Collections > Electronic Theses and Dissertations > Chemical Inhibitors of Phosphatidylinositol Transfer Proteins Enable Highly Selective Interference With Specific Pathways of Phosphoinositide Signaling in Cells
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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.