Optimizing Small Molecule Inhibitors of Soluble Adenylyl Cyclase (sAC) for Use as Male Contraceptives

• Main Author: Rossetti, Thomas K
• Format: Dissertation
• Language: English
• Published: ProQuest Dissertations & Theses 01-01-2023
• Subjects: Biology; Biomedical engineering; Developmental biology; Pharmaceutical sciences; Pharmacology
• ISBN: 9798371994929

Cyclic adenosine monophosphate (cAMP) is a ubiquitous second messenger that plays an important role in a wide variety of signal transduction pathways. The production of cAMP is catalyzed by enzymes known as adenylyl cyclases (ACs). Amongst the various ACs expressed in mammalian cells, the bicarbonate (HCO3 −)-regulated, nonmembrane-bound soluble AC (sAC; ADCY10) is essential in sperm for reproduction. Due to this important function, we hypothesized that pharmacological inhibition of sAC could act as an on-demand, non-hormonal form of male contraception. This hypothesis led to the initiation of a drug discovery program that sought to develop “drug-like” sAC inhibitors for use as male contraceptives; the data presented within this dissertation describes my contribution to this project.We employed the use of structure-based drug design to improve the potency of an existing sAC inhibitor (LRE1) by 20-fold, generating a lead compound (TDI-10229) with a 160 nM IC50. Despite this improved potency, TDI-10229 only partially blocked fertilization in an in vivo mating experiment. Additional optimization produced TDI-11861, a sAC inhibitor with a 1.7 nM IC50. In addition to potency, we focused on characterizing the binding kinetics of our sAC inhibitors, specifically the drug-target residence time, as we expected this property to play a major role in determining contraceptive efficacy. I used surface plasmon resonance to reveal that TDI-11861 had a residence time 230-fold longer than that of TDI-10229 (73 minutes vs. 20 seconds). When we tested TDI-11861 in vivo, it rendered male mice temporarily infertile and completely blocked fertilization.These proof-of-concept studies provide important in vivo evidence that sAC inhibitors can function as on-demand, non-hormonal male contraceptives. However, poor metabolic properties and a concerning safety profile preclude TDI-11861 from being our preclinical candidate, thus further optimization is required. To assist in these efforts, I characterized numerous sAC inhibitors using newly developed in vitro and in cellulo assays that focused on potency, residence times, and contraceptive efficacy. The insights gained from these assays will be used to develop a sAC inhibitor that fulfills our criteria for a preclinical candidate, which we hope to eventually move forward into the final phase of drug discovery: clinical trials.