembly are only partially understood. Peptidoglycan synthesis requires a suite of penicillin-binding proteins (PBPs), the individual roles of which are difficult to det. because each enzyme is often dispensable for growth perhaps due to functional redundancy. To address this challenge, the authors sought to generate tools that would enable selective examn. of a subset of PBPs. They designed and synthesized fluorescent and biotin derivs. of the β-lactam-contg. antibiotic cephalosporin C. These probes facilitated specific in vivo labeling of active PBPs in both Bacillus subtilis PY79 and an unencapsulated deriv. of D39 Streptococcus pneumoniae. Microscopy and gel-based anal. indicated that the cephalosporin C-based probes are more selective than BOCILLIN-FL, a com. available penicillin V analog, which labels all PBPs. Dual labeling of live cells performed by satn. of cephalosporin C-susceptible PBPs followed by tagging of the remaining PBP population with BOCILLIN-FL demonstrated that the two sets of PBPs are not co-localized. This suggests that even PBPs that are located at a particular site (e.g., septum) are not all intermixed, but rather that PBP subpopulations are discretely localized. Accordingly, the Ceph C probes represent new tools to explore a subset of PBPs and have the potential to facilitate a deeper understand of the roles of this crit. class of proteins. >> More from SciFinder https://chemport.cas.org/services/resolver?origin=ACS resolution=options coi=1%3ACAS%3A528%3ADC%252BC38Xht1eks7fP md5=5fbfeaaff9a0363111fe4f1689f30f9c(c) Zheng, X.; Sallium, U. W.; Verma, S.; Athar, H.; Evans, C. L.; Hasan, T. Exploiting a bacterial drug-resistance mechanism: A light-activated construct for the destruction of MRSA Angew. Chem., Int. Ed. 2009, 48, 2148– 2151[Crossref], [CAS], Google Scholar18cExploiting a bacterial drug-resistance mechanism: a light-activated construct for the destruction of MRSAZheng, Xiang; Sallum, Ulysses W.; Verma, Sarika; Athar, Humra; Evans, Conor L.; Hasan, TayyabaAngewandte Chemie, International Edition (2009), (12), 2148-2151CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH Co. KGaA) An enzyme-specific mol. construct exploits the overexpression of β-lactamase in several drug-resistant bacteria. Ed Specific photodynamic toxicity was detected towards β-lactam-resistant methicillin-resistant Staphylococcus aureus (MRSA), whereby the usual mechanism for antibiotic resistance (cleavage of the β-lactam ring) releases the phototoxic component from the prodrug. >> More from SciFinder https://chemport.cas.org/services/resolver?origin=ACS resolution=options coi=1%3ACAS%3A528%3ADC%252BD1MXktFegtr4%253D md5=aa3ee1067024a7174bae31b1b240fcde19Jones, R. N.; Wilson, H. W.; Novick, W. J., Jr.; Barry, A. L.; Thornberry, C. In vitro evaluation of CENTA, a new β-lactamase-susceptible chromogenic cephalosporin reagent J. Clin. Microbiol. 1982, 15, 954– 958[PubMed], [CAS], Google Scholar19In vitro evaluation of CENTA, a new beta-lactamase-susceptible chromogenic cephalosporin reagentJones, Ronald N.; Wilson, Harold W.; Novick, William J., Jr.; Barry, Arthur L.; Thornsberry, ClydeJournal of Clinical Microbiology (1982), (5), 954-8CODEN: JCMIDW; ISSN:0095-1137. CENTA is a newly synthesized, β-lactamase-labile, chromogenic cephalosporin reagent which changes color from light yellow (λ max. ∼340 nm) to chrome yellow (λ max. ∼405 nm) concomitant with hydrolysis of the β-lactam ring. This compd. offers promise as a diagnostic reagent comparable to other chromogens (PADAC and nitrocefin) for the early detection of β-lactamase-producing clin. isolates, while retaining some antimicrobial effect against Escherichia coli, Klebsiella, Proteus mirabilis, Staphylococcus aureus, and nonenterococcal Streptococcus. CENTA is relatively unaffected by commonly used microbiol. media and human serum. >> More from SciFinder https://chemport.cas.org/services/resolver?origin=ACS resolution=options coi=1%3ACAS%3A528%3ADyaL38XktVentrs%253D md5=4d2203ff80d47cab37a5e11ba775ce1120Jones, R. N.; Wilson, H. W.; Novick, Jr. In vitro evaluation of pyridine-2-azo-p-dimethylaniline cephalosporin, a new diagnostic chromogenic reagent, and comparison with nitrocefin, cephacetrile, and other β-lactam compounds J. Clin. Microbiol. 1982, 15, 677– 683[PubMed], [CAS], Google Scholar20In vitro evaluation of pyridine-2-azo-p-dimethylaniline cephalosporin, a new diagnostic chromogenic reagent, and comparison with nitrocefin, cephacetrile, and other beta-lactam compoundsJones, Ronald N.; Wilson, Harold W.; Novick, William J., Jr.Journal of Clinical Microbiology (1982), (4), 677-83CODEN: JCMIDW; ISSN:0095-1137. Pyridine-2-azo-p-dimethylanaline cephalosporin (PADAC), a chromogenic reagent which is purple and changes to yellow upon cleavage of its β-lactam ring, was evaluated in comparison with other chromogenic cephalosporins. PADAC exhibited little antimicrobial activity against gram-neg. bacteria, but it did have good activity (min. inhibitory concn., 0.12-0.5 μg/mL) against Staphylococcus aureus, a quality comparable to nitrocefin. Nitrocefin, however, demonstrated an unexpected and uniquely potent activity against Streptococcus faecalis (min. inhibitory concn., ≤0.06-0.12 μg/mL). The relative hydrolysis rate of PADAC when subjected to 6 different β-lactamases was substantially greater than that of cephacetrile but less than that of nitrocefin. The relative hydrolysis rates of PADAC and nitrocefin were comparable with type IIIa β-lactamase and that derived from Bacillus cereus. The inhibition of β-lactamase hydrolysis of the chromogenic cephalosporin substrates by 6 enzyme-stable inhibitors was generally greater with PADAC than with nitrocefin. Unlike nitrocefin, PADAC mixed with 50% human serum or various broth culture media showed no evidence of color change or degrdn. over several hours. The subsequent enzyme hydrolysis rates of such mixts. were the same as in phosphate buffer. β-Lactamase-contg. bacterial suspensions and clin. specimens contg. such bacteria produced pos. visual and spectrophotometric color changes when mixed with PADAC or nitrocefin. Although color changes occurred more slowly with PADAC than with nitrocefin, PADAC was not adversely influenced (nonenzyme-related color change) by the protein content of specimens. PADAC may be a promising alternative for β-lactamase diagnostic testing in the clin. and research microbiol. lab. >> More from SciFinder https://chemport.cas.org/services/resolver?origin=ACS resolution=options coi=1%3ACAS%3A528%3ADyaL38XitFCitLo%253D md5=d0c7f43594b9dc0cec5755406314424821Shannon, K.; Phillips, I. β-Lactamase detection by three simple methods: Intralactam, nitrocefin and acidimetric J. Antimicrob. Chemother. 1980, 6, 617– 621[Crossref], [PubMed], [CAS], Google Scholar21β-Lactamase detection by three simple methods: Intralactam, nitrocefin and acidimetricShannon, Kevin; Phillips, IanJournal of Antimicrobial Chemotherapy (1980), (5), 617-21CODEN: JACHDX; ISSN:0305-7453. Intralactam, an acidimetric paper-strip test for the detection of β-lactamase (I) [9073-60-3], was compared with the nitrocefin test and a tube-acidimetric method. Min. inhibitory concns. (MICs) of appropriate antibiotics were detd. for the organisms studied. All 3 methods detected the I-producing isolates of Neisseria gonorrhoeae and Haemophilus influenzae. Intralactam and nitrocefin detected I in highly carbenicillin (II) [4697-36-3]-resistant isolates of Pseudomonas aeruginosa (MICs 4096 mg/L) as did the tube acidimetric method when cells were subjected to ultrasonic disintegration. Other isolates of P. aeruginosa (II MICs, 32-512 mg/L) were neg. by all methods. The tests also detected benzylpenicillin [61-33-6]-resistant isolates of Staphylococcus aureus. When Enterobacteriaceae were tested, there were many discrepancies among the methods. The nitrocefin test, performed on disintegrated cell suspensions, was the most sensitive method. Intralactam was more sensitive than the tube acidimetric method. None of the methods reliably predicted sensitivity of Enterobacteriaceae to ampicillin [69-53-4] or cephaloridine [50-59-9], as assessed by MICs. >> More from SciFinder https://chemport.cas.org/services/resolver?origin=ACS resolution=options coi=1%3ACAS%3A528%3ADyaL3MXmsFE%253D md5=43b451e6f3f29ed249a97075c7af95c322(a) Gao, W.; Xing, B.; Tsien, R. Y.; Rao, J. Novel fluorogenic substrates for imaging β-lactamase gene expression J. Am. Chem. Soc. 2003, 125, 11146– 11147[ACS Full Text ], [CAS], Google Scholar22aNovel Fluorogenic Substrates for Imaging β-Lactamase Gene ExpressionGao, Wenzhong; Xing, Bengang; Tsien, Roger Y.; Rao, JianghongJournal of the American Chemical Society (2003), (37), 11146-11147CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society) A new class of small nonfluorescent fluorogenic substrates, based on release of a phenolic dye from a vinylogous cephalosporin, becomes brightly fluorescent after β-lactamase hydrolysis with up to 153-fold enhancement in the fluorescence intensity. Less than 500 fM of β-lactamase in cell lysates can be readily detected, and β-lactamase expression in living cells can be imaged with a red fluorescence deriv. These new fluorogenic substrates should find uses in clin. diagnostics and facilitate the applications of β-lactamase as a biosensor. >> More from SciFinder https://chemport.cas.org/services/resolver?origin=ACS resolution=options coi=1%3ACAS%3A528%3ADC%252BD3sXmsVelsrk%253D md5=3ff7d10e2798d1adb0174414da8cb98d(b) Rukavishnikov, A.; Gee, K. R.; Johnson, I.; Corry, S. Fluorogenic cephalosporin substrates for β-lactamase TEM-1 Anal. Biochem. 2011, 419, 9– 16Google ScholarThere is no corresponding record for this reference.(c) Zhang, Y.-L.; Xiao, J.-M.; Feng, J.-L.; Yang, K.-W.; Feng, L.; Zhou, L.-S.; Crowder, M. W. A novel fluorogenic substrate for dinuclear Zn(II)-containing metallo-β-lactamases Bioorg. Med. Chem. Lett. 2013, 23, 1676– 1679Google ScholarThere is no corresponding record for this reference.23Yao, H.; So, M.-K.; Rao, J.; Bioluminogenic, A. Substrate for in vivo imaging of β-lactamase activity Angew. Chem., Int. Ed. 2007, 46, 7031– 7034Google ScholarThere is no corresponding record for this reference.24(a) Watanabe, S.; Mizukami, S.; Hori, Y.; Kikuchi, K. Multicolor protein labeling in living cells using mutant β-lactamase-tag technology Bioconjugate Chem. 2010, 21, 2320– 2326[ACS Full Text ], [CAS], Google Scholar24aMulticolor Protein Labeling in Living Cells Using Mutant β-Lactamase-Tag TechnologyWatanabe, Shuji; Mizukami, Shin; Hori, Yuichiro; Kikuchi, KazuyaBioconjugate Chemistry (2010), (12), 2320-2326CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society) Protein labeling techniques using small mol. probes have become important as practical alternatives to the use of fluorescent proteins (FPs) in live cell imaging. These labeling techniques can be applied to more sophisticated fluorescence imaging studies such as pulse-chase imaging. Previously, the authors reported a novel protein labeling system based on the combination of a mutant β-lactamase (BL-tag) with coumarin-derivatized probes and its application to specific protein labeling on cell membranes. In this paper, the authors demonstrated the broad applicability of the authors\' BL-tag technol. to live cell imaging by the development of a series of fluorescence labeling probes for this technol., and the examn. of the functions of target proteins. These new probes have a fluorescein or rhodamine chromophore, each of which provides enhanced photophys. properties relative to coumarins for the purpose of cellular imaging. These probes were used to specifically label the BL-tag protein and could be used with other small mol. fluorescent probes. Simultaneous labeling using the authors\' new probes with another protein labeling technol. was effective. In addn., it was also confirmed that this technol. has a low interference with respect to the functions of target proteins in comparison to GFP. Highly specific and fast covalent labeling properties of this labeling technol. is expected to provide robust tools for investigating protein functions in living cells, and future applications can be improved by combining the BL-tag technol. with conventional imaging techniques. The combination of probe synthesis and mol. biol. techniques provides the advantages of both techniques and can enable the design of expts. that cannot currently be performed using existing tools. >> More from SciFinder https://chemport.cas.org/services/resolver?origin=ACS resolution=options coi=1%3ACAS%3A528%3ADC%252BC3cXhtlWhsr%252FP md5=3a0506ea01083309ef47f6989a2a25b0(b) Mizukami, S.; Watanabe, S.; Akimoto, Y.; Kikuchi, K. No-wash protein labeling with designed fluorogenic probes and application to real-time pulse-chase analysis J. Am. Chem. Soc. 2012, 134, 1623– 1629[ACS Full Text ], [CAS], Google Scholar24bNo-Wash Protein Labeling with Designed Fluorogenic Probes and Application to Real-Time Pulse-Chase AnalysisMizukami, Shin; Watanabe, Shuji; Akimoto, Yuri; Kikuchi, KazuyaJournal of the American Chemical Society (2012), (3), 1623-1629CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society) Small mol. labeling techniques for cellular proteins under physiol. conditions are very promising for revealing new biol. functions. The authors developed a no-wash fluorogenic labeling system by exploiting fluorescence resonance energy transfer (FRET)-based fluorescein-cephalosporin-azopyridinium probes and a mutant β-lactamase tag. Fast quencher elimination, hydrophilicity, and high resistance against autodegrdn. were achieved by rational refinement of the structure. By applying the probe to real-time pulse-chase anal., the trafficking of epidermal growth factor receptors between cell surface and intracellular region was imaged. In addn., membrane-permeable derivatization of the probe enabled no-wash fluorogenic labeling of intracellular proteins. >> More from SciFinder https://chemport.cas.org/services/resolver?origin=ACS resolution=options coi=1%3ACAS%3A528%3ADC%252BC38XktVGntw%253D%253D md5=da0f68ecab3c42879250946fefd12211(c) Shao, Q.; Xing, B. Enzyme responsive luminescent ruthenium(II) cephalosporin probe for intracellular imaging and photoinactivation of antibiotics resistant bacteria Chem. Commun. 2012, 48, 1739– 1741Google ScholarThere is no corresponding record for this reference.25References for plasmid production:(a) Griffin, D. H.; Richmond, T. K.; Sanchez, C.; Jon Moller, A.; Breece, R. M.; Tierney, D. L.; Bennett, B.; Crowder, M. W. Structural and kinetic studies on metallo-β-lactamase IMP-1 Biochemistry 2011, 50, 9125– 9134(IMP-1)[ACS Full Text ], Google ScholarThere is no corresponding record for this reference.(b) de Seny, D.; Prosperi-Meys, C.; Bebrone, C.; Maria Rossolini, G.; Page, M. I.; Noel, P.; Frère, J.-M.; Galleni, M. Mutational analysis of the two zinc-binding sites of the Bacillus cereus 569/H/9 metallo-β-lactamase Biochem. J. 2002, 363, 687– 696(Bc II)Google ScholarThere is no corresponding record for this reference.(c) Green, V. L.; Verma, A.; Owens, R. J.; Phillipsa, S. E. V.; Carr, S. B. Structure of New Delhi metallo-β-lactamase 1 (NDM-1) Acta Crystallogr. 2011, F67, 1160– 1164(NDM-1)Google ScholarThere is no corresponding record for this reference.26Berrow, N. S.; Alderton, D.; Sainsbury, S.; Nettleship, J.; Assenberg, R.; Rahman, N.; Stuart, D. I.; Owens, R. J. A versatile ligation-independent cloning method suitable for high-throughput expression screening applications Nucleic Acids Res. 2007, 35, e45Google ScholarThere is no corresponding record for this reference.27Bebrone, C.; Moali, C.; Mahy, F.; Rival, S.; Docquier, J.-D.; Maria Rossolini, G.; Fastrez, J.; Pratt, R. F.; Frère, J.-M.; Galleni, M. CENTA as a chromogenic substrate for studying β-lactamases Antimicrob. Agents Chemother. 2001, 45, 1868– 1871Google ScholarThere is no corresponding record for this reference.28For experimental details see the Supporting Information.There is no corresponding record for this reference.29Typically mCPBA oxidation to give the (S)-sulfoxide; see:Kaiser, G. V.; Cooper, R . D. G.; Koehler, R. E.; Murphy, C. F.; Webber, J. A.; Wright, I. G.; van Heyningen, E. M. Transformation of Δ2-cephem to Δ3-cephem by oxidation-reduction at sulfur J. Org. Chem. 1970, 35, 2430– 2433[ACS Full Text ], Google ScholarThere is no corresponding record for this reference.30Goddard, J.-P.; Reymond, J.-L. Enzyme assays for high-throughput screening Curr. Opin. Biotechnol. 2004, 15, 314– 322[Crossref], [PubMed], [CAS], Google Scholar30Enzyme assays for high-throughput screeningGoddard, Jean-Philippe; Reymond, Jean-LouisCurrent Opinion in Biotechnology (2004), (4), 314-322CODEN: CUOBE3; ISSN:0958-1669. (Elsevier Ltd.) A review. Assaying enzyme-catalyzed transformations in high-throughput is crucial to enzyme discovery, enzyme engineering and the drug discovery process. In enzyme assays, catalytic activity is detected using labeled substrates or indirect sensor systems that produce a detectable spectroscopic signal upon reaction. Recent advances in the development of high-throughput enzyme assays have identified new labels and chromophores to detect a wide range of enzymes activities. Enzyme activity profiling and fingerprinting have also been used as tools for identification and classification, while microarray formats have been devised to increase throughput. >> More from SciFinder https://chemport.cas.org/services/resolver?origin=ACS resolution=options coi=1%3ACAS%3A528%3ADC%252BD2cXmsVKms7c%253D md5=5f1248d6031258e0f91a8c4d3f3bee8d31Xie, H.; Mire, J.; Kong, Y.; Chang, M.; Hassounah, H. A.; Thornton, C. N.; Sacchettini, J. C.; Cirillo, J. D.; Rao, J. Rapid point-of-care detection of the tuberculosis pathogen using a BlaC-specific fluorogenic probe Nat. Chem. 2012, 4, 802– 809[Crossref], [PubMed], [CAS], Google Scholar31Rapid point-of-care detection of the tuberculosis pathogen using a BlaC-specific fluorogenic probeXie, Hexin; Mire, Joseph; Kong, Ying; Chang, Mi Hee; Hassounah, Hany A.; Thornton, Chris N.; Sacchettini, James C.; Cirillo, Jeffrey D.; Rao, JianghongNature Chemistry (2012), (10), 802-809CODEN: NCAHBB; ISSN:1755-4330. (Nature Publishing Group) Early diagnosis of tuberculosis can dramatically reduce both its transmission and the assocd. death rate. The extremely slow growth rate of the causative pathogen, Mycobacterium tuberculosis (Mtb), however, makes this challenging at the point of care, particularly in resource-limited settings. Here the authors report the use of BlaC (an enzyme naturally expressed/secreted by tubercle bacilli) as a marker and the design of BlaC-specific fluorogenic substrates as probes for Mtb detection. These probes showed an enhancement by 100-200 times in fluorescence emission on BlaC activation and a 1000-fold selectivity for BlaC over TEM-1 β-lactamase, an important factor in reducing false-pos. diagnoses. Insight into the BlaC specificity was revealed by successful co-crystn. of the probe/enzyme mutant complex. A refined green fluorescent probe (CDG-OMe) enabled the successful detection of live pathogen in less than ten minutes, even in unprocessed human sputum. This system offers the opportunity for the rapid, accurate detection of very low nos. of Mtb for the clin. diagnosis of tuberculosis in sputum and other specimens. >> More from SciFinder https://chemport.cas.org/services/resolver?origin=ACS resolution=options coi=1%3ACAS%3A528%3ADC%252BC38Xht12jtrnP md5=1ad9514e98464cc6b7940f0fda45a3bb32The optimal absorption wavelength for umbelliferone in HEPES buffer was found to be 330 nm with a second absorption band at 380 nm. Fluorogenic substrates FC3–FC5 did not show this second absorption band at 380 nm, allowing the specific excitation of umbelliferone at this wavelength.There is no corresponding record for this reference.33Kim, Y.; Tesar, C.; Mire, J.; Jedrzejczak, R.; Binkowski, A.; Babnigg, G.; Sacchettini, J.; Joachimiak, A. Structure of apo- and monometalated forms of NDM-1—A highly potent carbapenem-hydrolyzing metallo-β-lactamase PLoS One 2011, 6, e24621Google ScholarThere is no corresponding record for this reference.34Docquier, J.-D.; Lamotte-Brasseur, J.; Galleni, M.; Amicosante, G.; Frère, J.-M.; Maria Rossolini, G. On functional and structural heterogeneity of VIM-type metallo-β-lactamases J. Antimicrob. Chemother. 2003, 51, 257– 266[Crossref], [PubMed], [CAS], Google Scholar34On functional and structural heterogeneity of VIM-type metallo-β-lactamasesDocquier, Jean-Denis; Lamotte-Brasseur, Josette; Galleni, Moreno; Amicosante, Gianfranco; Frere, Jean-Marie; Rossolini, Gian MariaJournal of Antimicrobial Chemotherapy (2003), (2), 257-266CODEN: JACHDX; ISSN:0305-7453. (Oxford University Press) The VIM metallo-β-lactamases are emerging resistance determinants, encoded by mobile genetic elements, that have recently been detected in multidrug-resistant nosocomial isolates of Pseudomonas aeruginosa and other Gram-neg. pathogens. In this work a T7-based expression system for overprodn. of the VIM-2 enzyme by Escherichia coli was developed, which yielded ∼80 mg of protein per L of culture. The enzyme was mostly released into the medium, from which it was recovered at 99% purity by an initial ammonium sulfate pptn. followed by two chromatog. steps, with almost 80% efficiency. Detn. of kinetic parameters of VIM-2 under the same exptl. conditions previously used for VIM-1 (the first VIM-type enzyme detected in clin. isolates, which is 93% identical to VIM-2) revealed significant differences in Km values and/or turnover rates with several substrates, including penicillins, cephalosporins and carbapenems. Compared with VIM-1, VIM-2 is more susceptible to inactivation by chelators, indicating that the zinc ions of the latter are probably more loosely bound. These data indicated that at least some of the amino acid differences between the two proteins have functional significance. Mol. modeling of the two enzymes identified some amino acid substitutions, including those at positions 223, 224 and 228 (in the BBL numbering), that could be relevant to the changes in catalytic behavior. >> More from SciFinder https://chemport.cas.org/services/resolver?origin=ACS resolution=options coi=1%3ACAS%3A528%3ADC%252BD3sXovFehtA%253D%253D md5=136c3dfaac6f129c5006d9a9c344733735Laraki, N.; Franceschini, N.; Rossolini, G. M.; Santucci, P.; Meunier, C.; de Pauw, E.; Amicosante, G.; Frère, J.-M.; Galleni, M. Biochemical characterization of the Pseudomonas aeruginosa 101/1477 metallo-β-lactamase IMP-1 produced by Escherichia coli Antimicrob. Agents Chemother. 1999, 43, 902– 906Google ScholarThere is no corresponding record for this reference.36Murphy, T. A.; Simm, A. M.; Toleman, M. A.; Jones, R. N.; Walsh, T. R. Biochemical characterization of the acquired metallo-β-lactamase SPM-1 from Pseudomonas aeruginosa Antimicrob. Agents Chemother. 2003, 47, 582– 587Google ScholarThere is no corresponding record for this reference.37Paul-Soto, R.; Hernadez-Valladares, M.; Fonzé, E.; Goussard, S.; Courvalin, P.; Frère, J.-M. Mono- and binuclear Zn-β-lactamase from Bacteroides fragilis: Catalytic and structural roles of the zinc ions FEBS Lett. 1998, 438, 137– 140Google ScholarThere is no corresponding record for this reference.38Felici, A.; Amicosante, G. Kinetic Analysis of extension of substrate specificity with Xanthomonas maltophilia, Aeromonas hydrophila, and Bacillus cereus metallo-β-lactamases Antimicrob. Agents Chemother. 1995, 39, 192– 199[Crossref], [PubMed], [CAS], Google Scholar38Kinetic analysis of extension of substrate specificity with Xanthomonas maltophilia, Aeromonas hydrophila, and Bacillus cereus metallo-β-lactamasesFelici, Antonio; Amicosante, GianfrancoAntimicrobial Agents and Chemotherapy (1995), (1), 192-9CODEN: AMACCQ; ISSN:0066-4804. (American Society for Microbiology) Twenty β-lactam mols., including penicillins, cephalosporins, penems, carbapenems, and monobactams, were investigated as potential substrates for Xanthomonas maltophilia ULA-511, Aeromonas hydrophila AE036, and Bacillus cereus 5/B/6 metallo-β-lactamases. A detailed anal. of the kinetic parameters examd. confirmed these enzymes to be broad-spectrum β-lactamases with different ranges of catalyst efficiency. Cefoxitin and moxalactam, substrates for the β-lactamases from X. maltophilia ULA-511 and B. cereus 5/B/6, behaved as inactivators of the A. hydrophila AE036 metallo-β-lactamase, which appeared to be unique among the enzymes tested in this study. In addn., we report a new, faster, and reliable purifn. procedure for the B. cereus 5/B/6 metallo-β-lactamase, cloned in Escherichia coli HB101. >> More from SciFinder https://chemport.cas.org/services/resolver?origin=ACS resolution=options coi=1%3ACAS%3A528%3ADyaK2MXislKksbo%253D md5=23b390c06b4e125d01dffcdc36c70c2139Young, D.; Toleman, M. A.; Giske, G. C.; Cho, C. H.; Sundman, K.; Lee, K.; Walsh, T. R. Characterization of a new metallo-β-lactamase gene, blaNDM-1, and a novel erythromycin esterase gene carried on a unique genetic structure in Klebsiella pneumoniae sequence type 14 from India Antimicrob. Agents Chemother. 2009, 53, 5046– 5054Google ScholarThere is no corresponding record for this reference.40Badarau, A.; Llinás, A.; Laws, A. P.; Damblon, C.; Page, M. I. Inhibitors of metallo-β-lactamase generated from β-lactam antibiotics Biochemistry 2005, 44, 8578– 8589[ACS Full Text ], Google ScholarThere is no corresponding record for this reference.41Astrid Zervosen, A.; Hernandez Valladares, M.; Devreese, B.; Prosperi-Meys, C.; Adolph, H.-W.; Sandra Mercuri, P.; Vanhove, M.; Amicosante, G.; van Beeumen, J.; Frère, J.-M.; Galleni, M. Inactivation of Aeromonas hydrophila metallo-β-lactamase by cephamycins and moxalactam Eur. J. Biochem. 2001, 268, 3840– 3850[Crossref], [PubMed], Google ScholarThere is no corresponding record for this reference.42Fonseca, F.; Arthur, C. J.; Bromley, E. H. C.; Samyn, B.; Moerman, P.; Saavedra, M. J.; Correia, A.; Spencer, J. Biochemical characterization of Sfh-I, a subclass B2 metallo-β-lactamase from Serratia fonticola UTAD54 Antimicrob. Agents Chemother. 2011, 55, 5392– 5395Google ScholarThere is no corresponding record for this reference.43Low KM values can result in a low signal readout; in particular, when chromogenic substrates are used, this can decrease the sensitivity of the method (i.e., lead to high interexperimental error during kinetic measurements). Moreover, by using a high-affinity substrate (low KM), it is generally not possible to detect potential slow-binding inhibitors as, for example, in the case of NDM-1/nitrocefin, where the substrate presented a low micromolar range KM, with the enzyme concentration being in the same micromolar range (also see ref 44).There is no corresponding record for this reference.44Siemann, S.; Clarke, A. J.; Viswanatha, T.; Dmitrienko, G. I. Thiols as classical and slow-binding inhibitors of IMP-1 and other binuclear metallo-β-lactamases Biochemistry 2003, 42, 1673– 1683[ACS Full Text ], Google ScholarThere is no corresponding record for this reference.45(a) Badarau, A.; Llinas, A.; Laws, A. P.; Damblon, C.; Page, M. I. Inhibitors of metallo-β-lactamase generated from β-lactam antibiotics Biochemistry 2005, 44, 8578– 8589[ACS Full Text ], Google ScholarThere is no corresponding record for this reference.(b) Murphy, T. A; Catto, L. E.; Halford, S. E.; Hadfield, A. T.; Minor, W.; Walsh, T. R.; Spencer, J. Crystal structure of Pseudomonas aeruginosa SPM-1 provides insights into variable zinc affinity of metallo-β-lactamases J. Mol. Biol. 2006, 357, 890– 903Google ScholarThere is no corresponding record for this reference.46Zhang, H.; Hao, Q. Crystal structure of NDM-1 reveals a common β-lactam hydrolysis mechanism FASEB J. 2011, 25, 2574– 2582[Crossref], [PubMed], [CAS], Google Scholar46Crystal structure of NDM-1 reveals a common β-lactam hydrolysis mechanismZhang, Hong Min; Hao, QuanFASEB Journal (2011), (8), 2574-2582, 10.1096/fj.11-184036CODEN: FAJOEC; ISSN:0892-6638. (Federation of American Societies for Experimental Biology) Metallo-β-lactamases (MBLs) hydrolyze most β-lactam antibiotics, and bacteria contg. this kind of enzyme pose a serious threat to the public health. The newly identified New Delhi MBL (NDM-1) is a new member of this family that shows tight binding to penicillin and cephalosporins. The rapid dissemination of NDM-1 in clin. relevant bacteria has become a global concern. However, no clin. useful inhibitors against MBLs exist, partly due to the lack of knowledge about the catalysis mechanism of this kind of enzyme. Here we report the crystal structure of this novel enzyme in complex with a hydrolyzed ampicillin at its active site at 1.3-Å resoln. Structural comparison with other MBLs revealed a new hydrolysis mechanism applicable to all three subclasses of MBLs, which might help the design of mechanism-based inhibitors. >> More from SciFinder https://chemport.cas.org/services/resolver?origin=ACS resolution=options coi=1%3ACAS%3A528%3ADC%252BC3MXpvFersrk%253D md5=32c83d7babafe70cef7ff785b855e14247Garcia-Saez, I.; Docquier, J. D.; Rossolini, G. M.; Dideberg, O. The three-dimensional structure of VIM-2, a Zn-β-lactamase from Pseudomonas aeruginosa in its reduced and oxidised form J. Mol. Biol. 2008, 375, 604– 611[Crossref], [PubMed], [CAS], Google Scholar47The Three-Dimensional Structure of VIM-2, a Zn-β-Lactamase from Pseudomonas aeruginosa in Its Reduced and Oxidized FormGarcia-Saez, I.; Docquier, J.-D.; Rossolini, G. M.; Dideberg, O.Journal of Molecular Biology (2008), (3), 604-611CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.) The crystal structures of the universally widespread metallo-β-lactamase (MBL) Verona integron-encoded MBL (VIM)-2 from Pseudomonas aeruginosa have been solved in their native form as well as in an unexpected oxidized form. This carbapenem-hydrolyzing enzyme belongs to the so-called B1 subfamily of MBLs and shares the folding of αβ/βα sandwich, consisting of a core of β-sheet surrounded by α-helixes. Surprisingly, it showed a high tendency to be strongly oxidized at the catalytic cysteine located in the Cys site, Cys 221, which, in the oxidized structure, becomes a cysteinesulfonic residue. Its native structure was obtained only in the presence of Tris(2-carboxyethyl)phosphine. This oxidn. might be a consequence of a lower affinity for the second Zn located in the Cys site that would also explain the obsd. susceptibility of VIM-2 to chelating agents. This modification, if present in nature, might play a role in catalytic down-regulation. Comparison between native and oxidized VIM-2 and a predicted model of VIM-1 (which shows one residue different in the Cys site compared with VIM-2) is performed to explain the different activities and antibiotic specificities. >> More from SciFinder https://chemport.cas.org/services/resolver?origin=ACS resolution=options coi=1%3ACAS%3A528%3ADC%252BD2sXhsVOlt73K md5=8dfa1db3d5cb7727be63df010892101f48(a) Goodsell, D. S.; Morris, G. M.; Olson, A. J. Automated docking of flexible ligands: Applications of AutoDock J. Mol. Recognit. 1996, 9, 1– 5[Crossref], [PubMed], [CAS], Google Scholar48aAutomated docking of flexible ligands: application of AutoDockGoodsell, David S.; Morris, Garrett M.; Olson, Arthur J.Journal of Molecular Recognition (1996), (1), 1-5CODEN: JMORE4; ISSN:0952-3499. (Wiley) A review with 23 refs. AutoDock is a suite of C programs used to predict the bound conformations of a small, flexible ligand to a macromol. target of known structure. The technique combines simulated annealing for conformation searching with a rapid grid-based method of energy evaluation. This paper reviews recent applications of the technique and describes the enhancement included in the current release. >> More from SciFinder https://chemport.cas.org/services/resolver?origin=ACS resolution=options coi=1%3ACAS%3A528%3ADyaK28Xis1SqtrY%253D md5=8f8f3964ba0c51da8f8b351e65a40a59(b) Morris, G. M.; Huey, R.; Lindstrom, W.; Sanner, M. F.; Belew, R. K.; Goodsell, D. S.; Olson, A. J. AutoDock4 and AutoDockTools4: Automated docking with selective receptor flexibility J. Comput. Chem. 2009, 30, 2785– 2791[Crossref], [PubMed], [CAS], Google Scholar48bAutoDock and AutoDockTools: Automated docking with selective receptor flexibilityMorris, Garrett M.; Huey, Ruth; Lindstrom, William; Sanner, Michel F.; Belew, Richard K.; Goodsell, David S.; Olson, Arthur J.Journal of Computational Chemistry (2009), (16), 2785-2791CODEN: JCCHDD; ISSN:0192-8651. (John Wiley Sons, Inc.) We describe the testing and release of AutoDock4 and the accompanying graphical user interface AutoDockTools. AutoDock4 incorporates limited flexibility in the receptor. Several tests are reported here, including a redocking expt. with 188 diverse ligand-protein complexes and a cross-docking expt. using flexible sidechains in 87 HIV protease complexes. We also report its utility in anal. of covalently bound ligands, using both a grid-based docking method and a modification of the flexible sidechain technique. © 2009 Wiley Periodicals, Inc. J Comput Chem, 2009. >> More from SciFinder https://chemport.cas.org/services/resolver?origin=ACS resolution=options coi=1%3ACAS%3A528%3ADC%252BD1MXht1GitrnK md5=679ce22fc50e9291c9aa16e7a185584549(a) Gillet, V.; Johnson, A. P.; Mata, P.; Sike, S.; Williams, P. SPROUT: A program for structure generation J. Comput.-Aided Mol. Des. 1993, 7, 127– 153[Crossref], [PubMed], [CAS], Google Scholar49aSPROUT: A program for structure generationGillet, Valerie; Johnson, A. Peter; Mata, Pauline; Sike, Sandor; Williams, PhilipJournal of Computer-Aided Molecular Design (1993), (2), 127-53CODEN: JCADEQ; ISSN:0920-654X. SPROUT is a new computer program for constrained structure generation that is designed to generate mols. for a range of applications in mol. recognition. It uses artificial intelligence techniques to moderate the combinatorial explosion that is inherent in structure generation. The program is presented here for the design of enzyme inhibitors. Structure generation is divided into the following 2 phases: (i) primary structure generation to produce mol. graphs to fit the steric constraints; and (ii) secondary structure generation which is the process of introducing appropriate functionality to the graphs to produce mols. that satisfy the secondary constraints, e.g., electrostatics and hydrophobicity. Primary structure generation has been tested on 2 enzyme receptor sites, i.e., the p-amidinophenylpyruvate binding site of trypsin and the acetyl pepstatin binding site of HIV-1 protease. The program successfully generates structures that resemble known substrates and, more importantly, the predictive power of the program has been demonstrated by its ability to suggest novel structures. >> More from SciFinder https://chemport.cas.org/services/resolver?origin=ACS resolution=options coi=1%3ACAS%3A528%3ADyaK3sXmt1Ghtrk%253D md5=2664279a8e55d30e453810235f57837d(b) Gillet, V. J.; Newell, W.; Mata, P.; Myatt, G.; Sike, S.; Zsoldos, Z.; Johnson, A. P. SPROUT: Recent developments in the de novo design of molecules J. Chem. Inf. Comput. Sci. 1994, 34, 207– 217[ACS Full Text ], [CAS], Google Scholar49bSPROUT: Recent developments in the de novo design of moleculesGillet, Valerie J.; Newell, William; Mata, Paulina; Myatt, Glenn; Sike, Sandor; Zsoldos, Zsolt; Johnson, A. PeterJournal of Chemical Information and Computer Sciences (1994), (1), 207-17CODEN: JCISD8; ISSN:0095-2338. SPROUT is a computer program for constrained structure generation. It is designed to generate mols. for a range of applications in mol. recognition. The program uses a no. of approxns. that enable a wide variety of diverse structures to be generated. Practical use of the program is demonstrated in two examples. The first demonstrates the ability of the program to generate candidate inhibitors for a receptor site of known 3D structure, specifically the GDP binding site of p21. In the 2nd example, structures are generated to fit a pharmacophore hypothesis that models morphine agonists. >> More from SciFinder https://chemport.cas.org/services/resolver?origin=ACS resolution=options coi=1%3ACAS%3A528%3ADyaK2cXns1GqtQ%253D%253D md5=cb5c2f6abe6b8a0657968c46fabf784e50Cadag, E.; Vitalis, E.; Lennox, K. P.; Zhou, C. L. E.; Zemla, A. T. Computational analysis of pathogen-borne metallo β-lactamases reveals discriminating structural features between B1 types BMC Res. Notes 2012, 5, 96[Crossref], [CAS], Google Scholar50Computational analysis of pathogen-borne metallo β-lactamases reveals discriminating structural features between B1 typesCadag, Eithon; Vitalis, Elizabeth; Lennox, Kristin P.; Zhou, Carol L. Ecale; Zemla, Adam T.BMC Research Notes (2012), 96CODEN: BRNMAT; ISSN:1756-0500. (BioMed Central Ltd.) Background: Genes conferring antibiotic resistance to groups of bacterial pathogens are cause for considerable concern, as many once-reliable antibiotics continue to see a redn. in efficacy. The recent discovery of the metallo β-lactamase blaNDM-1 gene, which appears to grant antibiotic resistance to a variety of Enterobacteriaceae via a mobile plasmid, is one example of this distressing trend. The following work describes a computational anal. of pathogen-borne MBLs that focuses on the structural aspects of characterized proteins. Results: Using both sequence and structural analyses, we examine residues and structural features specific to various pathogen-borne MBL types. This anal. identifies a linker region within MBL-like folds that may act as a discriminating structural feature between these proteins and specifically resistance-assocd. acquirable MBLs. Recently released crystal structures of the newly emerged NDM-1 protein were aligned against related MBL structures using a variety of global and local structural alignment methods and the overall fold conformation is examd. for structural conservation. Conservation appears to be present in most areas of the protein, yet is strikingly absent within a linker region, making NDM-1 unique with respect to a linker-based classification scheme. Variability anal. of the NDM-1 crystal structure highlights unique residues in key regions as well as identifying several characteristics shared with other transferable MBLs. Conclusions: A discriminating linker region identified in MBL proteins is highlighted and examd. in the context of NDM-1 and primarily three other MBL types: IMP-1, VIM-2 and ccrA. The presence of an unusual linker region variant and uncommon amino acid compn. at specific structurally important sites may help to explain the unusually broad kinetic profile of NDM-1 and may aid in directing research attention to areas of this protein and possibly other MBLs, that may be targeted for inactivation or attenuation of enzymic activity. >> More from SciFinder https://chemport.cas.org/services/resolver?origin=ACS resolution=options coi=1%3ACAS%3A528%3ADC%252BC38XkvFektb0%253D md5=55c75b24400ec5bab78a49a6f383d8c751(a) Stubbs, C. J.; Loenarz, C.; Mecinović, J.; Kheng Yeoh, K.; Hindley, N.; Liénard, B. M.; Sobott, F.; Schofield, C. J.; Flashman, E. Application of a proteolysis/mass spectrometry method for investigating the effects of inhibitors on hydroxylase structure J. Med. Chem. 2009, 52, 2799– 2805[ACS Full Text ], Google ScholarThere is no corresponding record for this reference.(b) Tian, Y.-M.; Yeoh, K. K.; Lee, M. K.; Eriksson, T.; Kessler, B. M.; Kramer, H. B.; Edelmann, M. J.; Willam, C.; Pugh, C. W.; Schofield, C. J.; Ratcliffe, P. J. Differential sensitivity of hypoxia inducible factor hydroxylation sites to hypoxia and hydroxylase inhibitors J. Biol. Chem. 2011, 286, 13041– 13051Google ScholarThere is no corresponding record for this reference.52(a) Heinz, U.; Bauer, R.; Wommer, S.; Meyer-Klaucke, W.; Papamichaels, C.; Bateson, J.; Adolph, H.-W. Coordination geometries of metal ions in D- or L-captopril-inhibited metallo-β-lactamases J. Biol. Chem. 2003, 278, 20659– 20666Google ScholarThere is no corresponding record for this reference.(b) King, D. T.; Worrall, L. J.; Gruninger, R.; Strynadka, N. C. New Delhi metallo-β-lactamase: Structural insights into β-lactam recognition and inhibition J. Am. Chem. Soc. 2012, 134, 11362– 11365[ACS Full Text ], [CAS], Google Scholar52bNew Delhi metallo-β-lactamase: structural insights into β-lactam recognition and inhibitionKing, Dustin T.; Worrall, Liam J.; Gruninger, Robert; Strynadka, Natalie C. J.Journal of the American Chemical Society (2012), (28), 11362-11365CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society) The β-lactam antibiotics have long been a cornerstone for the treatment of bacterial disease. Recently, a readily transferable antibiotic resistance factor called New Delhi metallo-β-lactamase-1 (NDM-1) was been found to confer enteric bacteria resistance to nearly all β-lactams, including the heralded carbapenems, posing a serious threat to human health. The crystal structure of NDM-1 bound to meropenem showed for the 1st time the mol. details of how carbapenem antibiotics are recognized by di-Zn-contg. metallo-β-lactamases. Addnl., product complex structures of hydrolyzed benzylpenicillin-, methicillin-, and oxacillin-bound NDM-1 were solved to 1.8, 1.2, and 1.2 Å, resp., and represent the highest-resoln. structural data for any metallo-β-lactamase reported to date. Finally, the authors present the crystal structure of NDM-1 bound to the potent competitive inhibitor L-captopril, which revealed a unique binding mechanism. An anal. of the NDM-1 active site in these structures revealed key features important for the informed design of novel inhibitors of NDM-1 and other metallo-β-lactamases. >> More from SciFinder https://chemport.cas.org/services/resolver?origin=ACS resolution=options coi=1%3ACAS%3A528%3ADC%252BC38XoslGgsr8%253D md5=3388110aa1369787d2e91e04829a03b453Conversion of IC50 to Ki was performed according to Cer et al.:Cer, R. Z.; Mudunuri, U.; Stephens, R.; Lebeda, F. J. IC50-to-Ki: A Web-based tool for converting IC50 to Ki values for inhibitors of enzyme activity and ligand binding Nucleic Acids Res. 2009, 37, W441– W445Google ScholarThere is no corresponding record for this reference.54Guo, Y.; Wang, J.; Niu, G.; Shui, W.; Sun, Y.; Zhou, H.; Zhang, Y.; Yang, C.; Lou, Z.; Rao, Z. A structural view of the antibiotic degradation enzyme NDM-1 from a superbug Protein Cell 2011, 2, 384– 394[Crossref], [PubMed], [CAS], Google Scholar54A structural view of the antibiotic degradation enzyme NDM-1 from a superbugGuo, Yu; Wang, Jing; Niu, Guojun; Shui, Wenqing; Sun, Yuna; Zhou, Honggang; Zhang, Yaozhou; Yang, Cheng; Lou, Zhiyong; Rao, ZiheProtein Cell (2011), (5), 384-394CODEN: PCREFB; ISSN:1674-800X. (Higher Education Press) Gram-neg. Enterobacteriaceae with resistance to carbapenem conferred by New Delhi metallo-β-lactamase 1 (NDM-1) are a type of newly discovered antibiotic-resistant bacteria. The rapid pandemic spread of NDM-1 bacteria worldwide (spreading to India, Pakistan, Europe, America, and Chinese Taiwan) in less than 2 mo characterizes these microbes as a potentially major global health problem. The drug resistance of NDM-1 bacteria is largely due to plasmids contg. the blaNDM-1 gene shuttling through bacterial populations. The NDM-1 enzyme encoded by the blaNDM-1 gene hydrolyzes β-lactam antibiotics, allowing the bacteria to escape the action of antibiotics. Although biol. functions and structural features of NDM-1 have been proposed according to results from functional and structural investigation of its homologs, the precise mol. characteristics and mechanism of action of NDM-1 have not been clarified. Here, the authors report the 3-dimensional structure of NDM-1 with 2 catalytic Zn2+ ions in its active site. Biol. and mass spectroscopy results revealed that D-captopril could effectively inhibit NDM-1 by binding to its active site with high binding affinity. The unique features concerning the primary sequence and structural conformation of its active site distinguish NDM-1 from other reported metallo-β-lactamases (MBLs) and implicate its role in wide spectrum drug resistance. The authors also discuss the mol. mechanism of NDM-1 action and its essential role in the pandemic of drug-resistant NDM-1 bacteria. These results will provide helpful information for future drug discovery targeting drug resistance caused by NDM-1 and related metallo-β-lactamases. >> More from SciFinder https://chemport.cas.org/services/resolver?origin=ACS resolution=options coi=1%3ACAS%3A528%3ADC%252BC3MXntlahur0%253D md5=cbc06076c1714c860c353bb2bff35454PDB: 1JJTPDB: 3Q6XPDB: 1KO3Supporting InformationSupporting InformationARTICLE SECTIONSJump ToExperimental procedures, characterization of intermediates and target compounds, description of protein production and purification, biological assays, determination of residual activity measurements and IC50 values, and in silico docking data. This material is available free of charge via the Internet at http://pubs.acs.org.jm400769b_si_001.pdf (957.55 kb) Terms & ConditionsMost electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system:http://pubs.acs.org/page/copyright/permissions.html.Export articles to MendeleyGet article recommendations from ACS based on references in your Mendeley library.Export articles to MendeleyGet article recommendations from ACS based on references in your Mendeley library. Please note: If you switch to a different device, you may be asked to login again with only your ACS ID. Please note: If you switch to a different device, you may be asked to login again with only your ACS ID. Please note: If you switch to a different device, you may be asked to login again with only your ACS ID. 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