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

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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.


  • 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
Externa organisationer
  • Pfizer


Sidor (från-till)1885-96
Antal sidor12
TidskriftProtein Science
Utgåva nummer12
StatusPublished - 2012 dec
Peer review utfördJa
Externt publiceradJa