title: Identification of inhibitors of inositol 5-phosphatases through multiple screening strategies.
abstract: Phosphoinositides are low abundance membrane phospholipids that have key roles in signaling, membrane trafficking, and cytoskeletal dynamics in all cells. Until recently, strategies for robust and quantitative development of pharmacological tools for manipulating phosphoinositide levels have focused selectively on PI(3,4,5)P3 due to the importance of this lipid in growth factor signaling and cell proliferation. However, drugs that affect levels of other phosphoinositides have potential therapeutic applications and will be powerful research tools. Here, we describe methodology for the high-throughput screening of small molecule modulators of the inositol 5-phosphatases, which dephosphorylate PI(4,5)P2 (the precursor for PI(3,4,5)P3) and PI(3,4,5)P3). We developed three complementary in vitro activity assays, tested hit compounds on a panel of 5-phosphatases, and monitored efficacy toward various substrates. Two prominent chemical scaffolds were identified with high nanomolar/low micromolar activity, with one class showing inhibitory activity toward all 5-phosphatases tested and the other selective activity toward OCRL and INPP5B, which are closely related to each other. One highly soluble OCRL/INPP5B-specific inhibitor shows a direct interaction with the catalytic domain of INPP5B. The efficacy of this compound in living cells was validated through its property to enhance actin nucleation at the cell cortex, a PI(4,5)P2 dependent process, and to inhibit PI(4,5)P2 dephosphorylation by OCRL (both overexpressed and endogenous enzyme). The assays and screening strategies described here are applicable to other phosphoinositide-metabolizing enzymes, at least several of which have major clinical relevance. Most importantly, this study identifies the first OCRL/INPP5B specific inhibitor and provides a platform for the design of more potent inhibitors of this family of enzymes.
authors.affiliation: Department of Cell Biology, Howard Hughes Medical Institute and Program in Cellular Neuroscience, Neurodegeneration and Repair, Yale University School of Medicine , New Haven Connecticut 06510, United States.
authors.forename: Michelle
authors.initials: M
authors.lastname: Pirruccello
authors.forename: Ramiro
authors.initials: R
authors.lastname: Nandez
authors.forename: Olof
authors.initials: O
authors.lastname: Idevall-Hagren
authors.forename: Abel
authors.initials: A
authors.lastname: Alcazar-Roman
authors.forename: Laura
authors.initials: L
authors.lastname: Abriola
authors.forename: Shana Alexandra
authors.initials: SA
authors.lastname: Berwick
authors.forename: Louise
authors.initials: L
authors.lastname: Lucast
authors.forename: Dayna
authors.initials: D
authors.lastname: Morel
authors.forename: Pietro
authors.initials: P
authors.lastname: De Camilli
mesh_terms: D002478:Cells, Cultured; D020405:Dermis / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology; D024202:Electrophoretic Mobility Shift Assay; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology; D005454:Fluorescence Polarization; D057166:High-Throughput Screening Assays; D006801:Humans; D000072181:Inositol Polyphosphate 5-Phosphatases; D015394:Molecular Structure; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism*; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D012394:Rosaniline Dyes; D015398:Signal Transduction / Q000187:drug effects; D054852:Small Molecule Libraries / Q000737:chemistry / Q000494:pharmacology*; D013830:Thiadiazoles / Q000737:chemistry / Q000494:pharmacology*; D014230:Triazoles / Q000737:chemistry / Q000494:pharmacology*
chemical_list: D004791:Enzyme Inhibitors; D018129:Phosphatidylinositol Phosphates; D012394:Rosaniline Dyes; D054852:Small Molecule Libraries; D013830:Thiadiazoles; D014230:Triazoles; C000600630:YU142670; C005095:malachite green; D010744:Phosphoric Monoester Hydrolases; C075695:OCRL protein, human; D000072181:Inositol Polyphosphate 5-Phosphatases
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