Uncovering the mode of action of small molecule inhibitors against the RNA polymerase of SARS-CoV-2
by Florian Kabinger
Date of Examination:2024-08-26
Date of issue:2025-03-07
Advisor:Prof. Dr. Patrick Cramer
Referee:Prof. Dr. Patrick Cramer
Referee:Prof. Dr. Matthias Dobbelstein
Sponsor:Boehringer Ingelheim Fonds (BIF)
Persistent Address:
http://dx.doi.org/10.53846/goediss-11121
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Description:Doctoral thesis
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Abstract
English
Severe acute respiratory syndrome coronavirus 2 (SARS CoV 2) is the causative agent of coronavirus disease 2019 (COVID-19). The pandemic has claimed more than 7 million lives since its outbreak at the beginning of 2020, and the number of deaths is still raising. SARS CoV 2 is a positive-sense single-stranded RNA virus that encodes an RNA-dependent RNA polymerase (RdRp) for replication of its genome. Inhibition of this enzyme with small molecules blocks the viral life cycle and therefore presents a highly promising therapeutic strategy against COVID¬-19. Despite extensive efforts by the scientific community, potent and safe RdRp inhibitors are still lacking. Further improvement of potentially promising small molecules is often hampered by a lack of knowledge about their mode of action. In this doctoral thesis I present the biochemical and structural basis of RdRp inhibition by the nucleoside inhibitor Molnupiravir and by the non-nucleoside inhibitor HeE1-2Tyr. In order to perform quantitative analysis of compound-induced RdRp inhibition, I advanced the development of an existing minimal biochemical system recapitulating viral RNA replication in vitro. In addition, I established a fluorescence polarization assay that allows investigation of the interaction between RdRp and its RNA substrate. Combining these and other biochemical assays enabled us to quantify the inhibitory effects of small molecules on RdRp. To complement the biochemical data, single particle cryogenic electron microscopy (cryo-EM) was utilized to visualize the inhibitor bound to RdRp. I was able to solve cryo-EM structures of RdRp in the presence of Molnupiravir and HeE1-2Tyr with an overall resolution of 3 Å. This allowed us to visualize key drug-target interactions and to deduce the molecular mechanism of inhibition. Molnupiravir is an orally available antiviral drug that was in phase III clinical trials for treatment of COVID-19 patients at the time this research project was initiated. It was known that Molnupiravir increases the frequency of viral RNA mutations and impairs SARS CoV 2 replication in animal models and in humans, but the underlying mode of action remained elusive. We were able to establish the molecular mechanisms underlying Molnupiravir-induced RNA mutagenesis by RdRp. Biochemical assays show that the RdRp uses the active form of Molnupiravir, β-D-N4-hydroxycytidine (NHC) triphosphate, as a substrate instead of CTP or UTP. When the RdRp uses the resulting RNA as a template, NHC directs incorporation of either G or A, leading to mutated RNA products. In order to understand the mutagenesis on a molecular level, I solved two high-resolution cryo-EM structures of RdRp-RNA complexes containing the mutagenesis products. These show that NHC can form stable base pairs with either G or A in the RdRp active center, explaining how the polymerase escapes proofreading and synthesizes mutated RNA. This two-step mutagenesis mechanism likely applies to various viral polymerases and can explain the broad-spectrum antiviral activity of Molnupiravir. HeE1-2Tyr is a non-nucleoside inhibitor of the dengue virus RdRp, which was also shown to be effective against SARS CoV 2 in cell culture. However, it remains unclear how HeE1-2Tyr facilitates inhibition of the SARS CoV 2 RdRp. Combining biochemical and structural data, we were able to elucidate the mode of action of HeE1-2Tyr-mediated SARS CoV 2 RdRp inhibition. Biochemical assays confirm that HeE1-2Tyr inhibits RdRp with an IC50 of 5 µM and show that the compound interferes with RNA binding to RdRp in vitro. Structural analysis using cryo-EM reveals that a stack of three HeE1-2Tyr molecules binds to the RNA binding site of RdRp. The stack is stabilized by inter-compound π-π interactions and by a clamp of three arginine residues that are highly conserved across coronaviruses. This suggests that HeE1-2Tyr inhibits the RNA synthesis of SARS CoV 2 by sterically blocking the interaction between RdRp and RNA, which readily explains the biochemical data. In summary, my doctoral thesis contributes to the global drug development efforts to combat SARS CoV 2 in multiple ways. The here established biochemical assays provide a valuable tool for quantitative characterization of RdRp inhibition, and the applied cryo-EM strategy can be used for efficient structural elucidation of RdRp-inhibitor complexes. Understanding the two-step mutagenesis mechanism of Molnupiravir was highly relevant for its clinical usage and studying HeE1-2Tyr-mediated RdRp inhibition revealed a novel mechanism to inhibit RdRp. Altogether, this work has the potential to drive further development of potent pan-corona inhibitors adding to pandemic preparedness.
Keywords: SARS-CoV-2; cryo-EM; drug discovery; RNA-dependent RNA polymerase; non-nucleoside inhibitor; nucleoside analogue
