Inhibition of SARS-CoV-2 polymerase by nucleotide analogs: a single molecule perspective

  1. Mona Seifert
  2. Subhas Chandra Bera
  3. Pauline van Nies
  4. Robert N. Kirchdoerfer
  5. Ashleigh Shannon
  6. Thi-Tuyet-Nhung Le
  7. Xiangzhi Meng
  8. Hongjie Xia
  9. James M. Wood
  10. Lawrence D. Harris
  11. Flávia S. Papini
  12. Jamie J. Arnold
  13. Steven C. Almo
  14. Tyler L. Grove
  15. Pei-Yong Shi
  16. Yan Xiang
  17. Bruno Canard
  18. Martin Depken
  19. Craig E. Cameron
  20. David Dulin

  • Curated by eLife

    eLife logo

    Evaluation Summary:

    This work presented in this manuscript paper may advance our understanding of an important class of anti-viral drugs (nucleoside analogs) that target polymerase enzymes by being directly incorporated into the product strand. This class of drugs is known to be quite diverse in their precise mechanisms of action, yet many of the particular details have remained elusive, often due to experimental limitations. The current study employs a single-molecule magnetic-tweezers platform to provide a new paradigm for the mechanism of drug remdesivir against the SARS-CoV-2 polymerase target. The authors propose that remdesivir does not prevent the complete viral RNA synthesis but causes an increase of polymerase pausing and back tracking. This paper is of broad interest to readers and scientists working on SARS-CoV-2 and general nucleotide inhibitors. The work includes the single molecule characterization of other NAs.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

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Abstract

The nucleotide analog Remdesivir (RDV) is the only FDA-approved antiviral therapy to treat infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The physical basis for efficient utilization of RDV by SARS-CoV-2 polymerase is unknown. Here, we characterize the impact of RDV and other nucleotide analogs on RNA synthesis by the polymerase using a high-throughput, single-molecule, magnetic-tweezers platform. The location of the modification in the ribose or in the base dictates the catalytic pathway(s) used for its incorporation. We reveal that RDV incorporation does not terminate viral RNA synthesis, but leads the polymerase into deep backtrack, which may appear as termination in traditional ensemble assays. SARS-CoV-2 is able to evade the endogenously synthesized product of the viperin antiviral protein, ddhCTP, though the polymerase incorporates this nucleotide analog well. This experimental paradigm is essential to the discovery and development of therapeutics targeting viral polymerases.

Teaser

We revise Remdesivir’s mechanism of action and reveal SARS-CoV-2 ability to evade interferon-induced antiviral ddhCTP

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  1. eLife

    Author Response:

    Reviewer #1 (Public Review):

    This work investigated the mechanism of inhibition of SARS-CoV-2 polymerase by multiple nucleotide analogs using a high-throughput, single-molecule, magnetic tweezers platform. There was particular focus on the remdesivir (RDV) because it is the only FDA approved anti-coronavirus drug on the market at the time of this review. The study shows that remdesivir leads the polymerase to undergo a backtrack in which it moves back as much as 30 nucleotides from the last insertion. The results also show that RDV is not a chain terminator, which is consistent with prior work. In addition to RDV, the authors characterized other nucleotide analogs such as ddhCTP, 3'-dCTP, and Sofosbuvir-TP to propose that the location of the modification in the ribose or in the base dictates the catalytic pathway used for incorporation. The authors also propose that the use of magnetic tweezers is essential towards characterizing and discovering therapeutics that target viral polymerases.

    Strengths:

    A strength of the papers is the utilization of magnetic tweezers to characterize the polymerase at the single molecule level. This provides a unique method to capture less common or difficult to observe phenomena such as backtracking. Most bulk ensemble assays would have difficulty detecting these phenomena.

    The characterization of multiple different types of nucleotides analogs to investigate the different mechanisms by which they could inhibit the polymerase is a strength of the paper. The authors elegantly utilize their system to show different pause states and backtracking of the polymerase.

    In general, the paper is well written, and the data is clearly presented.

    The authors thank the Reviewer for the strong appraisal of our work!

    Weakness:

    The experiments performed with the magnetic tweezers appear to not have contained the exonuclease domain. This domain would presumably be involved in removing nucleotide analogs that have been inserted and may alter the pause states or backtracking prevalence. For example, does the prevalence of backtracking increase when the exonuclease domain is not present. This is particularly important in regard to the RDV experiments.

    To date, no laboratory has been able to couple the polymerase complex with the proofreading complex. Indeed, we have entire five-year R01 grant to pursue this objective. Just like all proofreading polymerases studied before this one, it is imperative to establish a baseline with exonuclease deficient state prior to adding that component. Even before we add the exonuclease, it will be important to add the helicase to determine if it can assist the polymerase with dsRNA, because its strand-displacement activity is weak.

    A major claim for this study is the utilization of the magnetic tweezers "experimental paradigm" as being essential to the discovery and development of therapeutics to viral polymerases. In addition the authors state this approach is superior to bulk ensemble studies. This reviewer found these conclusions to be an overstatement and unnecessary. The use of magnetic tweezers is not amenable to all laboratories or an easy technique to implement within the therapeutic drug development. In general, the authors also overstate the power and feasibility of the magnetic tweezers in comparison to bulk ensemble studies. All assays have limitations, and the magnetic tweezers is no different in regards to being purified proteins, an in vitro approach, limitations in regards to feasibility for all users, ability to detect the amount of active protein, and multiple other reasons. This is a minor weakness of the paper that can be easily addressed because it detracts from the novelty of the studies.

    We feel that it is important to avoid an either-or scenario. We apologize for evoking a negative reaction with our statement, as we were only trying to emphasize how illuminating the magnetic-tweezers approach can be. It was not our intention to rule out the need for bulk methods at the bench top or using quench-flow or stopped-flow devices.

    We have edited the text in l.83-87 to convey the following:

    “Magnetic tweezers permit the dynamics of an elongating polymerase/polymerase complex to be monitored in real time and the impact of nucleotide analogues to be monitored in the presence of all four natural nucleotides in their physiological concentration ranges. Here, we present a magnetic tweezers assay to provide insights into the mechanism and efficacy of current and underexplored NAs on the coronavirus polymerase.”

    Reviewer #2 (Public Review):

    This study investigates the impact of remdesivir (RDV) and other nucleotide analogs (NAs), 3'-dATP, 3'-dUTP, 3'-dCTP, Sofosbuvir-TP, ddhCTP, and T-1106-TP, on RNA synthesis by the SARS-CoV-2 polymerase using magnetic tweezer. This technique allows to directly quantify termination of viral synthesis, pausing or stalling of the polymerase, thus, defining the effect of these NAs on viral synthesis. The work includes good quality data and nicely stablishes an assay to follow the activity of the SARS-CoV-2 RNA-dependent RNA polymerase.

    The authors thank the Reviewer for her/his appreciation of our work!

    However, the basis of the assay and theory was largely presented before by the authors in Ref 22 and 23 (and other references therein).

    The main result here is that RDV incorporation does not prevent the complete viral RNA synthesis but causes an increase of pausing and back-tracking. This contrasts with a clear signature of synthesis termination induced by 3'-dATP. The work is complemented with the characterization of other NAs. Despite these results are of merit, I do not see this work to present a sufficient advance of our current knowledge.

    We acknowledge Reviewer #2 opinion. However, we believe that our work is highly novel and important, as noted by Reviewer #1: “This [utilization of magnetic tweezers] provides a unique method to capture less common or difficult to observe phenomena such as backtracking. Most bulk ensemble assays would have difficulty detecting these phenomena.”

    and Reviewer #3: “Overall, this manuscript constitutes a major advance in our understanding of chain termination in polymerases, and provides deep insights into the mechanism of action of remdesivir, which may contribute to further drug discovery efforts targeting this polymerase.”.

    How these results translate into more physiological conditions at zero force should be addressed.

    We show here that nucleotide analogs are incorporated via specific catalytic pathways (NAB, SNA, VSNA) depending on the nature of their modification (position and type in ribose, base). In the companion paper attached to this submission (https://doi.org/10.1101/2021.03.27.437309, currently in press), we show that the force has no effect on the probability to enter any catalytic pathways, and only affects the kinetics of a large conformational change occurring after chemistry. In conclusion, the force has no effect on nucleotide analog selection, as supported by our evaluation at both 25 and 35 pN. To clarify this, we have added in l.416-421:

    “The present study demonstrates that nucleotide analog selection and incorporation is not force-dependent (Figure 2–figure supplement 3), which further validates the utilization of high-throughput magnetic tweezers to study nucleotide analog mechanism of action. This result is in agreement with our recent SARS-CoV-2 polymerase mechanochemistry paper, where we showed that entry probability in NAB, SNA and VSNA was not force dependent, and that force mainly affected the kinetics of a large conformational subsequent to chemistry, i.e. after nucleotide selection and incorporation.”

    The rationale of testing other NAs apart from the mere systematic characterization of other compounds is unclear.

    We have tested 3’-dATP, a well-known chain terminator, with Remdesivir, which was claimed to be a delayed chain terminator, as both are ATP analogue. We monitored the incorporation of Sofosbuvir, a well-known inhibitor of HCV replication, with its 3’-dNTP homologue, i.e. 3’-dUTP. T-1106-TP is a compound that was recently tested for coronavirus because it has a proven efficacy against influenza. ddhCTP is an endogenously produced nucleotide analog and chain terminator, and we compared it to its 3’-dNTP homologue, 3’-dCTP. Furthermore, each of these nucleotide analogs have modification at specific position, i.e. either at the ribose or at the base, which helps to understand how the polymerase responds to each modification. We have added this sentence in introduction in l.83-84 for clarity:

    “We have therefore compared several analogs of the same natural nucleotide to determine how the nature of the modifications changes selection/mechanism of action.”

    Similarly, I do not see the benefits of adding cell experiments with three compounds and experiments with the nsp14 mutant to address proofreading because they were inconclusive.

    While we acknowledge Reviewer #2 opinion, Reviewer #3 has a different opinion and strongly appraises the importance of these results:

    “Interestingly, the ddhCTP didn't actually work in infected cells. However, the authors presented a few theories on why it didn't work and said they plan to follow up to elucidate why it didn't work in cells. I think those results will be very interesting for the larger community working in this area.”

    We share the opinion of Reviewer #3 and have therefore decided to keep these results in the revised manuscript.

    Reviewer #3 (Public Review):

    This manuscript focuses on understanding the mechanism of action of remdesivir in the inhibition of SARS-Cov2 polymerase, using single molecule methods. The findings are highly original, significant and surprising. The approach is highly robust and supported by a range of orthogonal studies. Overall, these findings should help those engaged directly in drug discovery by providing a critical foundational understanding for the action of remdesivir.

    The research described in this manuscript has several findings that significantly impact the broader field polymerase inhibition. First, the authors were able to show using single molecule methods that remdesivir-TP incorporation leads to polymerase backtrack. This is important because the pause is long enough that an ensemble assay could mistake this backtrack for a termination event. Secondly, the researchers found the effective incorporation of remdesivir-TP was determined by its absolute concentration. This suggests remdesivir-TP and similar nucleotide analogs incorporate via the SNA or VSNA pathway and would be more likely to add to the RNA chain when substrate concentration is low (independent of stoichiometry with the competing native nucleotide). Thirdly, the researchers found the effective incorporation rate of obligatory terminators was affected by the stoichiometry of their competing native nucleotide rather than their absolute concentration. This suggests that obligatory terminators are incorporated via the NAB pathway. The pausing that the researchers observed in the polymerase elongation kinetics have recently been demonstrated by two other groups. However, this study improved upon the assay conditions used by other researchers to recapitulate in vivo conditions and remove bias from kinetics measurements.

    The authors highlighted the issues with remdesivir, tested other nucleotide analogs, and proposed a better alternative based on their assays (ddhCTP). Interestingly, the ddhCTP didn't actually work in infected cells. However, the authors presented a few theories on why it didn't work and said they plan to follow up to elucidate why it didn't work in cells. I think those results will be very interesting for the larger community working in this area. It's clear that the authors made a substantial enough contribution on the mechanism of inhibition of SARS Cov2 polymerase to merit publication in eLife, independent of the work on the "improved" antiviral candidate.

    It would have been useful to clarify for the reader the pharmaceutical import of the putative delayed chain termination (or pausing) relative to actual chemical chain termination. In other words, I'm assuming that in both cases the viral genome is considered to be non-transcribed (in that a chemical agent has been incorporated into the growing strand). This is true for most compounds in this broad class of anti-virals. The issues are usually surrounding the width of the therapeutic index and the degree to which resistant mutants arise.

    Coronaviruses are unique among positive-strand RNA viruses in that they encode a proofreading exonuclease. Although it is unclear how the polymerase and exonuclease activities are coordinated, the current assumption is that errors are recognized when located at the terminus of nascent RNA. Therefore, nucleotide analogues which manifest their antiviral activity when embedded in nascent RNA should evade excision by the exonuclease.

    We have added text conveying this sentiment here in l.70:

    “The latter proofreads the terminus of the nascent RNA following synthesis by the polymerase and associated factors, a unique feature of coronaviruses relative to all other families of RNA viruses.”

    And in lines 75-77:

    “In other words, nsp14 adds another selection pressure on NAs: not only they must be efficiently incorporated by nsp12, they must also evade detection and excision by nsp14.”

    Overall, this manuscript constitutes a major advance in our understanding of chain termination in polymerases, and provides deep insights into the mechanism of action of remdesivir, which may contribute to further drug discovery efforts targeting this polymerase. Additionally, the authors have highlighted and addressed issues in the methodologies of previous mechanistic studies that led others to erroneous conclusions.

    We thank Reviewer #3 for her/his strong appraisal of our work.

  2. eLife

    Evaluation Summary:

    This work presented in this manuscript paper may advance our understanding of an important class of anti-viral drugs (nucleoside analogs) that target polymerase enzymes by being directly incorporated into the product strand. This class of drugs is known to be quite diverse in their precise mechanisms of action, yet many of the particular details have remained elusive, often due to experimental limitations. The current study employs a single-molecule magnetic-tweezers platform to provide a new paradigm for the mechanism of drug remdesivir against the SARS-CoV-2 polymerase target. The authors propose that remdesivir does not prevent the complete viral RNA synthesis but causes an increase of polymerase pausing and back tracking. This paper is of broad interest to readers and scientists working on SARS-CoV-2 and general nucleotide inhibitors. The work includes the single molecule characterization of other NAs.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

  3. eLife

    Reviewer #1 (Public Review):

    This work investigated the mechanism of inhibition of SARS-CoV-2 polymerase by multiple nucleotide analogs using a high-throughput, single-molecule, magnetic tweezers platform. There was particular focus on the remdesivir (RDV) because it is the only FDA approved anti-coronavirus drug on the market at the time of this review. The study shows that remdesivir leads the polymerase to undergo a backtrack in which it moves back as much as 30 nucleotides from the last insertion. The results also show that RDV is not a chain terminator, which is consistent with prior work. In addition to RDV, the authors characterized other nucleotide analogs such as ddhCTP, 3'-dCTP, and Sofosbuvir-TP to propose that the location of the modification in the ribose or in the base dictates the catalytic pathway used for incorporation. The authors also propose that the use of magnetic tweezers is essential towards characterizing and discovering therapeutics that target viral polymerases.

    Strengths:
    A strength of the papers is the utilization of magnetic tweezers to characterize the polymerase at the single molecule level. This provides a unique method to capture less common or difficult to observe phenomena such as backtracking. Most bulk ensemble assays would have difficulty detecting these phenomena.

    The characterization of multiple different types of nucleotides analogs to investigate the different mechanisms by which they could inhibit the polymerase is a strength of the paper. The authors elegantly utilize their system to show different pause states and backtracking of the polymerase.

    In general, the paper is well written, and the data is clearly presented.

    Weakness:

    The experiments performed with the magnetic tweezers appear to not have contained the exonuclease domain. This domain would presumably be involved in removing nucleotide analogs that have been inserted and may alter the pause states or backtracking prevalence. For example, does the prevalence of backtracking increase when the exonuclease domain is not present. This is particularly important in regard to the RDV experiments.

    A major claim for this study is the utilization of the magnetic tweezers "experimental paradigm" as being essential to the discovery and development of therapeutics to viral polymerases. In addition the authors state this approach is superior to bulk ensemble studies. This reviewer found these conclusions to be an overstatement and unnecessary. The use of magnetic tweezers is not amenable to all laboratories or an easy technique to implement within the therapeutic drug development. In general, the authors also overstate the power and feasibility of the magnetic tweezers in comparison to bulk ensemble studies. All assays have limitations, and the magnetic tweezers is no different in regards to being purified proteins, an in vitro approach, limitations in regards to feasibility for all users, ability to detect the amount of active protein, and multiple other reasons. This is a minor weakness of the paper that can be easily addressed because it detracts from the novelty of the studies.

  4. eLife

    Reviewer #2 (Public Review):

    This study investigates the impact of remdesivir (RDV) and other nucleotide analogs (NAs), 3'-dATP, 3'-dUTP, 3'-dCTP, Sofosbuvir-TP, ddhCTP, and T-1106-TP, on RNA synthesis by the SARS-CoV-2 polymerase using magnetic tweezer. This technique allows to directly quantify termination of viral synthesis, pausing or stalling of the polymerase, thus, defining the effect of these NAs on viral synthesis. The work includes good quality data and nicely stablishes an assay to follow the activity of the SARS-CoV-2 RNA-dependent RNA polymerase. However, the basis of the assay and theory was largely presented before by the authors in Ref 22 and 23 (and other references therein). The main result here is that RDV incorporation does not prevent the complete viral RNA synthesis but causes an increase of pausing and back-tracking. This contrasts with a clear signature of synthesis termination induced by 3'-dATP. The work is complemented with the characterization of other NAs. Despite these results are of merit, I do not see this work to present a sufficient advance of our current knowledge. How these results translate into more physiological conditions at zero force should be addressed. The rationale of testing other NAs apart from the mere systematic characterization of other compounds is unclear. Similarly, I do not see the benefits of adding cell experiments with three compounds and experiments with the nsp14 mutant to address proofreading because they were inconclusive.

  5. eLife

    Reviewer #3 (Public Review):

    This manuscript focuses on understanding the mechanism of action of remdesivir in the inhibition of SARS-Cov2 polymerase, using single molecule methods. The findings are highly original, significant and surprising. The approach is highly robust and supported by a range of orthogonal studies. Overall, these findings should help those engaged directly in drug discovery by providing a critical foundational understanding for the action of remdesivir.

    The research described in this manuscript has several findings that significantly impact the broader field polymerase inhibition. First, the authors were able to show using single molecule methods that remdesivir-TP incorporation leads to polymerase backtrack. This is important because the pause is long enough that an ensemble assay could mistake this backtrack for a termination event. Secondly, the researchers found the effective incorporation of remdesivir-TP was determined by its absolute concentration. This suggests remdesivir-TP and similar nucleotide analogs incorporate via the SNA or VSNA pathway and would be more likely to add to the RNA chain when substrate concentration is low (independent of stoichiometry with the competing native nucleotide). Thirdly, the researchers found the effective incorporation rate of obligatory terminators was affected by the stoichiometry of their competing native nucleotide rather than their absolute concentration. This suggests that obligatory terminators are incorporated via the NAB pathway. The pausing that the researchers observed in the polymerase elongation kinetics have recently been demonstrated by two other groups. However, this study improved upon the assay conditions used by other researchers to recapitulate in vivo conditions and remove bias from kinetics measurements.

    The authors highlighted the issues with remdesivir, tested other nucleotide analogs, and proposed a better alternative based on their assays (ddhCTP). Interestingly, the ddhCTP didn't actually work in infected cells. However, the authors presented a few theories on why it didn't work and said they plan to follow up to elucidate why it didn't work in cells. I think those results will be very interesting for the larger community working in this area. It's clear that the authors made a substantial enough contribution on the mechanism of inhibition of SARS Cov2 polymerase to merit publication in eLife, independent of the work on the "improved" antiviral candidate.

    It would have been useful to clarify for the reader the pharmaceutical import of the putative delayed chain termination (or pausing) relative to actual chemical chain termination. In other words, I'm assuming that in both cases the viral genome is considered to be non-transcribed (in that a chemical agent has been incorporated into the growing strand). This is true for most compounds in this broad class of anti-virals. The issues are usually surrounding the width of the therapeutic index and the degree to which resistant mutants arise.

    Overall, this manuscript constitutes a major advance in our understanding of chain termination in polymerases, and provides deep insights into the mechanism of action of remdesivir, which may contribute to further drug discovery efforts targeting this polymerase. Additionally, the authors have highlighted and addressed issues in the methodologies of previous mechanistic studies that led others to erroneous conclusions.

  6. Version published to 10.1101/2020.08.06.240325v2 on bioRxiv
  7. ScreenIT

    SciScore for 10.1101/2020.08.06.240325: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    NIH rigor criteria are not applicable to paper type.

    Table 2: Resources

    Experimental Models: Cell Lines
    SentencesResources
    The baculovirus was amplified through two passages in Sf9 cells, and then used to infect 1 L of Sf21 cells (Expression Systems) and incubated for 48 hrs at 27°C.
    Sf9
    suggested: None
    Software and Algorithms
    SentencesResources
    A custom written Labview routine 36 controlled the data acquisition and the (x-, y-, z-) positions analysis/tracking of both the magnetic and reference beads in real-time.
    Labview
    suggested: (LabView , RRID:SCR_014325)
    Maximum likelihood estimation (MLE) fitting routine: The above stochastic-pausing model was fit to the dwell time distributions using a custom Python 3.7 routine.
    Python
    suggested: (IPython, RRID:SCR_001658)

    Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

    Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

    Results from JetFighter: We did not find any issues relating to colormaps.

    Results from rtransparent:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
    • No protocol registration statement was detected.

    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  8. Version published to 10.1101/2020.08.06.240325v1 on bioRxiv