Allosteric Inhibition of PKMYT1 Induces a Unique, Inactive ATP Binding Site Conformation

Area of Science:

• Biochemistry
• Molecular Biology
• Oncology

Background:

• Protein kinase PKMYT1 is crucial for cell cycle checkpoints and DNA repair.
• In cancer, PKMYT1 promotes tumor survival by preventing mitotic catastrophe.
• PKMYT1 is a therapeutic target in breast, gastric, lung, and kidney cancers, but selective inhibitors are lacking.

Purpose of the Study:

• To discover and characterize novel small-molecule inhibitors of PKMYT1.
• To investigate the binding mode and mechanism of inhibition of new PKMYT1 inhibitors.
• To explore the utility of computational methods in identifying cryptic allosteric sites in kinases.

Main Methods:

• Discovery and characterization of PKMYT1 inhibitors P29 and P32.
• Structural and kinetic analyses of inhibitor-target interactions.
• Computational modeling including AlphaFold2, AlphaFold3, Boltz-2, and molecular dynamics (MD) simulations.

Main Results:

• A novel inhibitor, P29, was identified binding to a previously unknown allosteric site on PKMYT1.
• P29 inhibits PKMYT1 via a mixed ATP-competitive and noncompetitive mechanism, inducing conformational changes.
• Analogue P32 showed enhanced potency and selectivity but bound to the ATP pocket, demonstrating sensitivity to chemical modifications.
• Computational methods showed limitations in predicting the allosteric site and inhibitor-induced conformational changes.

Conclusions:

• An underexplored allosteric site in PKMYT1 offers a new strategy for selective kinase inhibitor design.
• Subtle chemical modifications can alter inhibitor binding modes and mechanisms.
• Integrating computational modeling with experimental validation is essential for discovering noncanonical kinase binding sites.