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

Cardoso, Rosa; Love, Robert; Nilsson, Carol L LU ; Bergqvist, Simon; Nowlin, Dawn; Yan, Jiangli; Liu, Kevin K-C; Zhu, Jing; Chen, Ping and Deng, Ya-Li, et al. (2012) In Protein Science 21(12). p.96-1885
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... (More)

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.

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keywords
Aryl Hydrocarbon Receptor Nuclear Translocator, Cysteine, Humans, Hypoxia-Inducible Factor 1, alpha Subunit, Models, Molecular, Protein Binding, Protein Conformation, Protein Interaction Domains and Motifs, Protein Interaction Maps, Protein Multimerization, Small Molecule Libraries, Journal Article
in
Protein Science
volume
21
issue
12
pages
12 pages
publisher
The Protein Society
external identifiers
  • scopus:84870354792
ISSN
1469-896X
DOI
10.1002/pro.2172
language
English
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no
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9edbdc34-2697-4c63-b090-a127497a9b73
date added to LUP
2017-05-16 10:28:28
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2017-07-23 05:28:15
@article{9edbdc34-2697-4c63-b090-a127497a9b73,
  abstract     = {<p>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.</p>},
  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},
  issn         = {1469-896X},
  keyword      = {Aryl Hydrocarbon Receptor Nuclear Translocator,Cysteine,Humans,Hypoxia-Inducible Factor 1, alpha Subunit,Models, Molecular,Protein Binding,Protein Conformation,Protein Interaction Domains and Motifs,Protein Interaction Maps,Protein Multimerization,Small Molecule Libraries,Journal Article},
  language     = {eng},
  number       = {12},
  pages        = {96--1885},
  publisher    = {The Protein Society},
  series       = {Protein Science},
  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},
  url          = {http://dx.doi.org/10.1002/pro.2172},
  volume       = {21},
  year         = {2012},
}