Chemical validation of a druggable site on Hsp27/HSPB1 using in silico solvent mapping and biophysical methods

Abstract

Destabilizing mutations in small heat shock proteins (sHsps) are linked to multiple diseases; however, sHsps are conformationally dynamic, lack enzymatic function and have no endogenous chemical ligands. These factors render sHsps as classically “undruggable” targets and make it particularly challenging to identify molecules that might bind and stabilize them. To explore potential solutions, we designed a multi-pronged screening workflow involving a combination of computational and biophysical ligand-discovery platforms. Using the core domain of the sHsp family member Hsp27/HSPB1 (Hsp27c) as a target, we applied mixed solvent molecular dynamics (MixMD) to predict three possible binding sites, which we confirmed using NMR-based solvent mapping. Using this knowledge, we then used NMR spectroscopy to carry out a fragment-based drug discovery (FBDD) screen, ultimately identifying two fragments that bind to one of these sites. A medicinal chemistry effort improved the affinity of one fragment by ~50-fold (16 µM), while maintaining good ligand efficiency (~0.32 kcal/mol/non-hydrogen atom). Finally, we found that binding to this site partially restored the stability of disease-associated Hsp27 variants, in a redox-dependent manner. Together, these experiments suggest a new and unexpected binding site on Hsp27, which might be exploited to build chemical probes.

Introduction

Small heat shock proteins (sHsps) are a recondite class of ATP-independent molecular chaperones1, 2 that aid in suppressing protein aggregation under stress conditions.³ It is thought that these chaperones bind to unfolded proteins, keeping them soluble until they can be refolded. Structurally, these chaperones are defined by a conserved α-crystallin core domain (ACD), flanked by disordered N- and C-terminal extensions.⁴ Each of these motifs are involved in extensive protein-protein interactions (PPIs). Specifically, the ACD is composed of six β-strands that form an immunoglobulin fold and two of these β-strands, associated in an antiparallel fashion, form a stable dimer interface. Additional contacts involving the N- and C-terminal extensions mediate higher-order assembly of these dimers into larger oligomers.5, 6, 7, 8 In theory, this architecture creates potential opportunities for drug discovery, such as targeting the intra- or inter-molecular PPI surfaces. However, the sHSP oligomers are polydisperse (typically composed of 10–20 dimers) and dynamic in response to cellular stress.5, 9 Thus, it is not clear which of the putative, transient pockets might allow chemical ligands to bind with good affinity.

To explore these questions, we focused on heat shock protein 27 (Hsp27/HSBP1). This member of the sHSP family is of interest because mutations in Hsp27’s ACD, including R127W and S135F, are closely linked to diseases, such as distal hereditary motor neuropathy and Charcot-Marie-Tooth diease.10, 11 These mutations are known to destabilize Hsp27 oligomers and aberrantly increase its chaperone activity.¹² This observation is consistent with an emerging model in which smaller, less stable Hsp27 oligomers are more active,5, 9 at least in some contexts. This idea is further supported by the fact that an intermolecular disulfide bond formed at Hsp27’s dimer interface is known to limit its conformational flexibility and, consistent with the model, decrease chaperone activity.13, 14, 15 Together, these observations suggest that one therapeutic objective, at least in a subset of Hsp27-related diseases, may be to identify drug-like molecules that bind and partially restore the stability of disease-associated mutations, such as R127W and S135F. However, there are no known binding pocket(s) for small molecules on Hsp27.

A number of techniques have been developed to identify binding sites on other “undruggable” targets, including computational methods for suggesting sites¹⁶ and fragment-based screening approaches.¹⁷ Here, we combine computational and experimental platforms towards the discovery of proof-of-principle ligands for Hsp27. Using this combination of approaches, we identified a promising and previously unexplored binding site within Hsp27’s ACD and found that a chemical fragment (K_d > 1 mM) could be matured through a medicinal chemistry campaign to bind with good affinity (K_d ~ 16 µM) and ligand efficiency (0.32 kcal/mol/non-hydrogen atom). Importantly, the resulting probe partially stabilized a disease-associated Hsp27 mutant in vitro, suggesting that this site might be tractable for further development. While additional work is required to increase affinity, these studies provide a starting point for Hsp27 drug discovery and, potentially, for exploration of other sHSPs.