Sema Oncel

Date of Award

December 2022

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biomedical Sciences

First Advisor

Marc D. Basson

Abstract

Gastrointestinal (GI) ulcers are common worldwide. The persistence of noxious agents such as gastric acid, pepsin, nonsteroidal anti-inflammatory drugs (NSAIDs), and inflammation caused by inflammatory bowel disease breaks down the mucosal barrier and causes mucosal injury in the GI tract. Currently available therapeutics such as proton pump inhibitors (PPIs), histamine-2 receptor antagonists (H2-antagonists), antibiotics, and corticosteroids all attempt to minimize injury by reducing the symptoms of GI pathology, however, they do not directly promote mucosal healing. NSAIDs increase the development of both upper and lower GI ulcers. PPIs ameliorate upper GI injury; however, PPIs actually worsen NSAID distal enteropathy by suppressing gastric acid secretion and changing the enteric microbiome. Therefore, there is an urgent need for a therapeutic agent that directly promotes both upper and lower GI healing.The non-receptor tyrosine kinase Focal Adhesion Kinase (FAK), 125 kDa, regulates the maturation and turnover of focal adhesion (FA) and thus, cell migration. We developed a series of small molecule FAK activators such as M64HCl, a derivative of a small molecule called ZINC40099027 (ZN27). To investigate the effects of these FAK activators, we utilized western blot, monolayer wound closure assays, co-immunoprecipitation, subcellular fractionation assays, and Immunofluorescent staining in a series of gastrointestinal epithelial cells and we used an ongoing aspirin-induced gastric ulcer model in vivo. In chapter one, we reviewed the importance of gut homeostasis, physiology of mucosal healing, protective factors for the gastrointestinal mucosa, drivers of mucosal injury, mucosal healing processes, restitution and quality of ulcer healing, regulators of mucosal healing, and potential new therapeutic targets. In chapter two, we, first, demonstrated that ZN27 (10 nM) stimulated the activation of FAK and monolayer wound closure in rat and human gastric cell lines. Then, in vivo studies revealed that four days of 900µg/kg/6h ZN27 ameliorates gastric injury similar to 10 mg/kg/day omeprazole positive control vs. vehicle controls in C57BL/6J mice treated with 300mg/kg/day aspirin for five days to induce ongoing gastric injury, without obvious toxicity, by a pathway involving FAK activation. In chapter three, we displayed that ZN27 increases intestinal epithelial monolayer wound closure by stimulating the autophosphorylation of FAK in the cytosol, distinct from long-established models of FAK activation. Its downstream substrates paxillin and ERK are phosphorylated right after phosphorylated FAK translocates to the membrane, leading to FA turnover and human intestinal epithelial cell migration. In chapter four, we demonstrated that once three-hour or twice two-hour treatment of M64HCl (100 nM) promotes sustained monolayer wound closure by stimulating prolonged activation of FAK and ERK1/2, suggesting that increasing its half-life to 2 to 3 hours might be beneficial to promote gastrointestinal mucosal healing in clinical settings. In conclusion, small molecule FAK activators showed promising results toward gut mucosal healing. M64HCl, specifically, is a promising novel compound with water-soluble and drug-like properties. Hence, designing and optimizing the next-generation small molecule with a longer half-life might be beneficial to ameliorate both upper and lower GI mucosal injuries.

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