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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 what is to our knowledge 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 that Sec14 is the sole essential NPPM target in yeast and 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 that 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.

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Figure 1: NPPMs specifically inactivate Sec14.
Figure 2: NPPM SARs.
Figure 3: Sec14 is the essential cellular target of bioactive NPPMs.
Figure 4: Sec14-active NPPMs exhibit compartment-specific inhibition of PtdIns-(4)-phosphate signaling.
Figure 5: NPPMs discriminate between Sec14- and Sfh4-mediated PtdIns-4-P signaling.
Figure 6: Model for NPPM-mediated inhibition of Sec14.

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Acknowledgements

This work was supported by the Robert A. Welch Foundation (V.A.B.) and grant GM44530 (V.A.B.) from the US National Institutes of Health (NIH). R.W.D. was supported by the NIH (HG003317). G. Giaever and C. Nislow were supported by the National Human Genome Research Institute (5RO1-003317-08) and the Canadian Cancer Society (020380). The Texas A&M Laboratory for Molecular Simulation provided software, support and computer time. G.E. Kellogg and eduSoft LC donated HINT software, U. Schlecht (Stanford) assisted with growth analyses, A. Holzenburg and R. Littleton (Texas A&M) assisted with electron microscopy. We are grateful to D. Lew (Duke University) for donating mss4ts alleles.

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A.H.N. was involved in all experimentation, design, data analysis, manuscript preparation and figure production. A. Tripathi, with assistance from B.T., D.T.P. and A. Tropsha, was involved in designing computational experiments, data analysis, manuscript preparation and figure production. P.Y. and C.J.M. performed electron microscopy and invertase and CPY experiments, and S.D.S. generated site-directed mutants and purified proteins. I.M.W., C.N., S.S. and R.P.S. selected initial SAR compounds, performed and designed chemogenomic profiling. R.W.D. and G.G. advised chemogenomic studies. V.A.B. was involved in all aspects of experimental design, data analysis and manuscript preparation.

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Correspondence to Ashutosh Tripathi or Vytas A Bankaitis.

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Supplementary Results, Supplementary Tables 1–3 and Supplementary Figures 1–25. (PDF 20340 kb)

Supplementary Note 1

Significant GIs observed (−2 ≤ S ≥ 2). (PDF 5387 kb)

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Nile, A., Tripathi, A., Yuan, P. et al. PITPs as targets for selectively interfering with phosphoinositide signaling in cells. Nat Chem Biol 10, 76–84 (2014). https://doi.org/10.1038/nchembio.1389

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