Shedding light on the interaction of human anti-apoptotic Bcl-2 protein with ligands through biophysical and in silico studies

João Ramos, Jayaraman Muthukumaran, Filipe Freire, João Paquete-Ferreira, Ana Rita Otrelo-Cardoso, Dmitri Svergun, Alejandro Panjkovich, Teresa Santos-Silva

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Abstract

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.

Original languageEnglish
Article number860
JournalInternational Journal of Molecular Sciences
Volume20
Issue number4
DOIs
Publication statusPublished - 2 Feb 2019

Keywords

  • Bcl-2
  • BH3 mimetics
  • Bioinformatics
  • Biophysical methods
  • Cell apoptosis
  • High throughput virtual screening
  • In silico
  • Protein-ligand interactions
  • Venetoclax

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