Discovery and characterization of a cryptic secondary binding site in the molecular chaperone HSP70

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

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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.
Original languageEnglish
Article number817
Number of pages22
Issue number3
Publication statusPublished - 26 Jan 2022

Bibliographical note

Funding Information:
Funding: This research was funded by the Mair and Franklyn Robinson Scholarship (S.O.), Cancer Research UK, grant number C309/A11566 (I.C., R.v.M., Y.-V.L.B., I.M.W. and M.L.); The Institute of Cancer Research (S.O., I.C. and R.v.M.) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior–Brasil (CAPES)–Finance Code 001 (research scholarships to A.P.R.P. and G.Z.).

Publisher Copyright:
© 2022 by the authors. Licensee MDPI, Basel, Switzerland.


  • Adenosine Triphosphate/metabolism
  • Binding Sites/drug effects
  • Drug Discovery
  • HSP70 Heat-Shock Proteins/antagonists & inhibitors
  • Humans
  • Ligands
  • Molecular Docking Simulation
  • Molecular Dynamics Simulation
  • Protein Domains/drug effects
  • Small Molecule Libraries/chemistry
  • HSP70
  • Fragment screen
  • Cryptic pocket
  • Virtual screen
  • Molecular dynamics

ASJC Scopus subject areas

  • Drug Discovery
  • Analytical Chemistry
  • Chemistry (miscellaneous)
  • Molecular Medicine
  • Physical and Theoretical Chemistry
  • Pharmaceutical Science
  • Organic Chemistry


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