The minor phospholipid, phosphatidylinositol 4-phosphate (PI4P), is emerging as a key regulator of lipid transfer in ER-membrane contact sites. Four different phosphatidylinositol 4-kinase (PI4K) enzymes generate PI4P in different membrane compartments supporting distinct cellular processes, many of which are crucial for the maintenance of cellular integrity but also hijacked by intracellular pathogens. While type III PI4Ks have been targeted by small molecular inhibitors, thus helping decipher their importance in cellular physiology, no inhibitors are available for the type II PI4Ks, which hinders investigations into their cellular functions. Here, we describe the identification of small molecular inhibitors of PI4K type II alpha (PI4K2A) by implementing a large scale small molecule high-throughput screening. A novel assay was developed that allows testing of selected inhibitors against PI4K2A in intact cells using a bioluminescence resonance energy transfer approach adapted to plate readers. The compounds disclosed here will pave the way to the optimization of PI4K2A inhibitors that can be used in cellular and animal studies to better understand the role of this enzyme in both normal and pathological states.

• Keywords
• endosome, phosphoinositide, vesicular traffic,
• MeSH
• 1-Phosphatidylinositol 4-Kinase antagonists & inhibitors   chemistry   metabolism   MeSH
• Biological Transport MeSH
• Chlorocebus aethiops MeSH
• COS Cells MeSH
• Endosomes drug effects   metabolism   MeSH
• Golgi Apparatus drug effects   metabolism   MeSH
• HEK293 Cells MeSH
• Enzyme Inhibitors metabolism   pharmacology   MeSH
• Protein Conformation MeSH
• Humans MeSH
• Drug Evaluation, Preclinical MeSH
• High-Throughput Screening Assays * MeSH
• Molecular Docking Simulation MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Intramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Names of Substances
• 1-Phosphatidylinositol 4-Kinase MeSH
• Enzyme Inhibitors MeSH

Phosphoinositides are a class of phospholipids generated by the action of phosphoinositide kinases with key regulatory functions in eukaryotic cells. Here, we present the atomic structure of phosphatidylinositol 4-kinase type IIα (PI4K IIα), in complex with ATP solved by X-ray crystallography at 2.8 Å resolution. The structure revealed a non-typical kinase fold that could be divided into N- and C-lobes with the ATP binding groove located in between. Surprisingly, a second ATP was found in a lateral hydrophobic pocket of the C-lobe. Molecular simulations and mutagenesis analysis revealed the membrane binding mode and the putative function of the hydrophobic pocket. Taken together, our results suggest a mechanism of PI4K IIα recruitment, regulation, and function at the membrane.

• Keywords
• Monte Carlo simulations, crystal structure, kinase, membrane, phosphatidyl inositol,
• MeSH
• Phosphatidylinositols chemistry   metabolism   MeSH
• Phosphotransferases (Alcohol Group Acceptor) chemistry   metabolism   ultrastructure   MeSH
• Inositol chemistry   MeSH
• Protein Conformation * MeSH
• Crystallography, X-Ray * MeSH
• Humans MeSH
• Membranes chemistry   MeSH
• Monte Carlo Method MeSH
• Signal Transduction MeSH
• Protein Binding MeSH
• Binding Sites MeSH
• Minor Histocompatibility Antigens MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Phosphatidylinositols MeSH
• Phosphotransferases (Alcohol Group Acceptor) MeSH
• Inositol MeSH
• phosphatidylinositol phosphate 4-kinase MeSH Browser
• Minor Histocompatibility Antigens MeSH