Hagen, Timothy J.
Ph.D. (Doctor of Philosophy)
Department of Chemistry and Biochemistry
There is an urgent need for new antifungal drugs. Acetyl CoA synthetases (ACSs) are Acyl-CoA/NRPS/Luciferase (ANL) superfamily enzymes that couple acetate with CoA to generate acetyl CoA, a key component of central carbon metabolism in eukaryotes and prokaryotes. Normal mammalian cells are not dependent on ACSs, while tumor cells, fungi, and parasites rely on acetate as a precursor for acetyl CoA. Consequently, ACSs have emerged as a potential drug target. This body of work focuses on the inhibition of the Acetyl CoA synthetase enzyme in fungal species. This work has involved designing and synthesizing compounds that are potential fungal ACS1 inhibitors. The first series of compounds focused on fragment-based strategies. Small molecule compounds were identified as a hit in an NMR-based screening, which was followed by validation with biochemical and structural characterization. Fragments were linked together to improve potency against the ACS1 enzyme, which eventually led to two compounds with antifungal activity of 4-32μg/mL. The Alkyl AMP esters have been found to be potent inhibitors of Acetyl CoA-synthetase. Previously reported syntheses of Alkyl AMP esters were performed in water that required reverse phase HPLC or ion-exchange chromatography to purify. To overcome these hurdles, the alkyl esters of AMP were synthesized using the corresponding alcohol as solvent and various coupling agents. Reaction conditions were optimized. The alkyl AMP esters were synthesized in excellent yield and purity. They were purified using Biotage® ZIP-Sphere cartridge packed with spherical silica (60 um, 50Å, 750 m2/g). The AMP alkyl esters were screened against ACS1 enzymes from four different fungal organisms. Several substrate analogs exhibited inhibition of ACS1 at nanomolar concentration. iii In a high throughput strategy screening of compounds in our library, we identify Otava 6057970 and 1876136 as a hit, with 68% and 62% inhibition against CnACS1 at 100 μM. Efforts were made to increase the potency of these compounds by making modifications to different positions of the compounds. Analogs synthesized were screened for their activity against the CnACS1 enzyme. AR-12/OSU-03012 is an antitumor celecoxib derivative that progressed to Phase I clinical trial as an anticancer agent and has activity against several infectious agents including fungi, bacteria, and viruses. Ar-12 showed activity against ACS1 enzyme Candida albicans, C. neoformans and Saccharomyces cerevisiae. Analogs of AR-12 were synthesized to overcome the challenges with AR-12 and some of the analogs showed improved potency and antifungal activity. In efforts to identify a non-substrate inhibitor of ACS1 enzyme, multicomponent reactions (MCRs) such as Mannich and Groebke-Blackburn-Bienaymé (GBB) reactions were also explored as a tool to synthesize ACS1 in inhibitors. Synthesized compounds were screened against Candida albicans, C. neoformans neoformans, and Aspergillus fumigatus. The second part of this work includes our efforts to identify SARS-CoV-2 3CLpro and PLpro inhibitors. Hundreds of compounds in our library were screened against SARS-CoV-2 3CLpro and PL pro. Several small-molecule compounds were identified as hits. The hit compounds were subjected to modification, including fragment merging and scaffold hopping. Some of the modifications resulted in similar or improved activity compared to initially reported compounds against SARS-CoV-2 3CLpro and PLpro.
Esan, Taiwo Elijah, "Discovery of Acetyl CoA Synthetase and SARS-CoV-2 Protease Inhibitors" (2023). Graduate Research Theses & Dissertations. 7141.
Northern Illinois University
Rights Statement 2
NIU theses are protected by copyright. They may be viewed from Huskie Commons for any purpose, but reproduction or distribution in any format is prohibited without the written permission of the authors.
Available for download on Monday, June 16, 2025