4'-Fluorouridine inhibits alphavirus replication and infection in vitro and in vivo.

Chikungunya virus (CHIKV) is an enveloped, positive-sense RNA virus that has re-emerged to cause millions of human infections worldwide. In humans, acute CHIKV infection causes fever and severe muscle and joint pain. Chronic and debilitating arthritis and joint pain can persist for months to years. To date, there are no approved antivirals against CHIKV. Recently, the ribonucleoside analog 4'-fluorouridine (4'-FlU) was reported as a highly potent orally available inhibitor of SARS-CoV-2, respiratory syncytial virus, and influenza virus replication. In this study, we assessed 4'-FlU's potency and breadth of inhibition against a panel of alphaviruses including CHIKV, and found that it broadly suppressed alphavirus production in cell culture. 4'-FlU acted on the viral RNA replication step, and the first 4 hours post-infection were the critical time for its antiviral effect. In vitro replication assays identified nsP4 as the target of inhibition. In vivo, treatment with 4'-FlU reduced disease signs, inflammatory responses, and viral tissue burden in mouse models of CHIKV and Mayaro virus infection. Treatment initiated at 2 hours post-infection was most effective; however, treatment initiated as late as 24-48 hours post-infection produced measurable antiviral effects in the CHIKV mouse model. 4'-FlU showed effective oral delivery in our mouse model and resulted in the accumulation of both 4'-FlU and its bioactive triphosphate form in tissues relevant to arthritogenic alphavirus pathogenesis. Together, our data indicate that 4'-FlU inhibits CHIKV infection in vitro and in vivo and is a promising oral therapeutic candidate against CHIKV infection.IMPORTANCEAlphaviruses including chikungunya virus (CHIKV) are mosquito-borne positive-strand RNA viruses that can cause various diseases in humans. Although compounds that inhibit CHIKV and other alphaviruses have been identified in vitro, there are no licensed antivirals against CHIKV. Here, we investigated a ribonucleoside analog, 4'-fluorouridine (4'-FlU), and demonstrated that it inhibited infectious virus production by several alphaviruses in vitro and reduced virus burden in mouse models of CHIKV and Mayaro virus infection. Our studies also indicated that 4'-FlU treatment reduced CHIKV-induced footpad swelling and reduced the production of pro-inflammatory cytokines. Inhibition in the mouse model correlated with effective oral delivery of 4'-FlU and accumulation of both 4'-FlU and its bioactive form in relevant tissues. In summary, 4'-FlU exhibits potential as a novel anti-alphavirus agent targeting the replication of viral RNA.

Modeling Heartland virus disease in mice and therapeutic intervention with 4'-fluorouridine.

Heartland virus (HRTV) is an emerging tick-borne bandavirus that causes a febrile illness of varying severity in humans, with cases reported in eastern and midwestern regions of the United States. No vaccines or approved therapies are available to prevent or treat HRTV disease. Here, we describe the genetic changes, natural history of disease, and pathogenesis of a mouse-adapted HRTV (MA-HRTV) that is uniformly lethal in 7- to 8-week-old AG129 mice at low challenge doses. We used this model to assess the efficacy of the ribonucleoside analog, 4'-fluorouridine (EIDD-2749), and showed that once-daily oral treatment with 3 mg/kg of drug, initiated after the onset of disease, protects mice against lethal MA-HRTV challenge and reduces viral loads in blood and tissues. Our findings provide insights into HRTV virulence and pathogenesis and support further development of EIDD-2749 as a therapeutic intervention for HRTV disease. IMPORTANCE: More than 60 cases of HRTV disease spanning 14 states have been reported to the United States Centers for Disease Control and Prevention. The expanding range of the Lone Star tick that transmits HRTV, the growing population of at-risk persons living in geographic areas where the tick is abundant, and the lack of antiviral treatments or vaccines raise significant public health concerns. Here, we report the development of a new small-animal model of lethal HRTV disease to gain insight into HRTV pathogenesis and the application of this model for the preclinical development of a promising new antiviral drug candidate, EIDD-2749. Our findings shed light on how the virus causes disease and support the continued development of EIDD-2749 as a therapeutic for severe cases of HRTV infection.

Therapeutic mitigation of measles-like immune amnesia and exacerbated disease after prior respiratory virus infections in ferrets.

Measles cases have surged pre-COVID-19 and the pandemic has aggravated the problem. Most measles-associated morbidity and mortality arises from destruction of pre-existing immune memory by measles virus (MeV), a paramyxovirus of the morbillivirus genus. Therapeutic measles vaccination lacks efficacy, but little is known about preserving immune memory through antivirals and the effect of respiratory disease history on measles severity. We use a canine distemper virus (CDV)-ferret model as surrogate for measles and employ an orally efficacious paramyxovirus polymerase inhibitor to address these questions. A receptor tropism-intact recombinant CDV with low lethality reveals an 8-day advantage of antiviral treatment versus therapeutic vaccination in maintaining immune memory. Infection of female ferrets with influenza A virus (IAV) A/CA/07/2009 (H1N1) or respiratory syncytial virus (RSV) four weeks pre-CDV causes fatal hemorrhagic pneumonia with lung onslaught by commensal bacteria. RNAseq identifies CDV-induced overexpression of trefoil factor (TFF) peptides in the respiratory tract, which is absent in animals pre-infected with IAV. Severe outcomes of consecutive IAV/CDV infections are mitigated by oral antivirals even when initiated late. These findings validate the morbillivirus immune amnesia hypothesis, define measles treatment paradigms, and identify priming of the TFF axis through prior respiratory infections as risk factor for exacerbated morbillivirus disease.

A Novel 7H-[1,2,4]Triazolo[3,4-b]thiadiazine-based Cystic Fibrosis Transmembrane Conductance Regulator Potentiator Directed toward Treatment of Cystic Fibrosis.

Cystic fibrosis (CF) is an autosomal genetic disorder caused by disrupted anion transport in epithelial cells lining tissues in the human airways and digestive system. While cystic fibrosis transmembrane conductance regulator (CFTR) modulator compounds have provided transformative improvement in CF respiratory function, certain patients exhibit marginal clinical benefit or detrimental effects or have a form of the disease not approved or unlikely to respond using CFTR modulation. We tested hit compounds from a 300,000-drug screen for their ability to augment CFTR transepithelial transport alone or in combination with the FDA-approved CFTR potentiator ivacaftor (VX-770). A subsequent SAR campaign led us to a class of 7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazines that in combination with VX-770 rescued function of G551D mutant CFTR channels to approximately 400% above the activity of VX-770 alone and to nearly wild-type CFTR levels in the same Fischer rat thyroid model system.

Design and Optimization of Novel Competitive, Non-peptidic, SARS-CoV-2 Mpro Inhibitors.

The SARS-CoV-2 main protease (Mpro) has been proven to be a highly effective target for therapeutic intervention, yet only one drug currently holds FDA approval status for this target. We were inspired by a series of publications emanating from the Jorgensen and Anderson groups describing the design of potent, non-peptidic, competitive SARS-CoV-2 Mpro inhibitors, and we saw an opportunity to make several design modifications to improve the overall pharmacokinetic profile of these compounds without losing potency. To this end, we created a focused virtual library using reaction-based enumeration tools in the Schrödinger suite. These compounds were docked into the Mpro active site and subsequently prioritized for synthesis based upon relative binding affinity values calculated by FEP+. Fourteen compounds were selected, synthesized, and evaluated both biochemically and in cell culture. Several of the synthesized compounds proved to be potent, competitive Mpro inhibitors with improved metabolic stability profiles.

Comparing molnupiravir and nirmatrelvir/ritonavir efficacy and the effects on SARS-CoV-2 transmission in animal models.

Therapeutic options against SARS-CoV-2 are underutilized. Two oral drugs, molnupiravir and paxlovid (nirmatrelvir/ritonavir), have received emergency use authorization. Initial trials suggested greater efficacy of paxlovid, but recent studies indicated comparable potency in older adults. Here, we compare both drugs in two animal models; the Roborovski dwarf hamster model for severe COVID-19-like lung infection and the ferret SARS-CoV-2 transmission model. Dwarf hamsters treated with either drug survive VOC omicron infection with equivalent lung titer reduction. Viral RNA copies in the upper respiratory tract of female ferrets receiving 1.25 mg/kg molnupiravir twice-daily are not significantly reduced, but infectious titers are lowered by >2 log orders and direct-contact transmission is stopped. Female ferrets dosed with 20 or 100 mg/kg nirmatrelvir/ritonavir twice-daily show 1-2 log order reduction of viral RNA copies and infectious titers, which correlates with low nirmatrelvir exposure in nasal turbinates. Virus replication resurges towards nirmatrelvir/ritonavir treatment end and virus transmits efficiently (20 mg/kg group) or partially (100 mg/kg group). Prophylactic treatment with 20 mg/kg nirmatrelvir/ritonavir does not prevent spread from infected ferrets, but prophylactic 5 mg/kg molnupiravir or 100 mg/kg nirmatrelvir/ritonavir block productive transmission. These data confirm reports of similar efficacy in older adults and inform on possible epidemiologic benefit of antiviral treatment.

4'-Fluorouridine mitigates lethal infection with pandemic human and highly pathogenic avian influenza viruses.

Influenza outbreaks are associated with substantial morbidity, mortality and economic burden. Next generation antivirals are needed to treat seasonal infections and prepare against zoonotic spillover of avian influenza viruses with pandemic potential. Having previously identified oral efficacy of the nucleoside analog 4'-Fluorouridine (4'-FlU, EIDD-2749) against SARS-CoV-2 and respiratory syncytial virus (RSV), we explored activity of the compound against seasonal and highly pathogenic influenza (HPAI) viruses in cell culture, human airway epithelium (HAE) models, and/or two animal models, ferrets and mice, that assess IAV transmission and lethal viral pneumonia, respectively. 4'-FlU inhibited a panel of relevant influenza A and B viruses with nanomolar to sub-micromolar potency in HAE cells. In vitro polymerase assays revealed immediate chain termination of IAV polymerase after 4'-FlU incorporation, in contrast to delayed chain termination of SARS-CoV-2 and RSV polymerase. Once-daily oral treatment of ferrets with 2 mg/kg 4'-FlU initiated 12 hours after infection rapidly stopped virus shedding and prevented transmission to untreated sentinels. Treatment of mice infected with a lethal inoculum of pandemic A/CA/07/2009 (H1N1)pdm09 (pdmCa09) with 4'-FlU alleviated pneumonia. Three doses mediated complete survival when treatment was initiated up to 60 hours after infection, indicating a broad time window for effective intervention. Therapeutic oral 4'-FlU ensured survival of animals infected with HPAI A/VN/12/2003 (H5N1) and of immunocompromised mice infected with pdmCa09. Recoverees were protected against homologous reinfection. This study defines the mechanistic foundation for high sensitivity of influenza viruses to 4'-FlU and supports 4'-FlU as developmental candidate for the treatment of seasonal and pandemic influenza.

The prophylactic and therapeutic efficacy of the broadly active antiviral ribonucleoside N4-Hydroxycytidine (EIDD-1931) in a mouse model of lethal Ebola virus infection.

The unprecedented magnitude of the 2013-2016 Ebola virus (EBOV) epidemic in West Africa resulted in over 11 000 deaths and spurred an international public health emergency. A second outbreak in 2018-2020 in DRC resulted in an additional >3400 cases and nearly 2300 deaths (WHO, 2020). These large outbreaks across geographically diverse regions highlight the need for the development of effective oral therapeutic agents that can be easily distributed for self-administration to populations with active disease or at risk of infection. Herein, we report the in vivo efficacy of N4-hydroxycytidine (EIDD-1931), a broadly active ribonucleoside analog and the active metabolite of the prodrug EIDD-2801 (molnupiravir), in murine models of lethal EBOV infection. Twice daily oral dosing with EIDD-1931 at 200 mg/kg for 7 days, initiated either with a prophylactic dose 2 h before infection, or as therapeutic treatment starting 6 h post-infection, resulted in 92-100% survival of mice challenged with lethal doses of EBOV, reduced clinical signs of Ebola virus disease (EVD), reduced serum virus titers, and facilitated weight loss recovery. These results support further investigation of molnupiravir as a potential therapeutic or prophylactic treatment for EVD.

Paxlovid-like nirmatrelvir/ritonavir fails to block SARS-CoV-2 transmission in ferrets.

Despite the continued spread of SARS-CoV-2 and emergence of variants of concern (VOC) that are capable of escaping preexisting immunity, therapeutic options are underutilized. In addition to preventing severe disease in high-risk patients, antivirals may contribute to interrupting transmission chains. The FDA has granted emergency use authorizations for two oral drugs, molnupiravir and paxlovid. Initial clinical trials suggested an efficacy advantage of paxlovid, giving it a standard-of-care-like status in the United States. However, recent retrospective clinical studies suggested a more comparable efficacy of both drugs in preventing complicated disease and case-fatalities in older adults. For a direct efficacy comparison under controlled conditions, we assessed potency of both drugs against SARS-CoV-2 in two relevant animal models; the Roborovski dwarf hamster model for severe COVID-19 in high-risk patients and the ferret model of upper respiratory tract disease and transmission. After infection of dwarf hamsters with VOC omicron, paxlovid and molnupiravir were efficacious in mitigating severe disease and preventing death. However, a pharmacokinetics-confirmed human equivalent dose of paxlovid did not significantly reduce shed SARS-CoV-2 titers in ferrets and failed to block virus transmission to untreated direct-contact ferrets, whereas transmission was fully suppressed in a group of animals treated with a human-equivalent dose of molnupiravir. Prophylactic administration of molnupiravir to uninfected ferrets in direct contact with infected animals blocked productive SARS-CoV-2 transmission, whereas all contacts treated with prophylactic paxlovid became infected. These data confirm retrospective reports of similar therapeutic benefit of both drugs for older adults, and reveal that treatment with molnupiravir, but not paxlovid, may be suitable to reduce the risk of SARS-CoV-2 transmission.

4'-Fluorouridine is an oral antiviral that blocks respiratory syncytial virus and SARS-CoV-2 replication.

The COVID-19 pandemic has underscored the critical need for broad-spectrum therapeutics against respiratory viruses. Respiratory syncytial virus (RSV) is a major threat to pediatric patients and older adults. We describe 4'-fluorouridine (4'-FlU, EIDD-2749), a ribonucleoside analog that inhibits RSV, related RNA viruses, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), with high selectivity index in cells and human airway epithelia organoids. Polymerase inhibition within in vitro RNA-dependent RNA polymerase assays established for RSV and SARS-CoV-2 revealed transcriptional stalling after incorporation. Once-daily oral treatment was highly efficacious at 5 milligrams per kilogram (mg/kg) in RSV-infected mice or 20 mg/kg in ferrets infected with different SARS-CoV-2 variants of concern, initiated 24 or 12 hours after infection, respectively. These properties define 4'-FlU as a broad-spectrum candidate for the treatment of RSV, SARS-CoV-2, and related RNA virus infections.

4'-Fluorouridine is a broad-spectrum orally efficacious antiviral blocking respiratory syncytial virus and SARS-CoV-2 replication.

The COVID-19 pandemic has underscored the critical need for broad-spectrum therapeutics against respiratory viruses. Respiratory syncytial virus (RSV) is a major threat to pediatric patients and the elderly. We describe 4'-fluorouridine (4'-FlU, EIDD-2749), a ribonucleoside analog that inhibits RSV, related RNA viruses, and SARS-CoV-2 with high selectivity index in cells and well-differentiated human airway epithelia. Polymerase inhibition in in vitro RdRP assays established for RSV and SARS-CoV-2 revealed transcriptional pauses at positions i or i +3/4 post-incorporation. Once-daily oral treatment was highly efficacious at 5 mg/kg in RSV-infected mice or 20 mg/kg in ferrets infected with SARS-CoV-2 WA1/2020 or variant-of-concern (VoC) isolate CA/2020, initiated 24 or 12 hours after infection, respectively. These properties define 4'-FlU as a broad-spectrum candidate for the treatment of RSV, SARS-CoV-2 and related RNA virus infections. ONE-SENTENCE SUMMARY: 4'-Fluorouridine is an orally available ribonucleoside analog that efficiently treats RSV and SARS-CoV-2 infections in vivo .

SARS-CoV-2 infection is effectively treated and prevented by EIDD-2801.

All coronaviruses known to have recently emerged as human pathogens probably originated in bats1. Here we use a single experimental platform based on immunodeficient mice implanted with human lung tissue (hereafter, human lung-only mice (LoM)) to demonstrate the efficient in vivo replication of severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), as well as two endogenous SARS-like bat coronaviruses that show potential for emergence as human pathogens. Virus replication in this model occurs in bona fide human lung tissue and does not require any type of adaptation of the virus or the host. Our results indicate that bats contain endogenous coronaviruses that are capable of direct transmission to humans. Our detailed analysis of in vivo infection with SARS-CoV-2 in human lung tissue from LoM showed a predominant infection of human lung epithelial cells, including type-2 pneumocytes that are present in alveoli and ciliated airway cells. Acute infection with SARS-CoV-2 was highly cytopathic and induced a robust and sustained type-I interferon and inflammatory cytokine and chemokine response. Finally, we evaluated a therapeutic and pre-exposure prophylaxis strategy for SARS-CoV-2 infection. Our results show that therapeutic and prophylactic administration of EIDD-2801-an oral broad-spectrum antiviral agent that is currently in phase II/III clinical trials-markedly inhibited SARS-CoV-2 replication in vivo, and thus has considerable potential for the prevention and treatment of COVID-19.

Orally efficacious broad-spectrum allosteric inhibitor of paramyxovirus polymerase.

Paramyxoviruses such as human parainfluenza virus type-3 (HPIV3) and measles virus (MeV) are a substantial health threat. In a high-throughput screen for inhibitors of HPIV3 (a major cause of acute respiratory infection), we identified GHP-88309-a non-nucleoside inhibitor of viral polymerase activity that possesses unusual broad-spectrum activity against diverse paramyxoviruses including respiroviruses (that is, HPIV1 and HPIV3) and morbilliviruses (that is, MeV). Resistance profiles of distinct target viruses overlapped spatially, revealing a conserved binding site in the central cavity of the viral polymerase (L) protein that was validated by photoaffinity labelling-based target mapping. Mechanistic characterization through viral RNA profiling and in vitro MeV polymerase assays identified a block in the initiation phase of the viral polymerase. GHP-88309 showed nanomolar potency against HPIV3 isolates in well-differentiated human airway organoid cultures, was well tolerated (selectivity index > 7,111) and orally bioavailable, and provided complete protection against lethal infection in a Sendai virus mouse surrogate model of human HPIV3 disease when administered therapeutically 48 h after infection. Recoverees had acquired robust immunoprotection against reinfection, and viral resistance coincided with severe attenuation. This study provides proof of the feasibility of a well-behaved broad-spectrum allosteric antiviral and describes a chemotype with high therapeutic potential that addresses major obstacles of anti-paramyxovirus drug development.

ATI-2173, a Novel Liver-Targeted Non-Chain-Terminating Nucleotide for Hepatitis B Virus Cure Regimens.

ATI-2173 is a novel liver-targeted molecule designed to deliver the 5'-monophosphate of clevudine for the treatment of chronic hepatitis B infection. Unlike other nucleos(t)ides, the active clevudine-5'-triphosphate is a noncompetitive, non-chain-terminating inhibitor of hepatitis B virus (HBV) polymerase that delivers prolonged reduction of viremia in both a woodchuck HBV model and in humans for up to 6 months after cessation of treatment. However, long-term clevudine treatment was found to exhibit reversible skeletal myopathy in a small subset of patients and was subsequently discontinued from development. ATI-2173 was designed by modifying clevudine with a 5'-phosphoramidate to deliver the 5'-monophosphate to the liver. Bypassing the first phosphorylation step of clevudine, the 5'-monophosphate is converted to the active 5'-triphosphate in the liver. ATI-2173 is a selective inhibitor of HBV with an anti-HBV 50% effective concentration (EC50) of 1.31 nM in primary human hepatocytes, with minimal to no toxicity in hepatocytes, skeletal muscle, liver, kidney, bone marrow, and cardiomyocytes. ATI-2173 activity was decreased by viral polymerase mutations associated with entecavir, lamivudine, and adefovir resistance, but not capsid inhibitor resistance mutations. A single oral dose of ATI-2173 demonstrated 82% hepatic extraction, no food effect, and greatly reduced peripheral exposure of clevudine compared with equimolar oral dosing of clevudine. Despite reduced plasma clevudine exposure, liver concentrations of the 5'-triphosphate were equivalent following ATI-2173 versus clevudine administration. By selectively delivering the 5'-monophosphate to the liver, while retaining the unique anti-HBV activity of the 5'-triphosphate, ATI-2173 may provide an improved pharmacokinetic profile for clinical use, reducing systemic exposure of clevudine and potentially eliminating skeletal myopathy.

An orally bioavailable broad-spectrum antiviral inhibits SARS-CoV-2 in human airway epithelial cell cultures and multiple coronaviruses in mice.

Coronaviruses (CoVs) traffic frequently between species resulting in novel disease outbreaks, most recently exemplified by the newly emerged SARS-CoV-2, the causative agent of COVID-19. Here, we show that the ribonucleoside analog ß-d-N4-hydroxycytidine (NHC; EIDD-1931) has broad-spectrum antiviral activity against SARS-CoV-2, MERS-CoV, SARS-CoV, and related zoonotic group 2b or 2c bat-CoVs, as well as increased potency against a CoV bearing resistance mutations to the nucleoside analog inhibitor remdesivir. In mice infected with SARS-CoV or MERS-CoV, both prophylactic and therapeutic administration of EIDD-2801, an orally bioavailable NHC prodrug (ß-d-N4-hydroxycytidine-5'-isopropyl ester), improved pulmonary function and reduced virus titer and body weight loss. Decreased MERS-CoV yields in vitro and in vivo were associated with increased transition mutation frequency in viral, but not host cell RNA, supporting a mechanism of lethal mutagenesis in CoV. The potency of NHC/EIDD-2801 against multiple CoVs and oral bioavailability highlights its potential utility as an effective antiviral against SARS-CoV-2 and other future zoonotic CoVs.

Analysis of the Potential for N4-Hydroxycytidine To Inhibit Mitochondrial Replication and Function.

N4-Hydroxycytidine (NHC) is an antiviral ribonucleoside analog that acts as a competitive alternative substrate for virally encoded RNA-dependent RNA polymerases. It exhibits measurable levels of cytotoxicity, with 50% cytotoxic concentration values ranging from 7.5 µM in CEM cells and up to >100 µM in other cell lines. The mitochondrial DNA-dependent RNA polymerase (POLRMT) has been shown to incorporate some nucleotide analogs into mitochondrial RNAs, resulting in substantial mitochondrial toxicity. NHC was tested in multiple assays intended to determine its potential to cause mitochondrial toxicity. NHC showed similar cytotoxicity in HepG2 cells incubated in a glucose-free and glucose-containing media, suggesting that NHC does not impair mitochondrial function in this cell line based on the Crabtree effect. We demonstrate that the 5'-triphosphate of NHC can be used by POLRMT for incorporation into nascent RNA chain but does not cause immediate chain termination. In PC-3 cells treated with NHC, the 50% inhibitory concentrations of mitochondrial protein expression inhibition were 2.7-fold lower than those for nuclear-encoded protein expression, but this effect did not result in selective mitochondrial toxicity. A 14-day incubation of HepG2 cells with NHC had no effect on mitochondrial DNA copy number or extracellular lactate levels. In CEM cells treated with NHC at 10 µM, a slight decrease (by ∼20%) in mitochondrial DNA copy number and a corresponding slight increase in extracellular lactate levels were detected, but these effects were not enhanced by an increase in NHC treatment concentration. In summary, the results indicate that mitochondrial impairment by NHC is not the main contributor to the compound's observed cytotoxicity in these cell lines.

Small-Molecule Antiviral ß-d-N4-Hydroxycytidine Inhibits a Proofreading-Intact Coronavirus with a High Genetic Barrier to Resistance.

Coronaviruses (CoVs) have emerged from animal reservoirs to cause severe and lethal disease in humans, but there are currently no FDA-approved antivirals to treat the infections. One class of antiviral compounds, nucleoside analogues, mimics naturally occurring nucleosides to inhibit viral replication. While these compounds have been successful therapeutics for several viral infections, mutagenic nucleoside analogues, such as ribavirin and 5-fluorouracil, have been ineffective at inhibiting CoVs. This has been attributed to the proofreading activity of the viral 3'-5' exoribonuclease (ExoN). ß-d-N4-Hydroxycytidine (NHC) (EIDD-1931; Emory Institute for Drug Development) has recently been reported to inhibit multiple viruses. Here, we demonstrate that NHC inhibits both murine hepatitis virus (MHV) (50% effective concentration [EC50] = 0.17 µM) and Middle East respiratory syndrome CoV (MERS-CoV) (EC50 = 0.56 µM) with minimal cytotoxicity. NHC inhibited MHV lacking ExoN proofreading activity similarly to wild-type (WT) MHV, suggesting an ability to evade or overcome ExoN activity. NHC inhibited MHV only when added early during infection, decreased viral specific infectivity, and increased the number and proportion of G:A and C:U transition mutations present after a single infection. Low-level NHC resistance was difficult to achieve and was associated with multiple transition mutations across the genome in both MHV and MERS-CoV. These results point to a virus-mutagenic mechanism of NHC inhibition in CoVs and indicate a high genetic barrier to NHC resistance. Together, the data support further development of NHC for treatment of CoVs and suggest a novel mechanism of NHC interaction with the CoV replication complex that may shed light on critical aspects of replication.IMPORTANCE The emergence of coronaviruses (CoVs) into human populations from animal reservoirs has demonstrated their epidemic capability, pandemic potential, and ability to cause severe disease. However, no antivirals have been approved to treat these infections. Here, we demonstrate the potent antiviral activity of a broad-spectrum ribonucleoside analogue, ß-d-N4-hydroxycytidine (NHC), against two divergent CoVs. Viral proofreading activity does not markedly impact sensitivity to NHC inhibition, suggesting a novel interaction between a nucleoside analogue inhibitor and the CoV replicase. Further, passage in the presence of NHC generates only low-level resistance, likely due to the accumulation of multiple potentially deleterious transition mutations. Together, these data support a mutagenic mechanism of inhibition by NHC and further support the development of NHC for treatment of CoV infections.

Characterization of orally efficacious influenza drug with high resistance barrier in ferrets and human airway epithelia.

Influenza viruses constitute a major health threat and economic burden globally, frequently exacerbated by preexisting or rapidly emerging resistance to antiviral therapeutics. To address the unmet need of improved influenza therapy, we have created EIDD-2801, an isopropylester prodrug of the ribonucleoside analog N 4-hydroxycytidine (NHC, EIDD-1931) that has shown broad anti-influenza virus activity in cultured cells and mice. Pharmacokinetic profiling demonstrated that EIDD-2801 was orally bioavailable in ferrets and nonhuman primates. Therapeutic oral dosing of influenza virus-infected ferrets reduced group pandemic 1 and group 2 seasonal influenza A shed virus load by multiple orders of magnitude and alleviated fever, airway epithelium histopathology, and inflammation, whereas postexposure prophylactic dosing was sterilizing. Deep sequencing highlighted lethal viral mutagenesis as the underlying mechanism of activity and revealed a prohibitive barrier to the development of viral resistance. Inhibitory concentrations were low nanomolar against influenza A and B viruses in disease-relevant well-differentiated human air-liquid interface airway epithelia. Correlating antiviral efficacy and cytotoxicity thresholds with pharmacokinetic profiles in human airway epithelium models revealed a therapeutic window >1713 and established dosing parameters required for efficacious human therapy. These data recommend EIDD-2801 as a clinical candidate with high potential for monotherapy of seasonal and pandemic influenza virus infections. Our results inform EIDD-2801 clinical trial design and drug exposure targets.

The prophylactic and therapeutic activity of a broadly active ribonucleoside analog in a murine model of intranasal venezuelan equine encephalitis virus infection.

The New World alphaviruses Venezuelan, Eastern, and Western equine encephalitis viruses (VEEV, EEEV and WEEV, respectively) commonly cause a febrile disease that can progress to meningoencephalitis, resulting in significant morbidity and mortality. To address the need for a therapeutic agent for the treatment of Alphavirus infections, we identified and pursued preclinical characterization of a ribonucleoside analog EIDD-1931 (ß-D-N4-hydroxycytidine, NHC), which has shown broad activity against alphaviruses in vitro and has a very high genetic barrier for development of resistance. To be truly effective as a therapeutic agent for VEEV infection a drug must penetrate the blood brain barrier and arrest virus replication in the brain. High plasma levels of EIDD-1931 are rapidly achieved in mice after oral dosing. Once in the plasma EIDD-1931 is efficiently distributed into organs, including brain, where it is rapidly converted to its active 5'-triphosphate. EIDD-1931 showed a good safety profile in mice after 7-day repeated dosing with up to 1000 mg/kg/day doses. In mouse model studies, EIDD-1931 was 90-100% effective in protecting mice against lethal intranasal infection when therapeutic treatment was started as late as 24 h post-infection, and partial protection was achieved when treatment was delayed for 48 h post-infection. These results support further preclinical development of EIDD-1931 as a potential anti-alphavirus drug.

Orally Efficacious Broad-Spectrum Ribonucleoside Analog Inhibitor of Influenza and Respiratory Syncytial Viruses.

Morbidity and mortality resulting from influenza-like disease are a threat, especially for older adults. To improve case management, next-generation broad-spectrum antiviral therapeutics that are efficacious against major drivers of influenza-like disease, including influenza viruses and respiratory syncytial virus (RSV), are urgently needed. Using a dual-pathogen high-throughput screening protocol for influenza A virus (IAV) and RSV inhibitors, we have identified N4-hydroxycytidine (NHC) as a potent inhibitor of RSV, influenza B viruses, and IAVs of human, avian, and swine origins. Biochemical in vitro polymerase assays and viral RNA sequencing revealed that the ribonucleotide analog is incorporated into nascent viral RNAs in place of cytidine, increasing the frequency of viral mutagenesis. Viral passaging in cell culture in the presence of an inhibitor did not induce robust resistance. Pharmacokinetic profiling demonstrated dose-dependent oral bioavailability of 36 to 56%, sustained levels of the active 5'-triphosphate anabolite in primary human airway cells and mouse lung tissue, and good tolerability after extended dosing at 800 mg/kg of body weight/day. The compound was orally efficacious against RSV and both seasonal and highly pathogenic avian IAVs in mouse models, reducing lung virus loads and alleviating disease biomarkers. Oral dosing reduced IAV burdens in a guinea pig transmission model and suppressed virus spread to uninfected contact animals through direct transmission. Based on its broad-spectrum efficacy and pharmacokinetic properties, NHC is a promising candidate for future clinical development as a treatment option for influenza-like diseases.