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

Rosa Cardoso, Robert Love, Carol L. Nilsson, Simon Bergqvist, Dawn Nowlin, Jiangli Yan, Kevin K C Liu, Jing Zhu, Ping Chen, Ya Li Deng, H. Jane Dyson, Michael J. Greig, Alexei Brooun

Research output: Contribution to journalArticle

32 Citations (Scopus)

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 small molecule modulators of protein function. Published by Wiley-Blackwell.

Original languageEnglish (US)
Pages (from-to)1885-1896
Number of pages12
JournalProtein Science
Volume21
Issue number12
DOIs
StatePublished - Dec 2012
Externally publishedYes

Fingerprint

Protein Interaction Domains and Motifs
Cysteine
Screening
Aryl Hydrocarbon Receptor Nuclear Translocator
Hypoxia-Inducible Factor 1
Molecules
Structural analysis
Modulators
Mass spectrometry
Conformations
Mass Spectrometry
Monomers
Crystal structure
Nuclear magnetic resonance
Proteins

Keywords

  • AlphaScreen
  • Covalent inhibitor
  • Crystal structure
  • Isothermal titration calorimetry (ITC)
  • Mass spectrometry
  • NMR spectroscopy
  • Protein-protein interaction (PPI)
  • Surface plasmon resonance (SPR)

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

Cite this

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.; 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.

In: Protein Science, Vol. 21, No. 12, 12.2012, p. 1885-1896.

Research output: Contribution to journalArticle

Cardoso, R, Love, R, Nilsson, CL, Bergqvist, S, Nowlin, D, Yan, J, Liu, KKC, Zhu, J, Chen, P, Deng, YL, Dyson, HJ, Greig, MJ & Brooun, A 2012, 'Identification of Cys255 in HIF-1α as a novel site for development of covalent inhibitors of HIF-1α/ARNT PasB domain protein-protein interaction', Protein Science, vol. 21, no. 12, pp. 1885-1896. https://doi.org/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. / Identification of Cys255 in HIF-1α as a novel site for development of covalent inhibitors of HIF-1α/ARNT PasB domain protein-protein interaction. In: Protein Science. 2012 ; Vol. 21, No. 12. pp. 1885-1896.
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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 small molecule modulators of protein function. Published by Wiley-Blackwell.",
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T1 - Identification of Cys255 in HIF-1α as a novel site for development of covalent inhibitors of HIF-1α/ARNT PasB domain protein-protein interaction

AU - Cardoso, Rosa

AU - Love, Robert

AU - Nilsson, Carol L.

AU - Bergqvist, Simon

AU - Nowlin, Dawn

AU - Yan, Jiangli

AU - Liu, Kevin K C

AU - Zhu, Jing

AU - Chen, Ping

AU - Deng, Ya Li

AU - Dyson, H. Jane

AU - Greig, Michael J.

AU - Brooun, Alexei

PY - 2012/12

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N2 - 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. Published by Wiley-Blackwell.

AB - 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. Published by Wiley-Blackwell.

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KW - Crystal structure

KW - Isothermal titration calorimetry (ITC)

KW - Mass spectrometry

KW - NMR spectroscopy

KW - Protein-protein interaction (PPI)

KW - Surface plasmon resonance (SPR)

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