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.
Highlights
Cancer is one of the leading causes of death in humans, owing to the plethora of events responsible for its activation, the lateness of diagnosis, and the ineffectiveness of treatments available
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
The significant binding affinity of the small molecule was corroborated by molecular docking calculations (∆G: −10.24 kcal/mol for physiological and −11.35 kcal/mol for chimeric forms of B-cell lymphoma 2 (Bcl-2)) and the interactions which yield the fundamental Bcl-2 specificity of venetoclax were identified
Summary
Cancer is one of the leading causes of death in humans, owing to the plethora of events responsible for its activation, the lateness of diagnosis, and the ineffectiveness of treatments available. In 2000, the hallmarks of cancer were postulated: sustaining proliferative signalling; evading growth suppressors; resisting cell death; enabling replicative immortality; inducing angiogenesis; activating invasion; and metastasis [1]. Evasion or resistance to cell death is a hallmark of cancer, and this happens due to genetic mutations that alter either the expression or function of proteins [4]. Programmed cell death (PCD), is a genetically defined mechanism that allows abnormal cells to commit suicide. It is a fundamental feature for multi-cellular organism survival, since it promotes the eradication of damaged cells which may interfere with the organism’s normal functioning and even promote tumor formation [5]. The apoptotic processes can be divided into two well-studied pathways, namely, intrinsic (or mitochondrial mediated) and extrinsic (or death receptor mediated)
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