Date of Award

Spring 4-15-2024

Degree Type

Thesis

Degree Name

Master of Science in Chemical Sciences

Department

Department of Chemistry and Biochemistry

Committee Chair/First Advisor

Mohammad A. Halim

Second Advisor

Chris Dockery

Third Advisor

Anthony Rojas

Abstract

The COVID-19 pandemic, caused by the SARS-CoV-2 virus, posed a substantial worldwide health threat, necessitating the development of effective treatments. To minimize the spread of this virus several vaccines and drugs have been approved for emergency use, such as Pfizer-BioNTech, Moderna, and remdesivir, paxlovid respectively, but their efficacy and accessibility remain limited. The SARS-CoV-2 main protease (Mpro) is essential for the replication of the virus, making it an appealing target for the development of antiviral drugs and peptides. In this study, various noncovalent staple peptides were developed from a potent frog-derived Temporin L-like peptide (TLP). For the staple peptide, α-methyl-L-phenylalanine (αF) was incorporated at crucial positions of the TLP1 to stabilize the peptide's helical shape and enhance Mpro interactions. Three stapled peptide analogs, TLP1M1, TLP3M2, and TLP4M3, and their corresponding linear TLP1, TLP3, and TLP4 peptides were synthesized and characterized. The TLP4M3 peptide displayed significant Mpro inhibition, with an IC50 value of 0.637μM in FRET and 0.284 μM in LC-MS-based protease assay. MD simulations show that TLP4M3's stable interactions with Mpro's active site residues (Thr24, His41, Ser46, Glu47, Asn142, Cys145, Glu166, and Gln189) involve hydrogen bonds, hydrophobic interactions, and π-π stacking. Moreover, TLP4M3 has a ~8-hour half-life in serum, indicating excellent stability. The subsequent approach conjugated the antimicrobial peptide TLP1/TL1 with cell-penetrating peptides (CPPs) to understand interactions, stability, and inhibition efficiency. Five TL1-CPP conjugated peptides (TL1-T2, L5a, PAS, RW9, and NLS) were identified by molecular docking and dynamics simulations. The non-bonded interaction investigations from MD simulations showed that the most prominent TL1-NLS conjugated peptide formed strong nonbonding interactions with key residues such as Thr25, His41, Thr44, Thr45, Ser45, Asn142, Cys145, Glu166, Pro168, Gln189 and Glu288 of Mpro. These peptides (TL1-T2, L5a, PAS, RW9, and NLS) were synthesized, characterized, and performed in vitro protease activity. TL1-NLS significantly inhibited Mpro, with an IC50 value of 5.62μM in FRET and 6.74μM in LC-MS. With a half-life of 7 hours, TL1-NLS was very stable in serum. Furthermore, covalent stapling and cyclization of these peptides are the future aims to develop orally active peptide therapeutics targeting the Mpro of SARS-CoV-2.

Available for download on Sunday, May 06, 2029

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