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-1a (hypoxia inducible factor)/HIF-b (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-1a PasB domain, we applied a cysteine-based reactomics ''hotspot identification'' strategy to locate regions of HIF1a 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-1a PasB domain. Biophysical characterization of the interaction between PasB domains of HIF-1a and ARNT revealed that covalent binding of COMPOUND 5 to Cys255 reduced binding affinity between HIF-1a and ARNT PasB domains approximately 10-fold. Detailed NMR structural analysis of HIF-1a-PasB-COMPOUND 5 conjugate showed significant local conformation changes in the HIF-1a associated with key residues involved in the HIF-1a/ARNT PasB domain interaction as revealed by the crystal structure of the HIF-1a/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.

Structural integration in hypoxia-inducible factors

“…Three 0X3-interacting residues differ in these two pockets: A277, S292 and S304 in HIF-2a are replaced by I275, T290 and T302 (which have bulkier side chains) in HIF-1a. As previously suggested 32 Fig. 5f, g).…”

“…Although no endogenous ligand has been identified for the HIF-a or ARNT proteins, a number of synthetic small molecules were discovered through screening campaigns [32][33][34][35][36] . Ligands that reduce the activity of HIF-a proteins are sought as potentially useful therapies in cancers where HIF-a overexpression correlates inversely with patient survival 9,11 .…”

On the acquisition and analysis of microscale thermophoresis data

“…820 [670, 990] nM (Table 3). We note that other researchers have obtained a K D of about 1.4 μM for the interaction of similar protein constructs [44]. Another point arguing in favor of the TJ analytical mode is that it presumably measures the effect of binding before temperature-induced changes in K D can fully manifest (see Section 4.2).…”

Covalent tethering of fragments for covalent probe discovery

“…Out of these, seven compounds were hyper-reactive and non-specifically labelled other nucleophilic residues on the protein surface and therefore were eliminated from the screen (88% false positives). 10 David J. Mann and coworkers reported the screen of a small acrylamide library (10 compounds) against thymidylate synthase and identified a covalent inhibitor of this enzyme, thus providing the first proof of concept studies for covalent tethering. 11 In addition, one compound ( 1 ) in their library was hyper-reactive (10% rate of potential false positive), and had to be discarded before screening.…”