PITPs as targets for selectively interfering with phosphoinositide signaling in cells

Abstract

Sec14-like phosphatidylinositol transfer proteins (PITPs) integrate diverse territories of intracellular lipid metabolism with stimulated phosphatidylinositol-4-phosphate production, and are discriminating portals for interrogating phosphoinositide signaling. Yet, neither Sec14-like PITPs, nor PITPs in general, have been exploited as targets for chemical inhibition for such purposes. Herein, we validate the first small molecule inhibitors (SMIs) of the yeast PITP Sec14. These SMIs are nitrophenyl(4-(2-methoxyphenyl)piperazin-1-yl)methanones (NPPMs), and are effective inhibitors in vitro and in vivo. We further establish Sec14 is the sole essential NPPM target in yeast, that NPPMs exhibit exquisite targeting specificities for Sec14 (relative to related Sec14-like PITPs), propose a mechanism for how NPPMs exert their inhibitory effects, and demonstrate NPPMs exhibit exquisite pathway selectivity in inhibiting phosphoinositide signaling in cells. These data deliver proof-of-concept that PITP-directed SMIs offer new and generally applicable avenues for intervening with phosphoinositide signaling pathways with selectivities superior to those afforded by contemporary lipid kinase-directed strategies.

Structural elements that govern Sec14-like PITP sensitivities to potent small molecule inhibitors

“…Computational docking simulations, coupled with rational mutagenesis experiments, indicate the latter binding interactions are crucial. The NPPM-activated aryl halide moiety is projected to engage residue Ser173, a key component of the PtdCho headgroup-coordinating substructure, via a halogen bond ( 14 ). Because four of the other fi ve yeast Sec14-like PITPs (Sfh2-Sfh5) do not conserve this PtdCho-coordinating unit ( 9 ), the NPPM resistance of those proteins is readily explained by their lack of the structural elements essential for NPPM binding.…”

“…Of the SMIs that target Sec14 activity, the nitrophenyl(4-(2-methoxyphenyl) piperazin-1-yl)methanones (NPPMs) are best understood ( 14,20,21 ). These SMIs inhibit Sec14 by loading into the phospholipid binding pocket of this PITP and preventing subsequent rounds of lipid exchange ( 14 ).…”

“…Briefl y, Escherichia coli BL21 (DE3) cells carrying the appropriate (PtdCho) in vitro, and it coordinates PtdIns-4-phosphate production with PtdCho metabolism so as to promote membrane traffi cking through trans-Golgi network and endosomal compartments in vivo (17)(18)(19). Of the SMIs that target Sec14 activity, the nitrophenyl(4-(2-methoxyphenyl) piperazin-1-yl)methanones (NPPMs) are best understood ( 14,20,21 ). These SMIs inhibit Sec14 by loading into the phospholipid binding pocket of this PITP and preventing subsequent rounds of lipid exchange ( 14 ).…”

“…Emerging evidence that PITPs instruct the biological outcomes of PtdIns kinase activities recommends these proteins as novel targets for chemical intervention with PIP signaling pathways in cells ( 11,14 ). The advantages of targeting PITPs for this purpose are that such interventions can be imposed with selectivities superior to those possible by popular strategies that either target individual PtdIns-kinase isoforms or individual PIP species ( 15,16 ).…”

“…We now appreciate that PtdIns transfer proteins (PITPs) play critical roles in functional compartmentalization of PIP signaling reactions by channeling PtdIns to PtdIns kinases and, subsequently, to distinct sets of effector proteins ( 9-11 ). The Sec14-like PITPs are best studied in this regard, and the multiplicity of Sec14-like PITPs expressed in even simple unicellular eukaryotes highlights the high degree of functional specifi cation for these proteins ( 12,13 ).Emerging evidence that PITPs instruct the biological outcomes of PtdIns kinase activities recommends these proteins as novel targets for chemical intervention with PIP signaling pathways in cells ( 11,14 ). The advantages of targeting PITPs for this purpose are that such interventions can be imposed with selectivities superior to those possible by popular strategies that either target individual PtdIns-kinase isoforms or individual PIP species ( 15,16 ).…”