Fulcrum Occupancy-Leverage Perturbation Strategy Enables Rapid Discovery of Potent CDK2-Cyclin A2 Interaction...

Traditional strategies for developing small-molecule inhibitors of protein-protein interactions (PPIs) are time-consuming and often yield low success rates due to the flat and dynamic interfaces of PPIs. To enable the rapid design of highly potent PPI inhibitors, we proposed a novel strategy named "Fulcrum Occupancy-Leverage Perturbation (FOLP)". In this strategy, high-affinity fragments serve as the "Fulcrum" by binding to the orthosteric pocket, while suitable moieties extend into allosteric sites near the PPI interface as "Leverage" to modulate the protein-protein interaction. As a proof of concept, the potent CDK2-Cyclin A2 PPI inhibitor LC-K2CAin-3, which fits the "FOLP" paradigm, was discovered with an IC₅₀ of 32.1 nM for inhibiting the interaction. Molecular dynamics simulations and cryptic pocket identification were employed, revealing the activation loop (A-loop) of CDK2 was flexible and targetable. X-ray crystallography and hydrogen deuterium exchange mass spectrometry (HDX-MS) analysis showed that LC-K2CAin-3 indeed bound to and stabilized the A-loop. LC-K2CAin-3 effectively inhibited the CDK2-Cyclin A2 interaction in CDK2 highly expressed melanoma cells, leading to cell cycle arrest and apoptosis and inhibition of CDK2 mediated signaling. In conclusion, the "FOLP" strategy offers a novel approach for PPI inhibitor discovery and could accelerate the development of PPI inhibitors.