Novel Modulation of Adenylyl Cyclase Type 2

Adenylyl cyclase isoforms are distinctly modulated by G protein subunits and are therefore hypothesized to be uniquely regulated by proteins that influence G protein signaling. Activator of G protein signaling 3 (AGS3) is a G protein modulator that binds Gαi subunits in the GDP-bound state, implicating AGS3 as an important regulatorof Gi-coupled receptor signaling. We studied the ability of AGS3 to modulate recombinant adenylyl cyclase(AC) type 1 and 2 signaling in HEK293 cells following both acute and persistent activation of the D 2Ldopamine receptor (D2L DR). AGS3 expression modestly enhanced the potency of acute quinpirole-induced D 2LDR modulation of AC1 or AC2 activity. AGS3 also promoted desensitization of D2L DR-mediated inhibition ofAC1, whereas desensitization of D2LDR-mediated AC2 activation was significantly attenuated. Additionally, AGS3 reduced D2L DR-mediated heterologous sensitization of AC1 and AC2. Our results suggest that AGS3 alters G protein signaling in a complex fashion that is effector-specific and dependent on the duration ofreceptor activation. The present work also addressed the role of Gβγ subunits in the development of D 2L DR-mediated sensitization of AC2. The molecular signaling components that contribute to the development ofheterologous sensitization are largely unknown, but G protein subunits are strongly implicated in this adaptive process. We utilized Gβγ subunit sequestering proteins, small molecule and peptide Gβγ signaling inhibitors, and pharmacological Gβγ effector and kinase inhibitors to study the role of Gβγ subunit signaling pathways in the development of AC2 sensitization in HEK293 cells. Our results suggest that Gβγ subunit signaling is necessary for D 2L DR-mediated sensitization of AC2. The multitude and diversity of Gβγ; signaling pathways that may underlie AC sensitization prompted us to develop a high-throughput cAMP assay platform to facilitate future unbiased approaches for the study of AC sensitization, such as siRNA library screening. As an intermediate step to the development of such assays, we addressed the lack of potent and selective small molecule modulators of AC. Identification of chemical probes for AC2 is particularly important because there are no published genetic deletion studies and few small molecule modulators. Therefore, we developed and executed an intact-cell small molecule screening approach and subsequent validation paradigm for the discovery of AC2 inhibitors. The NIH clinical collections I and II were screened for inhibitors of AC2 activity, using PMA-stimulated cAMP accumulation as a functional readout. Active compounds were subsequently confirmed and validated as direct AC2 inhibitors using orthogonal and counter screening assays. The screening effort identified SKF-83566 as a selective AC2 inhibitor with superior pharmacological properties for selectivemodulation of AC2 when compared to currently available AC inhibitors. The utility of SKF-83566 as a small molecule probe to study the function of endogenous ACs was demonstrated in C2C12 mouse skeletal muscle cells

Identification of novel therapeutics for the treatment of MMR deficient tumours using high-throughput screens.

PhDThe DNA Mismatch repair (MMR) pathway is responsible for the repair of base-base mismatches and insertion/deletion loops, formed during DNA replication. Mutations in MMR genes significantly increase the predisposition to cancer with MMR deficiency estimated to be present in 15-17 % of all colorectal cancers. 5-fluorouracil is the main treatment for advanced colorectal cancer however the majority of studies suggest that MMR deficient tumours are more resistant to 5-fluorouracil than MMR proficient tumours. Therefore, there is a critical clinical need to identify novel therapeutics to treat these tumours. To this end, we have performed a high-throughput compound screen, to identify compounds that cause selective lethality in MMR deficient cell lines. We identified the potassium-sparing diuretic drug, Triamterene, as selectively lethal in vitro and in vivo in MMR deficient cell lines. Our data suggest that this selectivity is through its antifolate activity, leading to the accumulation of reactive oxygen species and DNA double strand breaks in MMR deficient cells. Interestingly, we identified a requirement, for thymidylate synthase expression, the only de novo enzyme for dTTP synthesis for the Triamterene cytotoxicity. NRF2 and NRF2-induced antioxidants were regulated upon Triamterene treatment and thymidylate synthase silencing, therefore suggesting a role for the antioxidant response in Triamterene toxicity. Taken together, our results suggest Triamterene as a promising novel therapeutic for the treatment of MMR deficient cancers. In order to identify novel therapeutics to treat MMR deficient tumours, we have also performed a high-throughput siRNA screen, to identify genes that cause selective lethality in MMR deficient cell lines. We identified AURKA gene as synthetically lethal in MSH6 deficient cell lines which suggests AURKA as a promising novel therapeutic target for the treatment of MMR deficient cancers. Taken together, in this PhD thesis we have identified two novel therapeutic strategies for the treatment of MMR deficient cancers.Queen Mary University of Londo