Structural rearrangement in the binding pocket of KIT/PDGFRA revealed by covalent targeting of the DFG-1 Cysteine

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Covalent targeting of kinase cysteines is a widely used strategy to modulate potency and selectivity in drug discovery. In receptor tyrosine kinases KIT and PDGFRA, covalent engagement has been scarcely investigated, particularly with respect to DFG‑1 targeting, and both its feasibility and structural consequences remain unexplored. We investigated the covalent tractability of the conserved DFG-adjacent (DFG-1) cysteine in KIT and PDGFRA. A focused library of avapritinib-derived covalent type 1.5 inhibitors was designed to probe cysteine accessibility and binding geometry. Protein-mass spectrometry and biochemical analyses demonstrate that the DFG‑1 is covalently addressable for these kinases; protein X-ray crystallography provides the first structural insights of covalent modification at KIT-C809 and reveals that covalent tethering stabilizes an intermediate DFG-conformation through activation-loop reorganization. Notably, within the synthesized compound series, covalent engagement at KIT-C809 is not compatible with simultaneous occupation of the adjacent binding region engaged by avapritinib, consistent with mutually exclusive pharmacophoric requirements for covalent tethering versus noncovalent binding modes. These findings establish that the DFG-1 cysteine in KIT and PDGFRA can be covalently engaged, while also revealing structural constraints that govern productive binding in conformationally dynamic kinase states.

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