A large scale high-throughput screen identifies chemical inhibitors of phosphatidylinositol 4-kinase type II alpha

Abstract

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. 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. Phosphoinositides represent a small fraction of all phospholipids, but they regulate a whole range of cellular processes (1.Balla T. Phosphoinositides: tiny lipids with giant impact on cell regulation.Physiol. Rev. 2013; 93: 1019-1137Crossref PubMed Scopus (976) Google Scholar). These regulatory lipids are formed by sequential phosphorylation of phosphatidylinositol (PI) on its inositol ring at any of three positions (positions 3, 4, and 5). The different combination of these phosphorylations gives rise to the seven known PI species. There are specific enzymes for phosphorylation of each position and in most cases multiple enzymes catalyze the same reaction. A similar multiplicity of phosphatases exists, setting up an elaborate network of PI metabolism (1.Balla T. Phosphoinositides: tiny lipids with giant impact on cell regulation.Physiol. Rev. 2013; 93: 1019-1137Crossref PubMed Scopus (976) Google Scholar). Many of these enzymes have been linked to deregulation of PIP levels and human diseases. For example, numerous studies have investigated the role of PI3Ks and their lipid product, PI(3,4,5)P3, in cancer and immune regulations promoting the development of PI3K inhibitors currently in clinical trials (2.Shuttleworth S.J. Silva F.A. Cecil A.R. Tomassi C.D. Hill T.J. Raynaud F.I. Clarke P.A. Workman P. Progress in the preclinical discovery and clinical development of class I and dual class I/IV phosphoinositide 3-kinase (PI3K) inhibitors.Curr. Med. Chem. 2011; 18: 2686-2714Crossref PubMed Scopus (93) Google Scholar