ARV-825 is a BRD4 Inhibitor that recruits BRD4 to the E3 ubiquitin ligase cereblon, leading to fast, efficient, and prolonged degradation of BRD4 and sustained down-regulation of MYC.
Targets
BRD4 BD2 [1](Cell-free assay)
BRD4 BD1 [1](Cell-free assay)
28 nM(Kd)
90 nM(Kd)
In vitro
Compared with the BRD4 inhibitors, ARV-825 treatment results in a strikingly more pronounced effect on the levels of c-MYC, and downstream cell proliferation and apoptosis induction in BL (Burkitt’s Lymphoma) cell lines[1]. The IC50s of ARV-825 for all tested cell lines and primary AML cells at 72 hours are in the low nanomolar range (2-50 nM). ARV-825 treatment reduces PIM1 levels and phosphorylation of CXCR4 in AML cells while overexpression of PIM1 or Myc reverses the phenomena[3].
Assay
Methods
Test Index
PMID
Western blot
BRD1 / BRD2 / BRD3 / BRD4 / p21;
PubMed: 30903020
Temporal effects of ARV-825 and OTX015 (200 nM) on indicated proteins in LPS141 cells.
c-MYC;
PubMed: 26051217
Namalwa (left) and Ramos (right) cells were treated overnight with increasing doses of ARV-825 (up to 1.0 µM), or JQ1 (up to 10.0 µM), or OTX015 (up to 10.0 µM); lysates were analyzed by immunoblot for BRD4, c-MYC and actin.
PARP / cleaved PARP ;
PubMed: 26051217
Ramos and CA-46 cells were treated with increasing doses of ARV-825 (up to 1.0 µM), or JQ1 and OTX015 (up to 10.0 µM) for 48 hours. Lysates were collected and analyzed by immunoblot for PARP cleavage with actin as loading control
CDK6 / CDK4 ;
PubMed: 29581547
Effects of ARV-825 and ARV-763 on the expression levels of p21, CDK4, and CDK6 were examined by Western blotting of MM1.S cell extracts 24 hours after exposure to the indicated drug concentrations of drugs, with β-actin used as a loading control.
cleaved caspase 9 / cleaved caspase 3 ;
PubMed: 29581547
Programmed cell death activation was confirmed by preparing lysates of the indicated myeloma cell lines and subjecting them to Western blotting for cleaved PARP, Caspase-3 and -9, and β-actin as a loading control.
The effect of ARV-825 or ARV-763 on the expression levels of key intermediates in the PI3K-Akt-mTOR pathway signaling were probed by Western blotting of MM1.S (left panel) and RPMI 8226 (right panel) cells. These were exposed for 24 hours to the indicated䲧疝Ỵ疞㧀疜膉痘瘿⟸ᾰƌÐ㺣痖帉痖Ѐ瑖堘𢡄빢᎒Ð
SET-2 cells were treated with the indicated concentrations of ARV-825 or OTX015 for 18 hours. Total cell lysates were prepared and immunoblot analyses were conducted as indicated. The expression levels of β-Actin in the cell lysates served as the loading 䲧疝Ỵ疞
30903020 26051217 29581547 28042144
Immunofluorescence
BRD4 ;
PubMed: 28042144
SET2 cells were treated with the indicated concentrations of ARV-825 or OTX015 for 16 hours. Then, cells were cytospun onto glass slides and immunostained for BRD4 expression. Cells were counterstained with DAPI and images were acquired utilizing confocal䲧疝Ỵ疞㧀疜膉痘瘿⟸ᾰƌÐ㺣痖
28042144
Growth inhibition assay
Cell viability ;
PubMed: 26051217
Various BL cell lines were seeded at 50000 cells/100ul in 96-well plates and then treated with increasing doses of ARV-825, JQ1 and OTX015; relative proliferation was determined by CellTiter-Glow (CTG) assay 72 hours later.
26051217
In vivo
In a mouse model of human leukemia, the leukemia burdens are significantly lower in the ARV-825 treated mice as confirmed by luciferase imaging, flow cytometry, spleen size and survived longer compared to control mice[3].
