Discovery and Characterization of a Cryptic Secondary Binding Site in the Molecular Chaperone HSP70
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...
| Main Authors: | , , , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2022-01-01
|
| Series: | Molecules |
| Subjects: | |
| Online Access: | https://www.mdpi.com/1420-3049/27/3/817 |
| _version_ | 1797486069451587584 |
|---|---|
| author | Suzanne O’Connor Yann-Vaï Le Bihan Isaac M. Westwood Manjuan Liu Oi Wei Mak Gabriel Zazeri Ana P. R. Povinelli Alan M. Jones Rob van Montfort Jóhannes Reynisson Ian Collins |
| author_facet | Suzanne O’Connor Yann-Vaï Le Bihan Isaac M. Westwood Manjuan Liu Oi Wei Mak Gabriel Zazeri Ana P. R. Povinelli Alan M. Jones Rob van Montfort Jóhannes Reynisson Ian Collins |
| author_sort | Suzanne O’Connor |
| collection | DOAJ |
| 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 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. |
| first_indexed | 2024-03-09T23:28:55Z |
| format | Article |
| id | doaj.art-107fca8fd5c94697bed8fa0f96ed1083 |
| institution | Directory Open Access Journal |
| issn | 1420-3049 |
| language | English |
| last_indexed | 2024-03-09T23:28:55Z |
| publishDate | 2022-01-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Molecules |
| spelling | doaj.art-107fca8fd5c94697bed8fa0f96ed10832023-11-23T17:12:56ZengMDPI AGMolecules1420-30492022-01-0127381710.3390/molecules27030817Discovery and Characterization of a Cryptic Secondary Binding Site in the Molecular Chaperone HSP70Suzanne O’Connor0Yann-Vaï Le Bihan1Isaac M. Westwood2Manjuan Liu3Oi Wei Mak4Gabriel Zazeri5Ana P. R. Povinelli6Alan M. Jones7Rob van Montfort8Jóhannes Reynisson9Ian Collins10Cancer Research UK Cancer Therapeutics Unit, The Institute of Cancer Research, London SM2 5NG, UKCancer Research UK Cancer Therapeutics Unit, The Institute of Cancer Research, London SM2 5NG, UKCancer Research UK Cancer Therapeutics Unit, The Institute of Cancer Research, London SM2 5NG, UKCancer Research UK Cancer Therapeutics Unit, The Institute of Cancer Research, London SM2 5NG, UKSchool of Pharmacy and Bioengineering, Keele University, Keele ST5 5BG, UKSchool of Pharmacy, Institute of Clinical Sciences, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UKSchool of Pharmacy, Institute of Clinical Sciences, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UKSchool of Pharmacy, Institute of Clinical Sciences, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UKCancer Research UK Cancer Therapeutics Unit, The Institute of Cancer Research, London SM2 5NG, UKSchool of Pharmacy and Bioengineering, Keele University, Keele ST5 5BG, UKCancer Research UK Cancer Therapeutics Unit, The Institute of Cancer Research, London SM2 5NG, UKHeat 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.https://www.mdpi.com/1420-3049/27/3/817HSP70cryptic pocketfragment screenvirtual screenmolecular dynamics |
| spellingShingle | Suzanne O’Connor Yann-Vaï Le Bihan Isaac M. Westwood Manjuan Liu Oi Wei Mak Gabriel Zazeri Ana P. R. Povinelli Alan M. Jones Rob van Montfort Jóhannes Reynisson Ian Collins Discovery and Characterization of a Cryptic Secondary Binding Site in the Molecular Chaperone HSP70 Molecules HSP70 cryptic pocket fragment screen virtual screen molecular dynamics |
| title | 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_short | 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 |
| topic | HSP70 cryptic pocket fragment screen virtual screen molecular dynamics |
| url | https://www.mdpi.com/1420-3049/27/3/817 |
| work_keys_str_mv | AT suzanneoconnor discoveryandcharacterizationofacrypticsecondarybindingsiteinthemolecularchaperonehsp70 AT yannvailebihan discoveryandcharacterizationofacrypticsecondarybindingsiteinthemolecularchaperonehsp70 AT isaacmwestwood discoveryandcharacterizationofacrypticsecondarybindingsiteinthemolecularchaperonehsp70 AT manjuanliu discoveryandcharacterizationofacrypticsecondarybindingsiteinthemolecularchaperonehsp70 AT oiweimak discoveryandcharacterizationofacrypticsecondarybindingsiteinthemolecularchaperonehsp70 AT gabrielzazeri discoveryandcharacterizationofacrypticsecondarybindingsiteinthemolecularchaperonehsp70 AT anaprpovinelli discoveryandcharacterizationofacrypticsecondarybindingsiteinthemolecularchaperonehsp70 AT alanmjones discoveryandcharacterizationofacrypticsecondarybindingsiteinthemolecularchaperonehsp70 AT robvanmontfort discoveryandcharacterizationofacrypticsecondarybindingsiteinthemolecularchaperonehsp70 AT johannesreynisson discoveryandcharacterizationofacrypticsecondarybindingsiteinthemolecularchaperonehsp70 AT iancollins discoveryandcharacterizationofacrypticsecondarybindingsiteinthemolecularchaperonehsp70 |