Date of Award

6-2024

Degree Type

Thesis

Degree Name

Master of Science in Biomedical Sciences

First Advisor

Vicky Mody, Ph.D.

Second Advisor

Shashidharamurthy Taval, Ph.D.

Third Advisor

Valerie Cadet, Ph.D.

Abstract

The emergence of SARS-CoV-2, the virus responsible for the COVID-19 pandemic, has highlighted the critical need for effective antiviral treatments. Three significant coronaviruses—SARS-CoV-1, MERS-CoV, and SARS-CoV-2—have posed serious global health threats in the past two decades. Among them, SARS-CoV-2 has demonstrated the highest transmission and mortality rates, underscoring the urgency for innovative therapeutic strategies. This study delves into the potential of tanshinones, natural compounds derived from Salvia miltiorrhiza, as inhibitors of the SARS-CoV-2 PLpro enzyme. PLpro is a pivotal enzyme in the viral replication and an attractive target for antiviral drug development. By inhibiting PLpro, tanshinones may hinder viral replication and modulate the host immune response, offering a multifaceted approach to combating COVID-19.

Key Finding 1: In Chapter 3, we found that of 21 tanshinones used in this study, 19 of them showed more than 50% inhibitory activity of PLpro proteolytic activity. The dose-dependent curve for all 19 tanshinones revealed that the Tanshinone IIB has the lowest IC50 value of 0.14 µM, whereas Danshexinkun A has the highest IC50 value of 31.02 µM. The evaluation of the cytotoxicity of these agents shows that Tanshinone IIA Sulfonate, Tanshinone IIB, Cryptotanshinone, Tanshinone I, and Danshexinkun A are non-toxic and hence can be used for further viral challenge studies.

Key Finding 2: Among the 19 tanshinones that showed proteolytic activity, only eleven tanshinones were found to inhibit DUB activity by at least 50%. The IC50 values were calculated using a dose-dependent assay revealed that Danshexinkun A has the lowest IC50 value of 0.33 µM, whereas, Tanshinone I has the highest IC50 value of 61.19 µM.

Key Finding 3: The cytotoxicity studies of 19 tanshinones showed that 3-α Hydroxytanshinone IIA, 1,2-Didehydroisotanshinone, Dihydrotanshinone, Dihydrotanshinone II, Dihydroisotanshinone, and 1,2-Didehydrocryptotanshinone showed significant toxicity to CALU-3 cell lines with CC50 values of 3.23, 4.09, 4.93, 5.50, 5.56, and 6.25 µM, respectively after 24 hours.

Key Finding 4: In Chapter 4 the molecular modeling interactions showed clear hydrogen bonding interactions between 1,2-Didehydrocryptotanshinone, 3-α-Hydroxytanshinone II, Cryptotanshinone, Tanshinone IIB, Dihydroisotanshinone, Dihydroisotanshinone II, Dihydrotanshinone, Hydroxytanshinone II, Isocryptotanshinone, Isotanshinone I, and Isotanshinone II with the carbonyl group of Gln 269. Whereas Dihydroisotanshinone II, Dihydroisotanshinone II, Danshexinkun A, Dihydroisotanshinone II, Tanshinone I, and Tanshinone IIA shows strong hydrogen bonding interactions with both Tyr 268 and Gln 269. These interactions can easily explain the proteolytic activity of PLpro enzyme. However, no interactions were observed between the studied tanshinones and Glu167, the key amino acid responsible for DUB activity. Therefore, the mechanism by which tanshinones influence DUB activity cannot be elucidated through molecular modeling studies.

Future Research Directions: Future studies on this work will focus on the following key aspects.

Kinetics of SARS-CoV-2 PLpro Inhibition by Tanshinones: Investigate the detailed kinetics and binding mechanisms of how tanshinones inhibit the PLpro enzyme in SARS-CoV-2. This includes studying the rate of inhibition and the interaction dynamics at the molecular level.

Viral Challenge Studies: Conduct in vitro and in vivo viral challenge studies using Tanshinone IIA Sulfonate, Tanshinone IIB, Cryptotanshinone, Tanshinone I, and Danshexinkun A to assess their antiviral efficacy. These studies will determine the therapeutic potential of these compounds against SARS-CoV-2.

Pharmacokinetic Studies: Study the pharmacokinetics of Tanshinone IIA Sulfonate, Tanshinone IIB, Cryptotanshinone, Tanshinone I, and Danshexinkun A, focusing on their metabolic conversion, breakdown, and excretion profiles. This will help understand their bioavailability and potential side effects in the human body. This comprehensive approach aims to uncover new insights and pave the way for effective antiviral treatments, contributing to the global fight against COVID-19 and future coronavirus outbreaks. We hope this work inspires further research and development in the field of antiviral therapeutics, ultimately leading to better preparedness and response to emerging viral threats.

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