Breakthrough in Research of Small Molecule Drugs against SARS-CoV-2

Corona virus disease 2019 (COVID-19) caused by a novel single-stranded RNA coronavirus has swept the world. The International Committee on Taxonomy of Viruses (ICTV) named the virus SARS-CoV-2. The life cycle of SARS-CoV-2 consists mainly of viral attachment, membrane fusion, genome duplication and virion assembly and release. The current drug R&D for SARS-CoV-2 targets one or more events in the viral life cycle to prevent viral replication, such as blocking the binding of spike proteins to angiotensin-converting enzyme 2 (ACE2) and inhibiting viromembrane fusion with host cells or inhibiting 3CL-likeprotease (3CLpro) and papain (PLpro) or inhibiting RNA-dependent RNA polymerase (RdRp). At present, several therapeutic drugs have entered phase III clinical trials suggesting a breakthrough in oral small molecule drugs for SARS-CoV-2.

RNA polymerase (RdRp) inhibitors

1. Molnupiravir

Molnupiravir (MK-4482, EIDD-2801), approved for marketing by the UK Medicines and Healthcare Products Regulatory Agency (MHRA) on November 4, 2021, is the first oral inhibitor of RdRp for the treatment of mild to moderate COVID-19 infections in adults. Molnupiravir has demonstrated superior therapeutic effect and safety in the entire clinical trials (Phases I-III: NCT04392219, NCT04405570, NCT04405739, NCT04939428, NCT04575597, NCT04575584) in patients with COVID-19. The antiviral efficacy of molnupiravir in SARS-CoV-2-infected Vero cells was approximately EC50=0.3 μmol/L, and its antiviral replication potency in human respiratory epithelia was EC50=0.14 μmol/L. It is shown by in vitro toxicity data that a CC50 >10 μmol/L against Vero cells takes an ideal safe window for toxicity and efficacy so molnupiravir is selected as a clinical candidate compound. In the pharmacodynamic mechanism research, scientists found that molnupiravir can effectively insert into the RNA membrane of SARS-CoV-2 virus to promote mutation and end viral replication. It showed by preclinical data in cell culture that the number of viral RNAs with G to A and C to U transition mutations increased in a dose-dependent manner upon molnupiravir, which positively associated with the anti-coronaviral efficacy of molnupiravir. And molnupiravir is categorized as a mutagenic nucleotide analogue. With the RdRp protease purified by SARS-CoV-2, combined with the evaluation of viral RNA replication tests, the researchers investigated the potential biochemical mechanism of the drug, namely the metabolism of molnupiravir as a prodrug in plasma to N 4-Hydroxycytidine (EIDD-1931). As the active subject molecule, EIDD-1931 enters the host infected cells and is phosphorylated by kinase to form N-hydroxycytidine triphosphate as the active component of the drug, which participates in the process of viral RNA replication and induces the production of RNA replication mutants, ultimately leading to the termination of viral replication.

Phase II clinical results from Merck in March 2021 showed that patients with COVID-19 treated with the drug from day 5 achieved a 0% positive virus detection rate in all dose groups (compared to 24% in the placebo group) By the latest phase III clinical results, both indicators of mortality risk and hospitalization risk were reduced by 50% in molnupiravir subjects. The probability of hospitalization or death on day 29 after randomization grouping was 7.3% in patients treated with molnupiravir compared to 14.1% in placebo-treated patients. In terms of safety, the rate of subjects discontinuing treatment due to drug-related adverse events was approximately 1.3% in the molnupiravir-treated group compared with the rates of adverse reactions of any grade in the treatment and placebo groups were 35% and 40%.

2. Remdesivir

Remdesivir (GS-5734) is a nucleoside analogue precursor drug being developed by Gilead Sciences, Inc. that is obtained from a ribose derivative synthesized in a multi-step reaction and effectively binds to the nascent RNA chain via RdRp, resulting in premature termination of viral replication. Remdesivir has a broad-spectrum antiviral activity against various viruses of the family Viridae, including filoviruses and coronaviruses, with half-maximal effect concentration (EC50) values ranging from 0.0035 to 0.75 μmol-L-1. It has demonstrated by in vitro tests that the remdesivir has effective antiviral activity against SARS-CoV-2. Remdesivir is readily recognized in plasma by intravenous administration over 30 to 120 min of infusion and reaches peak concentrations at the end of the infusion, followed by rapid disappearance (median time 0.5 to 1 h). In vitro tests showed that SARS-CoV-2 could be inhibited by remdesivir (EC50=0.77 μmol/L in Vero E6 cells). However, clinical research on the effectiveness of the remdesivir has had conflicting results. The U.S. FDA has approved the drug for the treatment of inpatients with COVID-19 based on the results of the adaptive clinical design trial for COVID-19. However, the World Health Organization, based on the Solidarity Clinical Trial, concluded that the drug did not have a significant impact on improving patient survival or reducing the need for oxygen.

Remdesivir is the first drug approved for the treatment of COVID-19 that has been registered and approved for the treatment of patients with COVID-19 in several countries around the world, including Japan, Australia, Korea, Canada and Israel. The mechanism of drug action and the available results of research indicate that remdesivir is poorly effective in heavy/critical patients and should be applied early and promptly in clinical treatment. However, the restrictions of its administration route limit the early clinical drug use.

3. Favipiravir

Favipiravir (T-705) is prescribed for the novel or recurrent influenza. Industrially, it is mainly produced from 3-aminopyrazine-2-carboxylic acid by hydroxylation, esterification, ammonolysis, nitration, reduction, diazotization and other reactions with low yield and high production cost. As a guanine analogue, Favipiravir, a prodrug, enters into infected cells and competitively inhibits RdRp by mimicking guanosine triphosphate (GTP) embedding in the extended RNA strand, thus inhibiting the replication and transcription of the viral genome to obtain anti-RNA viruses (e.g., influenza virus, Ebola virus, yellow fever virus, Chikungunya virus, norovirus, and enterovirus). In vitro test, favipiravir showed high inhibitory activity against SARS-CoV-2(EC50=61.88 μmol/L in Vero E6 cells). In May 2020, Famipiravir was the first drug to receive an 'emergency use authorization' in India to treat COVID-19, followed by the Russian health regulator granting an emergency use authorization for the drug in patients with COVID-19. In Japan, phase III clinical trials of favipiravir which are in the 'pre-registration' have been completed in critically ill patients with COVID-19. What remains controversial are the results obtained for the clinical phase of favipiravir. However, cryo-electron microscopic structural research of the RdRp complex with SARS-CoV-2 in the pre-catalytic state revealed that the presence of favipiravir in the RNA product does not immediately terminate the extension of the growing chain unless a repetitive pyrimidine residue is encountered in the template RNA. Moreover, favipiravir can escape the proofreading of the replication and transcription complex (RTC) leading to mutations in the progeny viral genome and thus exerting antiviral effects. It explains that the drug does not substantially inhibit the synthesis of RNA in vitro.

At present, oral treatment with favipiravir has been recommended in guidelines for patients with mild to moderate covid-19 in Japan, Russia, Saudi Arabia, Thailand, Kenya, etc. It is notable that favipiravir should be used with caution in patients with reproductive needs, a history of gout and high uric acid. It is contraindicated in patients with allergies, severe liver damage and renal impairment. However, with the promising results of the drug's mechanism of action and several clinical trials, favipiravir is still expected to be a new option for SARS-CoV-2 antiviral therapy.

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Protease inhibitor

1. 3C-like protease (3CLpro) inhibitor

3C-like protease (3CLpro, Mpro, nsp5), also known as M protease, is an essential functional protein in viral replication. The protease ensures the production of nsp4-16, a non-structural protein critical for viral transcription and replication, by hydrolyzing at least 11 conserved sites in the polyproteins pp1a and pp1ab including RdRp, decapping enzymes and Mpro itself. Mpro is highly homologous among different strains and lacks homology with human host cells. M-protease is also considered to be an essential target for antiviral drug development.

Pfizer researchers have designed and screened a clinical candidate, PF-07321332, with potent viral inhibition with SARS-CoV-2 Mpro as a target. In in vitro tests, the drug showed an enzyme inhibition constant (Ki) of 3.11 nmol/L and enzyme inhibitory activity of IC50=18.0 nmol/L against SARS-CoV-2 Mpro, however, the antiviral activity against SARS-CoV-2 virus infection was EC50=74-93 nmol/L. In preclinical trials, PF-07321332 had a low oral bioavailability. In pharmacokinetic experiments on rats and dogs, the bioavailability was maintained at 20%-25% because PF-07321332 is a metabolic substrate for the drug metabolizing enzyme CYP3A4 in vivo. As a result, clinical trials have combined with ritonavir (NCT04756531) that is a potent inhibitor of CYP3A4 to improve the half-life and bioavailability of the drug in vivo. It showed by the crystal structure of the drug with SARS-CoV-2 that the CN substituent in the drug molecule is bound to the Cys145 residue in the protease covalently, but the covalent binding mode was shown to be a reversible mode in non-reversible SARS-CoV-2 inhibitor competition experiments.

It has been completed that phase III clinical trials and research of PF-07321332 implemented in mild and moderate SARS-CoV-2 infected patients. It showed that after administration of this inhibitor within 3 d after the onset of symptoms of COVID-19, such as fever and dry cough, the hospitalization rate decreased to 0.8% and the mortality rate to 0% in 1219 adult patients who were not vaccinated COVID-19 vaccine compared with 7% and 1.8% respectively in the placebo group. In November 2021, the drug applied for emergency use authorization from the U.S. FDA.

2. Papain-like protease (PLpro) inhibitor

PLpro is one of the key proteases that shears the polyproteins pp1a and pp1ab expressed via the host cell translation machinery (PLpro is responsible for cleaving nsp1, nsp2 and nsp3). It can cleave Lys48-linked polyubiquitin and ubiquitin-like molecule interferon-stimulated gene 15 (ISG15) modifications with high activity to achieve immune escape Theoretically, the viral load can be reduced lower as well as the host innate immune system can be restored after PLpro being suppressed, The multiple roles of PLpro in viral replication and host cell control have led it to be considered as a potential antiviral target.

When the SARS outbroke in 2003, GRL0617 was the originally developed PLpro inhibitor. For PLpro of SARS-CoV-2 sharing 82% sequence similarity and 90% structural similarity with SARS-CoV PLpro, researchers conducted a SARS-CoV-2 antiviral activity screen for compounds wit SARS-CoV inhibitory activity at prophase. It is suggested by the research that 100 μmol/L of GRL0617 effectively inhibited the intracellular replication of SARS-CoV-2 in SARS-CoV-2-infected Vero E6 cells with an inhibition rate of over 50% To verify the relevance of the antiviral replication effect of the compounds to the target, researchers performed in vitro cellular tests and obtained the eutectic structure of the drug with PLpro. In vitro tests shows that the inhibitor has strong inhibitory activity (IC50=2.1 μmol/L) against the PLpro protease of SARS-CoV-2, and there is no obvious cytotoxicity of the drug in Vero E6 cells at concentrations up to 100 μmol/L.

With the global COVID-19 pandemic and the continued mutation of SARSCoV-2, the development and widespread application of effective and safe antiviral drugs are important initiatives to reduce the global burden of disease. The advantages of small molecule drugs are their easy absorption, small molecular size, ability to penetrate cell membranes and ease of mass industrial production. It is expected that these drugs will be widely used at prophase for reducing the rate of severe morbidity and mortality. It will make a significant contribution to overcoming the COVID-19.

References:

[1] Zhao Zhe, Zhang Qing, Ge Ziruo, Zhang Wei, Chen Zhihai. Giant progress in small molecule antiviral drugs for SARS-CoV-2 [J]. Chinese Journal of Pharmacovigilance, 2022, 19 (01): 1-6.DOI: 10.19803/j.1672-8629.2022.01.01.

[2] Zhang Ru, Sun Ziru, Liu Shengnan, Gao Qingzhi. Current status and perspectives of research on COVID-19 therapeutic drugs [J/OL]. Chinese Science Bulletin: 1-15[2022-02-13].https://kns-cnki-net.wvpn.ncu.edu.cn/kcms/detail/11.1784.N.20220107.2020.006.html.

About the Author: 

Little Sand, a food science and technology worker, master of food science, graduated from the School of Food Science and Engineering of South China University of Technology, now working in a drug R&D corporation in China and engaging in the development and research of nutritional food.