Phosphatidylinositol phosphate kinases (PIPKs) produce lipid signaling molecules and have been attracting increasing attention as drug targets for cancer and viral infection. Given the potential cross-inhibition of kinases and other ATP-utilizing enzymes by ATP-competitive inhibitors, targeting the unique lipid substrate binding site represents a superior strategy for PIPK inhibition. Here, by taking advantage of the nearly identical stereochemistry between myo-inositol and D-galactose, we designed and synthesized a panel of D-galactosyl lysophospholipids, one of which was found to be a selective substrate of phosphatidylinositol 4-phosphate 5-kinase. Derivatization of this compound led to the discovery of a human PIKfyve inhibitor with an apparent IC50 of 6.2 ÎơM, which significantly potentiated the inhibitory effect of Apilimod, an ATPcompetitive PIKfyve inhibitor under clinical trials against SARS-CoV-2 infection and amyotrophic lateral sclerosis. Our results provide the proof of concept that D-galactose-based phosphoinositide mimetics can be developed into artificial substrates and new inhibitors of PIPKs. In addition, the activity loops of PIPKs were disordered in all crystal structures because of the membrane sensing mechanism. Here, through conjugation of a zebrafish PIP5KÎł with maleimide functionalized lipid, the protein can be immobilized on nanodisc for structural characterization.
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