Phospholipase Cβ (PLCβ) enzymes are peripheral membrane proteins required for normal cardiovascular function. PLCβ hydrolyzes phosphatidylinositol 4,5-bisphosphate, producing second messengers that increase intracellular Ca2+ level and activate protein kinase C. Under basal conditions, PLCβ is autoinhibited by its C-terminal domains and by the X–Y linker, which contains a stretch of conserved acidic residues required for interfacial activation. Following stimulation of G protein-coupled receptors, the heterotrimeric G protein subunit Gαq allosterically activates PLCβ and helps orient the activated complex at the membrane for efficient lipid hydrolysis. However, the molecular basis for how the PLCβ X–Y linker, its C-terminal domains, Gαq, and the membrane coordinately regulate activity is not well understood. Using compressed lipid monolayers and atomic force microscopy, we found that a highly conserved acidic region of the X–Y linker is sufficient to regulate adsorption. Regulation of adsorption and activity by the X–Y linker also occurs independently of the C-terminal domains. We next investigated whether Gαq-dependent activation of PLCβ altered interactions with the model membrane. Gαq increased PLCβ adsorption in a manner that was independent of the PLCβ regulatory elements and targeted adsorption to specific regions of the monolayer in the absence of the C-terminal domains. Thus, the mechanism of Gαq-dependent activation likely includes a spatial component.

Phospholipase C (PLC) is a member of the PLC family of enzymes, which hydrolyze phosphatidylinositol lipids following the activation of G protein coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs). PLC is unique among the PLC superfamily as it contains an Nterminal CDC25 domain, which has a guanine nucleotide exchange factor (GEF) activity for the small G protein Rap1A, and two C-terminal Ras association (RA) domains that bind scaffolding proteins and activated G proteins. PLC activity plays an important role in cardiomyocyte contractility and survival. The best-characterized pathway of PLC activation is mediated by adrenergic (-AR) receptors. Stimulation of these receptors culminates in the activation of the small GTPase Rap1A, which binds to PLC at the sarcoplasmic reticulum. There, PLC hydrolyzes phosphatidylinol-4-phosphate (PI4P) to produce diacylglycerol (DAG). Prolonged activation of this pathway results in increased Ca 2+ -induced Ca 2+ release (CICR) and increased expression of hypertrophy-related genes. However, the structural basis of PLC basal activity, and the mechanism of Rap1A activation are largely unknown. We have now obtained the first highresolution structure of PLC. These studies, together with biochemical validation of our structurebased hypotheses, provide the first molecular insights into this enzyme. 9 CHAPTER 1.

Soluble epoxide hydrolase (sEH) is a novel therapeutic target for the treatment neurodegenerative diseases, including Parkinson’s disease and Alzheimer’s disease. Although many sEH inhibitors exhibit sub‐nanomolar potency, they suffer from poor blood‐brain barrier penetration and drug‐like properties. As high in vivolevel is required to achieve clinical efficacy for sEH inhibitors for other therapeutic applications, low CNS penetration represents a significant barrier for success for CNS diseases. Therefore, sEH inhibitors with high blood‐brain barrier penetration are needed. Although significant structure‐activity relationships (SAR) have been reported, little data exist exploring the features that overcome the blood‐brain barrier. Most SAR studies have focused on terminal substitutions on both ends of the reported inhibitors with little focus on the “linker” or core of the inhibitors. In this presentation, we will show how the “linker” of urea‐based sEH inhibitors affects their potency, physical properties, and ability to penetrate the blood‐brain barrier. Based on our computational model and the crystal structure of our lead compound, we have prepared new sEH inhibitors with linker containing various ring sizes, bicyclic structures, and simple methyl or fluorine substituents. Perhaps not unexpectantly, the linker of sEH inhibitors has substantial effects on the inhibitor’s potency, solubility, and blood‐brain barrier penetration. In some cases, we observed an increase in blood‐brain penetration by 4‐fold and improvements in solubility by almost 10‐fold. In addition, unexpectedly, our data provides new insights that the linker of the sEH inhibitor affects the binding kinetics without improving the potency of the inhibitors. Our results will create a new direction for the design of novel sEH inhibitors, and we are in the process of leveraging the existing SAR to create much‐improved sEH inhibitors to treat neurodegenerative diseases.