Compounds mimicking the Michaelis-Menten transition state of the phosphatidylinositol 4-kinase

Phosphatidylinositol 4-kinases (PI4Ks) are crucial enzymes in lipid signaling responsible for generating phosphatidylinositol-4-phosphate (PI4P). Although the ATP-binding site of PI4Ks has been extensively studied, the structural characterization of their natural substrate, phosphatidylinositol (PI), bound to the enzyme remains elusive. In this study, we synthesized novel non-hydrolyzable Michaelis-Menten transition state mimetics in which the ADP molecule is covalently linked to inositol-4-phosphate or simple phosphatidylinositol-4-phosphate via a methylene or ethylene bridge. During our synthesis efforts, we successfully addressed the significantly limited reactivity at position 4 of inositols by utilizing P(III) phosphorus reagents, which proved crucial for the synthesis of these mimetics. For the simplest ADP-C-4PI analogue, we obtained a crystal structure of PI4K2B, which can be used to understand how these phospholipids are phosphorylated on membrane surfaces.