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Title: Identification of Cys255 in HIF-1α as a novel site for development of covalent inhibitors of HIF-1α/ARNT PasB domain protein-protein interaction

Abstract

The heterodimer HIF-1α (hypoxia inducible factor)/HIF-β (also known as ARNT-aryl hydrocarbon nuclear translocator) is a key mediator of cellular response to hypoxia. The interaction between these monomer units can be modified by the action of small molecules in the binding interface between their C-terminal heterodimerization (PasB) domains. Taking advantage of the presence of several cysteine residues located in the allosteric cavity of HIF-1α PasB domain, we applied a cysteine-based reactomics “hotspot identification” strategy to locate regions of HIF-1α PasB domain critical for its interaction with ARNT. COMPOUND 5 was identified using a mass spectrometry-based primary screening strategy and was shown to react specifically with Cys255 of the HIF-1α PasB domain. Biophysical characterization of the interaction between PasB domains of HIF-1α and ARNT revealed that covalent binding of COMPOUND 5 to Cys255 reduced binding affinity between HIF-1α and ARNT PasB domains approximately 10-fold. Detailed NMR structural analysis of HIF-1α-PasB-COMPOUND 5 conjugate showed significant local conformation changes in the HIF-1α associated with key residues involved in the HIF-1α/ARNT PasB domain interaction as revealed by the crystal structure of the HIF-1α/ARNT PasB heterodimer. Our screening strategy could be applied to other targets to identify pockets surrounding reactive cysteines suitable for development of smallmore » molecule modulators of protein function.« less

Authors:
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  1. Pfizer
  2. (
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
Sponsoring Org.:
INDUSTRY
OSTI Identifier:
1064490
Resource Type:
Journal Article
Journal Name:
Protein Science
Additional Journal Information:
Journal Volume: 21; Journal Issue: 12; Journal ID: ISSN 0961-8368
Publisher:
The Protein Society
Country of Publication:
United States
Language:
ENGLISH

Citation Formats

Cardoso, Rosa, Love, Robert, Nilsson, Carol L, Bergqvist, Simon, Nowlin, Dawn, Yan, Jiangli, Liu, Kevin K.-C., Zhu, Jing, Chen, Ping, Deng, Ya-Li, Dyson, H Jane, Greig, Michael J, Brooun, Alexei, and Scripps). Identification of Cys255 in HIF-1α as a novel site for development of covalent inhibitors of HIF-1α/ARNT PasB domain protein-protein interaction. United States: N. p., 2012. Web. doi:10.1002/pro.2172.
Cardoso, Rosa, Love, Robert, Nilsson, Carol L, Bergqvist, Simon, Nowlin, Dawn, Yan, Jiangli, Liu, Kevin K.-C., Zhu, Jing, Chen, Ping, Deng, Ya-Li, Dyson, H Jane, Greig, Michael J, Brooun, Alexei, & Scripps). Identification of Cys255 in HIF-1α as a novel site for development of covalent inhibitors of HIF-1α/ARNT PasB domain protein-protein interaction. United States. doi:10.1002/pro.2172.
Cardoso, Rosa, Love, Robert, Nilsson, Carol L, Bergqvist, Simon, Nowlin, Dawn, Yan, Jiangli, Liu, Kevin K.-C., Zhu, Jing, Chen, Ping, Deng, Ya-Li, Dyson, H Jane, Greig, Michael J, Brooun, Alexei, and Scripps). Fri . "Identification of Cys255 in HIF-1α as a novel site for development of covalent inhibitors of HIF-1α/ARNT PasB domain protein-protein interaction". United States. doi:10.1002/pro.2172.
@article{osti_1064490,
title = {Identification of Cys255 in HIF-1α as a novel site for development of covalent inhibitors of HIF-1α/ARNT PasB domain protein-protein interaction},
author = {Cardoso, Rosa and Love, Robert and Nilsson, Carol L and Bergqvist, Simon and Nowlin, Dawn and Yan, Jiangli and Liu, Kevin K.-C. and Zhu, Jing and Chen, Ping and Deng, Ya-Li and Dyson, H Jane and Greig, Michael J and Brooun, Alexei and Scripps)},
abstractNote = {The heterodimer HIF-1α (hypoxia inducible factor)/HIF-β (also known as ARNT-aryl hydrocarbon nuclear translocator) is a key mediator of cellular response to hypoxia. The interaction between these monomer units can be modified by the action of small molecules in the binding interface between their C-terminal heterodimerization (PasB) domains. Taking advantage of the presence of several cysteine residues located in the allosteric cavity of HIF-1α PasB domain, we applied a cysteine-based reactomics “hotspot identification” strategy to locate regions of HIF-1α PasB domain critical for its interaction with ARNT. COMPOUND 5 was identified using a mass spectrometry-based primary screening strategy and was shown to react specifically with Cys255 of the HIF-1α PasB domain. Biophysical characterization of the interaction between PasB domains of HIF-1α and ARNT revealed that covalent binding of COMPOUND 5 to Cys255 reduced binding affinity between HIF-1α and ARNT PasB domains approximately 10-fold. Detailed NMR structural analysis of HIF-1α-PasB-COMPOUND 5 conjugate showed significant local conformation changes in the HIF-1α associated with key residues involved in the HIF-1α/ARNT PasB domain interaction as revealed by the crystal structure of the HIF-1α/ARNT PasB heterodimer. Our screening strategy could be applied to other targets to identify pockets surrounding reactive cysteines suitable for development of small molecule modulators of protein function.},
doi = {10.1002/pro.2172},
journal = {Protein Science},
issn = {0961-8368},
number = 12,
volume = 21,
place = {United States},
year = {2012},
month = {11}
}