Discovery and Characterization of a Cryptic Secondary Binding Site in the Molecular Chaperone HSP70
Autor(a) principal: | |
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Data de Publicação: | 2022 |
Outros Autores: | , , , , , , , , , |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | Repositório Institucional da UNESP |
Texto Completo: | http://dx.doi.org/10.3390/molecules27030817 http://hdl.handle.net/11449/230279 |
Resumo: | 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 micromo-lar 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. |
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Discovery and Characterization of a Cryptic Secondary Binding Site in the Molecular Chaperone HSP70Cryptic pocketFragment screenHSP70Molecular dynamicsVirtual screenHeat 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 micromo-lar 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.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Cancer Research UK Cancer Therapeutics Unit The Institute of Cancer ResearchSchool of Pharmacy and Bioengineering Keele UniversityDepartment of Biochemistry Albert Einstein College of MedicineSchool of Pharmacy Institute of Clinical Sciences College of Medical and Dental Sciences University of Birmingham, EdgbastonDepartamento de Física Instituto de Biociências Letras e Ciências Exatas (IBILCE) UNESP, Rua Cristovão Colombo 2265Departamento de Física Instituto de Biociências Letras e Ciências Exatas (IBILCE) UNESP, Rua Cristovão Colombo 2265CAPES: 001The Institute of Cancer ResearchKeele UniversityAlbert Einstein College of MedicineUniversity of BirminghamUniversidade Estadual Paulista (UNESP)O’connor, SuzanneLe Bihan, Yann-VaïWestwood, Isaac M.Liu, ManjuanMak, Oi WeiZazeri, Gabriel [UNESP]Povinelli, Ana P. R. [UNESP]Jones, Alan M.van Montfort, RobReynisson, JóhannesCollins, Ian2022-04-29T08:38:50Z2022-04-29T08:38:50Z2022-02-01info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articlehttp://dx.doi.org/10.3390/molecules27030817Molecules, v. 27, n. 3, 2022.1420-3049http://hdl.handle.net/11449/23027910.3390/molecules270308172-s2.0-85123550511Scopusreponame:Repositório Institucional da UNESPinstname:Universidade Estadual Paulista (UNESP)instacron:UNESPengMoleculesinfo:eu-repo/semantics/openAccess2022-04-29T08:38:50Zoai:repositorio.unesp.br:11449/230279Repositório InstitucionalPUBhttp://repositorio.unesp.br/oai/requestopendoar:29462024-08-05T20:55:11.139384Repositório Institucional da UNESP - Universidade Estadual Paulista (UNESP)false |
dc.title.none.fl_str_mv |
Discovery and Characterization of a Cryptic Secondary Binding Site in the Molecular Chaperone HSP70 |
title |
Discovery and Characterization of a Cryptic Secondary Binding Site in the Molecular Chaperone HSP70 |
spellingShingle |
Discovery and Characterization of a Cryptic Secondary Binding Site in the Molecular Chaperone HSP70 O’connor, Suzanne Cryptic pocket Fragment screen HSP70 Molecular dynamics Virtual screen |
title_short |
Discovery and Characterization of a Cryptic Secondary Binding Site in the Molecular Chaperone HSP70 |
title_full |
Discovery and Characterization of a Cryptic Secondary Binding Site in the Molecular Chaperone HSP70 |
title_fullStr |
Discovery and Characterization of a Cryptic Secondary Binding Site in the Molecular Chaperone HSP70 |
title_full_unstemmed |
Discovery and Characterization of a Cryptic Secondary Binding Site in the Molecular Chaperone HSP70 |
title_sort |
Discovery and Characterization of a Cryptic Secondary Binding Site in the Molecular Chaperone HSP70 |
author |
O’connor, Suzanne |
author_facet |
O’connor, Suzanne Le Bihan, Yann-Vaï Westwood, Isaac M. Liu, Manjuan Mak, Oi Wei Zazeri, Gabriel [UNESP] Povinelli, Ana P. R. [UNESP] Jones, Alan M. van Montfort, Rob Reynisson, Jóhannes Collins, Ian |
author_role |
author |
author2 |
Le Bihan, Yann-Vaï Westwood, Isaac M. Liu, Manjuan Mak, Oi Wei Zazeri, Gabriel [UNESP] Povinelli, Ana P. R. [UNESP] Jones, Alan M. van Montfort, Rob Reynisson, Jóhannes Collins, Ian |
author2_role |
author author author author author author author author author author |
dc.contributor.none.fl_str_mv |
The Institute of Cancer Research Keele University Albert Einstein College of Medicine University of Birmingham Universidade Estadual Paulista (UNESP) |
dc.contributor.author.fl_str_mv |
O’connor, Suzanne Le Bihan, Yann-Vaï Westwood, Isaac M. Liu, Manjuan Mak, Oi Wei Zazeri, Gabriel [UNESP] Povinelli, Ana P. R. [UNESP] Jones, Alan M. van Montfort, Rob Reynisson, Jóhannes Collins, Ian |
dc.subject.por.fl_str_mv |
Cryptic pocket Fragment screen HSP70 Molecular dynamics Virtual screen |
topic |
Cryptic pocket Fragment screen HSP70 Molecular dynamics Virtual screen |
description |
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 micromo-lar 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. |
publishDate |
2022 |
dc.date.none.fl_str_mv |
2022-04-29T08:38:50Z 2022-04-29T08:38:50Z 2022-02-01 |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/article |
format |
article |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
http://dx.doi.org/10.3390/molecules27030817 Molecules, v. 27, n. 3, 2022. 1420-3049 http://hdl.handle.net/11449/230279 10.3390/molecules27030817 2-s2.0-85123550511 |
url |
http://dx.doi.org/10.3390/molecules27030817 http://hdl.handle.net/11449/230279 |
identifier_str_mv |
Molecules, v. 27, n. 3, 2022. 1420-3049 10.3390/molecules27030817 2-s2.0-85123550511 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
Molecules |
dc.rights.driver.fl_str_mv |
info:eu-repo/semantics/openAccess |
eu_rights_str_mv |
openAccess |
dc.source.none.fl_str_mv |
Scopus reponame:Repositório Institucional da UNESP instname:Universidade Estadual Paulista (UNESP) instacron:UNESP |
instname_str |
Universidade Estadual Paulista (UNESP) |
instacron_str |
UNESP |
institution |
UNESP |
reponame_str |
Repositório Institucional da UNESP |
collection |
Repositório Institucional da UNESP |
repository.name.fl_str_mv |
Repositório Institucional da UNESP - Universidade Estadual Paulista (UNESP) |
repository.mail.fl_str_mv |
|
_version_ |
1808129264226140160 |