Chemical Inhibitors of Phosphatidylinositol Transfer Proteins Enable Highly Selective Interference With Specific Pathways of Phosphoinositide Signaling in Cells

Nile, Aaron Hugh

Chemical Inhibitors of Phosphatidylinositol Transfer Proteins Enable Highly Selective Interference With Specific Pathways of Phosphoinositide Signaling in Cells

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March 19, 2019

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Nile, Aaron Hugh
- Affiliation: School of Medicine, Department of Cell Biology and Physiology

Abstract

Phosphatidylinositol phosphates (PIP) are phosphorylated derivatives of phosphatidylinositol (PtdIns) that signal to and regulate diverse cellular functions including membrane trafficking, cytokinesis, cell cycle regulation and DNA repair. PIP-signaling is regulated by a variety of proteins through degradation, phosphorylation and dephosphorylation. Members of the Sec14-like phosphatidylinositol transfer protein superfamily (Sec14-PITPs) have at least two functions which include lipid-binding platforms and/or `nanoreactors' that direct PtdIns-OH kinase activity to generate discrete PIP-pools. In Chapter 1, I outline the current literature on the Sec14-superfamily and the structurally unrelated START-like PITPs with special emphasis on mammalian PITPs, and how their disruption results in a number of inherited mammalian diseases. Neither Sec14-like or START-like PITPs have been targeted for chemical intervention using small molecule inhibitors (SMIs). The development of PITP-directed SMIs provide applications not only as tool compounds, but also as therapeutic agents that inhibit a number of pathogenic organisms and potentially as activators of defective PITPs. As proof-of-concept, I developed the first PITP-directed SMIs that specifically inhibit the prototype Sec14-like PITP from Saccharomyces cerevisiae. In yeast, Sec14 connects the production of phosphatidylinositol 4-phosphate (PtdIns(4)P) and phosphatidylcholine (PtdCho) metabolism with trafficking through the trans-Golgi/endosomal network. In Chapter 2, I describe the development of the Sec14-directed SMI, 4-chloro-3-nitrophenyl)(4-(2-methoxyphenyl) piperazin-1-yl)methanones or NPPM. These SMIs specifically and directly inhibit Sec14 through its hydrophobic cavity, likely by a halogen-bonding mechanism. Based on my work in Chapter 2, I developed a routine for the rapid validation of novel PITP-directed SMIs from a variety of organisms that will streamline future SMI-identification. Together, these data deliver proof-of-concept that PITP-directed SMIs offer new and generally applicable avenue for intervening with phosphoinositide signaling pathways with selectivities superior to those afforded by contemporary lipid kinase-directed strategies. Finally, the study of PIPs has been advanced through the development of multiple methodologies that both detect and modify PIPs in vivo. In Chapter 3, I discuss current methods used to monitor and manipulate PIP-signaling pathways with special emphasis on SMIs that target PIP-modifying enzymes.

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