Targeting the anti-apoptotic Bcl-2 family proteins: machine learning virtual screening and biological evaluation of new small molecules

Abstract

Bcl-2 family anti-apoptotic proteins are overexpressed in several hematological and solid tumors, and contribute to tumor formation, progression, and resistance to therapy. They represent a promising therapeutic avenue to explore for cancer treatment. Venetoclax, a Bcl-2 inhibitor is currently used for hematological malignancies or is undergoing clinical trials for either hematological or solid tumors. Despite these progresses, ongoing efforts are focusing on the identification and development of new molecules targeting Bcl-2 protein and/or other family members. Methods: Machine learning guided virtual screening followed by surface plasmon resonance, molecular docking and pharmacokinetic analyses were performed to identify new inhibitors of anti-apoptotic members of Bcl-2 family and their pharmacokinetic profile. The sensitivity of cancer cells from different origin to the identified compounds was evaluated both in in vitro (cell survival, apoptosis, autophagy) and in vivo (tumor growth in nude mice) preclinical models. Results: IS20 and IS21 were identified as potential new lead compounds able to bind Bcl-2, Bcl-xL and Mcl-1 recombinant proteins. Molecular docking investigation indicated IS20 and IS21 could bind into the Beclin-1 BH3 binding site of wild type Bcl-2, Bcl-xL and Mcl-1 proteins. In particular, although the IS21 docked conformation did not show a unique binding mode, it clearly showed its ability in flexibly adapting to either BH3 binding sites. Moreover, both IS20 and IS21 reduced cell viability, clonogenic ability and tumor sphere formation, and induced apoptosis in leukemic, melanoma and lung cancer cells. Autophagosome formation and maturation assays demonstrated induction of autophagic flux after treatment with IS20 or IS21. Experiments with z-VAD-fmk, a pan-caspase inhibitor, and chloroquine, a late-stage autophagy inhibitor, demonstrated the ability of the two compounds to promote apoptosis by autophagy. IS21 also reduced in vivo tumor growth of both human leukemia and melanoma models. Conclusion: Virtual screening coupled with in vitro and in vivo experimental data led to the identification of two new promising inhibitors of anti-apoptotic proteins with good efficacy in the binding to recombinant Bcl-2, Bcl-xL and Mcl-1 proteins, and against different tumor histotypes.

Bcl-2 family inhibitors sensitize human cancer models to target therapy

“…Moreover, docking experiments indicated that IS21 is able to displace more effectively BH3 helix from Bcl-2 and Bcl-xL and with lower potency from Mcl-1. All these data con rmed the docking analysis and are in good agreement with the Surphace Plasmon Resonance experimental data previously published [41], indicating that the main targets of IS21 are Bcl-2 and Bcl-xL. Also, Mcl-1, although endowed with less a nity towards IS21 than Bcl-xL and Bcl-2, may represent an important player dictating the IS21 response in our biological settings.…”

“…IS21 and ABT-199 sensitize BRAF mutant melanoma cells to MAPKi in vivo IS21 and ABT-199, administered as single agents, were previously reported to reduce in vivo tumor growth of melanoma models [41]. To evaluate their effect in combinatorial regimes and to validate our results in vivo, mice carrying A375luc tumors were treated with double (dabrafenib + trametinib), or triple (dabrafenib + trametinib + IS21 or dabrafenib + trametinib + ABT-199) combinations (Fig.…”

“…We rstly assessed the e cacy of IS21, a compound able to bind Bcl-2, Bcl-xL and Mcl-1 proteins [41], in tumor histotypes in which Bcl-2 family proteins play a key role, such as T-ALL [48], melanoma [49], ovarian cancer [27], pancreatic cancer [50] and NSCLC [51].…”

“…Speci c inhibition of Bcl-xL has also been reported to improve ovarian cancer's response to taxane-based therapy and to poly-ADP ribose polymerase inhibitors (PARPi) [29,[38][39][40]. We have recently identi ed IS21, a pan-inhibitor of anti-apoptotic proteins with antitumor activity in different histotypes, including melanoma [41]. Here, by using in vitro and in vivo approaches, we explored the sensitivity of cell lines from different tumor types to IS21 and ABT-199 as single agents, and their ability to affect the sensitivity of human melanoma and ovarian carcinoma models to the standard-of-care agents.…”

A Systematic Review of Apoptosis in Correlation With Cancer: Should Apoptosis Be the Ultimate Target for Cancer Treatment?

“…Cancer cells often over-express different proteins that have a major contribution in resisting the cascade of apoptosis. Multiple mechanisms induced by cancer cells rescue them from programmed cell death, especially through the over-expression of the anti-apoptotic molecules [ 7 ]. In fact, the majority of apoptosis signaling research is dependent on B-cell lymphoma 2 homology 3 (BH3) proteins [ 8 ].…”