Allosteric site engagement and cooperativity mechanism by PHI1 for BRAF kinase inhibition.

With the ability to reveal allosteric sites, Ponatinib and Ponatinib Hybrid Inhibitor 1 (PHI1) are novel inhibitors of BRAF, a potent oncogene that activates the MAPK pathway. PHI1 also exhibits unique positive cooperativity, with enhanced inhibition on the other monomer when one monomer of the BRAF dimer bound to an inhibitor. The abovementioned properties lack rigorous theoretical verification, so this study compared the interaction mechanisms of four inhibitor types and explored the source of the cooperativity of PHI1 via various computational methods. Results revealed that residues on the αC-helix formed hydrogen bonds with inhibitors, shifting the position of the αC-helix. PHI1 induced binding pocket contraction through contact with allosteric sites. Entropy contributions were considerably weakened when both BRAF monomers were occupied, thereby increasing the binding ability of PHI1, indicating that entropy contributions were the main source of PHI1 cooperativity. The change in overall motion intensity tightened the binding pocket, increasing the binding abilities of hotspot residues, including Arg575 and Leu567. Moreover, three key hydrogen bonds formed between PHI1 and BRAF in the dimer system were conducive to the binding. The insights derived from this study are expected to advance the development of inhibitors targeting BRAF kinase.