1. Preclinical Models and New Therapeutic Agents Unit, IRCCS Regina Elena National Cancer Institute, Via Elio Chianesi 53, Rome, Italy.
2. Department of Chemistry, Sapienza University of Rome, Piazzale Aldo Moro 5, Rome, Italy.
3. Sarcomas and Rare Tumours Departmental Unit- IRCCS Regina Elena National Cancer Institute, Via Elio Chianesi 53, Rome, Italy.
4. Rome Center for Molecular Design, Department of Drug Chemistry and Technology, Sapienza University of Rome, Piazzale Aldo Moro 5, Rome, Italy.
5. Department of Drug Chemistry, Sapienza University of Rome, Piazzale Aldo Moro 5, Rome, Italy.
6. Pasteur Institute, Cenci Bolognetti Foundation, Sapienza University of Rome, Piazzale Aldo Moro 5, Rome, Italy.
7. Institute of Molecular Biology and Pathology, Italian National Research Council, Piazzale A.Moro 5, 20 Rome, Italy.
* Equal contribution as first authors: Elisabetta Valentini, Simona D'Aguanno, Marta Di Martile
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.
Keywords: Bcl-2 family, Bcl-2 family inhibitors, virtual screening, apoptosis, autophagy