Discovery and Characterization of a Cryptic Secondary Binding Site in the Molecular Chaperone HSP70.

Suzanne O’Connor,Isaac M Westwood,Jóhannes Reynisson,Alan M Jones,Manjuan Liu,Oi Wei Mak,Rob Van Montfort,Ian Collins,Yann-Vaï Le Bihan,Gabriel Zazeri,Ana P R Povinelli

doi:10.3390/molecules27030817

Abstract

Heat Shock Protein 70s (HSP70s) are key molecular chaperones that are overexpressed in many cancers and often associated with metastasis and poor prognosis. It has proven difficult to develop ATP-competitive, drug-like small molecule inhibitors of HSP70s due to the flexible and hydrophilic nature of the HSP70 ATP-binding site and its high affinity for endogenous nucleotides. The aim of this study was to explore the potential for the inhibition of HSP70 through alternative binding sites using fragment-based approaches. A surface plasmon resonance (SPR) fragment screen designed to detect secondary binding sites in HSP70 led to the identification by X-ray crystallography of a cryptic binding site in the nucleotide-binding domain (NBD) of HSP70 adjacent to the ATP-binding site. Fragment binding was confirmed and characterized as ATP-competitive using SPR and ligand-observed NMR methods. Molecular dynamics simulations were applied to understand the interactions with the protein upon ligand binding, and local secondary structure changes consistent with interconversion between the observed crystal structures with and without the cryptic pocket were detected. A virtual high-throughput screen (vHTS) against the cryptic pocket was conducted, and five compounds with diverse chemical scaffolds were confirmed to bind to HSP70 with micromolar affinity by SPR. These results identified and characterized a new targetable site on HSP70. While targeting HSP70 remains challenging, the new site may provide opportunities to develop allosteric ATP-competitive inhibitors with differentiated physicochemical properties from current series.

Highlights

We have previously reported a fragment screening approach to Heat Shock Protein 70s (HSP70s) that identified a series of quinazoline ligands binding in the ATP site [8]
When HSP72-nucleotide-binding domain (NBD) was added to 1, a significant reduction (58%) of the intensity of the 1H NMR signal of the thiazole proton was observed in comparison to the 1H NMR spectrum of 1 alone, confirming that the compound was binding to the protein (Figure 4)
Our results demonstrate theeffectiveness effectiveness ofaaparallel parallel surface plasmon resonance (SPR) fragment fragment screening strategy

Summary

Introduction

Heat Shock Protein 70s (HSP70s) are a family of molecular chaperones with diverse functions, including directly assisting in the correct folding of nascent polypeptides, preventing protein aggregation and aiding the translocation of newly folded proteins to their correct location in the cell [1]. HSP70s have been described as nanomachines that can change the conformation of their substrate polypeptides [2]. This can occur during de novo protein synthesis at the ribosome, with aggregation prone protein intermediates, with stress-denatured proteins or during the assembly and disassembly of protein complexes. HSP70s interact with almost all newly synthesized, partially folded proteins and are able to recognize such a diverse range of proteins by interacting with short motifs of five amino acids, enriched with hydrophobic residues, which are found in practically all polypeptides [3]

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