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

ABSTRACT: The heterodimer HIF-1? (hypoxia inducible factor)/HIF-? (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-1? PasB domain, we applied a cysteine-based reactomics "hotspot identification" strategy to locate regions of HIF-1? 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-1? PasB domain. Biophysical characterization of the interaction between PasB domains of HIF-1? and ARNT revealed that covalent binding of COMPOUND 5 to Cys255 reduced binding affinity between HIF-1? and ARNT PasB domains approximately 10-fold. Detailed NMR structural analysis of HIF-1?-PasB-COMPOUND 5 conjugate showed significant local conformation changes in the HIF-1? associated with key residues involved in the HIF-1?/ARNT PasB domain interaction as revealed by the crystal structure of the HIF-1?/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.

SUBMITTER: Cardoso R 

PROVIDER: S-EPMC3575918 | biostudies-literature | 2012 Dec

REPOSITORIES: biostudies-literature