Targeted isolation of diverse human protective broadly neutralizing antibodies against SARS-like viruses.

The emergence of current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) and potential future spillovers of SARS-like coronaviruses into humans pose a major threat to human health and the global economy. Development of broadly effective coronavirus vaccines that can mitigate these threats is needed. Here, we utilized a targeted donor selection strategy to isolate a large panel of human broadly neutralizing antibodies (bnAbs) to sarbecoviruses. Many of these bnAbs are remarkably effective in neutralizing a diversity of sarbecoviruses and against most SARS-CoV-2 VOCs, including the Omicron variant. Neutralization breadth is achieved by bnAb binding to epitopes on a relatively conserved face of the receptor-binding domain (RBD). Consistent with targeting of conserved sites, select RBD bnAbs exhibited protective efficacy against diverse SARS-like coronaviruses in a prophylaxis challenge model in vivo. These bnAbs provide new opportunities and choices for next-generation antibody prophylactic and therapeutic applications and provide a molecular basis for effective design of pan-sarbecovirus vaccines.

Broadly neutralizing anti-S2 antibodies protect against all three human betacoronaviruses that cause deadly disease.

Pan-betacoronavirus neutralizing antibodies may hold the key to developing broadly protective vaccines against novel pandemic coronaviruses and to more effectively respond to SARS-CoV-2 variants. The emergence of Omicron and subvariants of SARS-CoV-2 illustrates the limitations of solely targeting the receptor-binding domain (RBD) of the spike (S) protein. Here, we isolated a large panel of broadly neutralizing antibodies (bnAbs) from SARS-CoV-2 recovered-vaccinated donors, which targets a conserved S2 region in the betacoronavirus spike fusion machinery. Select bnAbs showed broad in vivo protection against all three deadly betacoronaviruses, SARS-CoV-1, SARS-CoV-2, and MERS-CoV, which have spilled over into humans in the past two decades. Structural studies of these bnAbs delineated the molecular basis for their broad reactivity and revealed common antibody features targetable by broad vaccination strategies. These bnAbs provide new insights and opportunities for antibody-based interventions and for developing pan-betacoronavirus vaccines.

Validating Enterovirus D68-2Apro as an Antiviral Drug Target and the Discovery of Telaprevir as a Potent D68-2Apro Inhibitor.

Enterovirus D68 (EV-D68) is a viral pathogen that leads to severe respiratory illness and has been linked with the development of acute flaccid myelitis (AFM) in children. No vaccines or antivirals are currently available for EV-D68 infection, and treatment options for hospitalized patients are limited to supportive care. Here, we report the expression of the EV-D68 2A protease (2Apro) and characterization of its enzymatic activity. Furthermore, we discovered that telaprevir, an FDA-approved drug used for the treatment of hepatitis C virus (HCV) infections, is a potent antiviral against EV-D68 by targeting the 2Apro enzyme. Using a fluorescence resonance energy transfer-based substrate cleavage assay, we showed that the purified EV-D68 2Apro has proteolytic activity selective against a peptide sequence corresponding to the viral VP1-2A polyprotein junction. Telaprevir inhibits EV-D68 2Apro through a nearly irreversible, biphasic binding mechanism. In cell culture, telaprevir showed submicromolar-to-low-micromolar potency against several recently circulating neurotropic strains of EV-D68 in different human cell lines. To further confirm the antiviral drug target, serial viral passage experiments were performed to select for resistance against telaprevir. An N84T mutation near the active site of 2Apro was identified in resistant viruses, and this mutation reduced the potency of telaprevir in both the enzymatic and cellular antiviral assays. Collectively, we report for the first time the in vitro enzymatic activity of EV-D68 2Apro and the identification of telaprevir as a potent EV-D68 2Apro inhibitor. These findings implicate EV-D68 2Apro as an antiviral drug target and highlight the repurposing potential of telaprevir to treat EV-D68 infection.IMPORTANCE A 2014 EV-D68 outbreak in the United States has been linked to the development of acute flaccid myelitis in children. Unfortunately, no treatment options against EV-D68 are currently available, and the development of effective therapeutics is urgently needed. Here, we characterize and validate a new EV-D68 drug target, the 2Apro, and identify telaprevir-an FDA-approved drug used to treat hepatitis C virus (HCV) infections-as a potent antiviral with a novel mechanism of action toward 2Apro 2Apro functions as a viral protease that cleaves a peptide sequence corresponding to the VP1-2A polyprotein junction. The binding of telaprevir potently inhibits its enzymatic activity, and using drug resistance selection, we show that the potent antiviral activity of telaprevir was due to 2Apro inhibition. This is the first inhibitor to selectively target the 2Apro from EV-D68 and can be used as a starting point for the development of therapeutics with selective activity against EV-D68.

Development of broad-spectrum enterovirus antivirals based on quinoline scaffold.

Non-polio enteroviruses such as enterovirus A71 (EV-A71), EV-D68, and coxsackievirus B3 (CVB3) are significant human pathogens with disease manifestations ranging from mild flu-like symptoms to more severe encephalitis, myocarditis, acute flaccid paralysis/myelitis, and even death. There is currently no effective antivirals to prevent or treat non-polio enterovirus infection. In this study, we report our progress in developing potent and broad-spectrum antivirals against these non-polio enteroviruses. Starting from our previously developed lead compounds that had potent antiviral activity against EV-D68, we synthesized 43 analogs and profiled their broad-spectrum antiviral activity against additional EV-D68, EV-A71, and CVB3 viruses. Promising candidates were also selected for mouse microsomal stability test to prioritize lead compounds for future in vivo mouse model studies. Collectively, this multi-parameter optimization process revealed a promising lead compound 6aw that showed single-digit to submicromolar EC50 values against two EV-D68 strains (US/KY and US/MO), two EV-A71 strains (Tainan and US/AK), and one CVB3 strain, with a high selectivity index. Encouragingly, 6aw was stable in mouse microsomes with a half-life of 114.7 min. Overall, 6aw represents one of the most potent broad-spectrum antiviral against non-polio enteroviruses, rendering it a promising lead candidate for non-polio enteroviruses with translational potential.

The L46P mutant confers a novel allosteric mechanism of resistance toward the influenza A virus M2 S31N proton channel blockers.

The Food and Drug Administration-approved influenza A antiviral amantadine inhibits the wild-type (WT) AM2 channel but not the S31N mutant predominantly found in circulating strains. In this study, serial viral passages were applied to select resistance against a newly developed isoxazole-conjugated adamantane inhibitor that targets the AM2 S31N channel. This led to the identification of the novel drug-resistant mutation L46P located outside the drug-binding site, which suggests an allosteric resistance mechanism. Intriguingly, when the L46P mutant was introduced to AM2 WT, the channel remained sensitive toward amantadine inhibition. To elucidate the molecular mechanism, molecular dynamics simulations and binding free energy molecular mechanics-generalized born surface area (MM-GBSA) calculations were performed on WT and mutant channels. It was found that the L46P mutation caused a conformational change in the N terminus of transmembrane residues 22-31 that ultimately broadened the drug-binding site of AM2 S31N inhibitor 4, which spans residues 26-34, but not of AM2 WT inhibitor amantadine, which spans residues 31-34. The MM-GBSA calculations showed stronger binding stability for 4 in complex with AM2 S31N compared with 4 in complex with AM2 S31N/L46P, and equal binding free energies of amantadine in complex with AM2 WT and AM2 L46P. Overall, these results demonstrate a unique allosteric resistance mechanism toward AM2 S31N channel blockers, and the L46P mutant represents the first experimentally confirmed drug-resistant AM2 mutant that is located outside of the pore where drug binds. SIGNIFICANCE STATEMENT: AM2 S31N is a high-profile antiviral drug target, as more than 95% of currently circulating influenza A viruses carry this mutation. Understanding the mechanism of drug resistance is critical in designing the next generation of AM2 S31N channel blockers. Using a previously developed AM2 S31N channel blocker as a chemical probe, this study was the first to identify a novel resistant mutant, L46P. The L46P mutant is located outside of the drug-binding site. Molecular dynamics simulations showed that L46P causes a dilation of drug-binding site between residues 22 and 31, which affects the binding of AM2 S31N channel blockers, but not the AM2 WT inhibitor amantadine.

Chemical Genomics Approach Leads to the Identification of Hesperadin, an Aurora B Kinase Inhibitor, as a Broad-Spectrum Influenza Antiviral.

Influenza viruses are respiratory pathogens that are responsible for annual influenza epidemics and sporadic influenza pandemics. Oseltamivir (Tamiflu®) is currently the only FDA-approved oral drug that is available for the prevention and treatment of influenza virus infection. However, its narrow therapeutic window, coupled with the increasing incidence of drug resistance, calls for the next generation of influenza antivirals. In this study, we discovered hesperadin, an aurora B kinase inhibitor, as a broad-spectrum influenza antiviral through forward chemical genomics screening. Hesperadin inhibits multiple human clinical isolates of influenza A and B viruses with single to submicromolar efficacy, including oseltamivir-resistant strains. Mechanistic studies revealed that hesperadin inhibits the early stage of viral replication by delaying the nuclear entry of viral ribonucleoprotein complex, thereby inhibiting viral RNA transcription and translation as well as viral protein synthesis. Moreover, a combination of hesperadin with oseltamivir shows synergistic antiviral activity, therefore hesperadin can be used either alone to treat infections by oseltamivir-resistant influenza viruses or used in combination with oseltamivir to delay resistance evolution among oseltamivir-sensitive strains. In summary, the discovery of hesperadin as a broad-spectrum influenza antiviral offers an alternative to combat future influenza epidemics and pandemics.

Effects of transportation stress and addition of salt to transport water on the skin mucosal homeostasis of rainbow trout (Oncorhynchus mykiss).

Transportation of live fish is a common practice among aquaculture facilities. Many studies have previously reported how transport elicits physiological stress responses and increases disease susceptibility in farmed fish. The aim of this work is to investigate the changes that the skin of rainbow trout (Oncorhynchus mykiss) experiences due to stress. Since NaCl is commonly added to transport water as a stress mitigator, the effects of salt addition on the skin mucosa and skin-associated bacteria were also examined. Three experimental groups (Control, post-transport no salt (PTNS) and post-transport with salt (PTS)) were analyzed in a 5-hour transport acute stress model. Results indicate that the skin mucosa and the skin-associated bacteria are affected by transport stress. Total numbers of culturable skin-associated bacteria increased by ~10-fold and ~50-fold in the PTS and PTNS groups, respectively. Compared to controls, MUC2 expression was increased by 5-fold and 2-fold in the PTNS and PTS groups, respectively. Claudin-7, 8d and 12 expression levels were higher in both PTNS and PTS groups whereas antimicrobial peptide gene expression was lower than controls. Expression of the anti-inflammatory cytokine TGF-ß but not IL-1ß, IL-6 and TNF-α was up-regulated 2-3 fold in both the PTS and PTNS groups. The addition of salt diminished some of the physiological responses measured including the numbers of skin-associated bacteria. The responses recorded here appeared to be efficient at controlling bacterial translocation since stress did not lead to significant presence of bacteria in the liver or spleen of rainbow trout. When examining the ability of skin mucus to inhibit or promote growth of the bacterial pathogen Vibrio anguillarum, the skin mucus of PTS trout was more efficient at inhibiting V. anguillarum growth (20% inhibition) compared to control or PTNS mucus (11-12% inhibition). Our data clearly indicate the skin and skin microbiota of rainbow trout undergo important physiological responses during stress. The reduction in the magnitude of the skin responses recorded when salt was added to the transport water explains a new mechanism by which salt is an effective stress mitigator in some fish species. Aquaculture specialists will benefit from the present study by taking into consideration the importance of skin health during live transport.

Deep repertoire mining uncovers ultra-broad coronavirus neutralizing antibodies targeting multiple spike epitopes.

The development of vaccines and therapeutics that are broadly effective against known and emergent coronaviruses is an urgent priority. We screened the circulating B cell repertoires of COVID-19 survivors and vaccinees to isolate over 9,000 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific monoclonal antibodies (mAbs), providing an expansive view of the SARS-CoV-2-specific Ab repertoire. Among the recovered antibodies was TXG-0078, an N-terminal domain (NTD)-specific neutralizing mAb that recognizes diverse alpha- and beta-coronaviruses. TXG-0078 achieves its exceptional binding breadth while utilizing the same VH1-24 variable gene signature and heavy-chain-dominant binding pattern seen in other NTD-supersite-specific neutralizing Abs with much narrower specificity. We also report CC24.2, a pan-sarbecovirus neutralizing antibody that targets a unique receptor-binding domain (RBD) epitope and shows similar neutralization potency against all tested SARS-CoV-2 variants, including BQ.1.1 and XBB.1.5. A cocktail of TXG-0078 and CC24.2 shows protection in vivo, suggesting their potential use in variant-resistant therapeutic Ab cocktails and as templates for pan-coronavirus vaccine design.

Deep repertoire mining uncovers ultra-broad coronavirus neutralizing antibodies targeting multiple spike epitopes.

Development of vaccines and therapeutics that are broadly effective against known and emergent coronaviruses is an urgent priority. Current strategies for developing pan-coronavirus countermeasures have largely focused on the receptor binding domain (RBD) and S2 regions of the coronavirus Spike protein; it has been unclear whether the N-terminal domain (NTD) is a viable target for universal vaccines and broadly neutralizing antibodies (Abs). Additionally, many RBD-targeting Abs have proven susceptible to viral escape. We screened the circulating B cell repertoires of COVID-19 survivors and vaccinees using multiplexed panels of uniquely barcoded antigens in a high-throughput single cell workflow to isolate over 9,000 SARS-CoV-2-specific monoclonal Abs (mAbs), providing an expansive view of the SARS-CoV-2-specific Ab repertoire. We observed many instances of clonal coalescence between individuals, suggesting that Ab responses frequently converge independently on similar genetic solutions. Among the recovered antibodies was TXG-0078, a public neutralizing mAb that binds the NTD supersite region of the coronavirus Spike protein and recognizes a diverse collection of alpha- and beta-coronaviruses. TXG-0078 achieves its exceptional binding breadth while utilizing the same VH1-24 variable gene signature and heavy chain-dominant binding pattern seen in other NTD supersite-specific neutralizing Abs with much narrower specificity. We also report the discovery of CC24.2, a pan-sarbecovirus neutralizing mAb that targets a novel RBD epitope and shows similar neutralization potency against all tested SARS-CoV-2 variants, including BQ.1.1 and XBB.1.5. A cocktail of TXG-0078 and CC24.2 provides protection against in vivo challenge with SARS-CoV-2, suggesting potential future use in variant-resistant therapeutic Ab cocktails and as templates for pan-coronavirus vaccine design.

Broadly neutralizing antibodies targeting a conserved silent face of spike RBD resist extreme SARS-CoV-2 antigenic drift.

Developing broad coronavirus vaccines requires identifying and understanding the molecular basis of broadly neutralizing antibody (bnAb) spike sites. In our previous work, we identified sarbecovirus spike RBD group 1 and 2 bnAbs. We have now shown that many of these bnAbs can still neutralize highly mutated SARS-CoV-2 variants, including the XBB.1.5. Structural studies revealed that group 1 bnAbs use recurrent germline-encoded CDRH3 features to interact with a conserved RBD region that overlaps with class 4 bnAb site. Group 2 bnAbs recognize a less well-characterized "site V" on the RBD and destabilize spike trimer. The site V has remained largely unchanged in SARS-CoV-2 variants and is highly conserved across diverse sarbecoviruses, making it a promising target for broad coronavirus vaccine development. Our findings suggest that targeted vaccine strategies may be needed to induce effective B cell responses to escape resistant subdominant spike RBD bnAb sites.

Broadly neutralizing antibodies to SARS-related viruses can be readily induced in rhesus macaques.

To prepare for future coronavirus (CoV) pandemics, it is desirable to generate vaccines capable of eliciting broadly neutralizing antibody responses to CoVs. Here, we show that immunization of macaques with SARS-CoV-2 spike (S) protein with a two-shot protocol generated potent serum receptor binding domain cross-neutralizing antibody responses to both SARS-CoV-2 and SARS-CoV-1. Furthermore, responses were equally effective against most SARS-CoV-2 variants of concern (VOCs) and some were highly effective against Omicron. This result contrasts with human infection or many two-shot vaccination protocols where responses were typically more SARS-CoV-2 specific and where VOCs were less well neutralized. Structural studies showed that cloned macaque neutralizing antibodies, particularly using a given heavy chain germline gene, recognized a relatively conserved region proximal to the angiotensin converting enzyme 2 receptor binding site (RBS), whereas many frequently elicited human neutralizing antibodies targeted more variable epitopes overlapping the RBS. B cell repertoire differences between humans and macaques appeared to influence the vaccine response. The macaque neutralizing antibodies identified a pan-SARS-related virus epitope region less well targeted by human antibodies that could be exploited in rational vaccine design.

Broadly neutralizing anti-S2 antibodies protect against all three human betacoronaviruses that cause severe disease.

Pan-betacoronavirus neutralizing antibodies may hold the key to developing broadly protective vaccines against coronaviruses that cause severe disease, for anticipating novel pandemic-causing viruses, and to respond more effectively to SARS-CoV-2 variants. The emergence of the Omicron variant of SARS-CoV-2 has illustrated the limitations of solely targeting the receptor binding domain (RBD) of the envelope Spike (S)-protein. Here, we isolated a large panel of broadly neutralizing antibodies (bnAbs) from SARS-CoV-2 recovered-vaccinated donors that target a conserved S2 region in the fusion machinery on betacoronavirus spikes. Select bnAbs show broad in vivo protection against all three pathogenic betacoronaviruses, SARS-CoV-1, SARS-CoV-2 and MERS-CoV, that have spilled over into humans in the past 20 years to cause severe disease. The bnAbs provide new opportunities for antibody-based interventions and key insights for developing pan-betacoronavirus vaccines.

Targeted isolation of panels of diverse human protective broadly neutralizing antibodies against SARS-like viruses.

The emergence of current SARS-CoV-2 variants of concern (VOCs) and potential future spillovers of SARS-like coronaviruses into humans pose a major threat to human health and the global economy 1-7 . Development of broadly effective coronavirus vaccines that can mitigate these threats is needed 8, 9 . Notably, several recent studies have revealed that vaccination of recovered COVID-19 donors results in enhanced nAb responses compared to SARS-CoV-2 infection or vaccination alone 10-13 . Here, we utilized a targeted donor selection strategy to isolate a large panel of broadly neutralizing antibodies (bnAbs) to sarbecoviruses from two such donors. Many of the bnAbs are remarkably effective in neutralization against sarbecoviruses that use ACE2 for viral entry and a substantial fraction also show notable binding to non-ACE2-using sarbecoviruses. The bnAbs are equally effective against most SARS-CoV-2 VOCs and many neutralize the Omicron variant. Neutralization breadth is achieved by bnAb binding to epitopes on a relatively conserved face of the receptor binding domain (RBD) as opposed to strain-specific nAbs to the receptor binding site that are commonly elicited in SARS-CoV-2 infection and vaccination 14-18 . Consistent with targeting of conserved sites, select RBD bnAbs exhibited in vivo protective efficacy against diverse SARS-like coronaviruses in a prophylaxis challenge model. The generation of a large panel of potent bnAbs provides new opportunities and choices for next-generation antibody prophylactic and therapeutic applications and, importantly, provides a molecular basis for effective design of pan-sarbecovirus vaccines.

Discovery of Potent and Broad-Spectrum Pyrazolopyridine-Containing Antivirals against Enteroviruses D68, A71, and Coxsackievirus B3 by Targeting the Viral 2C Protein.

The enterovirus genus of the picornavirus family contains many important human pathogens. EV-D68 primarily infects children, and the disease manifestations range from respiratory illnesses to neurological complications such as acute flaccid myelitis (AFM). EV-A71 is a major pathogen for the hand, foot, and mouth disease (HFMD) in children and can also lead to AFM and death in severe cases. CVB3 infection can cause cardiac arrhythmias, acute heart failure, as well as type 1 diabetes. There is currently no FDA-approved antiviral for any of these enteroviruses. In this study, we report our discovery and development of pyrazolopyridine-containing small molecules with potent and broad-spectrum antiviral activity against multiple strains of EV-D68, EV-A71, and CVB3. Serial viral passage experiments, coupled with reverse genetics and thermal shift binding assays, suggested that these molecules target the viral protein 2C. Overall, the pyrazolopyridine inhibitors represent a promising class of candidates for the urgently needed nonpolio enterovirus antivirals.

Discovery of M2 channel blockers targeting the drug-resistant double mutants M2-S31N/L26I and M2-S31N/V27A from the influenza A viruses.

Influenza virus infections are a persistent threat to human health due to seasonal outbreaks and sporadic pandemics. Amantadine and rimantadine are FDA-approved influenza antiviral drugs and work by inhibiting the viral M2 proton channel. However, the therapeutic potential for the antiviral amantadine/rimantadine was curtailed by the emergence of drug-resistant mutations in its target protein M2. In this study, we identified four amantadine-resistant M2 mutants among avian and human influenza A H5N1 strains circulating between 2002 and 2019: the single S31N and V27A mutants, and the S31N/L26I and S31N/V27A double mutants. Herein, utilizing two-electrode voltage clamp (TEVC) assays, we screened a panel of structurally diverse M2 inhibitors against these single and double mutant channels. Three compounds 6, 7, and 15 were found to significantly block all three M2 mutants: M2-S31N, M2-S31N/L26I, and M2-S31N/V27A. Using recombinant viruses generated from reverse genetics, we further showed that these compounds also inhibited the replication of recombinant viruses harboring either the single S31N or double S31N/L26I and S31N/V27A mutants. This work represents the first example in developing antivirals by targeting the drug-resistant double mutants of M2 proton channels.

Enterovirus D68 Antivirals: Past, Present, and Future.

Enterovirus D68 (EV-D68) is an RNA virus that causes respiratory illnesses mainly in children. In severe cases, it can lead to neurological complications such as acute flaccid myelitis (AFM). EV-D68 belongs to the enterovirus genera of the Picornaviridae family, which also includes many other significant human pathogens such as poliovirus, enterovirus A71, and rhinovirus. There are currently no vaccines or antivirals against EV-D68. In this review, we present the current understanding of the link between EV-D68 and AFM, the mechanism of viral replication, and recent progress in developing EV-D68 antivirals by targeting various viral proteins and host factors that are essential for viral replication. The future directions of EV-D68 antiviral drug discovery and the criteria for drugs to reach clinical trials are also discussed.

Investigation of the Drug Resistance Mechanism of M2-S31N Channel Blockers through Biomolecular Simulations and Viral Passage Experiments.

Recent efforts in drug development against influenza A virus (IAV) M2 proton channel S31N mutant resulted in conjugates of amantadine linked with aryl head heterocycles. To understand the mechanism of drug resistance, we chose a representative M2-S31N inhibitor, compound 3, as a chemical probe to identify resistant mutants. To increase the possibility of identifying novel resistant mutants, serial viral passage experiments were performed with multiple strains of H1N1 and H3N2 viruses in different cell lines. This approach not only identified M2 mutations around the drug-binding site, including the pore-lining residues (V27A, V27F, N31S, and G34E) and an interhelical residue (I32N), but also a new allosteric mutation (R45H), in addition to L46P previously identified, located at the C-terminus of M2 that is more than 10 Å away from the drug-binding site. The effects of each mutation were next investigated using electrophysiology, recombinant viruses, and molecular dynamics (MD) simulations. The reduced sensitivity in channel blockage correlated with increased drug resistance in antiviral assays using recombinant viruses. The MD simulations show that the V27A, V27F, G34E, and R45H mutations increase the diameter and hydration state of the pore in complex with compound 3. The Molecular Mechanics Generalized Born (MM-GBSA) calculations result in more positive binding free energies for the complexes of resistant M2 (V27A, V27F, G34E, R45H) with compound 3 compared to the stable complexes (S31N and I32N). Overall, this is the first systematic study of the drug resistance mechanism of M2-S31N channel blockers using multiple viruses in different cell lines.

Focusing on the Influenza Virus Polymerase Complex: Recent Progress in Drug Discovery and Assay Development.

Influenza viruses are severe human pathogens that pose persistent threat to public health. Each year more people die of influenza virus infection than that of breast cancer. Due to the limited efficacy associated with current influenza vaccines, as well as emerging drug resistance from small molecule antiviral drugs, there is a clear need to develop new antivirals with novel mechanisms of action. The influenza virus polymerase complex has become a promising target for the development of the next-generation of antivirals for several reasons. Firstly, the influenza virus polymerase, which forms a heterotrimeric complex that consists of PA, PB1, and PB2 subunits, is highly conserved. Secondly, both individual polymerase subunit (PA, PB1, and PB2) and inter-subunit interactions (PA-PB1, PB1- PB2) represent promising drug targets. Lastly, growing insight into the structure and function of the polymerase complex has spearheaded the structure-guided design of new polymerase inhibitors. In this review, we highlight recent progress in drug discovery and assay development targeting the influenza virus polymerase complex and discuss their therapeutic potentials.

A Novel Capsid Binding Inhibitor Displays Potent Antiviral Activity against Enterovirus D68.

Enterovirus D68 (EV-D68) is a respiratory viral pathogen that primarily infects children under the age of 8. Although EV-D68 infection typically leads to moderate to severe respiratory illnesses, recent years have seen increasing cases of EV-D68 triggered neurological complications such as acute flaccid myelitis (AFM). There is currently no vaccine or antiviral available for EV-D68; we therefore aimed to develop potent and specific small molecule antivirals against EV-D68. In this study, we report our discovery of a viral capsid inhibitor R856932 that inhibits multiple contemporary EV-D68 strains with single-digit to submicromolar efficacy. Mechanistic studies have shown that the tetrazole compound R856932 binds to the hydrophobic pocket of viral capsid protein VP1, thereby preventing viral uncoating and release of viral genome in the infected cells. The mechanism of action of R856932 was confirmed by time-of-addition, Western blot, RT-qPCR, viral heat inactivation, serial viral passage, and reverse genetics experiments. A single mutation located at VP1, A129V, confers resistance against R856932. However, a recombination virus encoding VP1-A129V appeared to have compromised fitness of replication compared to the wild-type EV-D68 virus as shown by the competition growth assay. Overall, the hit compound identified in this study, R856932, represents a promising starting point with a confirmed mechanism of action that can be further developed into EV-D68 antivirals.

Identification of NMS-873, an allosteric and specific p97 inhibitor, as a broad antiviral against both influenza A and B viruses.

Influenza virus infection causes substantial morbidity and mortality worldwide. The limited efficacy of oseltamivir in delayed treatment, coupled with the increasing incidences of oseltamivir-resistant strains, calls for next-generation of antiviral drugs. In this study, we discovered NMS-873, an allosteric and specific p97 inhibitor, as a broad-spectrum influenza antiviral through forward chemical genomics screening. NMS-873 shows potent antiviral activity with low-nanomolar EC50s against multiple human influenza A and B viruses, including adamantine-, oseltamivir-, or double resistant strains. Our data further showed that silencing of p97 via siRNA or inhibiting p97 by NMS-873 both inhibited virus replication and retained viral ribonucleoproteins (vRNPs) in the nucleus, confirming p97 is the drug target. Mechanistic studies have shown that the nuclear retention of vRNP with NMS-873 treatment is a combined result of two effects: the reduced viral M1 protein level (indirect effect), and the disruption of p97-NP interactions (direct effect). Taken together, our results suggest that p97 could be a novel antiviral target and its inhibitor, NMS-873, is a promising antiviral drug candidate.