Indiana University-Purdue University Indianapolis (IUPUI) / The DNA Base Excision Repair (BER) pathway repairs DNA damaged by endogenous and exogenous agents including chemotherapeutic agents. Removal of the damaged base by a DNA glycosylase creates an apurinic / apyrimidinic (AP) site. AP endonuclease1 (Ape1), a critical component in this pathway, hydrolyzes the phosphodiester backbone 5’ to the AP site to facilitate repair. Additionally, Ape1 also functions as a redox factor, known as Ref-1, to reduce and activate key transcription factors such as AP-1 (Fos/Jun), p53, HIF-1α and others. Elevated Ape1 levels in cancers are indicators of poor prognosis and chemotherapeutic resistance, and removal of Ape1 via methodology such as siRNA sensitizes cancer cell lines to chemotherapeutic agents. However, since Ape1 is a multifunctional protein, removing it from cells not only inhibits its DNA repair activity but also impairs its other functions. Our hypothesis is that a small molecule inhibitor of the DNA repair activity of Ape1 will help elucidate the importance (role) of its repair function in cancer progression as wells as tumor drug response and will also give us a pharmacological tool to enhance cancer cells’ sensitivity to chemotherapy. In order to discover an inhibitor of Ape1’s DNA repair function, a fluorescence-based high-throughput screening (HTS) assay was used to screen a library of drug-like compounds. Four distinct compounds (AR01, 02, 03 and 06) that inhibited Ape1’s DNA repair activity were identified. All four compounds inhibited the DNA repair activity of purified Ape1 protein and also inhibited Ape1’s activity in cellular extracts. Based on these and other in vitro studies, AR03 was utilized in cell culture-based assays to test our hypothesis that inhibition of the DNA repair activity of Ape1 would sensitize cancer cells to chemotherapeutic agents. The SF767 glioblastoma cell line was used in our assays as the chemotherapeutic agents used to treat gliobastomas induce lesions repaired by the BER pathway. AR03 is cytotoxic to SF767 glioblastoma cancer cells as a single agent and enhances the cytotoxicity of alkylating agents, which is consistent with Ape1’s inability to process the AP sites generated. I have identified a compound, which inhibits Ape1’s DNA repair activity and may have the potential in improving chemotherapeutic efficacy of selected chemotherapeutic agents as well as to help us understand better the role of Ape1’s repair function as opposed to its other functions in the cell.

The homologous recombination (HR) pathway of DNA repair is essential for the faithful repair of double-stranded DNA breaks (DSBs) in all organisms and as such helps maintain genomic stability. Furthermore, HR is instrumental in the cellular response to exogenous DNA damaging agents such as those used in the clinic for chemo- and radiotherapy. HR in humans is a complex, incompletely understood process involving numerous stages and diverse biochemical activities. Advancing our knowledge of the HR pathway in humans aids the understanding of how chemo- and radiotherapies act and may be used to develop novel therapeutic strategies. Recent studies have identified inhibition of HR as one of the mechanisms via which a number of recently developed chemotherapeutics have their effect. Accordingly, the clinical potential of HR inhibitors is under investigation. My work has centred around the identification of both novel HR proteins and novel, small molecule HR inhibitors. To further these aims, I have successfully employed high-throughput RNAi and small molecule screening strategies. RNAi screens are commonly used to identify genes involved in a given cellular process via genetic loss of function, whilst small molecule, cell based screens are a powerful tool in the drug discovery process.

Cross-coupling reactions, where a transition metal catalyst facilitates the formation of a new carbon-carbon or carbon-heteroatom bond between two coupling partners, has become one of the most widely used, reliable, and robust family of transformations for the construction of molecules. The Nobel Prize was awarded to pioneers in this field who primarily used aryl iodides, bromides, and triflates as electrophilic coupling partners. The expansion of the reaction scope to non-traditional electrophiles is an ongoing challenge to enable an even greater number of useful products to be made from simple starting materials. The major goal of this thesis research is to improve and expand upon this field by using esters as electrophiles via the activation of the strong C(acyl)−O bond. Esters are particularly robust in comparison to other carboxylic acid derivatives used in cross-coupling reactions. Success on the activation of such inert functional group using catalysis has both fundamental and practical value. By discovering new reaction modes of this abundant functional group, synthetic routes to access novel or industrially important molecules can be improved. Chapter 1 of this thesis describes a literature overview of what has been accomplished in the field of cross coupling reactions using carboxylic acid derivatives as electrophilic coupling partners. Chapter 2 discloses the first palladium Suzuki-Miyaura couplings of phenyl esters to produce ketones. The method is efficient and robust, giving good yields of useful products. The reaction is proposed to proceed via an oxidative addition to the strong C(acyl)−O bond of the ester. In contrast to previous efforts in this field that use traditional catalysts such as Pd(PPh3)4, the developed reaction requires use of an electron-rich, bulky N-heterocyclic carbene ligand, which facilitates the strong bond activation. Furthermore, a palladium-catalyzed cross-coupling between aryl esters and anilines is reported, enabling access to diverse amides. The reaction takes place via a similar activation of the C−O bond by oxidative addition with a Pd−NHC complex, which enables the use of relatively non-nucleophilic anilines that otherwise require stoichiometric activation with strong bases to react. Chapter 3 discloses a nickel-catalyzed amide bond formation using unactivated and abundant esters. In this transformation, an accessible nickel catalyst can facilitate the activation of diverse aliphatic and aromatic esters to enable direct amide bond formation with amines as nucleophiles. No stoichiometric base, acid, or other activating agent is needed, providing exceptional functional group tolerance and producing only methanol as a by-product. This reaction is of both fundamental and practical importance because it is the first to demonstrate that simple conditions can enable Ni to cleave the C–O bond of an ester to make an oxidative addition product, which can be subsequently coupled with amines. This discovery contrasts industrially-common and wasteful methods that still require stoichiometric activating agents or multistep synthesis. Chapter 4 describes the evaluation of different types of cross-coupling reactions using methyl esters as electrophilic coupling partner. A high-throughput screening technique has been applied to this project. A combination between specific ligands, known by their efficiency to activate strong C−O bonds, and literature-based conditions has been designed for the chosen transformations. Using this strategy, two promising hits have been obtained using the same NHC ligand: a decarbonylative Suzuki-Miyaura and a decarbonylative borylation reaction.

Thesis advisor: Charles S. Hoffman / In this thesis, I describe the development of a screening platform for detecting PDE8A inhibitors using the cAMP-dependent glucose sensing pathway of the fission yeast Schizosaccharomyces pombe, which led us to discover several PDE8A selective inhibitors. In this system, the only PDE of the fission yeast is replaced with mammalian PDE8A1 in strains that have been engineered such that PDE inhibition is required to allow cell growth. Using this system, I screened 56 compounds obtained from PDE4 and PDE7 high throughput screens (HTSs) and identified a PDE4-PDE8 dual specificity inhibitor. Using this as a positive control, I developed a robust high-throughput screen (HTS) for PDE8A inhibitors and screened 240,267 compounds at the Harvard Medical School ICCB Screening Facility. Approximately 0.2 % of the screened compounds were potential PDE8A inhibitors with 0.03% displaying significant potency. Secondary assays of 367 of the most effective compounds against strains expressing PDE8A (both full length and catalytic domain), PDE4A and PDE7A or PDE7B led to the selection of structurally diverse compounds for further testing. To profile the selectivity of twenty-eight of these compounds, dose response assays were conducted using 16 yeast strains that express different PDE isoforms (representing all PDE families with the exception of the PDE6 family). These assays identified compounds with different patterns of inhibition, including structurally-distinct PDE8A-specific inhibitors. By evaluating the effects of these compounds for steroid production in mouse Leydig cells, biologically active compounds that can elevate steroid production were identified. / Thesis (PhD) — Boston College, 2011. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Many congenital skeletal muscle disorders including muscular dystrophies are caused by genetic mutations that lead to a dysfunction in myocytes effectively binding to the extracellular matrix. This leads to a chronic and continuous cycle of breakdown and regeneration of muscle tissue, ultimately resulting in loss of muscle function and patient mortality. Such disorders lack effective clinical treatments and challenge researchers to develop new therapeutics. The current drug development process often yields ineffective therapeutics due to the lack of genetic homology between pre-clinical animal models and humans. In addition current engineered tissue models using human cells fail to properly emulate native muscle morphology and function due to necrotic tissue cores and an abundance undigested ECM protein. Thus, a more precise benchtop model of 3D engineered human muscle tissue could serve as a better platform for translation to a disease model and could better predict candidate drug efficacy during pre- clinical development. This work presents the methodology for generating a high-content system of contiguous skeletal muscle tissue constructs produced entirely from human cells by using a non-adhesive hydrogel micro-molding technique. Subsequent culture and mold modifications confirmed by morphological and contractile protein analysis improve tissue longevity and myocyte maturation. Finally, mechanical strength and contractile force measurements confirmed that such modulations resulted in skeletal muscle microtissues that were more mimetic of human muscle tissue. This cell self-assembly technique yielded tissues approximately 150um in diameter with cell densities approaching that of native muscle. Modifications including seeding pre-differentiated myoblasts and the addition of ECM producing fibroblasts improved both tissue formation efficiency and cell alignment. Further culture modifications including supplementation of the culture medium with 50ug/ml ascorbic acid and 100ng/ml Insulin-like growth factor-1 coupled with a mold redesign that allowed tissue to passively contract during maturation while still remaining anchored under tension further improved ECM production, myogenic differentiation, and long-term longevity in culture. Further confirmation of the culture improvements were demonstrated by increases in mechanical strength and contractile force production. In conclusion, this approach overcomes cell density limitations with exogenous ECM-based methods and provides a platform for producing 3D models of human skeletal muscle by making tissue entirely using cells. Future work will attempt to translate the methodology used for tissue generation and long-term culture to create benchtop models of disease models of skeletal muscle, streamlining pre- clinical benchtop testing to better predict candidate drug efficacy for skeletal muscle diseases and disorders along with elucidating side effects of non-target drugs.

La disponibilité à la fois des données à grande échelle du transcriptome et du génome de tumeurs permet maintenant d'identifier assez facilement des oncogènes candidats, gènes qui sont surexprimés en conséquence de l'amplification d'ADN. Ces oncogènes candidats doivent alors être fonctionnellement validés et leur rôle dans la cellule normale et tumorale doit être étudié.Dans cette étude, nous nous sommes principalement focalisés sur le cancer du sein, le cancer le plus fréquent chez les femmes et la deuxième cause de décès par cancer chez les femmes à travers le monde. En France, 52.000 nouveaux cas avec 12.000 décès dus au cancer du sein ont été estimés en 2010 représentant 34% de tous les nouveaux cas de cancer chez les femmes. Les chromosomes les plus fréquemment altérés dans le cancer du sein sont les chromosomes 8, 11 et 17, qui contiennent les amplicons 17q12 (ERBB2) et 11q13 (CCND1). Le développement de «l 'herceptine" contre ERBB2 illustre le potentiel de la génomique fonctionnelle du cancer pour l'identification de cibles thérapeutiques. Plusieurs études ont identifié d'autres amplicons avec des oncogènes candidats. Cependant très peu d'études ont rapporté la validation fonctionnelle des candidats identifiés, mettant ainsi en évidence la nécessité des analyses fonctionnelles à grande échelle des différents amplicons dans le cancer du sein pour identifier de nouveaux gènes pilotes qui pourraient ensuite être utilisés pour le développement de stratégies thérapeutiques pour le cancer du sein. Ces dernières années, l'ARNi est devenu un outil de choix pour le criblage à haut débit pour caractériser la fonction des gènes dans des lignées cellulaires. Dans cette étude, nous avons effectué un criblage fonctionnel à moyen-débit basé sur l’utilisation de l'ARNi de 127 gènes amplifiés et surexprimés appartenant à 11 amplicons majeurs sur les chromosomes 8, 11 et 17 dans le cancer du sein. Ce crible à permis l'identification de 8 oncogènes au sein de 5 amplicons différents. En outre, la validation fonctionnelle de 5 de ces gènes a permis de démontrer que 4 gènes, RAD21, EIF3H, TANC2 et CHRAC1 au sein de 3 amplicons, régulent l'apoptose, la prolifération et la transformation cellulaire de cellule dérivées de carcinomes mammaires. Les régions d'altération génétique dans un cancer peuvent être également modifiées dans de multiples types d’autres cancers. L'amplicon 8p11-p12 a par exemple été décrit dans le cancer du sein, du pancréas, du poumon et de la vessie. Ces amplicons communs dans différents cancers peuvent contenir des oncogènes « pilote » communs. Pour vérifier cette hypothèse, nous avons évalué l'implication possible dans des lignées cellulaires dérivées de cancer du pancréas et du poumon présentant un amplicon en 8p11-p12 de deux oncogènes à savoir, PPAPDC1B et WHSC1L1 qui ont été décrits comme des gènes « driver » de l'amplicon 8p11-p12 dans le cancer du sein et également dans le cancer du poumon pour WHSC1L1. L'inhibition de ces deux gènes réduit la survie cellulaire et la croissance indépendante de l'ancrage à un support de lignées tumorales du pancréas et du poumon présentant une amplification en 8p11-p12. Cette constatation met en évidence l'importance de ces deux gènes dans de multiples cancers et l'intérêt thérapeutique potentiel d'inhiber ces enzymes dans les cancers présentant un amplicon en 8p11-p12. Des modèles de souris transgéniques permettent d’étudier la fonction de gènes candidats in vivo. Pour évaluer in vivo le rôle de PPAPDC1B, nous avons établi des souris transgéniques sur-exprimant PPAPDC1B sous la dépendance du promoteur de la kératine 5 permettant de cibler les épithéliums pluri ou pseudo stratifiés. Les souris transgéniques développent deux phénotypes inattendus, le développement de poils le long des incisives et une inflammation aiguë des glandes salivaires, des ganglions lymphatiques, de la vessie et du pancréas. / Availability of both large scale transcriptomic and genomic data of tumours now allows to identify relatively easily candidate oncogenes that are over-expressed as a consequence of DNA amplification. These candidate oncogenes have then to be functionally validated and studied for their role in the normal and cancer cell.In this study, we mainly focused on breast cancer, the most common cancer among women and the second leading cause of cancer deaths in women around the world. In France, 52,000 new cases with 12,000 deaths of breast cancer were estimated in 2010 accounting for 34% of all new cases of cancer in women. In breast cancer the main altered chromosomes include chromosome 8, 11 and 17 which contain the 17q12 (ERBB2) and the 11q13 (CCND1) amplicons. Development of “herceptin” against ERBB2 illustrates the potential of cancer genomics in identifying therapeutic targets. Several studies have identified other amplicons with candidate oncogenes. However very few studies reported functional validation of identified candidates, thus highlighting the need of large scale functional analyses of different amplicons in breast cancer to identify new driver genes which may be used for development of therapeutic strategies for breast cancer. In recent years, RNAi has become a tool of choice for high-throughput screening to characterize gene function in cultured cells. In this study we performed high-throughput RNAi based functional screening of 127 amplified and over-expressed genes from 11 major amplicons on chromosome 8, 11 and 17 in breast cancer. This resulted in the identification of 8 driver genes from 5 amplicons. Further functional validation of 5 of these genes demonstrated that 4 genes, RAD21, EIF3H, TANC2 and CHRAC1 from 3 amplicons, regulate breast cancer cell proliferation, apoptosis and transformation. Regions of genetic alteration in one cancer may be altered in multiple cancer types. One such example includes the 8p11-p12 amplicon which has been reported to be amplified in breast, pancreatic, lung and bladder cancer. Also common amplicons from different cancers may harbor common driver oncogenes. To investigate this hypothesis we evaluated the possible involvement in 8p11-12 amplified pancreatic and lung cancer cell lines of two oncogenes namely, PPAPDC1B and WHSC1L1 that have been described to be driver genes of the 8p11-12 amplicon in breast cancer and furthermore in lung cancer for WHSC1L1. Inhibition of both genes reduced cell survival and anchorage independent growth in amplified pancreatic and lung cancer cell lines. This finding highlights the importance of these two genes in multiple cancers and therapeutic potential interest to inhibit these enzymes in multiple cancers with 8p11-p12 amplification.Transgenic mouse models play an important role to investigate in vivo function of candidate genes. To evaluate in vivo role of PPAPDC1B, we established a transgenic mouse model over-expressing PPAPDC1B under the Keratin 5 promoter. Transgenic mice developed two unexpected phenotypes including development of hair follicles along front teeth and acute inflammation of salivary glands, lymph nodes, bladder and pancreas. This is an ongoing study that may help to understand the mechanism of action of PPAPDC1B in vivo.

The identification of drug treatments that are useful in diverse therapeutic settings is a significant driving force in biomedical research [Macarron et al., 2011], [Poureetezadi et al., 2014], [Lamb, 2007]. Typical means for measuring the efficacy of a drug for a given clinical application include protein-protein interactions, cell death, mitochondrial respiration and cell growth as well as broader measurements of absorption, distribution, metabolism, excretion and toxicity (ADMET), specifically related the the drug or drugs being tested [Szakcs et al., 2008]. A wide array of methods are routinely employed to perform these screens, from ligand binding assays [Wagner et al., 2016] to high-throughput proteomics [Verheul, 2014]. One method that is currently underutilized in small-molecule drug screens and drug discovery is high-throughput transcriptome sequencing, such as RNA-Seq. Although RNA-Seq is routinely used to profile patterns of genetic changes following perturbations such as drug treatment [Young et al., 2014], it has not, to my knowledge, yet been used as the primary readout of a drug screen.

To identify novel therapeutic targets for basal-like breast cancer (BLBC) subtype, we investigated several DNA repair mechanisms associated with maintenance of high genomic instability for cell survival in cancer cells. We identified that the mismatch repair proteins, MSH2 and MSH6 (referred to as MSH2/6 hereafter), are highly elevated across BLBC samples. High expression level of MSH2/6 in BLBC is associated with worse prognosis and survivability for patients. Therefore, we knocked out MSH2 in BLBC cell lines and performed in vivo xenograft and syngeneic mice model studies to find significant attenuation of tumor growth in MSH2 KO group. Also, MSH2-deficient BLBC cells have increased rate of new mutations. Additionally, we tested the efficacy of conventional chemotherapeutics and radiation treatment that would further tip the genomic instability in MSH2-deficient BLBC cells towards cell death, but found them to be ineffective. Next, we performed high-throughput screening of 1280 FDA-approved compounds to discover that calcium channel blockers preferentially kill MSH2-deficient BLBC cells. This was likely due to association of significantly mutated pathways that involved calcium ion binding and calmodulin binding sites. Here we provide evidence of an alternative therapeutic strategy targeting DNA repair genes in BLBC patients utilizing bioinformatics analysis, high-throughput drug screening, in vitro,and vivoexperimentalmodels.

Regulator of G-protein Signaling (RGS) proteins temporally regulate the G protein signaling cascades initiated by GPCR activation. Reports have established dysregulation of RGS expression in a variety of disease states including several cancers. Additionally, use of genetic ablation techniques has implicated RGS proteins in a variety of other disease states through the native action of the RGS i.e. not a consequence of dysregulation of RGS expression. Therefore identification and optimization of small molecule lead compounds that alter RGS protein function has emerged as a promising therapeutic strategy. In this thesis, we use high throughput screening to interrogate small molecule libraries targeting two RGS proteins, RGS6 and RGS17. RGS6 has been reported as an essential mediator of doxorubicin induced cardiotoxicity, alcohol induced cardio and hepatotoxicity, anxiety, depression, and alcohol dependence. RGS17 has largely been implicated in a variety of cancer pathogenesis, with reported over expression in prostate, lung, breast, and hepatocellular carcinomas. Chapter 2 of this work focuses on the screening efforts targeting RGS6. Three separate screening campaigns interrogating over 20K compounds led to the identification of 3 small molecules that inhibit the RGS6: Gαo protein protein interaction with appreciable selectivity over control assays. The development of a cell based protein interaction assay is discussed, and the compounds were investigated using this system. All compounds tested did not appreciably alter signal over control, meaning that the cellular activity of these compounds remains ambiguous. Chapter 3 details the screening and follow up efforts targeting RGS17. The primary screening and/or follow up of four separate screening campaigns interrogating over 110K compounds is discussed. In total, 10 identified leads and a panel of analogs were subjected to significant follow up evaluation. All compounds were found to be cysteine dependent. The second generation RGS17 inhibitors (UI series) were determined to be both cytostatic and cytotoxic against lung and prostate cancer cell lines in culture, although whether this is due to RGS17 dependent mechanisms or due to general promiscuity of the compounds remains to be determined. Lead compounds from a library provided by the NCI were found to have cellular activity and were subjected to an investigation of structure activity relationships via commercially available compounds. The active form of three of these compounds was found to be a degradation product, which is likely due to decomposition of furan or methyl furan moieties that these compounds shared. One compound demonstrated robust SAR which allowed for the generation of schemes detailing putative inhibitory mechanisms. Finally, the role of RGS17 in the transition from epithelial to mesenchymal phenotypes is investigated. RGS17 was found to cause a sub population of PC3 cells to shift to mesenchymal phenotype, indicating that RGS17 may indeed play a role in this transition. Chapter 4 focuses on efforts to investigate variable potencies of published RGS4 inhibitors against a panel of RGS proteins, with the goal of gleaning insight in to structural characteristics that influence the inhibitability of RGS proteins. Most compounds tested were found to be more potent inhibitors of RGS14 rather than RGS4 in biochemical assays. We developed the NanoBit protein complementation assay to assess the interaction of RGS proteins with either Gαi1 or Gαq in a cellular context, and used this system to investigate compound selectivity in a cellular context. The compounds tested showed selectivity for RGS2, RGS4, and RGS14 over the other RGS proteins tested. The structural differences between the RGS proteins is discussed. Chapter 5 focuses on the future directions the lab may take with respect to the projects outlined in the previous chapters. This includes the screening of more targeted libraries or even virtual screening for RGS6, the development of in vivo assessment tools for RGS17, and an expanded structural examination of RGS proteins including NMR and crystal structure analysis. Additionally, the development of the NanoBit system to interrogate RGS protein interactions that are not RGS: Gα interactions is discussed.

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.