Shedding Light on the Interaction of Human Anti-Apoptotic Bcl-2 Protein with Ligands through Biophysical and in Silico Studies

• Published in: International journal of molecular sciences Vol. 20; no. 4; p. 860
• Main Authors: Ramos, Joao, Muthukumaran, Jayaraman, Freire, Filipe, Paquete-Ferreira, João, Otrelo-Cardoso, Ana Rita, Svergun, Dmitri, Panjkovich, Alejandro, Santos-Silva, Teresa
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 16.02.2019; MDPI
• Subjects: Apoptosis; Apoptosis - genetics; Binding Sites; Biophysical Phenomena; Bridged Bicyclo Compounds, Heterocyclic - chemistry; Cancer; Computer Simulation; Cytochrome; Homeostasis; Humans; Kinases; Ligands; Lymphoma; Mitochondria; Molecular Docking Simulation; Molecular Dynamics Simulation; NMR; Nuclear magnetic resonance; Physiology; Polymorphism, Single Nucleotide - genetics; Protein Binding; Protein Conformation; Proteins; Proto-Oncogene Proteins c-bcl-2 - chemistry; Proto-Oncogene Proteins c-bcl-2 - genetics; Sulfonamides - chemistry; Bcl-2; protein-ligand interactions; biophysical methods; bioinformatics; high throughput virtual screening; cell apoptosis; in silico; venetoclax; BH3 mimetics
• ISSN: 1422-0067; 1661-6596; 1422-0067
• DOI: 10.3390/ijms20040860

Bcl-2 protein is involved in cell apoptosis and is considered an interesting target for anti-cancer therapy. The present study aims to understand the stability and conformational changes of Bcl-2 upon interaction with the inhibitor venetoclax, and to explore other drug-target regions. We combined biophysical and in silico approaches to understand the mechanism of ligand binding to Bcl-2. Thermal shift assay (TSA) and urea electrophoresis showed a significant increase in protein stability upon venetoclax incubation, which is corroborated by molecular docking and molecular dynamics simulations. An 18 °C shift in Bcl-2 melting temperature was observed in the TSA, corresponding to a binding affinity multiple times higher than that of any other reported Bcl-2 inhibitor. This protein-ligand interaction does not implicate alternations in protein conformation, as suggested by SAXS. Additionally, bioinformatics approaches were used to identify deleterious non-synonymous single nucleotide polymorphisms (nsSNPs) of Bcl-2 and their impact on venetoclax binding, suggesting that venetoclax interaction is generally favored against these deleterious nsSNPs. Apart from the BH3 binding groove of Bcl-2, the flexible loop domain (FLD) also plays an important role in regulating the apoptotic process. High-throughput virtual screening (HTVS) identified 5 putative FLD inhibitors from the Zinc database, showing nanomolar affinity toward the FLD of Bcl-2.