The drug is designed to suppress the rapid replication of the SARS-CoV-2 virus in human cells by blocking the viral copying machine, called RNA polymerase.
Researchers at the Max Planck Institute (MPI) for Biophysical Chemistry in Göttingen and the University of Würzburg have now elucidated how remdesivir interferes with the viral polymerase during copying and why it does not inhibit it completely.
“After complicated studies, we come to a simple conclusion,” Max Planck Director Patrick Cramer says. “Remdesivir does interfere with the polymerase while doing its work, but only after some delay. And the drug does not fully stop the enzyme.”
At the pandemic’s beginning, Cramer’s team at the MPI for Biophysical Chemistry had elucidated how the coronavirus duplicates its RNA genome. For the pathogen this is a colossal task as its genome comprises around 30,000 RNA building blocks, making it particularly long.
To elucidate remdesivir’s mechanism of action, Cramer’s team collaborated with Claudia Höbartner’s group. The latter produced special RNA molecules for the structural and functional studies.
“Remdesivir’s structure resembles that of RNA building blocks,” explains Höbartner, a professor of chemistry at the University of Würzburg. The polymerase is thereby misled and integrates the substance into the growing RNA chain.
Pausing instead of blocking
After remdesivir had been incorporated into the viral genome, the researchers examined the polymerase-RNA complexes using biochemical methods and cryo-electron microscopy. They discovered that the copying process pauses precisely when three more building blocks have been added after remdesivir was incorporated into the RNA chain.
“The polymerase does not allow the installation of a fourth one. This pausing is caused by only two atoms in the structure of remdesivir that get hooked at a specific site on the polymerase. However, remdesivir does not fully block RNA production.
Often, the polymerase continues its work after correcting the error,” explains Goran Kokic, a research associate in Cramer’s lab, who together with Hauke Hillen, Dimitry Tegunov, Christian Dienemann, and Florian Seitz, had conducted the crucial experiments. They all are first authors of the publication about this work recently published in the scientific magazine Nature Communications.
Understanding how remdesivir works opens up new opportunities for scientists to tackle the virus. “Now that we know how remdesivir inhibits the corona polymerase, we can work on improving the substance and its effect.
In addition, we want to search for new compounds that stop the viral copying machine,” Max Planck Director Cramer says. “The vaccinations now underway are essential to bring the pandemic under control. But we also need to develop effective drugs that mitigate COVID-19 disease progression in the event of infection.”
DISCOVERY OF REMDESIVIR (GS-5734)
Nucleoside Analogs as Antiviral Agents
Nucleoside and nucleotide analogs as small-molecule-based antivirals have been explored for many years and form the backbone of treatment against viral infections, including HIV, hepatitis B virus, and herpesvirus infections (8,–10). In 2013, the nucleotide analog sofosbuvir was approved by the FDA for the treatment of chronic hepatitis C virus infections.
The novel compound that targets the RNA-dependent viral polymerase (NS5B) revolutionized HCV treatment, as it is able to cure the formerly lifelong chronic progressive disease when combined with other antivirals (11). In the past years, nucleoside/nucleotide analogs were increasingly recognized as potential antivirals targeting other positive-stranded RNA viruses such as members of the Flaviviridae, Picornaviridae, Caliciviridae, and Coronaviridae families, as they share relevant amino acid sequences with HCV (12), and the RNA-dependent polymerases are closely related phylogenetically (13, 65).
This supported the assembly of antiviral compound libraries that could be screened against emerging RNA viruses. In the past years, several pharmacological advances in the development of nucleoside analogs were made based on structure-to-activity relationship (SAR) studies that improved pharmacokinetics, antiviral activity, and selectivity (14,–16).
A comprehensive overview of the medicinal chemistry and pharmacological evolution of antiviral nucleoside analogs can be found elsewhere (17, 18). Nucleoside analogs require intracellular activation by phosphorylation in order to become their active metabolites. One of the most important milestones was the addition of a monophosphate prodrug to the nucleoside, which significantly improved intracellular delivery and activation (19,–21). This so-called ProTide approach, developed by McGuigan et al. (22, 23), was also used to optimize the precursor of remdesivir named GS-441524.
A Broad-Spectrum Antiviral Inhibits Ebolavirus
The parent molecule of remdesivir, GS-441524, was derived from a small-molecule library of around 1,000 diverse nucleoside and nucleoside phosphonate analogs that were assembled over many years of antiviral research based on their potential ability to target emerging RNA viruses such as SARS-CoV and MERS-CoV of the Coronaviridae or Zika and dengue viruses of the Flaviviridae family (20).
Following the ebolavirus (EBOV) epidemic in West Africa from 2013 to 2016, a selection of promising leads from this library underwent intensive testing against different types of EBOV in collaboration with the Centers for Disease Control and Prevention (CDC) and the U.S. Army Medical Research Institute of Infectious Diseases (USAMRIID), which included studies in nonhuman primates (NHPs) (24). These efforts finally led to the identification of GS-5734, a monophosphate prodrug version of GS-441524, as the most promising lead against EBOV. GS-5734, later renamed remdesivir, had a broad antiviral spectrum, including EBOV, Marburg virus, respiratory syncytial virus (RSV), HCV, and several paramyxoviruses (20, 21, 24), in vitro.
In addition, it demonstrated activity against MERS-CoV (24,–26) and SARS-CoV (25, 26). Favorable in vitro results stimulated further evaluation in EBOV-infected macaques, where remdesivir suppressed viral replication and improved survival, clinical signs of the disease, and pathophysiological blood markers (24).
After its discovery, remdesivir was administered under compassionate use to patients with ebolavirus disease (EVD) but stopped after an interim analysis of the first randomized controlled clinical trial (RCT) showed an inferiority of remdesivir to treatments with monoclonal antibodies (MAb114 and REGN-EB3). The trial evaluated the efficacies of different investigational therapeutics against EVD. Following the interim analysis, the remdesivir arm was halted for the remainder of the trial (27).
Lead Candidate against COVID-19
In December 2019, a novel coronavirus, SARS-CoV-2, emerged and caused a pandemic that is still ongoing. There were strong arguments for the antiviral effect of remdesivir against coronaviruses emerging from multiple cell-based in vitro models, including primary human airway epithelial (HAE) cell cultures (25), and, for MERS-CoV, from a mouse model of pulmonary infection (28).
In addition, in a rhesus macaque model of MERS-CoV infection, remdesivir demonstrated strong prophylactic properties, and administration was associated with clinical benefits for treated subjects (29). The global hazards caused by the pandemic with the novel SARS-CoV-2 prompted the identification of potential treatment options. Given the solid preclinical data, remdesivir was considered one of the most promising candidates that went into clinical testing against COVID-19.
MECHANISM OF ACTION
Remdesivir is a monophosphoramidate nucleoside prodrug that undergoes intracellular metabolic conversion to its active metabolite nucleoside triphosphate (NTP). As described for several other direct-acting antivirals, the active metabolite of remdesivir (remdesivir triphosphate [remdesivir-TP] or GS-443902) subsequently targets the machinery responsible for the replication of the viral RNA genome, a highly conserved element of the viral life cycle.
Nucleoside analogs are synthetic compounds that work by competition with endogenous natural nucleoside pools for incorporation into replicating viral RNA. While these compounds mimic their physiological counterparts, the incorporation of the analog molecule disrupts subsequent molecular processes.
The drug target and the exact processes that lead to the inhibition of viral replication have been studied extensively in ebolavirus (24, 30). The suggested drug target, the EBOV RNA-dependent RNA polymerase (RdRp) complex, was only recently biochemically purified, which allowed for in-depth molecular analyses.
Viral RdRp is the target protein for the active metabolite remdesivir-TP. Remdesivir-TP acts as the substrate for RdRp where it competes with ATP for incorporation into new strands. Inhibition of EBOV RdRp most probably results from delayed chain termination, a mechanism that is known from approved antivirals against human immunodeficiency virus type 1 (HIV-1) and HBV (31,–34).
In the case of EBOV, the incorporation of remdesivir-TP into replicating RNA was observed to cause chain termination predominantly at five positions downstream (i + 5) (30). Importantly, the activity of human RNA polymerase is not inhibited in the presence of remdesivir-TP (24).
In SARS-CoV and MERS-CoV, remdesivir-TP interferes with the nsp12 polymerase, which is a multisubunit RNA synthesis complex of viral nonstructural proteins (nsp’s) produced as cleavage products of viral polyproteins. As nsp12 is highly conserved across the coronavirus family, it is most likely that the mechanism of action (MOA) of remdesivir does not differ significantly among CoVs (35, 36).
Like in EBOV, remdesivir-TP efficiently inhibits the replication of SARS-CoV and MERS-CoV by causing delayed chain termination when being incorporated into the replicating RNA (26). A recent biochemical analysis revealed that in SARS-CoV-2, remdesivir-TP causes the termination of RNA synthesis at three positions after the position where it is incorporated (i + 3).
This mechanism was nearly identical in RdRps of SARS-CoV and MERS-CoV (37). The premature termination of RNA synthesis ultimately abrogates further transcriptional and translational processes needed for the generation of new virions (Fig. 2). The resulting antiviral effects of remdesivir have been studied in different cell-based models.
Intracellular activation of remdesivir (GS-5734) and inhibition of coronavirus replication. Passage through the cell membrane by remdesivir is facilitated by the prodrug component attached to the nucleoside core. Upon entering the target cell, the pronucleotide undergoes further phosphorylation steps to become the active triphosphate metabolite that effectively inhibits viral RNA replication. Delayed chain termination is caused by the following processes: (i) misintegration of nucleoside triphosphate (NTP) into replicating RNA by RdRp, (ii) prevention of further chain elongation after NTP plus 3 additional nucleosides, and (iii) premature termination of RNA synthesis.
reference link: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7566896/
More information: Goran Kokic et al. Mechanism of SARS-CoV-2 polymerase stalling by remdesivir, Nature Communications (2021). DOI: 10.1038/s41467-020-20542-0
