Identification of a novel BCL2‐specific inhibitor that binds predominantly to the BH1 domain

Abstract

The antiapoptotic protein BCL2 is overexpressed in several cancers and contributes to prolonged cell survival and chemoresistance, lending itself as an excellent target for cancer therapy. Here, we report the design, synthesis, and characterization of Disarib, a novel BCL2 inhibitor. Disarib showed selective cytotoxicity in BCL2 high cancer cell lines, and CLL patient primary cells, as compared to BCL2 low cell lines. BCL2 knock down in cells rendered remarkable resistance to Disarib, while sensitivity was regained upon its ectopic expression, establishing target specificity. In silico, biochemical and biophysical studies demonstrated strong affinity of Disarib to BCL2, but not to other antiapoptotic BCL2 family members viz., BCL-xL, BCL2A1 etc. Interestingly, biophysical studies showed that BH1 domain deletion mutant demonstrated~67-fold reduction in BCL2-Disarib interaction, while it was only~20-fold in the case of BH3 deletion mutant, suggesting predominant involvement of the BH1 domain for Disarib binding. Thus, we report identification of a novel BCL2 inhibitor with a unique mechanism of BCL2 inhibition, as opposed to the well-studied BH3 domain targeting.

An allosteric inhibitor of Mycobacterium tuberculosis ArgJ: Implications to a novel combinatorial therapy

“…To experimentally determine the binding site of PRK and SRB, we designed an ANS‐based fluorescence titration assay (Iyer et al , ). The Mt ArgJ, saturated with ANS, gives a characteristically high fluorescence intensity at 470 nm, and any molecule that competes for ANS binding site should result in a dose‐dependent decrease in fluorescence.…”

“…To further establish the binding affinity of PRK and SRB to Mt ArgJ, we used thermal shift assay (TSA), a method orthologous to isothermal titration calorimetry (ITC; Niesen et al , ; Iyer et al , ) and is being productively used for drug discovery (Renaud et al , ). Mt ArgJ with varying concentration of PRK/SRB was subjected to gradually increasing temperature, and the shift in melting temperature ( T m ) was calculated.…”

“…Further, the ANS‐ Mt ArgJ complex was titrated against increasing concentrations of PRK/SRB in separate experiments. The decrease in fluorescence due to the displacement of ANS by PRK/SRB from hydrophobic pocket on the protein was monitored (Iyer et al , ). To determine the dissociation constant, net change in the relative fluorescence unit (RFU) was plotted against increasing PRK/SRB concentration (at constant protein and ANS concentrations).…”

“…DMSO was kept constant at 2% throughout the screens. According to the experimental protocol, samples were heated at 0.5°C per minute, ranging from 10 to 95°C, and the fluorescence intensity was measured at the interval of 0.5°C (Niesen et al , ; Iyer et al , ). Cy5 filter with red‐orange color intensity was selected for the SYPRO Orange detection.…”

A Benzothiazole Derivative (5g) Induces DNA Damage And Potent G2/M Arrest In Cancer Cells

“…In order to assay induction of apoptosis in 5g treated Nalm6 cells, Hoechst staining was performed as described before 42 . Briefly, Nalm6 cells (50,000/ml) were seeded in 6 well plates and treated with increasing concentrations of 5g (10 and 30 µM) for a period of 48 h. Cells were harvested and washed with PBS, stained with Hoechst (1 µg/ml) for 10′ at room temperature and imaged using a Zeiss Apotome Fluorescence Microscope (Zeiss, Germany).…”