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 small molecule modulators of protein function.
authors.affiliation: Oncology Chemistry, Worldwide Research and Development, Pfizer Inc., San Diego, California 92121, USA.
authors.forename: Rosa
authors.initials: R
authors.lastname: Cardoso
authors.forename: Robert
authors.initials: R
authors.lastname: Love
authors.forename: Carol L
authors.initials: CL
authors.lastname: Nilsson
authors.forename: Simon
authors.initials: S
authors.lastname: Bergqvist
authors.forename: Dawn
authors.initials: D
authors.lastname: Nowlin
authors.forename: Jiangli
authors.initials: J
authors.lastname: Yan
authors.forename: Kevin K-C
authors.initials: KK
authors.lastname: Liu
authors.forename: Jing
authors.initials: J
authors.lastname: Zhu
authors.forename: Ping
authors.initials: P
authors.lastname: Chen
authors.forename: Ya-Li
authors.initials: YL
authors.lastname: Deng
authors.forename: H Jane
authors.initials: HJ
authors.lastname: Dyson
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Greig
authors.forename: Alexei
authors.initials: A
authors.lastname: Brooun
mesh_terms: D051784:Aryl Hydrocarbon Receptor Nuclear Translocator / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D003545:Cysteine / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D011485:Protein Binding / Q000187:drug effects; D011487:Protein Conformation / Q000187:drug effects; D054730:Protein Interaction Domains and Motifs / Q000187:drug effects*; D060066:Protein Interaction Maps / Q000187:drug effects; D055503:Protein Multimerization / Q000187:drug effects; D054852:Small Molecule Libraries / Q000737:chemistry* / Q000494:pharmacology*
chemical_list: D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D054852:Small Molecule Libraries; D051784:Aryl Hydrocarbon Receptor Nuclear Translocator; D003545:Cysteine