BMS-345541 is a highly selective inhibitor of the catalytic subunits of IKK-2 and IKK-1 with IC50 of 0.3 μM and 4 μM in cell-free assays, respectively.
Features
Allosteric IKK inhibitor with anti-inflammatory activity.
Targets
IKK2 [1](Cell-free assay)
IKK1 [1](Cell-free assay)
0.3 μM
4 μM
In vitro
BMS-345541 dose-dependently inhibits the TNF-α-stimulated phosphorylation of IκBα in THP-1 monocytic cells with an IC50 of ~4 μM. BMS-345541 inhibits lipopolysaccharide-stimulated tumor necrosis factorα, interleukin-1β, interleukin-8, and interleukin-6 in THP-1 cells with IC50 values in the 1- to 5 μM range. BMS-345541 binds in a mutually exclusive manner with respect to a peptide inhibitor corresponding to amino acids 26 - 42 of IκBα with Ser-32 and Ser-36 changed to aspartates and in a non-mutually exclusive manner with respect to ADP. BMS-345541 binds to similar allosteric sites on IKK-1 and IKK-2, which then affects the active sites of the subunits differently. [1] BMS-345541 affects several mitotic cell cycle transitions, including mitotic entry, prometaphase to anaphase progression and cytokinesis. Adding BMS-345541 to the cells released form arrest in G-phase blocks the activation of Aurora A, B, and C, Cdk1 activation and histone H3 phosphorylation. Treatment of the mitotic cells with BMS-345541 results in precocious cyclin B1 and securin degradation, defective chromosome separation and improper cytokinesis. BMS-345541 is also found to override the spindle checkpoint in nocodazole-arrested cells. These effects are not primarily due to a direct inhibitory effect of BMS-345541 on mitotic kinases such as Cdk1, Aurora A or B, Plk1 or NEK2. [2] BMS-345541 (10 μM) inhibits growth of Normal human epidermal melanocytes, and metastatic melanoma cells (SK-MEL-5, A375, and Hs 294T) by 96% and 99% at 72h, respectively. Application of 100 μM of BMS-345541 to SK-MEL-5 cell culture results in 87% apoptotic cells at 24 h through caspase-independent and AIF-dependent mitochondria-mediated manner. BMS-345541 treatment (10 μM) results in 76% and 95% reduction in IKK activities and NF-kB activity, as well as CXCL1 production. [3] BMS-345541 inhibits the growth of T-cell acute lymphoblastic leukemia (T-ALL) cells line BE-13, RPMI-8402 and DND-41, all three harboring a Notch1 mutation, and T-ALL primary cells from pediatric patients, with IC50 of 2-6 μM. 5 μM BMS-345541 induces an arrest in the G2/M phase of the cell cycle in BE-13 and DND-41 cells, and sub-G1 peak increase in RPMI-8402 cells. 5 μM BMS-345541 treated for 16 h leads to an increase in apoptotic cells in all these cell, accompanied by a time-dependent cleavage of procaspase-8, procaspase-3 and poly (ADP-ribose) polymerase (PARP). BMS-34554 (5 μM) induces a time dependent dephosphorylation of IκBα and p65. T-ALL cells treated with BMS-345541 displays nuclear translocation of FOXO3a and restoration of its functions, including control of p21Cip1 expression levels. [4] BMS-345541 inhibits the TNFα-induced expression of both ICAM-1 and VCAM-1 in human umbilical vein endothelial cells with IC50 of 5 μM. [5]
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Assay
Methods
Test Index
PMID
Western blot
caspase-3 (p17) / PARP / cleaved PARP ;
PubMed: 18544167
BMS-345541 induced caspase-3 and PARP cleavage C8166. MT-2, C8166, and CEM cells were treated with BMS-345541 at 0.1, 0.5, 1, and 5 μM for 48 hr. Total cell extracts were subjected to Western blot analysis for caspase-3 and PARP. β-actin Western blot was used as internal control. The results of caspase-3 were quantitated and normalized with β-actin. The ratio of c/un PARP was calculated by dividing cleaved PARP to un-cleaved PARP (data not shown).
IkB / p-IkB / p65 / p-p65 / p50 / p52 / Tax ;
PubMed: 18544167
Effect of BMS-345541 on inhibition of IκB and p65 phosphorylation in vivo. MT-2, C8166, and CEM cells were treated with BMS-345541 at 0.1, 0.5, 1, and 5 μM for 48 hr. Total cell extracts were collected and subjected to Western blot analysis using anti-IκB, phospho IκB (ser 32), p65, phospho p65 (ser 536), p50, p52, Tax and actin. Twenty five microgram of each extract was used to separate on a 4–20% SDS/PAGE. Levels of total IκB and p65 did not change between cell types, however there was a dramatic increase of phosphor-IκB and phosphor-p65 in HTLV-1 infected cells and their suppression by BMS-345541 which inhibits IKKβ activity in vivo.
18544167
Immunofluorescence
NF-κB-p65;
PubMed: 28409156
HEp-2 cells were pretreated with 20 μM CA15 and 20 μM BMS for 1 h followed by incubation with TNF-α(5 ng/ml) for 60 min. Cells were then incubated with NF-κB-p65 antibody and PE-conjugated secondary antibody (red), and the nuclei were stained with DAPI (blue). The photographs were obtained by fluorescence microscope (magnification ×200). TNF-α: tumor necrosis factor-α; Cur: curcumin; BMS: BMS-345541.
Cyclin D1 / p-Cyclin D1 ;
PubMed: 27546592
(D) Platelet releasates-mediated nuclear localization of cyclin D1 was measured in EA.hy926 cells co-incubated with ERK1/2 inhibitor (PD-98059), mTORC1 inhibitor (rapamycin), IKKα and IKKβ inhibitors (BAY11-7082 and BMS-345541 respectively). (E) The same as D except that platelet releasates-mediated inhibition of cyclin D1 phosphorylation was monitored. The scale for the microscopic figure is 20µm.
28409156 27546592
In vivo
BMS-345541 effectively inhibits melanoma tumor growth in a dose-dependent manner. Tumor-bearing mice treated with 75 mg/kg of BMS-345541 shows effective inhibition of growth of SK-MEL-5, A375, and Hs 294T tumors by 86 %, 69% and 67%, respectively, when compared with control animals treated with vehicle alone.[3]BMS-345541 administered orally at doses of 100 mg/kg, reduces the severity of dextran sulfate sodium-induced colitis in mice with weight ratio, clinical scoring of colons, mean injury score and mean inflammation score of 0.86 (vs 0.77 of vehicle group), 1.0 (vs 2.5 of vehicle group), 5.66 (vs 8.52 of vehicle group), 6.82 (vs 12.33 of vehicle group), respectively. [6] BMS-345541 (100 mg/kg), when administered by oral gavage in water once daily beginning at the time of the first collagen immunization, inhibits clinical signs of disease in the murine CIA model (0 vs ~8 of vehicle group), accompanied by reduced paw swelling. BMS-345541 (100 mg/kg) reduces cumulative arthritis injury score from 4.4 to 0, accompanied by lower degrade of tibiotarsal joints and severity of inflammation, synovial hyperplasia, bone resorption, and cartilage erosion. No discernible injury is observed in the joints of animals, which is histologically indistinguishable from those from age-matched, disease-free control animals. BMS-345541 dose-dependently inhibits IL-1β message, with animals in the 100 mg/kg dose group showing levels comparable with those of disease-free control animals. [7]
