RNA-dependent RNA polymerase inhibitors in COVID-19 therapy

By Dr. Liji Thomas, MDOct 10 2022Reviewed by Danielle Ellis, B.Sc.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pathogen emerged in Wuhan at the end of 2019 to trigger the latest and most devastating coronavirus (CoV) pandemic of the last hundred years. With a toll of six million deaths and counting, it triggered intense scientific efforts to discover effective and safe antivirals.

RNA-dependent RNA polymerase (RdRp) is a key enzyme for SARS-CoV-2 genomic replication and transcription and, thus, for viral replication. A new paper reviews current knowledge of RdRp inhibitors that could be developed further to provide broadly effective antivirals against SARS-CoV-2 in particular and CoVs in general.

Introduction

SARS-CoV-2 is a beta-CoV with a single-stranded ribonucleic acid (RNA) genome with ~30,000 bases encoding 27 different proteins on 14 open reading frames (ORFs). The 3’ end encodes structural proteins, comprising the immunodominant spike protein, envelope, membrane, and nucleocapsid proteins. At the 5’ end, ORF1a/1b encode two large polyproteins that undergo cleavage to yield 16 non-structural proteins.

One of these is the RdRp, which forms part of the replication-transcription complex (RTC) responsible for synthesizing genomic and subgenomic RNAs required for successful infection. Its catalytic component, the nonstructural protein (nsp) 12, forms a subcomplex with nsp7 and nsp8, before it can display adequate enzymatic activity. This is the holoenzyme RdRp (holo-RdRp).  

Emerging virus variants have displayed mutations in the spike protein that enhance virulence, transmissibility, or immune evasion capabilities. Therefore, antivirals remain a highly desirable goal, despite the rollout of numerous vaccines against the virus.

When developing broad-spectrum antivirals, the target must be a highly conserved viral protein. RdRp is the most conserved protein among CoVs. Importantly, there is no human homolog. The current study, published in Biochemical Pharmacology, summarizes current RdRp inhibitors and the mechanisms by which they interact with the viral RdRp, to provide a foundation for future drug research in this area.

Nucleoside inhibitors

There are two main types of RdRp inhibitors. The first comprises nucleoside analog inhibitors that bind to the substrate (nucleoside)-binding site on the enzyme. The other type, non-nucleoside inhibitors, show allosteric inhibition, binding at non-substrate sites to hinder nucleoside binding at the substrate site.

Nucleoside analog inhibitors (NAIs) have received a lot of attention. They fall into three classes:  those which induce mutations after incorporation into the elongating RNA strand; obligate chain terminators; and delayed chain terminators.

All three types are activated by phosphorylation. This allows them to be inserted into the nascent RNA.

Remdesivir (RDV) was developed to fight the Ebola virus back in 2014 but was repurposed against the current coronavirus disease 2019 (COVID-19). It is a prodrug that is first metabolized to adenine before subsequent phosphorylation to form remdesivir triphosphate (RTP), the artificial substrate that inhibits further RNA synthesis.

It was extensively employed during the ongoing pandemic, with mild effect against mortality in patients without moderate or severe disease. It also cut recovery time by 30%, according to preliminary results. It is the only COVID-19 drug approved by the Food and Drug Administration for use in such situations.

Other drugs include favipiravir (FPV), an anti-influenza drug with broad antiviral activity; and molnupiravir, which showed encouraging early results and has received emergency use authorization (EUA) from the Medicines and Healthcare products Regulatory Agency (MHRA) and FDA.

Molnupiravir is mutagenic in its antiviral action. However, since it may induce mutations in the host cell also, as observed in cell culture models, further study is required.

Galidesivir, an adenosine analog that, when phosphorylated, has higher affinity for the viral RNA polymerase than the host cell polymerase. The result is a change in its electrostatic nature, causing premature replication termination.

Ribavirin is over four decades old and is a guanosine analog that can pair with either cytidine or uridine triphosphate, producing lethal mutagenesis but also inhibiting de novo synthesis of guanine nucleotides. Both mechanisms contribute to the antiviral effect. Fetal toxicity, potential calcium- and magnesium-lowering effects, and hemolytic anemia are all concerning adverse effects.

Sofosbuvir and daclatasvir are used against hepatitis C virus infection. This virus shares a similar mechanism of replication with CoVs, and therefore these drugs might be potentially useful against the latter too.

Some in vitro evidence show cooperative inhibition of SARS-CoV-2 replication with a combination of these drugs. A small randomized clinical trial in moderate-severe COVID-19 patients supports their utility, with reduced hospital stays compared to standard COVID-19 care.

Tenofovir, a drug with activity against the human immunodeficiency virus and hepatitis B virus, is another potentially useful molecule, as is the new orally available RDV derivatives VV11 6 and GS-621763. AT-527 is another broad-spectrum inhibitor, being a guanosine nucleotide analog.

The problem with any NAI is the presence of SARS-CoV-2 nsp14, which is a proofreading exonuclease enzyme capable of editing out the incorporated missense nucleotide analogs, thus restoring replicative capacity.

Non-nucleoside inhibitors (NNIs)

NNIs have the advantage of not depending on their incorporation into the nucleotide chain disruption for their anti-replicative effects. In addition, they cover a wider chemical range and are probably orally bioavailable. In most cases, these drugs cause allosteric effects on RdRp-substrate binding.

Their additional anti-inflammatory, antioxidant, immunomodulatory, and cardioprotective functions increase their potential usefulness in COVID-19.

The most promising of these compounds include suramin, an old drug used to treat many parasitic and viral infections, as well as cancer, snakebite, and autism. It has multiple actions on viral infection, including inhibition of viral attachment and entry and virion release from the host cells.

Cell culture assays have shown its ability to inhibit SARS-CoV-2 replication by preventing viral entry into the cell, with a 2-3-fold reduction in viral load. It also inhibits RdRp, binding at two different sites, one causing direct and the other allosteric hindrance.

“This unique binding pattern is consistent with suramin and its derivatives being at least 20-fold more potent than the triphosphate form of remdesivir (RDV-TP) in polymerase activity inhibition assays in vitro.”

Other mechanisms are also being observed, such as electrostatic binding to RNA-binding proteins required for RNA replication and perhaps a synergistic effect with quinacrine on the SARS-CoV-2 main protease.

The ability of suramin to inhibit multiple important viral proteins makes it very attractive, but it also has frequent and potentially serious adverse effects such as proteinuria, adrenal impairment, and corneal damage. Large, well-designed trials are essential to arrive at a proper cost-benefit assessment of this compound.

Other NNIs include quinoline derivatives, corilagin, lycorine, and phenolic plant compounds like baicalein.

Corilagin is of herbal origin, with reported anti-tumor and anti-inflammatory activity. It also inhibits RdRp activity and SARS-CoV-2 infection in cell culture, probably by preventing the necessary conformational changes in the enzyme. It has additive effects when used with RDV, without toxicity in preclinical experiments.

Lycorine is a natural alkaloid with many biological effects. It inhibits SARS-CoV-2 infection in a dose-dependent fashion at low nanomolar concentrations, with a higher binding affinity for RdRp than RDV.

What are the conclusions?

With the emergence of Omicron, which has the largest ever number of mutations of all known SARS-CoV-2 variants, the pandemic gained new life. The mutations included many that conferred immune evasion capability on the virus, causing breakthrough infections and reinfections at a higher rate than ever before.

Moreover, SARS-CoV-2 is the third pathogenic CoV to cause a potential or actual pandemic in the last two decades, indicating increased potential for more unknown zoonotic agents to emerge and disrupt world health in the coming years.

This lends urgency to the search for effective, broadly acting antivirals. In this, RdRp appears to be a most attractive drug target, being critical to viral replication, non-homologous in humans, and highly conserved among CoVs. The best approach seems to be NAIs, provided that nsp14 exoribonuclease screening is introduced to prevent escape from such therapy by this proofreading enzyme.

NAIs should be designed to either escape recognition by this enzyme or to have such a high rate of incorporation that the excision enzyme cannot keep up. Inhibitors of exoribonuclease activity are another potential strategy to achieve this goal.

NNIs resist nsp14 activity. These require further trials to confirm and define their efficacy.

Combinations of these drugs, along with phytochemicals, may be the best approach to treating COVID-19 without the rise of drug resistance by mutations. Such a “cocktail of inhibitors in the near future may have significant implications for alleviating the current global public health threat.”