2024 AIChE Annual Meeting

Investigating Stereoisomers of the Nucleotide Analog Tenofovir As Inhibitors of Sars-Cov-2 RdRp and Exonuclease

Severe Acute Respiratory Syndrome Coronavirus 2 or SARS-CoV-2, the causative agent of COVID-19, is a member of the Nidovirales order of positive-strand RNA viruses. SARS-CoV-2 possesses two protein complexes responsible for viral genome replication and transcription: RNA-dependent RNA polymerase (RdRp) for replicating viral RNA and 3'-5' exonuclease (ExoN) for proofreading. While several nucleoside and nucleotide analogs have been developed as inhibitors of RdRp with antiviral activity, the proofreader ExoN removes these incorporated analogs, which reduces the drug efficacy. Discovery and optimization of nucleotide analogs which not only inhibit RNA replication by RdRp, but also resist degradation by ExoN, would improve the efficacy of antiviral drugs.

The FDA-approved HIV drug Tenofovir, an acyclic nucleotide lacking a ribose moiety, is a promising candidate to resist ExoN excision. However, another study proved that Tenofovir is inactive against SARS-CoV-2 due to its low incorporation activity with RdRp. Cryo-electron microscopy and X-ray crystallography revealed that the methyl group in (R)-Tenofovir pointing towards the active site of RdRp may cause a spatial clash with the amino group and lead to poor incorporation activity. Therefore, we propose that (S)-Tenofovir with methyl group pointing away from the active site can improve the incorporation activity with RdRp while maintaining the ExoN resistance ability.

To test this hypothesis, we synthesized (S)-Tenofovir diphosphate (DP) and performed RNA extension reactions with SARS-CoV-2 RdRp to assess its incorporation efficiency. Exonuclease reactions were conducted to evaluate the ExoN resistance of (S)-Tenofovir-DP, with MALDI-TOF MS used to quantify incorporation and excision resistance. Results supported our hypothesis, demonstrating that (S)-Tenofovir-DP improves RdRp incorporation and resists ExoN degradation. However, its incorporation efficiency remains lower than that of natural nucleotides.

While (S)-Tenofovir-DP shows promise as an RdRp inhibitor with ExoN resistance, further chemical modifications are necessary to enhance its antiviral efficacy. These findings highlight the potential of Tenofovir derivatives as antiviral agents against SARS-CoV-2 and other coronaviruses, given the conservation of RdRp and ExoN across coronaviruses.