Induction of apoptosis and anti-tumor activity of BL-K49: A novel and selective small molecule inhibitor of Bcl-2 Free

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Inhibition of the anti-apoptotic function of Bcl-2 represents an attractive new strategy for developing anticancer drugs. The anti-apoptotic function of Bcl-2 is attributed in part by its ability to heterodimerize with pro-apoptotic members such as Bak, Bad or Bax. This BH3 hydrophobic binding pocket in Bcl-2 is essential for its anti-apoptotic function since mutations at this site can abolish this function. Thus, a small molecule that binds to this BH3 site in Bcl-2 may block the binding of Bcl-2 with pro-apoptotic members such as Bak, Bad or Bax. This blocking in turn may inhibit the anti-apoptotic function of Bcl-2 and induce apoptosis in cancer cells with Bcl-2 overexpression. Using a structure-based strategy, we have discovered several classes of novel small molecule inhibitors of BH3/Bcl-2 complex. A small molecule BL-K49 is designed to inhibit the binding of BH3 peptides to this pocket. This molecule could compete a fluorescence-labeled Bad peptide binding to Bcl-2 protein with a high affinity (IC₅₀=0.45 μM) as detected by a fluorescence polarization based binding assay, but not with Bcl-xL protein. BL-K49 preferably inhibits the growth of cancer cells overexpressing Bcl-2 in a dose-dependent manner. BL-K49 inhibits potently the growth of cancer cells with high expression of Bcl-2 protein such as leukemia cell line HL-60 with IC₅₀ at 0.27 μM, and breast cancer cell MDA-231 with IC₅₀ at 0.64 μM. However, BL-K49 is inactive for cancer cells with low expression of Bcl-2 protein, such as T47D cells, with IC₅₀ at 37 μM. There is a reduction of mitochondrial transmembrane potential following 1.25 μM of BL-K49 at 8 hours of treatment using DiOC6₍₃₎ staining. This molecule induces apoptosis in MDA-231 cell in a dose-dependent manner as detected by Annexin-V Propidium iodide flow cytometry analysis, or activation of caspase-3 and PARP following BL-K49 treatment in MDA-231 cells. We further demonstrate that BL-K49 has anti-tumor activity in three xenograft tumor models and is dose-dependent. As a monotherapy at dose of 10 mg/kg (i.p., qdx5, 2 cycles), BL-K49 resulted in 55% tumor growth inhibition in MDA-361 xenograft. At dose of 10 mg/kg and 20 mg/kg, BL-K49 caused 23%, and 56% tumor growth inhibition in prostate cancer PC-3 xenografts, respectively. At dose of 20 mg/kg, the same regimen, only 37% tumor growth inhibition was observed in a fast growing tumor model MDA-231. The anti-tumor efficacy of monotherapy appears related to the growth rate of xenograft models in vivo. Further studies with combination therapy are currently underway. These results warrant further development of a small molecule targeting directly at apoptosis machinery of BH3/Bcl-2 complex as a novel therapy for the treatment of cancers with Bcl-2 overexpression.