New preprint on SARS-CoV-2 replication and anti-viral drugs!
We are very excited to share our latest results on how antiviral drugs stop replication of SARS-CoV-2, the virus responsible for the COVID19 pandemic. Check our preprint on bioRxiv!
Nucleotide analogues, such as Remdesivir, are our best strategy to cure SARS-CoV-2 infection. The genome of SARS-CoV-2 is made of RNA, which building blocks are the four natural nucleotides A, U, C and G. SARS-CoV-2 encodes in its genome enzymes responsible to synthesize all viral RNA essential for the virus to multiply. Nucleotide analogues are modified natural nucleotides (also called NTPs) that either stop viral RNA synthesis or induce a lethal increase in mutations when incorporated in the viral RNA. In the cellular environment, nucleotide analogues compete with natural large amount NTPs to be incorporated, and only the ones that can outcompete NTPs can stop infection. We need to understand what makes a nucleotide analogue good at competing with NTPs to rationally design them. Nucleotide analogue incorporation generates a kinetic signature (pause, termination) in viral replication. In the presence of competing NTPs, nucleotide analogue incorporation is rare and stochastic and therefore impossible to observe with standard bulk assay as their temporal signature is diluted in the signal of the much more common NTP incorporation. To evaluate how good a nucleotide analogue really is, we need single molecule techniques to monitor their incorporation and high throughput data acquisition to acquire a sufficient statistics of these rare events.
We established a single molecule, high throughtput magnetic tweezers assay to monitor the replication activity of the SARS-CoV-2 replicase.
- SARS-CoV-2 replicase is the fastest RNA polymerase observed to date:170 nt/s at 37°C (others: ~50 nt/s)! We tested several nucleotide analogues using 1kb template and high NTPs conc:
- We found that Remdesivir is extremely well incorporated by SARS-CoV-2 , and induces pauses in replication traces. #Remdesivir is NOT a terminator at physiological concentration of NTPs. Also true for the original SARS-CoV-1.
- T-1106, a Favipiravir analogue used against flu, is poorly incorporated: 10-20 time less than Remdesivir. It may explain its poor efficacy in vivo. T-1106 does not induce termination, but makes SARS-CoV-2 replication pause.
- We found that Sofosbuvir, an analogue used against hepatitis C virus, competes poorly against UTP. Endogenously produced nucleotide analogue ddhCTP is well incorporated at physiological CTP concentration: this is potentially a new avenue to cure coronavirus infection!
Conclusion: we are able to characterize nucleotide analogue incorporation and mode of action for SARS-CoV-2 in physiological settings. Ready to help designing the new generation of nucleotide analogues!
Congrats to Mona, Subhas, Pauline and Flavia for their hard and great work. Thanks also to the collaborators that made it possible: Cameron lab, Depken lab, Kirchdoerfer lab, Canard lab, Almo lab and Joy Feng from Gilead Sciences.