Although some of these agents have clinical usefulness e. Antiviral agents inhibit viral replication at the cellular level, interrupting one or more steps in the life cycle of the virus.

These agents have a limited spectrum of activity and, because most of them also interrupt host cell function, they are toxic to various degrees. The emergence of drug resistant viruses may occur during clinical use that further limits the effectiveness of various antivirals.

Immunomodulators such as interferons that alter the host immune responses to infection could, in principle, be protective, and several are under investigation. A number of antiviral agents with demonstrated effectiveness are now available TABLE These antiviral agents improve the clinical course of disease, but typically have important limitations especially as therapeutics for chronic or latent infections.

For example, the four nucleoside analog drugs now available for the therapy of HIV-1 do not prevent the ultimate worsening of disease. The concept of a targeted approach is now practical since information concerning the structure and replication of viruses and the spatial configuration and function of their proteins is available.

Such data may be useful in identifying specific target sites for antiviral agents. Since the mids, scientists have recognized that under certain circumstances one virus can interfere with another. In , Isaacs and Lindenman made a dramatic discovery that explained the mechanism of resistance.

They found that virus-infected cells can elaborate a protein substance called interferon, which, when added to normal cells in culture, protects them from viral infection.

Other microbial agents such as rickettsiae and bacteria and natural and synthetic polypeptides were later shown to induce interferon. There are three types of interferon: alpha, beta and gamma. Interferon alpha is produced by leukocytes, interferon beta is produced predominantly by fibroblasts and interferon gamma is produced by activated lymphocytes.

Interferons tend to exhibit species specificity mouse cell interferon protects mouse cells to a much greater extent than human cells and are inhibitory to numerous viruses. For many years it was not possible to obtain sufficient quantities of interferons to conduct major studies.

However, recombinant DNA technology and cell culture technology led to the production of adequate supplies of interferons and the subsequent conduct of extensive clinical trials. Although broadly antiviral in some animal models, interferon alpha has proven effective in a limited number of viral illnesses of humans, including chronic hepatitis B and C and refractory condylomata acuminata.

In addition, interferons have been effective in the treatment of other diseases. For instance interferon alpha is effective for hairy cell leukemia and AIDS-related Kaposi's sarcoma in a selected group of individuals; interferon beta for relapsing-remitting multiple sclerosis; and interferon gamma for reducing the frequency and severity of serious infections associated with chronic granulomatous disease.

The improved basic science knowledge base of viruses combined with the urgent need for improved therapeutics, especially for HIV-1, has given considerable impetus to the search for new approaches.

Some approaches under investigation that may lead to future approved therapies are described here:. The use of multiple drugs with different mechanisms of action is being studied as a method of improving clinical effectiveness. Such combinations may offer advantages over monodrug therapy such as improved antiviral activity, preventing or delaying the development of drug resistance, and use of lower, less toxic doses.

Combinations of various antiviral agents have been extensively studied for HIV. In addition, approaches investigated for HIV have included combining a cytokine with one or more antiviral agents.

Combination therapy has been effective in the treatment of diseases caused by other infectious agents e. New drugs with novel mechanisms of action are being sought and developed. Some of these have displayed considerable antiviral activity in human clinical trials, e.

Interleukin-2, a cytokine currently approved for treating renal cell carcinoma, has shown considerable immunomodulatory activity in some HIV-1 infected patients in early human studies. Turn recording back on.

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Show details Baron S, editor. Galveston TX : University of Texas Medical Branch at Galveston ; Search term. Chapter 51 Control of Viral Infections and Diseases Karen L.

General Concepts Immunoprophylaxis Immunoprophylaxis against viral illnesses includes the use of vaccines or antibody-containing preparations to provide immune protection against a specific disease.

Active Prophylaxis Vaccines Active immunization involves administering a virus preparation that stimulates the body's immune system to produce its own specific immunity. Passive Prophylaxis Passive immunity is conferred by administering antibodies formed in another host.

Sanitation and Vector Control Many viral diseases are controlled by reducing exposure to the virus by 1 eliminating nonhuman reservoirs, 2 eliminating the vector, and 3 improving sanitation. Antiviral Chemotherapy There are three types of antiviral agents: 1 virucidal agents, which directly inactivate viruses, 2 antiviral agents, which inhibit viral replication, and 3 immunomodulators, which boost the host immune response.

Interferons Virus-infected cells and cells induced with other agents, e. Cytokines Cytokines are molecules produced by cells which modify the biological responses of the same or other cells. Introduction Viral diseases range from trivial infections to plagues that alter the course of history.

Immunoprophylaxis Immunoprophylaxis against viral illnesses includes the use of vaccines or antibody-containing preparations to provide a susceptible individual with immunologic protection against a specific disease.

Active Prophylaxis Vaccines The viral vaccines currently approved for use in the United States are listed in TABLE These products are of three types: Table Viral Vaccines Approved for Use in the United States.

Immune Response to Vaccines Vaccination evokes an antibody response which is, in turn, a measure of the effectiveness of the vaccine in stimulating B lymphocytes. Vaccine Production Because viruses are obligate intracellular parasites, all viral vaccines contain substances derived from the cells or living tissues used in virus production.

Developing new vaccines The past success with developing highly effective viral vaccines has been considerable. Passive Prophylaxis The use of immunoglobulin preparations remains a mainstay of passive prophylaxis and occasionally of therapy for viral illnesses.

Table Approved Products Currently Used for Passive Immunization and Immunotherapy Against Viral Disease in the United States. Sanitation and Vector Control Several early approaches to virus control deserve recognition even though they are less dramatic than vaccination. Antiviral Chemotherapy Antiviral chemotherapeutic agents can be divided into three categories: virucidal agents, antiviral agents, and immunomodulators.

Table Approved Antiviral Agents in the U. Interferons: Cytokines With Antiviral Activity Since the mids, scientists have recognized that under certain circumstances one virus can interfere with another. Identifying New Effective Therapeutics The improved basic science knowledge base of viruses combined with the urgent need for improved therapeutics, especially for HIV-1, has given considerable impetus to the search for new approaches.

Some approaches under investigation that may lead to future approved therapies are described here: Combination Therapy The use of multiple drugs with different mechanisms of action is being studied as a method of improving clinical effectiveness.

Discovering New Drugs New drugs with novel mechanisms of action are being sought and developed. Evaluating Available Drugs for New Indications Interleukin-2, a cytokine currently approved for treating renal cell carcinoma, has shown considerable immunomodulatory activity in some HIV-1 infected patients in early human studies.

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Accessed July 7th , Download references. Department of Basic Sciences, Loma Linda University School of Medicine, Campus Street, Alumni Hall, Loma Linda, CA, , USA. You can also search for this author in PubMed Google Scholar.

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Download PDF. Review Open access Published: 23 July Keep out! Abstract The COVID pandemic has put healthcare infrastructures and our social and economic lives under unprecedented strain. Introduction COVID, the disease caused by the novel coronavirus SARS-CoV-2, was declared a pandemic and global emergency shortly after it began in late Full size image.

Viral entry inhibitors and their translational relevance The availability of several effective vaccines against SARS-CoV-2 has given hope to billions of people across the globe [ 21 , 22 ]. Table 1 Prominent examples of viral entry inhibitors that have demonstrated therapeutic or prophylactic efficacy in cross-neutralization, suppression of escape mutants and broad activity against circulating variants and sarbecoviruses Full size table.

Host proteases and endosome acidification inhibitors Although S1 and S2 mediate viral attachment and membrane fusion to enable the virus to unload its genetic cargo, function of these two subunits is enabled by the participation of at least 3 types of host proteases: furins, cathepsins and surface serine proteases.

Furin and TMPRSS2 inhibition Furin inhibitors have previously been reported as possible targets in the context of other viruses such as influenza, and may also be relevant for SARS-CoV Cathepsin inhibition A number of cathepsin inhibitors against coronaviruses have also been reported in various studies.

Conclusions and future perspectives COVID is now understood as a biphasic illness, with an early viral phase and a more dangerous host-immune response phase.

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In particular, the recent emergence of a novel, heavily mutated VOC, B. Discovering novel classes of antiviral compounds—including both direct-acting antivirals DAAs and host-directed antivirals HDAs —and intensive in cellulo and in vivo studies of their antiviral profiles as mono- or combination therapies against emerging SARS-CoV-2 VOCs are critical for developing preventive and therapeutic strategies to combat COVID 13 , At present, three antivirals have been approved for clinical use against SARS-CoV Remdesivir is a DAA that targets the viral RNA-dependent RNA polymerase that catalyses the synthesis of viral RNA Remdesivir is administered intravenously to hospitalized individuals with COVID Paxlovid targets the main protease of the coronavirus 3CL pro , also known as M pro , an essential protease that is involved in processing viral replicase polyproteins, whereas molnupiravir is a ribonucleoside analogue that inhibits viral replication 17 , Alternatively, HDAs also termed indirect-acting antivirals are under investigation and may offer a complement to DAAs.

Emerging SARS-CoV-2 VOCs are less likely to develop resistance to HDAs than to DAAs because, unlike viral genes, host genes have a low propensity to mutate 5 , Camostat mesylate Cm , for example, is a broad spectrum serine protease inhibitor used to treat pancreatitis that has been repositioned as a clinical candidate for treating COVID 4 , 5 , Accumulating evidence has shown that SARS-CoV-2 is dependent on host pathways, including the hijacking of TMPRSS2-related proteases for viral entry; this suggests that TTSPs could be therapeutic targets to prevent SARS-CoV-2 infection 5 , 6 , The SARS-CoV-2 lifecycle begins with attachment and entry into respiratory epithelium via the angiotensin-converting enzyme 2 ACE2 receptor 4 , 8.

This is mediated by the major viral surface glycoprotein, spike S , which must undergo two sequential proteolytic cleavages by host proteases before it can mediate fusion of the virus with host cell membranes, a requirement for subsequent viral replication 3 , 21 , This cleavage, which triggers the fusion event, is mediated by host TTSPs such as TMPRSS2 and TMPRSS13, which cleave after specific single arginine or lysine residues 4 , The KhACE2 mouse model transgenic expression of human ACE2 hACE2 under a cytokeratin 18 promoter offers a stringent system for testing the efficacy of DAAs and HDAs against severe disease and mortality after SARS-CoV-2 infection So far, only a few studies have tested antiviral efficacy in this animal model, with only one DAA reported as protective against lethal SARS-CoV-2 infection in this model 25 , Here we report on the design and testing of peptidomimetics for their inhibitory activity against TMPRSS2 and related TTSPs.

We then investigated the antiviral activities of the peptidomimetics against an ancestral strain of SARS-CoV-2 lineage B, VIDO and four variants—B. Finally, we tested our top highly potent antiviral, N, against SARS-CoV-2 lineage A strain and SARS-CoV-2 Delta-induced morbidity and mortality in KhACE2 mice.

We found that N provides a high level of protection and a therapeutic benefit after either multiple administrations or a single administration in this model of severe disease. Thus, N is an antiviral with a high potential for use against COVID We previously designed first-generation peptidomimetic tetrapeptide compounds with ketobenzothiazole warheads, and they exhibited inhibitory activity against a host TTSP, matriptase 27 , These compounds act as slow tight-binding inhibitors in vitro but their potency in cellular systems was modest against influenza A virus To improve their stability and potency, we modified their N terminus either by capping or through the synthesis of desamino moieties 29 Fig.

Moreover, these compounds exhibited low nanomolar efficacies when tested in H1N1 models of influenza A virus infection 28 , a , Peptidomimetic compounds used in this study along with their respective sequences. The structures of N-terminal caps, the ketobenzothiazole warhead and the alcohol ketobenzothiazole are shown on the right.

H Arg, desamino arginine; kbt, ketobenzothiazol. Relative TMPRSS2 activity was measured using the mock-subtracted fluorescence and is reported as the percentage of residual activity relative to the vehicle-treated cells 0. c , Dose—response curves were generated for the indicated compounds using the assay described in b , and IC 50 values were determined using nonlinear regression analysis.

d , Specificity of selected compounds toward other serine proteases. e , Main image, docking of N green; warhead in purple in the binding pocket of TMPRSS2 homology model. Residues of the catalytic triad are shown in cyan. Inset, interaction of N with TMPRSS2 residues.

N forms a covalent bond with the catalytic triad residue Ser Source data. Expanding on that work here, we developed a small library of peptidomimetic compounds Fig. We included in this screen our first-generation tetrapeptide 28 , N, which lacks an N-terminal stabilizing group, along with three desamino tetrapeptide analogues.

We also tested four tripeptides containing different N-terminal capping groups. To evaluate the efficacies of these compounds, we set up a cellular assay to measure TMPRSS2-dependent pericellular inhibition of proteolytic activity. We expressed the full-length, wild-type TMPRSS2 or an inactive form of the protease in which the serine residue of the catalytic triad was replaced by alanine TMPRSS2 SA in Vero E6 cells.

Using this assay, we show that, as expected, the SA substitution completely abrogated the proteolytic activity of TMPRSS2. The first-generation peptidomimetic, N, did not inhibit TMPRSS2 activity under these conditions.

Notably, several peptidomimetic compounds were more efficient than Cm at reducing the activity of TMPRSS2 Fig.

We then investigated the dose response of the four most promising peptidomimetics N, N, N and N The half-maximal inhibitory concentration IC 50 of Cm was To confirm the contribution of the ketobenzothiazole warhead to the inhibitory activity of the molecule, the ketone functional group of N was replaced with an alcohol group to generate N OH Fig.

We also confirmed the efficacy of N against mouse TMPRSS2 with an IC 50 of Next, we sought to determine the selectivity profile of these inhibitors by measuring the inhibition constant K i on selected recombinant serine proteases, including three members of the TTSP family matriptase, hepsin and DESC1 as well as furin, thrombin, and cathepsin L.

All four of the tested peptidomimetic compounds behaved as low nanomolar inhibitors for the TTSPs, but they were inactive or showed only weak inhibition against the other proteases Fig.

Overall, these data show that TTSP-targeting peptidomimetics containing a ketobenzothiazole warhead inhibit TMPRSS2-dependent pericellular activity in a cellular assay and preferentially inhibit other members of the TTSP family.

To understand the mode of binding and the main interactions of our inhibitors and how these compounds achieve their high inhibitory potential, we built a homology model of TMPRSS2 using the crystal structure of matriptase Protein Data Bank PDB : 6N4T Docking of N was modelled to this structure Fig.

As predicted and previously published 32 , the catalytic triad Ser catalytic triad: Ser, His, and Asp; Fig. Several key interactions can be observed in the binding pocket. As in all TTSP inhibitors possessing a guanidine group on the sidechain, a strong hydrogen bond network stabilizes this pharmacophore deep within the binding pocket Fig.

This includes Asp and Gly, as well as Gln via a water molecule. Gln is also involved in another hydrogen bond of this same water molecule to the oxygen of the main-chain ketone group.

This ketone also acts as a hydrogen bond acceptor with Gly The N-terminal mesylate forms two hydrogen bonds—one intramolecular with the side-chain amide of the Gln residue of N, and another with Gly Finally, the oxygen of the newly formed hemiacetal is stabilized by two hydrogen bond donors from the Gly and Ser amines.

A portion of the ketobenzothiazole warhead and the aromatic ring from the phenylalanine are exposed to the solvents, which could allow us to further optimize the design of this second-generation inhibitor, leading to an improved pharmacokinetic profile.

The peptidomimetic compounds that we screened against TMPRSS2 were subsequently tested for their efficacy at preventing SARS-CoV-2 infection. Cells were fixed and immunofluorescently stained for double-stranded RNA dsRNA , a marker of viral replication 33 , and for the viral nucleocapsid, a marker of viral entry and translation 6 Extended Data Fig.

Fluorescent high-content imaging and relative quantification of virally infected cells showed consistent inhibitory profiles across dsRNA and nucleocapsid staining, which mirrored the inhibitory profile observed in the TMPRSS2 proteolytic activity assay Fig. Thus, TMPRSS2-inhibiting peptidomimetics are also inhibitors of SARS-CoV-2 replication and translation in Calu-3 cells, and the stabilizing N-terminal caps and the ketobenzothiazole warhead are likely to be essential for compound stability and antiviral potency.

Intracellular infection was relatively quantified using N staining. Representative fluorescent images of colonoids subjected to the indicated treatments are shown Hoechst in blue, nucleocapsid in red and dsRNA in green.

Error bars, s. The half-maximal effective concentration EC 50 of Cm was Thus, the selectivity index for these compounds N, N, N and N was between 8.

Overall, these results confirm that two TTSP-targeted peptidomimetic compounds N and N are extremely potent low nanomolar inhibitors of SARS-CoV-2 infection in human lung epithelial cells. a , Representative fluorescent images of SARS-CoVinfected Calu-3 cells. Calu-3 cells infected with the indicated SARS-CoV-2 variants and mock infected are shown.

b , Representative images from a 3D volume rendering of Delta-infected cells. In a , b , Hoechst is shown in blue, nucleocapsid N in red, dsRNA in green and actin in cyan; images were captured with a Leica TCS SP8 3× STED microscope. We next examined the effects of Cm, N and N OH on the extracellular release of SARS-CoV-2 infectious virions from Calu-3 cells.

These results confirm that N, which targets TMPRSS2, is a potent inhibitor of SARS-CoV-2 infectivity in Calu-3 cells and that the ketobenzothiazole warhead is required for N antiviral potency. Although Calu-3 cells represent a scalable and clinically relevant system of antiviral screening for SARS-CoV-2 inhibitors, they are an immortalized cell line.

To evaluate the effectiveness of N in a primary human cell-based model, we examined SARS-CoV-2 infection in donor-derived human colonoids 7 , SARS-CoV-2 initially causes a respiratory infection, but many infected individuals also experience gastrointestinal symptoms that are frequently linked with increased disease duration and severity A recent report identified TMPRSS2 as an essential co-factor for SARS-CoV-2 infection in colonoids We first relatively quantified the mRNA expression of ACE2 and TMPRSS2 in colonoids and Calu-3 cells using quantitative PCR qPCR.

ACE2 showed comparable levels of expression in colonoids compared to Calu-3 cells, whereas TMPRSS2 had much higher expression levels in colonoids compared to Calu-3 cells Extended Data Fig. We then investigated the susceptibility of colonoid monolayers to SARS-CoV-2 infection.

Consistent with previous work, the colonoids were susceptible to infection, as evidenced by dsRNA and nucleocapsid staining Fig. N and N OH were then tested for their efficacy at preventing SARS-CoV-2 infection in colonoids. These results align with observations in Calu-3 cells and confirm the nanomolar potency of N against SARS-CoV-2 in primary human cells.

To our knowledge, mutations in the TMPRSS2 cleavage site have not been identified in SARS-CoV-2 variants, which suggests that N should retain high potency against SARS-CoV-2 VOCs First, we confirmed the infectivity of four VOCs in Calu-3 cells: B.

Confocal imaging of infected cells confirmed the infectivity of these variants, as demonstrated by nucleocapsid and dsRNA staining Fig. Although the viral marker staining patterns were relatively consistent in Calu-3 cells infected with a lineage B isolate VIDO , B.

We then evaluated the efficacy of N for preventing infection with SARS-CoV-2 VOCs in Calu-3 cells. The EC 50 of N against all VOCs was in the low nanomolar range, ranging from 2. This underscores the potential of N to act as a pan-variant, host-directed antiviral against emerging SARS-CoV-2 VOCs.

After establishing the efficacy of N in vitro and in cellulo, we tested whether intranasal administration would improve morbidity and survival in vivo, using KhACE2 mice 36 , 37 , an established mouse model of severe COVID Dosing regimens and drug concentrations were chosen on the basis of preliminary studies performed in a mouse model of influenza A virus infection, which showed antiviral efficacy at 7.

Ten mice per group five females and five males were administered a single daily intranasal dose of 7. Surviving mice were euthanized at the study end-point. b — d , Weight change of mice treated with saline control b , N OH c or N d. f , Probability of survival.

Uninfected mice tissues i, ii, ix, x were normal. Challenged mice iii—viii, xi—xvi developed perivascular infiltrates of inflammatory cells arrowheads. Severe inflammatory changes including alveolar fibrin and oedema asterisks were found only in the saline group iii, iv, xi, xii. Perivascular inflammatory cell infiltrates arrowheads were more widespread in saline iii, xi and control N OH v, xiii compared to N mice vii, xv.

Surviving N mice vii, viii, xv, xvi had smaller and fewer perivascular inflammatory infiltrates arrowheads and occasional type II pneumocyte hyperplasia red arrows. Saline-treated mice i, ii, ix, x developed perivascular cuffs of inflammatory cells asterisks , necrotic neurons arrows , gliosis and meningeal infiltrates arrowheads.

Brain lesions were reduced in N OH mice v, vi, xiii, xiv and absent in surviving N mice vii, viii, xv, xvi. The magnified areas were selected to best represent the presence of inflammatory cells and pathological changes.

Compared to Ntreated mice, control saline-treated mice frequently had additional histological changes including alveolar oedema, alveolar fibrin and inflammatory cells within alveoli. Of the mice that survived up to the study end-point, three had focal areas of fibrosis, type II pneumocyte hyperplasia and occasionally lymphoid hyperplasia.

However, most of the mice that survived showed little to no pathological signs in the lungs Fig. Histological lesions in the brain included multifocal perivascular cuffs of inflammatory cells, reactive glial cells, neutrophils and lymphocytes in the adjacent neuroparenchyma gliosis , infiltration of the meninges with inflammatory cells, and neuronal necrosis characterized by shrunken neuron bodies with hypereosinophilic cytoplasm and pyknotic or karyorrhectic nuclei.

No lesions were observed in the brains of mice that survived to the study end-point Fig. Although samples obtained at different time points are not directly comparable, the amounts of antigen and viral titres were lower in mice treated with N, particularly in those that survived to the study end-point Extended Data Fig.

No infectious virus was detected in the lung of Ntreated mice at the study end-point, or in the two saline-treated mice that survived to the end-point. This demonstrates the effectiveness of N in blocking SARS-CoV-2 infection and improving disease outcomes and survival using a short, early treatment regimen.

b , Probability of survival. c , Weight change of saline control mice. d , Weight change of Ntreated mice.

Two-tailed unpaired t -test was used to determine significance. Plaque assays were performed twice per sample from each mouse and the average was used to determine the PFU per g. Two-tailed Mann—Whitney test was used to determine significance.

Statistical analysis was not performed as samples are from different time points. logarithmic scale precludes negative values being shown.

Next, we further investigated the treatment window of N as well as the pan-variant effectiveness against SARS-CoV-2 B. Weight was monitored for six days after infection.

N showed significant protection against infection-associated weight loss Fig. Similarly, total pathology scores of lung tissue assessed using IHC sections were improved by approximately 1. Together, the in vivo data strongly suggest that N considerably prevents morbidity and mortality and reduces viral burden in the KhACE2 mouse model of severe SARS-CoV-2 infection, when used as a prophylactic or therapeutic treatment.

Two-tailed Mann Whitney test was used to determine significance. In this study, we report on N—a potent small-molecule protease inhibitor of human TMPRSS2 and a SARS-CoV-2 pan-variant HDA that is effective in vivo against the B.

N acts as an inhibitor of the TTSP-dependent proteolytic activation of virus spike protein, a critical step in permitting viral—cell membrane fusion and entry into target cells 4. The nanomolar potency of N against SARS-CoV-2 infection in human Calu-3 cells and patient-derived colonoids without detectable toxicity yields a selectivity index of greater than 10 6.

These data suggest that N may provide an effective early treatment option against emerging SARS-CoV-2 VOCs. We have previously shown how peptidomimetic-based compounds with ketobenzothiazole warheads exhibit potent antiviral efficacy in impeding the infection of Calu-3 cells with influenza A H1N1 virus, through inhibition of TTSPs The activation of the influenza A virus surface glycoprotein hemagglutinin is notably similar to that of the SARS-CoV-2 spike protein, in that both are viral surface protein homotrimers cleaved by proteolytic enzymes of the TTSP family that are expressed by host epithelial cells 14 , TTSPs are attractive broad-spectrum, HDA drug targets because of i their central role in mediating viral entry 5 ; ii their accessibility on the surface of nasal and pulmonary epithelial cells 41 , 42 ; and iii their demonstrated therapeutic potential for combating viruses such as SARS-CoV-2 and other human coronaviruses, as well as influenza viruses 14 , 40 , When we screened selected TMPRSS2 inhibitors for antiviral activity against SARS-CoV-2, a similar inhibitory profile was observed against TMPRSS2 expressed in Vero E6 cells compared to SARS-CoV-2 infection in Calu-3 cells.

N, the lead antiviral candidate, showed potent inhibition of SARS-CoV-2 infection in Calu-3 cells, with an EC 50 of 2. The potency of N was validated using two viral biomarkers of intracellular infection as well as by measuring the release of infectious viral particles. This complements a recent report that showed that peptidomimetic compounds targeting TMPRSS2 have high potency against SARS-CoVinduced cytopathic effects, as well as excellent stability and safety in mice The usefulness of N needs to be considered in the context of circulating SARS-CoV-2 variants.

VOCs such as B. In , B. We hypothesized that the efficacy of N against four SARS-CoV-2 VOCs B. Our results confirmed the low nanomolar pan-variant antiviral activity of N against these four SARS-CoV-2 VOCs in human cells.

Previous work has shown that the KhACE2 mouse model used in our studies is an ideal model for recapitulating the pathology of severe COVID in humans as well as its high morbidity and mortality. SARS-CoV-2 challenge in this model leads to high viral titres in lung and brain tissues with commensurate high morbidity and mortality, weight loss and cytokine and chemokine production 36 , Therefore, this model is ideal for testing SARS-CoV-2 therapeutic agents, owing to its severe disease burden as compared to other animal models including mouse-attenuated SARS-CoV-2 in wild-type mice or wild-type SARS-CoV-2 in golden Syrian hamsters, which exhibit milder symptoms.

Protection in an animal model with high levels of hACE2, such as the KhACE2 mouse model, is thus indicative of the high promise of anti-SARS-CoV-2 antivirals The mouse TMPRSS2 protein contains amino acids and shares Intranasal administration has several advantages for the prevention and treatment of SARS-CoV-2 and other viral diseases, including ease of self-administration.

SARS-CoV-2 mainly enters the human body through ACE2- and TMPRSS2-positive nasal epithelial cells 47 , 48 , Intranasal drug delivery maximizes airway and lung exposure while limiting systemic exposure.

For example, intranasal administration of a membrane fusion inhibitory lipopeptide prevented the transmission of SARS-CoV-2 in ferrets 50 ; however, the efficacy of intranasal delivery of a small molecule inhibitor has not to our knowledge been shown.

Under our conditions, intranasal administration of N markedly reduced morbidity and mortality in the KhACE2 mouse model of severe COVID pathology. This is indicative of the effective reduction of virus propagation by N in this animal model.

Although further studies are needed to understand the ideal time points for N administration, we have shown that N may also contribute therapeutic efficacy against SARS-CoV-2 VOCs. Antiviral candidates for SARS-CoV-2 infection are under investigation in clinical trials and in animal models, but at present, only one study on the DAA GC has reported protection against lethal SARS-CoV-2 infection in the KhACE2 model Plitidepsin, a naturally occurring HDA, protected against lung pathology in the KhACE2 model; however, the effect on mortality was not reported Cm and nafamostat mesylate are also HDAs that target serine proteases including host TTSPs and these are also undergoing human trials against SARS-CoV-2; however, no significant protection against infection was observed in the adenovirus hACE2 model Cm of SARS-CoV-2 infection 4 , Clinical trial data for the treatment of hospitalized patients with COVID with Cm showed that Cm had no effect on the time to recovery and incidence of death after SARS-CoV-2 infection Antivirals will probably need to be administered during the very early phase of COVID to be effective in lowering the risk of disease progression, consistent with our short early treatment regimen in KhACE2 mice infected with SARS-CoV Overall, we have developed and characterized N, a highly potent inhibitor of TMPRSS2-like proteases that blocks SARS-CoV-2 VOCs B.

In addition, we have shown that N provides an effective early treatment option against SARS-CoV-2 and the B. Moreover, N analogues may have broader applications in combating other widespread respiratory viruses that usurp TMPRSS2-related proteases for viral entry, including other established coronaviruses, influenza viruses and additional viruses that depend on TTSPs for entering host cells 4 , 30 , We envision a practical use of N for unvaccinated individuals or those with high risk of exposure or severe disease outcome related to SARS-CoV-2 VOCs and future emerging pathogens.

Practically, TTSP inhibitors should be administered as soon as possible after exposure to SARS-CoV-2 for maximal effect and may possibly act synergistically when used in multi-drug combinations with replication inhibitors such as remdesivir, paxlovid and molnupiravir to reduce the risk of antiviral resistance mutations.

Calu-3 cells 52 ATCC HTB were cultured according to ATCC recommendations. All experiments were performed in these cells below passage Cell density was kept between 0. Cm was obtained from MilliporeSigma. The SARS-CoV-2 nucleocapsid antibody HL GTX was provided by Genetex; mouse anti-dsRNA antibody J was purchased from Scions English and Scientific Consulting 33 ; Hoechst and secondary antibodies goat anti-mouse IgG Alexa Fluor A and goat anti-rabbit IgG Alexa Fluor A were obtained from Invitrogen.

Cell lines were screened for mycoplasma contamination using MycoAlert Mycoplasma Detection Kit Lonza. Preparation of the compounds using a mixed approach of solution and solid-phase synthesis is described in the Supplementary Information, in addition to a synthetic scheme of analogues, nuclear magnetic resonance NMR , high-resolution mass spectrometry HRMS , ultraperformance liquid chromatography—mass spectrometry UPLC—MS retention time, structure, purity, and molecular formula strings of compounds.

Amino acids and coupling reagents were obtained from Chem-Impex International and used as received. All other reagents and solvents were purchased from Sigma-Aldrich or Thermo Fisher Scientific. Tetrahydrofuran THF was dried over sodium benzophenone ketyl; dichloromethane over P 2 O 5 ; methanol over magnesium.

Celite AW Standard Super-Cel NF was obtained from Sigma-Aldrich. Chlorotrityl chloride CTC resin was obtained from Matrix Innovation and generally used with a loading of 1. Purity was analysed on a Waters UPLC H-Class with UV detection PDA equipped with an Acquity UPLC CSH C18 1. The final model was refined and minimized using the AmberExtended Huckel Theory EHT force field.

After drawing the structure, all protein—ligand complexes were prepared using the Protonate 3D tool; then the partial charges were calculated, and the ligands were energy-minimized. Molecules were docked in the protein-binding site with the software MOE All atoms were fixed, and the ligands were allowed to be flexible.

The carbon of the ketone making the reversible covalent bond with the protein was fixed at 3. The guanidine of the arginine in P1 was also fixed through two key interactions in the binding site. Conformational search using LowModeMD was made with AMBEREHT as a molecular mechanics force field with default parameters rejection limit: ; RMS gradient: 0.

Finally, a second round of energy minimization was performed around the ligand-binding site. The low energy conformations of the inhibitor-protein complexes were analysed for their binding interactions.

Vero E6 cells were transfected with mock pcDNA3. For the mouse TMPRSS2 assay, empty vector pCMV6-Entry, Origene PS and TMPRSS2-Myc-DDK Origene MR were used. IC 50 values were determined after generating a nonlinear regression analysis from a log [Compound] versus a proteolytic activity plot using GraphPad Prism software v.

of at least three independent experiments. All infections were carried out in a Biosafety Level 3 BSL3 facility UBC FINDER in accordance with the Public Health Agency of Canada and UBC FINDER regulations UBC BSL3 Permit B to F.

SARS-CoV-2 VOCs B. SARS-CoV-2 VOCs were first isolated in Vero-TMPRSS2 cells passage 1 and then passaged in Vero E6 cells passage 2. Viral stocks used in the experiments passage 3 were propagated in Vero E6 cells For experiments, passage three of the virus was used with a determined viral titre of 1.

Calu-3 cells were seeded at a concentration of 10, cells per well in well plates the day before infection. SARS-CoV-2 stocks were diluted in cell-specific medium to a multiplicity of infection MOI of 2. The fixative was removed, and cells were washed with PBS, permeabilized with 0.

Three-dimensional 3D volume rendering was done with LAS-X. Two-dimensional images were exported into tiff format.

Monitoring of the total number of cells based on nuclei staining and the number of virus-infected cells based on dsRNA and nucleocapsid staining was performed using the CellInsight CX7 HCS platform Thermo Fisher Scientific , as previously described 55 , Finally, the software HCS Studio Cell Analysis Software, v.

The fluorescence measured within each cell circle is then added and quantified for each well. Nine fields were sampled from each well. Nuclei stain Hoechst was also used to quantify cell loss owing to cytotoxicity or loss of adherence and to verify that the changes in viral infection did not result from a decrease in cell numbers.

Viral infection was detected by staining for dsRNA or nucleocapsid signal and quantified as described above. EC 50 experiments were repeated at least three times for each compound with three technical replicates in each experiment.

Intracellular nucleocapsid levels were interpolated to negative control 0. Calu-3 and Vero E6 cells or 10, cells for samples, 80—20, cells for standard curve were seeded in well plates. Cellular viability was assessed using Cell Titer-Glo 2. The number of viable cells was extrapolated using the standard curve.

Recombinant human matriptase, hepsin and DESC1 were expressed and purified as described previously 57 , Recombinant human furin, human cathepsin L Bio-Techne , and human thrombin MilliporeSigma were obtained from commercial sources. K i values were determined using steady-state velocities as previously reported 27 , To measure proteolytic activity, protease 0.

The preferred model was used for K i determination. Intestinal biopsy-derived colonoids from healthy donors were obtained from the Johns Hopkins Conte Digestive Disease Basic and Translational Research Core Center NIH NIDDK PDK and grown as described previously In brief, human colonoid monolayers were generated by combining the colonoids from one Matrigel dome around or more colonoids in a µl dome.

Cells were fed every two days and were used for experiments after they were fully confluent four to five days.

RNA quality and the presence of contaminating genomic DNA were verified as described previously RNA integrity was assessed with an Agilent Bioanalyzer Agilent Technologies. Reverse transcription was performed on 1. RNA was isolated and reverse-transcribed using Quanta Biosciences qScript cDNA SuperMix.

Relative expression levels were calculated according to the qBASE framework 60 with YWHAZ, PUM1 and MRPL19 as housekeeping genes for normalization. Primer design and validation were evaluated as described elsewhere In every qPCR run, a no-template control was performed for each primer pair; these were consistently negative.

All qPCR assays were performed by the RNomics Platform of the Université de Sherbrooke. Animal studies were performed in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All protocols were performed under approved BSL-3 conditions and approved by the Institutional Animal Care and Use Committee at Cornell University IACUC mouse protocol and BSL3 IBC MUA Intranasal virus and antiviral treatments were performed under anaesthesia, and all efforts were made to minimize animal suffering.

Since inhibitors such as Ruxolitinib can be administered in vivo , they may also prove useful in studies designed to investigate the importance of the IFN response in controlling virus infections in animal models. We thank Dan Young for expert technical assistance and Zoe Gage for proof reading the manuscript.

Conceived and designed the experiments: CSA RER CES. Performed the experiments: CSA CSE. Analyzed the data: CSA CES RER. Wrote the paper: CSA RER. Browse Subject Areas? Click through the PLOS taxonomy to find articles in your field.

Article Authors Metrics Comments Media Coverage Reader Comments Figures. Abstract Virus replication efficiency is influenced by two conflicting factors, kinetics of the cellular interferon IFN response and induction of an antiviral state versus speed of virus replication and virus-induced inhibition of the IFN response.

Materials and Methods Inhibitors, viruses and cells Inhibitors of the IFN response BX, MRT, MRT, TPCA-1, Cyt, AZD, Ruxolitinib, Tofacitinib were prepared as 10 mM stocks in dimethyl sulfoxide DMSO and used at the indicated concentrations. Virus plaque assays and growth kinetics Standard plaque assays were conducted in the appropriate cells using a 0.

Results and Discussion Eight small molecules that have previously been described to inhibit the cellular IFN response were obtained; four inhibitors that target components of the IFN induction pathway: TBK1 inhibitors BX, MRT, MRT [23] , [24] and the IKK-2 inhibitor TPCA-1 [25] , plus four inhibitors that target JAK1 a component of the IFN signaling pathway: Cyt, AZD, Ruxolitinib and Tofacitinib [26] — [29].

Download: PPT. Figure 1. Verification of IFN inhibitors ability to block IFN induction or IFN signaling. Figure 2. Effect of a panel of IFN inhibitors on BUNΔNSs virus growth in A cells.

Figure 3. Effect of a combination of different IFN inhibitors on BUNΔNSs growth in A and Vero cells. Figure 4.

Effect of Ruxolitinib RUX on BUNΔNSs and BUN-WT wildtype plaque formation in cell-lines derived from different mammalian species. Figure 5. Effect of Ruxolitinib RUX on plaque formation of a selection of viruses.

Figure 6. Inhibitory activity of Ruxolitinib and TPCA-1 is stable over time in cell culture. Acknowledgments We thank Dan Young for expert technical assistance and Zoe Gage for proof reading the manuscript.

Author Contributions Conceived and designed the experiments: CSA RER CES. References 1. Randall RE, Goodbourn S Interferons and viruses: an interplay between induction, signalling, antiviral responses and virus countermeasures.

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