DOI

10.17077/etd.9hm9v05b

Document Type

Dissertation

Date of Degree

Fall 2017

Degree Name

PhD (Doctor of Philosophy)

Degree In

Pharmaceutical Sciences and Experimental Therapeutics

First Advisor

Roman, David L.

First Committee Member

Fisher, Rory A.

Second Committee Member

Doorn, Jonathan A.

Third Committee Member

Rice, Kevin G.

Fourth Committee Member

Duffel, Michael W.

Abstract

G protein-coupled receptors (GPCRs) play a central role in numerous biological processes, from olfaction to vision to neurotransmission and more, leading to their classification as important drug targets. Signaling through GPCRs is carried out by a number of intracellular effector proteins, such as the α subunits of heterotrimeric G proteins and Adenylyl Cyclases (AC). In this work, inhibition of protein-protein interactions to reduce GPCR-mediated cAMP formation is explored to identify compounds with potential therapeutic value.

Regulators of G Protein Signaling (RGS) inhibit Gαi/o signaling, resulting in persistent AC-mediated cAMP formation. Fragment-based screening against RGS17, which has been implicated in a number of cancers, identified seven hits that bind RGS17, though only one leads to inhibition of RGS17 function in vitro. Importantly, these compounds represent a starting point for future structure-based drug discovery targeting RGS17 for the development of new chemotherapeutics.

As different RGS proteins serve different functions throughout the body, achieving isoform selectivity is important for reducing potentially deleterious side effects. Analysis of the selectivity of previously described RGS4 inhibitors revealed that several are more potent inhibitors of RGS14. This work also identified the RGS proteins for which inhibitors are likely to be found (RGS14, RGS4, RGS1) and those which are likely to be more difficult drug targets (RGS6 and RGS7).

Finally, inhibition of AC8 as a means to decrease intracellular levels of cAMP is explored. AC1 and AC8 are robustly stimulated by CaM, so inhibition of the AC/CaM interaction was explored as a novel mechanism of AC inhibition. Biochemical and cell-based assays were developed, and a pilot screen of 1,000 FDA-approved compounds identified six capable of inhibiting the AC8/CaM interaction. This ultimately resulted in reduced AC8 activity and cAMP accumulation, validating this interaction as a druggable target. The compounds identified were not selective for AC8 over AC1, but they have pre-clinical utility in evaluating the biology of CaM-stimulated AC activity.

Pages

x, 119 pages

Bibliography

Includes bibliographical references (pages 107-119).

Comments

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Copyright

Copyright © 2017 Michael Patrick Hayes

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