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Fragment screening using capillary electrophoresis (CEfrag) for hit identification of heat shock protein 90 ATPase inhibitors

Fragment screening using capillary electrophoresis (CEfrag) for hit identification of heat shock protein 90 ATPase inhibitors
Fragment screening using capillary electrophoresis (CEfrag) for hit identification of heat shock protein 90 ATPase inhibitors

CEfrag is a new fragment screening technology based on affinity capillary electrophoresis (ACE). Here we report on the development of a mobility shift competition assay using full-length human heat shock protein 90α (Hsp90α), radicicol as the competitor probe ligand, and successful screening of the Selcia fragment library. The CEfrag assay was able to detect weaker affinity (IC(50) >500 µM) fragments than were detected by a fluorescence polarization competition assay using FITC-labeled geldanamycin. The binding site of selected fragments was determined by co-crystallization with recombinant Hsp90α N-terminal domain and X-ray analysis. The results of this study confirm that CEfrag is a sensitive microscale technique enabling detection of fragments binding to the biological target in near-physiological solution.

Adenosine Triphosphatases, Adenosine Triphosphate, Binding Sites, Crystallography, X-Ray, Drug Discovery, Drug Evaluation, Preclinical, Electrophoresis, Capillary, Electrophoretic Mobility Shift Assay, HSP90 Heat-Shock Proteins, Humans, Macrolides, Protein Binding, Protein Structure, Tertiary, Small Molecule Libraries, Journal Article
1087-0571
868-76
Austin, Carol
883c7d26-5eae-45c8-a86a-6080d210d741
Pettit, Simon N
d9d90fdd-04cf-4495-a370-ebf52405b748
Magnolo, Sharon K
5c1e7740-6380-41c4-874a-ed93cf67dab5
Sanvoisin, Jonathan
9effd554-5ca8-44d0-b7a9-69b83c5653dc
Chen, Wenjie
eba18e04-075b-4477-9e60-020314078f82
Wood, Stephen P
d7187a5c-6d5a-48e1-9934-b9fb69e3f43c
Freeman, Lauren D
102441a6-b145-428d-8b34-36dc26347594
Pengelly, Reuben J
af97c0c1-b568-415c-9f59-1823b65be76d
Hughes, Dallas E
49b7acb7-cfb1-4692-bbef-498463d4529b
Austin, Carol
883c7d26-5eae-45c8-a86a-6080d210d741
Pettit, Simon N
d9d90fdd-04cf-4495-a370-ebf52405b748
Magnolo, Sharon K
5c1e7740-6380-41c4-874a-ed93cf67dab5
Sanvoisin, Jonathan
9effd554-5ca8-44d0-b7a9-69b83c5653dc
Chen, Wenjie
eba18e04-075b-4477-9e60-020314078f82
Wood, Stephen P
d7187a5c-6d5a-48e1-9934-b9fb69e3f43c
Freeman, Lauren D
102441a6-b145-428d-8b34-36dc26347594
Pengelly, Reuben J
af97c0c1-b568-415c-9f59-1823b65be76d
Hughes, Dallas E
49b7acb7-cfb1-4692-bbef-498463d4529b

Austin, Carol, Pettit, Simon N, Magnolo, Sharon K, Sanvoisin, Jonathan, Chen, Wenjie, Wood, Stephen P, Freeman, Lauren D, Pengelly, Reuben J and Hughes, Dallas E (2012) Fragment screening using capillary electrophoresis (CEfrag) for hit identification of heat shock protein 90 ATPase inhibitors. Journal of Biomolecular Screening, 17 (7), 868-76. (doi:10.1177/1087057112445785).

Record type: Article

Abstract

CEfrag is a new fragment screening technology based on affinity capillary electrophoresis (ACE). Here we report on the development of a mobility shift competition assay using full-length human heat shock protein 90α (Hsp90α), radicicol as the competitor probe ligand, and successful screening of the Selcia fragment library. The CEfrag assay was able to detect weaker affinity (IC(50) >500 µM) fragments than were detected by a fluorescence polarization competition assay using FITC-labeled geldanamycin. The binding site of selected fragments was determined by co-crystallization with recombinant Hsp90α N-terminal domain and X-ray analysis. The results of this study confirm that CEfrag is a sensitive microscale technique enabling detection of fragments binding to the biological target in near-physiological solution.

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More information

Published date: August 2012
Keywords: Adenosine Triphosphatases, Adenosine Triphosphate, Binding Sites, Crystallography, X-Ray, Drug Discovery, Drug Evaluation, Preclinical, Electrophoresis, Capillary, Electrophoretic Mobility Shift Assay, HSP90 Heat-Shock Proteins, Humans, Macrolides, Protein Binding, Protein Structure, Tertiary, Small Molecule Libraries, Journal Article
Organisations: Human Development & Health

Identifiers

Local EPrints ID: 409201
URI: https://eprints.soton.ac.uk/id/eprint/409201
ISSN: 1087-0571
PURE UUID: d3792495-ee9b-4bca-8db2-519020cca85e
ORCID for Reuben J Pengelly: ORCID iD orcid.org/0000-0001-7022-645X

Catalogue record

Date deposited: 28 May 2017 04:07
Last modified: 14 Mar 2019 01:34

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Contributors

Author: Carol Austin
Author: Simon N Pettit
Author: Sharon K Magnolo
Author: Jonathan Sanvoisin
Author: Wenjie Chen
Author: Stephen P Wood
Author: Lauren D Freeman
Author: Dallas E Hughes

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