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

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). PI 4-kinases (PI4Ks) phosphorylate the 4-position on the ring and have long been viewed only as enzymes that produce an intermediate for the synthesis of PI(4,5)P2, a plasma membrane (PM) lipid of great significance (1.Balla T. Phosphoinositides: tiny lipids with giant impact on cell regulation.Physiol. Rev. 2013; 93: 1019-1137Crossref PubMed Scopus (976) Google Scholar, 3.Boura E. Nencka R. Phosphatidylinositol 4-kinases: function, structure, and inhibition.Exp. Cell Res. 2015; 337: 136-145Crossref PubMed Scopus (89) Google Scholar). However, research in the last 10 years revealed that the four distinct PI4Ks make PI 4-phosphate (PI4P) in various intracellular compartments, such as the Golgi and endosomes, where the lipid regulates trafficking of various cargos (3.Boura E. Nencka R. Phosphatidylinositol 4-kinases: function, structure, and inhibition.Exp. Cell Res. 2015; 337: 136-145Crossref PubMed Scopus (89) Google Scholar). There is now a better understanding of the cellular functions and importance of type III PI4Ks: PI4KB plays a role in the organization of the Golgi and trafficking through this compartment (4.D'Angelo G. Vicinanza M. Wilson C. De Matteis M.A. Phosphoinositides in Golgi complex function.Subcell. Biochem. 2012; 59: 255-270Crossref PubMed Scopus (23) Google Scholar); whereas, PI4KA has functions at the ER and the PM at junctional sites where the two kinds of membranes are juxtaposed (5.Nakatsu F. Baskin J.M. Chung J. Tanner L.B. Shui G. Lee S.Y. Pirruccello M. Haio M. Ingolia N.T. Wenk M.R. et al.PtdIns4P synthesis by PI4KIIIa at the plasma membrane and its impact on plasma membrane identity.J. Cell Biol. 2012; 199: 1003-1016Crossref PubMed Scopus (185) Google Scholar). Much less is known about the type II PI4Ks [PI4K type II alpha (PI4K2A) and PI4K2B] that are present in endomembranes, including the trans-Golgi network, and they have been shown to contribute to the regulation of trafficking through various endocytic compartments (6.Minogue S. The many roles of type II phosphatidylinositol 4-kinases in membrane trafficking: new tricks for old dogs.Bioessays. 2018; 40doi:10.1002/bies.201700145Crossref PubMed Scopus (17) Google Scholar). The relevance of PI4Ks has largely increased lately after the discovery that hepatitis C virus (HCV) replication requires the host PI4KA enzyme, whereas several enteropathogenic viruses utilize PI4KB for their replication (7.Altan-Bonnet N. Balla T. Phosphatidylinositol 4-kinases: hostages harnessed to build panviral replication platforms.Trends Biochem. Sci. 2012; 37: 293-302Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). Based on these discoveries, several pharmaceutical companies are developing inhibitors that target the type III PI4Ks. These efforts have been greatly aided by the fact that type III PI4Ks are structural relatives of PI3Ks, and isoform-specific PI3K inhibitors have already been pursued for treatment of cancer and various inflammatory conditions (8.Vanhaesebroeck B. Stephens L. Hawkins P. PI3K signalling: the path to discovery and understanding.Nat. Rev. Mol. Cell Biol. 2012; 13: 195-203Crossref PubMed Scopus (674) Google Scholar). Interestingly, several drugs that have been in the pipeline to inhibit HCV (9.Bianco A. Reghellin V. Donnici L. Fenu S. Alvarez R. Baruffa C. Peri F. Pagani M. Abrignani S. Neddermann P. et al.Metabolism of phosphatidylinositol 4-kinase IIIalpha-dependent PI4P is subverted by HCV and is targeted by a 4-anilino quinazoline with antiviral activity.PLoS Pathog. 2012; 8: e1002576Crossref PubMed Scopus (103) Google Scholar) or enteroviruses (10.Arita M. Kojima H. Nagano T. Okabe T. Wakita T. Shimizu H. Phosphatidylinositol 4-kinase III beta is a target of enviroxime-like compounds for antipoliovirus activity.J. Virol. 2011; 85: 2364-2372Crossref PubMed Scopus (119) Google Scholar) without solid knowledge of their mechanism of action turned out to inhibit the type III PI4Ks. It is also notable that various HCV strains vary in their PI4K needs; some use exclusively PI4KA, whereas some others use PI4KB and some enteroviruses already show resistance to drugs that we now know inhibit type III PI4Ks of the host cell (10.Arita M. Kojima H. Nagano T. Okabe T. Wakita T. Shimizu H. Phosphatidylinositol 4-kinase III beta is a target of enviroxime-like compounds for antipoliovirus activity.J. Virol. 2011; 85: 2364-2372Crossref PubMed Scopus (119) Google Scholar, 11.Thibaut H.J. van der Schaar H.M. Lanke K.H. Verbeken E. Andrews M. Leyssen P. Neyts J. van Kuppeveld F.J. Fitness and virulence of a coxsackievirus mutant that can circumnavigate the need for phosphatidylinositol 4-kinase class III beta.J. Virol. 2014; 88: 3048-3051Crossref PubMed Scopus (7) Google Scholar). These observations suggest that viruses can adapt to utilizing different PI4Ks to generate their replication platform, and mutations will allow them to switch enzymes under selection pressure. Because type II PI4Ks belong to a different family of enzymes with no close relatives within the kinase network, they are not targeted by any of the type III PI4K or PI3K inhibitors (12.Balla A. Kim Y.J. Varnai P. Szentpetery Z. Knight Z. Shokat K.M. Balla T. Maintenance of hormone-sensitive phosphoinositide pools in the plasma membrane requires phosphatidylinositol 4-kinase III{alpha}.Mol. Biol. Cell. 2008; 19: 711-721Crossref PubMed Scopus (146) Google Scholar). Because of their localization and functional properties, these enzymes assume some overlapping functions with the type III PI4Ks, especially PI4KB, and will be available for viruses that need PI4P when type III enzymes are inhibited. Modulators of the type II enzymes would provide a better understanding of their physiological functions and their involvement in human disease, and it might trigger an interest in them as targets for development of therapeutics. Independently, type II PI4Ks, but not the type III enzymes, were found to be needed for Wnt signaling H. L. L. L. C. L. et of phosphatidylinositol regulates 2008; PubMed Scopus Google Scholar, L. of phosphatidylinositol and metabolism by and Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, J. V. G. V. 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J. et a specific of the of cancer Res. PubMed Scopus Google which in cancer cell used and to which was found to with or and not with J. Z. L. H. P. J. J. et a specific of the of cancer Res. PubMed Scopus Google Scholar). studies have used a different the of a small molecule of compounds using a The inhibitors in have a different of with with the of the lipid was by the to in the A. G. M. M. Balla T. of type II phosphatidylinositol 4-kinase of the enzymes with Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The PI4P bioluminescence resonance energy transfer was to a with an PI4P a such that PI4P on would the bioluminescence energy For a was in multiple a was using the in Balla T. A novel for phosphatidylinositol 4-phosphate multiple pools the Cell Biol. 2014; PubMed Scopus Google by the of this with using the of the two the the was by using a a and a the and an The was used as after an this in G. Balla A. L. Balla T. 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Balla T. are plasma membrane that to Cell Sci. 2015; PubMed Scopus Google For was in cells and in a assay using a with The the PI4K2A enzyme were for kinase using the in enzyme assay with no enzyme or no The a the of inhibitors the enzymes cells and that the can inhibit the enzyme in a same as the of the inhibitors on the enzyme within the in kinase were in cells using by phosphoinositide and the of the of PI4KA and PI4KB enzymes that generate a large of PI4P within the treatment of the cells with 10 of for 10 was used treatment with the inhibitors for in cell to of the inhibitors and a of these and the of two is shown in a of the compounds was with the and also revealed that some inhibitors also the of that they kinase Based on these two and were selected for The of the two selected inhibitors were in cell of cells with a the different cellular PI4P pools Balla T. A novel for phosphatidylinositol 4-phosphate multiple pools the Cell Biol. 2014; PubMed Scopus Google Scholar). PI4P is by different PI4Ks in specific intracellular compartments on studies on the localization of the enzyme (6.Minogue S. The many roles of type II phosphatidylinositol 4-kinases in membrane trafficking: new tricks for old dogs.Bioessays. 2018; 40doi:10.1002/bies.201700145Crossref PubMed Scopus (17) Google PI4K2A was to be for the and for the Golgi of shown in 10 of a in the Golgi and PI4P However, no of the in the PI4P in the is of the of cell of the and the for the a of cells to have a is not for the of a of compounds for The cell assay was used to the of the compounds to and but it was not to and the in these A assay was developed to the PI4P in the various in a assay to the testing of the of the compounds and their for of and were to PI4P levels in For we the of the to the and the to an of the and the were a by the The of PI4P in the in to the in the of the that can be in a plate a whole cell P. G. M. N. Balla T. lipid in various membrane compartments using lipid PubMed Scopus Google Scholar). the of the in the we used an of the PI4KA enzyme, which of the large PM of of the lipid available in the to the PI4P pools It is to that the PI4KA not inhibit PI4K2A or N. J. G. R. M. R. A. Wilson S. et and of its role in plasma membrane and Biol. Chem. 2014; Full Text Full Text PDF PubMed Scopus (103) Google Scholar). in for pools and PI4P present in and that of PI4K2A of the PI4P by this et These have that the assay the PI4P was a to PI4K2A the all inhibitors were using this The that the PI4P in the compartment by of its in the enzyme the to show any in intact that this have a the an that was in of the to its or The in the ring to be to or the of in this ring in a of was no structural J. Z. L. H. P. J. J. et a specific of the of cancer Res. PubMed Scopus Google Scholar) and any of the compounds in this The of three a a and a the the of the the was or its to such as of a different of similar or The was with or their and the was in to and type of at of the new compounds were the in selected is shown in these efforts to the structure, we a and of the these compounds to similar in the enzyme to the of the of the function, they were found in of cell The of was to their only by using different of were by the treatment of a with or was at small molecular inhibitors of The role of this enzyme and its enzyme, in cellular functions is the the four PI4K While most studies found PI4K2A for the of the PI4P pools R. S. L. De P. and through regulation of Full Text Full Text PDF PubMed Scopus Google Scholar) on its structural to M. A. H. M. Nencka R. E. The of phosphatidylinositol 4-kinase and the of phosphatidylinositol 4-kinase a provide a structural for isoform-specific Biol. 2015; PubMed Scopus Google a functional these enzymes A small molecule would most target both enzymes of their cellular functions under after their efforts have compounds that can be for studies that of we not any of to their we not studies at this to the of these against the these inhibitors are specific the PI4Ks to the type II but they For the same we not to any studies compounds with better are found that would be of efforts are pursued in A in this was the development of a that allows the of PI4K2A inhibitors in a cellular using the in a plate has to be an to of inhibitors without the need of lipid testing PI4K2A inhibitors in the The are for the by the Varnai of was at the and of the of and of with the of and with bioluminescence resonance energy transfer hepatitis C virus high-throughput phosphatidylinositol phosphatidylinositol 4-kinase phosphatidylinositol 4-kinase type II alpha plasma membrane phosphatidylinositol 4-phosphate high-throughput