<p>Recent advances in structural biology and biochemistry have identified proteins downstream of G protein-coupled receptors (GPCRs) as promising drug targets. These proteins are highly regulated to ensure proper physiological responses from extracellular stimuli. Dysregulation of these signaling enzymes can have detrimental consequences, including cardiovascular disease and cancer. Understanding how these proteins are regulated from a structural and biochemical standpoint can therefore be exploited to develop new therapeutics.</p> <p>In this work, the molecular mechanism of regulation of two different proteins downstream of GPCRs is investigated. The first protein, Regulator of G Protein Signaling 17 (RGS17), is involved in numerous processes throughout the body, including the development and progression of lung cancer. This work presents the crystal structure of RGS17 bound to Ca²⁺. Ca²⁺ was found to bind to the same site as the predicted Ga binding surface and increases interactions between RGS17 and Ga_o. Therefore, Ca²⁺ positively regulates RGS17, supporting a mechanism in which Ca²⁺ increases the GTPase activating function of the RZ-family of RGS proteins to ultimately downregulate Ca²⁺ signaling.</p> <p>The second protein, phospholipase Ce (PLCe), has been implicated in cardiac hypertrophy through its production of second messengers. This process is regulated by the small GTPase Rap1A. This work presents insight into the molecular mechanism of Rap1A-dependent activation of PLCe, in which four conserved, hydrophobic residues on the surface of the RA2 domain of PLCe play an essential role. Furthermore, small angle X-ray scattering studies show that binding of Rap1A induces conformational changes in PLCe, resulting in a more compact activated complex. This supports a mechanism in which Rap1A is an allosteric activator of PLCe, inducing conformational changes in PLCe that increase lipid hydrolysis by relieving autoinhibitory interactions and/or by promoting interactions with the cell membrane.</p>

G–Protein Coupled Receptors are one of the most important targets in drug development, making up over 60% of drug targets. Recent studies have implicated a role of Regulator of G–Protein Signaling (RGS) proteins in the development and progression of pathologies, including some cancers. RGS17, the most–recently identified family member of the RZ family of RGS proteins, has been implicated in the growth, proliferation, metastasis and migration of prostate tumors as well as small–cell and non–small cell lung cancers. In neoplastic tumor tissues RGS17 is up–regulated 13 fold over patient–matched normal tissues in prostate cancer. Studies have shown that RGS17 RNAi knockdown inhibits colony formation and decreases tumorigenesis in nude mice. Based on these findings, this thesis explores the research undertaken to develop small molecule inhibitors of the RGS17: Gαo protein: protein interaction. In this thesis, we implemented AlphaScreen® technology to develop a high–throughput screening method for interrogating small molecule libraries for inhibitors of RGS17. Chapter 3 focuses on the initial results of the AlphaScreen® in 384–well format. The screen utilizes a measurement of the Gα: RGS17 protein: protein interaction (PPI) and with an excellent Z–score exceeding 0.73, a signal to noise ratio >70 and a screening time of 1,100 compounds per hour. Chapter 3 presents the development, validation and initial high–throughput screening for inhibitors of Gα: RGS17 interaction as well as preliminary characterization of the RL series of hits. In this pilot screen the NCI Diversity Set II was interrogated, yielding 35 initial hits of which 16 were confirmed after screening against controls. The 16 compounds exhibited IC50 <10 ΜM in dose–response experiments for inhibiting the Gα: RGS17 interaction. Four exhibited IC50 values <6 ΜM while inhibiting the Gα: RGS17 interaction >50% when compared to a biotinylated GST control (TrueHits). Compounds RL–1 and RL–2 were confirmed by flow cytometry protein interaction assay (FCPIA) while RL–3 and RL–4 were unable to disrupt this PPI in FCPIA. All four compounds were tested using the differential scanning fluorimetry (DSF) method, which is based on energetic coupling between ligand binding and protein unfolding and found compounds RL–1 to RL–4 all slightly increased protein stability upon ligand binding. Chapter 4 focuses on the miniaturization and optimization of AlphaScreen® to a 1536–well format and screening of the MicroSource SPECTRUM and NDL3000 small molecule libraries. This increased throughput 11–fold and decreased our working volumes from 45 ΜL to 10 ΜL, which reduced reagent cost. After optimization, we retained in an excellent Z–factor ≥0.70 with S/N>5.77 and increased the screening rate to more than 12,000 compounds per hour. In this format, the initial screening of the SPECTRUM and NDL3000 libraries was completed and filtered the initial hits by counter screening and PAINs filtering as well as developing four powerful orthogonal assays for the characterization of potential lead molecules. Chapter 6 focuses on the future directions, which include the screening the in–house 50,000 compound library in the University of Iowa HTS Core facility as well as the development of cell based assays to determine the activity of these leads in the cellular milieu. These screens are the first step to developing novel pharmacophores for further optimization of structure with the focus on RGS17 activity in enzymatic, whole cell, xenograft and whole animal models as well as providing new avenues for the development of anticancer therapies.