Structure-guided design of direct-acting antivirals that exploit the gem-dimethyl effect and potently inhibit 3CL proteases of severe acute respiratory syndrome Coronavirus-2 (SARS-CoV-2) and middle east respiratory syndrome coronavirus (MERS-CoV).

The high morbidity and mortality associated with SARS-CoV-2 infection, the etiological agent of COVID-19, has had a major impact on global public health. Significant progress has been made in the development of an array of vaccines and biologics, however, the emergence of SARS-CoV-2 variants and breakthrough infections are an ongoing major concern. Furthermore, there is an existing paucity of small-molecule host and virus-directed therapeutics and prophylactics that can be used to counter the spread of SARS-CoV-2, and any emerging and re-emerging coronaviruses. We describe herein our efforts to address this urgent need by focusing on the structure-guided design of potent broad-spectrum inhibitors of SARS-CoV-2 3C-like protease (3CLpro or Main protease), an enzyme essential for viral replication. The inhibitors exploit the directional effects associated with the presence of a gem-dimethyl group that allow the inhibitors to optimally interact with the S4 subsite of the enzyme. Several compounds were found to potently inhibit SARS-CoV-2 and MERS-CoV 3CL proteases in biochemical and cell-based assays. Specifically, the EC50 values of aldehyde 1c and its corresponding bisulfite adduct 1d against SARS-CoV-2 were found to be 12 and 10 nM, respectively, and their CC50 values were >50 µM. Furthermore, deuteration of these compounds yielded compounds 2c/2d with EC50 values 11 and 12 nM, respectively. Replacement of the aldehyde warhead with a nitrile (CN) or an α-ketoamide warhead or its corresponding bisulfite adduct yielded compounds 1g, 1eand1f with EC50 values 60, 50 and 70 nM, respectively. High-resolution cocrystal structures have identified the structural determinants associated with the binding of the inhibitors to the active site of the enzyme and, furthermore, have illuminated the mechanism of action of the inhibitors. Overall, the high Safety Index (SI) (SI=CC50/EC50) displayed by these compounds suggests that they are well-suited to conducting further preclinical studies.

Roles of host proteases in the entry of SARS-CoV-2.

The spike protein (S) of SARS-CoV-2 is responsible for viral attachment and entry, thus a major factor for host susceptibility, tissue tropism, virulence and pathogenicity. The S is divided with S1 and S2 region, and the S1 contains the receptor-binding domain (RBD), while the S2 contains the hydrophobic fusion domain for the entry into the host cell. Numerous host proteases have been implicated in the activation of SARS-CoV-2 S through various cleavage sites. In this article, we review host proteases including furin, trypsin, transmembrane protease serine 2 (TMPRSS2) and cathepsins in the activation of SARS-CoV-2 S. Many betacoronaviruses including SARS-CoV-2 have polybasic residues at the S1/S2 site which is subjected to the cleavage by furin. The S1/S2 cleavage facilitates more assessable RBD to the receptor ACE2, and the binding triggers further conformational changes and exposure of the S2' site to proteases such as type II transmembrane serine proteases (TTPRs) including TMPRSS2. In the presence of TMPRSS2 on the target cells, SARS-CoV-2 can utilize a direct entry route by fusion of the viral envelope to the cellular membrane. In the absence of TMPRSS2, SARS-CoV-2 enter target cells via endosomes where multiple cathepsins cleave the S for the successful entry. Additional host proteases involved in the cleavage of the S were discussed. This article also includes roles of 3C-like protease inhibitors which have inhibitory activity against cathepsin L in the entry of SARS-CoV-2, and discussed the dual roles of such inhibitors in virus replication.

Broad-Spectrum Cyclopropane-Based Inhibitors of Coronavirus 3C-like Proteases: Biochemical, Structural, and Virological Studies.

The advent of SARS-CoV-2, the causative agent of COVID-19, and its worldwide impact on global health, have provided the impetus for the development of effective countermeasures that can be deployed against the virus, including vaccines, monoclonal antibodies, and direct-acting antivirals (DAAs). Despite these efforts, the current paucity of DAAs has created an urgent need for the creation of an enhanced and diversified portfolio of broadly acting agents with different mechanisms of action that can effectively abrogate viral infection. SARS-CoV-2 3C-like protease (3CLpro), an enzyme essential for viral replication, is a validated target for the discovery of SARS-CoV-2 therapeutics. In this report, we describe the structure-guided utilization of the cyclopropane moiety in the design of highly potent inhibitors of SARS-CoV-2 3CLpro, SARS-CoV-1 3CLpro, and MERS-CoV 3CLpro. High-resolution cocrystal structures were used to identify the structural determinants associated with the binding of the inhibitors to the active site of the enzyme and unravel the mechanism of action. Aldehydes 5c and 11c inhibited SARS-CoV-2 replication with EC50 values of 12 and 11 nM, respectively. Furthermore, the corresponding aldehyde bisulfite adducts 5d and 11d were equipotent with EC50 values of 13 and 12 nM, respectively. The safety index (SI) values for compounds 5c / 11c and 5d / 11d ranged between 7692 and 9090. Importantly, aldehydes 5c / 11c and bisulfite adducts 5d / 11d potently inhibited MERS-CoV 3CLpro with IC50 values of 80 and 120 nM, and 70 and 70 nM, respectively. Likewise, compounds 5c / 11c and 5d / 11d inhibited SARS-CoV-1 with IC50 values of 960 and 350 nM and 790 and 240 nM, respectively. Taken together, these studies suggest that the inhibitors described herein have low cytotoxicity and high potency and are promising candidates for further development as broad-spectrum direct-acting antivirals against highly pathogenic coronaviruses.

SARS-CoV-2 Omicron (B.1.1.529) Infection of Wild White-Tailed Deer in New York City.

There is mounting evidence of SARS-CoV-2 spillover from humans into many domestic, companion, and wild animal species. Research indicates that humans have infected white-tailed deer, and that deer-to-deer transmission has occurred, indicating that deer could be a wildlife reservoir and a source of novel SARS-CoV-2 variants. We examined the hypothesis that the Omicron variant is actively and asymptomatically infecting the free-ranging deer of New York City. Between December 2021 and February 2022, 155 deer on Staten Island, New York, were anesthetized and examined for gross abnormalities and illnesses. Paired nasopharyngeal swabs and blood samples were collected and analyzed for the presence of SARS-CoV-2 RNA and antibodies. Of 135 serum samples, 19 (14.1%) indicated SARS-CoV-2 exposure, and 11 reacted most strongly to the wild-type B.1 lineage. Of the 71 swabs, 8 were positive for SARS-CoV-2 RNA (4 Omicron and 4 Delta). Two of the animals had active infections and robust neutralizing antibodies, revealing evidence of reinfection or early seroconversion in deer. Variants of concern continue to circulate among and may reinfect US deer populations, and establish enzootic transmission cycles in the wild: this warrants a coordinated One Health response, to proactively surveil, identify, and curtail variants of concern before they can spill back into humans.

Potent Protease Inhibitors of Highly Pathogenic Lagoviruses: Rabbit Hemorrhagic Disease Virus and European Brown Hare Syndrome Virus.

Rabbit hemorrhagic disease (RHD) and European brown hare syndrome (EBHS) are highly contagious diseases caused by lagoviruses in the Caliciviridae family. These infectious diseases are associated with high mortality and a serious threat to domesticated and wild rabbits and hares, including endangered species such as riparian brush rabbits (Sylvilagus bachmani riparius). In the United States (U.S.), only isolated cases of RHD had been reported until Spring 2020. However, RHD caused by GI.2/rabbit hemorrhagic disease virus (RHDV)2/b was unexpectedly reported in April 2020 in New Mexico and has subsequently spread to several U.S. states, infecting wild rabbits and hares and making it highly likely that RHD will become endemic in the U.S. Vaccines are available for RHD; however, there is no specific treatment for this disease. Lagoviruses encode a 3C-like protease (3CLpro), which is essential for virus replication and a promising target for antiviral drug development. We have previously generated focused small-molecule libraries of 3CLpro inhibitors and demonstrated the in vitro potency and in vivo efficacy of some protease inhibitors against viruses encoding 3CLpro, including caliciviruses and coronaviruses. Here, we report the development of the enzyme and cell-based assays for the 3CLpro of GI.1c/RHDV, recombinant GI.3P-GI.2 (RHDV2/b), and GII.1/European brown hare syndrome virus (EBHSV) as well as the identification of potent lagovirus 3CLpro inhibitors, including GC376, a protease inhibitor being developed for feline infectious peritonitis. In addition, structure-activity relationship study and homology modeling of the 3CLpro and inhibitors revealed that lagovirus 3CLpro share similar structural requirements for inhibition with other calicivirus 3CLpro. IMPORTANCE Rabbit hemorrhagic disease (RHD) and European brown hare syndrome (EBHS) are viral diseases that affect lagomorphs with significant economic and ecological impacts. RHD vaccines are available, but specific antiviral treatment for these viral infections would be a valuable addition to the current control measures. Lagoviruses encode 3C-like protease (3CLpro), which is essential for virus replication and an attractive target for antiviral drug discovery. We have screened and identified potent small-molecule inhibitors that block lagovirus 3CLpro in the enzyme- and cell-based assays. Our results suggest that these compounds have the potential for further development as antiviral drugs for lagoviruses.

Structure-Guided Design of Potent Spirocyclic Inhibitors of Severe Acute Respiratory Syndrome Coronavirus-2 3C-like Protease.

The worldwide impact of the ongoing COVID-19 pandemic on public health has made imperative the discovery and development of direct-acting antivirals aimed at targeting viral and/or host targets. SARS-CoV-2 3C-like protease (3CLpro) has emerged as a validated target for the discovery of SARS-CoV-2 therapeutics because of the pivotal role it plays in viral replication. We describe herein the structure-guided design of highly potent inhibitors of SARS-CoV-2 3CLpro that incorporate in their structure novel spirocyclic design elements aimed at optimizing potency by accessing new chemical space. Inhibitors of both SARS-CoV-2 3CLpro and MERS-CoV 3CLpro that exhibit nM potency and high safety indices have been identified. The mechanism of action of the inhibitors and the structural determinants associated with binding were established using high-resolution cocrystal structures.

Effects of Spike Mutations in SARS-CoV-2 Variants of Concern on Human or Animal ACE2-Mediated Virus Entry and Neutralization.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a zoonotic agent capable of infecting humans and a wide range of animal species. Over the duration of the pandemic, mutations in the SARS-CoV-2 spike (S) protein have arisen, culminating in the spread of several variants of concern (VOCs) with various degrees of altered virulence, transmissibility, and neutralizing antibody escape. In this study, we used pseudoviruses that express specific SARS-CoV-2 S protein substitutions and cell lines that express angiotensin-converting enzyme 2 (ACE2) from nine different animal species to gain insights into the effects of VOC mutations on viral entry and antibody neutralization capability. All animal ACE2 receptors tested, except mink, support viral cell entry for pseudoviruses expressing the ancestral prototype S at levels comparable to human ACE2. Most single S substitutions did not significantly change virus entry, although 614G and 484K resulted in a decreased efficiency. Conversely, combinatorial VOC substitutions in the S protein were associated with increased entry of pseudoviruses. Neutralizing titers in sera from various animal species were significantly reduced against pseudoviruses expressing the S proteins of Beta, Delta, or Omicron VOCs compared to the parental S protein. Especially, substitutions in the S protein of the Omicron variant significantly reduced the neutralizing titers of the sera. This study reveals important insights into the host range of SARS-CoV-2 and the effect of recently emergent S protein substitutions on viral entry, virus replication, and antibody-mediated viral neutralization. IMPORTANCE The ongoing coronavirus disease 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), continues to have devastating impacts on global health and socioeconomics. The recent emergence of SARS-CoV-2 variants of concern, which contain mutations that can affect the virulence, transmission, and effectiveness of licensed vaccines and therapeutic antibodies, are currently becoming the common strains circulating in humans worldwide. In addition, SARS-CoV-2 has been shown to infect a wide variety of animal species, which could result in additional mutations of the SARS-CoV-2 virus. In this study, we investigate the effect of mutations present in SARS-CoV-2 variants of concern and determine the effects of these mutations on cell entry, virulence, and antibody neutralization activity in humans and a variety of animals that might be susceptible to SARS-CoV-2 infection. This information is essential to understand the effects of important SARS-CoV-2 mutations and to inform public policy to create better strategies to control the COVID-19 pandemic.

Structure-Guided Design of Potent Inhibitors of SARS-CoV-2 3CL Protease: Structural, Biochemical, and Cell-Based Studies.

The COVID-19 pandemic is having a major impact on public health worldwide, and there is an urgent need for the creation of an armamentarium of effective therapeutics, including vaccines, biologics, and small-molecule therapeutics, to combat SARS-CoV-2 and emerging variants. Inspection of the virus life cycle reveals multiple viral- and host-based choke points that can be exploited to combat the virus. SARS-CoV-2 3C-like protease (3CLpro), an enzyme essential for viral replication, is an attractive target for therapeutic intervention, and the design of inhibitors of the protease may lead to the emergence of effective SARS-CoV-2-specific antivirals. We describe herein the results of our studies related to the application of X-ray crystallography, the Thorpe-Ingold effect, deuteration, and stereochemistry in the design of highly potent and nontoxic inhibitors of SARS-CoV-2 3CLpro.

Postinfection treatment with a protease inhibitor increases survival of mice with a fatal SARS-CoV-2 infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection continues to be a serious global public health threat. The 3C-like protease (3CLpro) is a virus protease encoded by SARS-CoV-2, which is essential for virus replication. We have previously reported a series of small-molecule 3CLpro inhibitors effective for inhibiting replication of human coronaviruses including SARS-CoV-2 in cell culture and in animal models. Here we generated a series of deuterated variants of a 3CLpro inhibitor, GC376, and evaluated the antiviral effect against SARS-CoV-2. The deuterated GC376 displayed potent inhibitory activity against SARS-CoV-2 in the enzyme- and the cell-based assays. The K18-hACE2 mice develop mild to lethal infection commensurate with SARS-CoV-2 challenge doses and were proposed as a model for efficacy testing of antiviral agents. We treated lethally infected mice with a deuterated derivative of GC376. Treatment of K18-hACE2 mice at 24 h postinfection with a derivative (compound 2) resulted in increased survival of mice compared to vehicle-treated mice. Lung virus titers were decreased, and histopathological changes were ameliorated in compound 2-treated mice compared to vehicle-treated mice. Structural investigation using high-resolution crystallography illuminated binding interactions of 3CLpro of SARS-CoV-2 and SARS-CoV with deuterated variants of GC376. Taken together, deuterated GC376 variants have excellent potential as antiviral agents against SARS-CoV-2.

Structure-Guided Design of Conformationally Constrained Cyclohexane Inhibitors of Severe Acute Respiratory Syndrome Coronavirus-2 3CL Protease.

A series of nondeuterated and deuterated dipeptidyl aldehyde and masked aldehyde inhibitors that incorporate in their structure a conformationally constrained cyclohexane moiety was synthesized and found to potently inhibit severe acute respiratory syndrome coronavirus-2 3CL protease in biochemical and cell-based assays. Several of the inhibitors were also found to be nanomolar inhibitors of Middle East respiratory syndrome coronavirus 3CL protease. The corresponding latent aldehyde bisulfite adducts were found to be equipotent to the precursor aldehydes. High-resolution cocrystal structures confirmed the mechanism of action and illuminated the structural determinants involved in binding. The spatial disposition of the compounds disclosed herein provides an effective means of accessing new chemical space and optimizing pharmacological activity. The cellular permeability of the identified inhibitors and lack of cytotoxicity warrant their advancement as potential therapeutics for COVID-19.

3C-like protease inhibitors block coronavirus replication in vitro and improve survival in MERS-CoV-infected mice.

Pathogenic coronaviruses are a major threat to global public health, as exemplified by severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and the newly emerged SARS-CoV-2, the causative agent of coronavirus disease 2019 (COVID-19). We describe herein the structure-guided optimization of a series of inhibitors of the coronavirus 3C-like protease (3CLpro), an enzyme essential for viral replication. The optimized compounds were effective against several human coronaviruses including MERS-CoV, SARS-CoV, and SARS-CoV-2 in an enzyme assay and in cell-based assays using Huh-7 and Vero E6 cell lines. Two selected compounds showed antiviral effects against SARS-CoV-2 in cultured primary human airway epithelial cells. In a mouse model of MERS-CoV infection, administration of a lead compound 1 day after virus infection increased survival from 0 to 100% and reduced lung viral titers and lung histopathology. These results suggest that this series of compounds has the potential to be developed further as antiviral drugs against human coronaviruses.