Abstract
Src homology 2 domain-containing inositol phosphate phosphatase 2 (SHIP2) is one of the 10 human inositol phosphate 5-phosphatases. One of its physiological functions is dephosphorylation of phosphatidylinositol 3,4,5-trisphosphate, PtdIns(3,4,5)P3. It is therefore a therapeutic target for pathophysiologies dependent on PtdIns(3,4,5)P3 and PtdIns(3,4)P2. Therapeutic interventions are limited by the dearth of crystallographic data describing ligand/inhibitor binding. An active site-directed fluorescent probe facilitated screening of compound libraries for SHIP2 ligands. With two additional orthogonal assays, several ligands including galloflavin were identified as low micromolar Ki inhibitors. One ligand, an oxo-linked ethylene-bridged dimer of benzene 1,2,4-trisphosphate, was shown to be an uncompetitive inhibitor that binds to a regulatory site on the catalytic domain. We posit that binding of ligands to this site restrains L4 loop motions that are key to interdomain communications that accompany high catalytic activity with phosphoinositide substrate. This site may, therefore, be a future druggable target for medicinal chemistry.
Highlights
Multiple mammalian enzymes share a conserved 5phosphatase domain, and these include INPP5A, INPP5B, INPP5E, INPP5J, OCRL, SKIP, Synaptojanin 1, Synaptojanin 2, SHIP1, and SHIP2.1,2 While the physiological substrates of these enzymes are not all clearly defined some, for example, SHIP1 and SHIP2, that act physiologically on PtdIns(3,4,5)P3 have become therapeutic targets, not least because of the restricted expression of SHIP1 in blood cell lineages.[3]
In an attempt to confirm active-site binding of 2-FAM-InsP5, 2-FAM-InsP5 Binds to SHIP2 at the Catalytic Site. 2FAM-InsP5 has been shown to be a promising active-site probe of both inositol pentakisphosphate 2-kinase[19] and human SHIP2.16 Figure 1A compares the structure of 2-FAM-InsP5 with the physiological substrate PtdIns(3,4,5)P3, its watersoluble head group, Ins(1,3,4,5)P4, and Ins(1,3,4,5,6)P5
We explore the binding of 2-FAM-InsP5 to the catalytic domain of human SHIP2 in greater detail, hereafter SHIP2 catalytic domain (SHIP2cd)
Summary
Multiple mammalian enzymes share a conserved (inositol) 5phosphatase domain, and these include INPP5A, INPP5B, INPP5E, INPP5J, OCRL, SKIP, Synaptojanin 1, Synaptojanin 2, SHIP1, and SHIP2.1,2 While the physiological substrates of these enzymes are not all clearly defined some, for example, SHIP1 and SHIP2, that act physiologically on PtdIns(3,4,5)P3 have become therapeutic targets, not least because of the restricted expression of SHIP1 in blood cell lineages.[3]. Irrespective of the mode of inhibition or activation of inositide- or phosphoinositide-metabolizing enzymes, the efficacy of inhibitors or activators reflects binding and subsequent catalytic processing of the bound inositide/ phosphoinositide substrate. We note prior description of measurement of lipid phosphatase reaction products by indirect end-point competition assays employing FP probes.[12,17] By the use of orthogonal approaches, several lead compounds are identified. Simple phosphate release and HPLC assays confirm the lead compounds to be potent inhibitors of SHIP2. The HPLC approach, itself, obviates the use of radiolabeled substrate and is able to confirm inhibition of the 5-phosphatase activity of SHIP2. Using a structural biology approach, we solve the structure of a SHIP2 catalytic domain in complex with a bound dimeric ligand and potent inhibitor. Our study reveals a potentially druggable allosteric site on the enzyme
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