Yunjeong Kim and Kyeong-Ok “KC” Chang, virologists in the College of Veterinary Medicine at Kansas State University, have published a study showing a possible therapeutic treatment for COVID-19.
Pathogenic coronaviruses are a major threat to global public health, as shown by severe acute respiratory syndrome coronavirus, or SARS-CoV; Middle East respiratory syndrome coronavirus, known as MERS-CoV; and the newly emerged SARS-CoV-2, the virus that causes COVID-19 infection.
The study, “3C-like protease inhibitors block coronavirus replication in vitro and improve survival in MERS-CoV-infected mice,” appears in the Aug. 3 issue of the prestigious medical journal Science Translational Medicine.
It reveals how small molecule protease inhibitors show potency against human coronaviruses. These coronavirus 3C-like proteases, known as 3CLpro, are strong therapeutic targets because they play vital roles in coronavirus replication.
“Vaccine developments and treatments are the biggest targets in COVID-19 research, and treatment is really key,” said Chang, professor of diagnostic medicine and pathobiology.
“This paper describes protease inhibitors targeting coronavirus 3CLpro, which is a well-known therapeutic target.”
The study demonstrates that this series of optimized coronavirus 3CLpro inhibitors blocked replication of the human coronaviruses MERS-CoV and SARS-CoV-2 in cultured cells and in a mouse model for MERS. These findings suggest that this series of compounds should be investigated further as a potential therapeutic for human coronavirus infection.
Chang and Kim have been using National Institutes of Health grants to develop antiviral drugs to treat MERS and human norovirus infections. Their work extends to other human viruses such as rhinoviruses and SARS-CoV-2.
“The work that this group of collaborators has been doing on antivirals and inhibitors for SARS and MERS at K-State for a number of years has been vital to their ability to quickly pivot to emphasize research on SARS-CoV-2 virus and therapeutics,” said Peter K. Dorhout, vice president for research at K-State.
Co-collaborators on the research include teams lead by Bill Groutas at Wichita State University, Stanley Perlman at the University of Iowa and Scott Lovell at the University of Kansas.
“Drs. Groutas, Perlman and Lovell brought decades of experience to our research team,” Chang said. “We would not have been able to come this far without important collaborations with our colleagues at other institutions.”
“Getting things published right now is very important for the scientific community,” Kim said. “I think we are adding valuable information to the antiviral field.”
The outbreak of coronavirus SARS-CoV-2 in Wuhan, China in December 2019, the cause of Corona Virus Disease of 2019 (COVID-19), represents a pandemic threat to global health [1,2]. The WHO declared COVID-19 as a pandemic on March 11th 2020.
The outbreak has spread to more than 185 countries with more than 3,200,000 confirmed cases, more than 230,000 confirmed deaths and more than 1,000,000 total recoveries worldwide as of May 1st 2 2020 [3].
Hundreds of millions of lives have been affected as a result of mandatory isolations/quarantines. This pandemic has the potential to overwhelm national healthcare systems, and have major consequences on global economy if SARS-CoV-2 spread and virulence is not contained, or effective treatments are not developed.Coronaviruses are grouped into alpha, beta, gamma, and delta classes. Coronaviruses can infect both humans and animals.
The source of the beta coronavirus SARS-CoV-2 is believed to be bats, which carry the virus with no signs of disease [4]. Beta coronaviruses caused earlier outbreaks of severe acute respiratory syndromes (SARS), including SARS-CoV (2002/2003 in Guangdong, China) and Middle East Respiratory Syndrome virus MERS-CoV (2012 in Saudi Arabia) [5].
Beta coronaviruses are pathogenic for humans and have a single stranded RNA genome, encapsulated by a membrane envelope [6]. The coronavirus crown-like (“corona”) morphology is created by transmembrane spike glycoproteins (S proteins) that form homotrimers protruding from the viral surface [7].
The S proteins of SARS-CoV and SARS-CoV-2 display structural homology and conserved ectodomains, so earlier strategies employed to prevent binding of SARS-CoV to its host cell receptor angiotensin-converting enzyme 2 (ACE2) may be relevant, since SARS-CoV-2 also employs ACE2 for cell entry [8,9].
ACE2, an exopeptidase expressed on epithelial cells of the respiratory tract, may constitute a pharmacological target to limit cell entry of SARS-CoV-2. The established antimalarial drugs chloroquine and hydroxychloroquine have been shown to inhibit terminal phosphorylation of ACE2 and to elevate the pH in endosomes, respectively.
Chloroquine and hydroxychloroquine constitute candidate drugs against SARS-CoV-2 infection and COVID-19 disease, and are now investigated for their therapeutic efficacy in international clinical trials with COVID-19 patients (i. e. SOLIDARITY Trial).
The glycosylated S protein of SARS-CoV-2 is highly immunogenic to the host immune system, and murine polyclonal antibodies against SARS-Co-V S protein potently inhibit SARS-CoV-2 S-mediated cell entry, indicating that cross-neutralizing antibodies targeting conserved S epitopes can be elicited upon vaccination [9].
Similar to the earlier SARS and MERS beta coronaviruses, SARS-CoV-2 primarily infects alveolar epithelial cell of the lung, leading to a severe bilateral peripheral pneumonia with ground glass opacity in CT images (COVID-19 disease), with a mortality rate of 2 % to 5 % [10].
SARS-CoV-2 also can contribute to multiple organ failure, affecting heart, liver, kidney, central nervous system and gastrointestinal tract [11]. Epidemiology thus far suggests that SARS-CoV-2 is more contagious than SARS-CoV or MERS-CoV [12].
Multiple mechanisms now identified in the infective and replication processes of SARS-CoV-2 offer targets for pharmacological interventions. Infection of pneumocytes, macrophages and pulmonary mast cells requires viral S protein.
This invasion process which involves attachment of S protein to the ACE2 receptor is facilitated by host cell derived serine protease TMPRSS2 [8]. Agents that inhibit TMPRSS2, such as camostat mesilate, may be useful in blocking viral host cell entry.
After host cell entry, the viral single-stranded positive RNA is released for replication of virus RNA and translation of virus polyproteins that are finally cleaved into mature effector proteins by virus proteases [13].
The S protein interaction with ACE2 on host cell cytoplasmic membrane initiates viral infection. Strategies capable of disrupting S protein interaction with ACE2 could be of significant therapeutic value, because the binding affinity of SARS-CoV-2 S protein to ACE2 is 10−20-fold higher than for the S protein of SARS-CoV which may contribute to the higher contagiousness of SARS-CoV-2 as compared to SARS-CoV [12].
Although SARS-CoV and SARS-CoV-2 have only 79 % genomic sequence similarity, they share a highly conserved receptor binding domain for their S proteins [1]. There is also potential for targeting other highly conserved proteins associated with SARS-CoV and SARS-Co-V-2, including RdRp and 3Clpro (also termed Mpro), which share over 95 % similarity between the two viruses, despite only 79 % genomic sequence sharing.
RdRp is an RNA-dependent RNA polymerase required for replicating the viral genome within the host cell. 3Clpro and Plpro are both viral proteases which break down viral polyprotein into functional units within host cells that are finally assembled into new viruses. The 3Clpro sequences between the two viruses are 96 % similar, the Plpro sequence identity is 83 %, and their active sites show a high degree of conservation [14].
Drugs that have recently been shown to target MERS-CoV in mice [15], and to inhibit Ebola virus RdRp and SARS-CoV-2 proteases in humans, such as remdesivir and ritonavir/lopinavir, also constitute candidate drugs against SARS-CoV-2 and are now investigated for their therapeutic efficacy in COVID-19 patients in 2 international clinical trials (SOLIDARITY Trial and DisCoVeRy Trial).
Finally, certain phytochemicals and natural products with high antiviral activity should be considered for treatment of SARS-CoV-2 infection and COVID-19 disease.
Inhibitors of SARS-CoV-2 3Clpro protease
3Clpro (also termed Mpro) constitutes the main protease of beta coronaviruses that is essential for processing of polyproteins translated from the viral RNA [125]. Recently, the X-ray structures of the unligated SARS-CoV-2 3Clpro and its complex with α-ketoamides designed as specific inhibitors of 3Clpro were reported [126].
Two pyridine-containing α-ketoamides, designated 13a and 13b, displayed favorable pharmacokinetic properties in mice and were detected at sufficient concentrations in lung tissue and broncheo-alveolar lavage fluid within 4 hours–24 hours after subcutaneous administration, demonstrating lung tropism of the compounds [126].
Besides subcutaneous administration, inhalation of nebulized 13b by mice resulted in high and long-lasting (24 h) concentrations in lung tissue, without any adverse effects [126], pointing out a role of pyridine-containing α-ketoamides in COVID-19 therapy.
In a recent study that employed combined structure-assisted drug design, virtual drug screening and high-throughput screening, a mechanism-based inhibitor of 3Clpro, termed N3, was identified by computer-aided drug design [127].
N3, a Michael acceptor inhibitor that can inhibit the 3Clpros of SARS-CoV and MERS-CoV, was shown to form a covalent bond with and to be an irreversible inhibitor of SARS-CoV-2 3Clpro [127].
Further, in a high-throughput screening approach for identifying inhibitors of SARS-CoV-2 3Clpro, ebselen, an organoselenium compound with anti- inflammatory, anti-oxidant and cytoprotective properties, was identified [127].
In a plaque-reduction assay with simian Vero cells infected with SARS-CoV-2, N3 and ebselen displayed antiviral and cell protection efficacy at EC50 values of 16.77 μM and 4.67 μM, respectively [127], ultimately demonstrating their antiviral potential against SARS-CoV-2.
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More information: “3C-like protease inhibitors block coronavirus replication in vitro and improve survival in MERS-CoV-infected mice,” Science Translational Medicine (2020). DOI: 10.1126/scitranslmed.abc5332 , stm.sciencemag.org/content/ear … scitranslmed.abc5332
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