Michael G. Natchus

Potent Host-Directed Small-Molecule Inhibitors of Myxovirus RNA-Dependent RNA-Polymerases

Therapeutic targeting of host cell factors required for virus replication rather than of pathogen components opens new perspectives to counteract virus infections. Anticipated advantages of this approach include a heightened barrier against the development of viral resistance and a broadened pathogen target spectrum. Myxoviruses are predominantly associated with acute disease and thus are particularly attractive for this approach since treatment time can be kept limited. To identify inhibitor candidates, we have analyzed hit compounds that emerged from a large-scale high-throughput screen for their ability to block replication of members of both the orthomyxovirus and paramyxovirus families. This has returned a compound class with broad anti-viral activity including potent inhibition of different influenza virus and paramyxovirus strains. After hit-to-lead chemistry, inhibitory concentrations are in the nanomolar range in the context of immortalized cell lines and human PBMCs. The compound shows high metabolic stability when exposed to human S-9 hepatocyte subcellular fractions. Antiviral activity is host-cell species specific and most pronounced in cells of higher mammalian origin, supporting a host-cell target. While the compound induces a temporary cell cycle arrest, host mRNA and protein biosynthesis are largely unaffected and treated cells maintain full metabolic activity. Viral replication is blocked at a post-entry step and resembles the inhibition profile of a known inhibitor of viral RNA-dependent RNA-polymerase (RdRp) activity. Direct assessment of RdRp activity in the presence of the reagent reveals strong inhibition both in the context of viral infection and in reporter-based minireplicon assays. In toto, we have identified a compound class with broad viral target range that blocks host factors required for viral RdRp activity. Viral adaptation attempts did not induce resistance after prolonged exposure, in contrast to rapid adaptation to a pathogen-directed inhibitor of RdRp activity.

An Orally Available, Small-Molecule Polymerase Inhibitor Shows Efficacy Against a Lethal Morbillivirus Infection in a Large Animal Model

An orally available, shelf-stable pan-morbillivirus inhibitor targeting a viral polymerase effectively treats ferrets infected with a lethal dose of a measles-like virus. A Boon for Measles Eradication Measles virus causes about 150,000 deaths per year globally despite the existence of an effective vaccine. Insufficient vaccination coverage and vaccine refusal cause gaps in population immunity that challenge current measles virus eradication goals. In their new work, Krumm et al. explore whether an antiviral therapeutic blocking the measles virus RNA polymerase can aid in solving this problem by suppressing measles-like disease. The authors show that the drug efficiently inhibits the RNA polymerase of canine distemper virus (CDV), which is closely related to the measles virus and causes lethal, measles-like disease in ferrets. They demonstrated that prophylactic treatment of CDV-infected ferrets by mouth reduced viral load and prolonged survival. When treatment was initiated 3 days after infection, disease was completely suppressed. All of the animals survived the infection and developed immunity against the virus, which protected against later challenge with a lethal virus dose. Next, the authors generated drug-resistant CDV strains and demonstrated that resistant viruses caused milder disease than did the parent strain and were transmitted less efficiently between animals. These results suggest that this drug may be useful in the future for preemptive treatment of unprotected human contacts of measles cases. Measles virus is a highly infectious morbillivirus responsible for major morbidity and mortality in unvaccinated humans. The related, zoonotic canine distemper virus (CDV) induces morbillivirus disease in ferrets with 100% lethality. We report an orally available, shelf-stable pan-morbillivirus inhibitor that targets the viral RNA polymerase. Prophylactic oral treatment of ferrets infected intranasally with a lethal CDV dose reduced viremia and prolonged survival. Ferrets infected with the same dose of virus that received post-infection treatment at the onset of viremia showed low-grade viral loads, remained asymptomatic, and recovered from infection, whereas control animals succumbed to the disease. Animals that recovered also mounted a robust immune response and were protected against rechallenge with a lethal CDV dose. Drug-resistant viral recombinants were generated and found to be attenuated and transmission-impaired compared to the genetic parent virus. These findings may pioneer a path toward an effective morbillivirus therapy that could aid measles eradication by synergizing with vaccination to close gaps in herd immunity due to vaccine refusal.

Non-nucleoside inhibitors of the measles virus RNA-dependent RNA polymerase: synthesis, structure-activity relationships, and pharmacokinetics.

The measles virus (MeV), a member of the paramyxovirus family, is an important cause of pediatric morbidity and mortality worldwide. In an effort to provide therapeutic treatments for improved measles management, we previously identified a small, non-nucleoside organic inhibitor of the viral RNA-dependent RNA polymerase by means of high-throughput screening. Subsequent structure-activity relationship (SAR) studies around the corresponding pyrazole carboxamide scaffold led to the discovery of 2 (AS-136a), a first generation lead with low nanomolar potency against life MeV and attractive physical properties suitable for development. However, its poor water solubility and low oral bioavailability (F) in rat suggested that the lead could benefit from further SAR studies to improve the biophysical characteristics of the compound. Optimization of in vitro potency and aqueous solubility led to the discovery of 2o (ERDRP-00519), a potent inhibitor of MeV (EC(50) = 60 nM) with an aqueous solubility of approximately 60 μg/mL. The agent shows a 10-fold exposure (AUC/C(max)) increase in the rat model relative to 2, displays near dose proportionality in the range of 10-50 mg/kg, and exhibits good oral bioavailability (F = 39%). The significant solubility increase appears linked to the improved oral bioavailability.

Discovery of Tetrahydroisoquinoline-Based CXCR4 Antagonists

A de novo hit-to-lead effort involving the redesign of benzimidazole-containing antagonists of the CXCR4 receptor resulted in the discovery of a novel series of 1,2,3,4-tetrahydroisoquinoline (TIQ) analogues. In general, this series of compounds show good potencies (3-650 nM) in assays involving CXCR4 function, including both inhibition of attachment of X4 HIV-1IIIB virus in MAGI-CCR5/CXCR4 cells and inhibition of calcium release in Chem-1 cells. Series profiling permitted the identification of TIQ-(R)-stereoisomer 15 as a potent and selective CXCR4 antagonist lead candidate with a promising in vitro profile. The drug-like properties of 15 were determined in ADME in vitro studies, revealing low metabolic liability potential. Further in vivo evaluations included pharmacokinetic experiments in rats and mice, where 15 was shown to have oral bioavailability (F = 63%) and resulted in the mobilization of white blood cells (WBCs) in a dose-dependent manner.

Novel Synthesis and Biological Evaluation of Enigmols as Therapeutic Agents for Treating Prostate Cancer

Enigmol is a synthetic, orally active 1-deoxysphingoid base analogue that has demonstrated promising activity against prostate cancer. In these studies, the pharmacologic roles of stereochemistry and N-methylation in the structure of enigmols were examined. A novel enantioselective synthesis of all four possible 2S-diastereoisomers of enigmol (2-aminooctadecane-3,5-diols) from l-alanine is reported, which features a Liebeskind-Srogl cross-coupling reaction between l-alanine thiol ester and (E)-pentadec-1-enylboronic acid as the key step. In vitro biological evaluation of the four enigmol diastereoisomers and 2S,3S,5S-N-methylenigmol against two prostate cancer cell lines (PC-3 and LNCaP) indicates that all but one diastereomer demonstrate potent oncolytic activity. In nude mouse xenograft models of human prostate cancer, enigmol was equally effective as standard prostate cancer therapies (androgen deprivation or docetaxel), and two of the enigmol diastereomers, 2S,3S,5R-enigmol and 2S,3R,5S-enigmol, also caused statistically significant inhibition of tumor growth. A pharmacokinetic profile of enigmol and N-methylenigmol is also presented.

Asymmetric synthesis of host-directed inhibitors of myxoviruses

Summary High-throughput screening (HTS) previously identified benzimidazole 1 (JMN3-003) as a compound with broad antiviral activity against different influenza viruses and paramyxovirus strains. In pursuit of a lead compound from this series for development, we sought to increase both the potency and the aqueous solubility of 1. Lead optimization has achieved compounds with potent antiviral activity against a panel of myxovirus family members (EC50 values in the low nanomolar range) and much improved aqueous solubilities relative to that of 1. Additionally, we have devised a robust synthetic strategy for preparing 1 and congeners in an enantio-enriched fashion, which has allowed us to demonstrate that the (S)-enantiomers are generally 7- to 110-fold more potent than the corresponding (R)-isomers.

Water-Soluble Progesterone Analogues Are Effective, Injectable Treatments in Animal Models of Traumatic Brain Injury

After more than 30 years of research and 30 failed clinical trials with as many different treatments, progesterone is the first agent to demonstrate robust clinical efficacy as a treatment for traumatic brain injuries. It is currently being investigated in two, independent phase III clinical trials in hospital settings; however, it presents a formidable solubility challenge that has so far prevented the identification of a formulation that would be suitable for emergency field response use or battlefield situations. Accordingly, we have designed and tested a novel series of water-soluble analogues that address this critical need. We report here the synthesis of C-20 oxime conjugates of progesterone as therapeutic agents for traumatic brain injuries with comparable efficacy in animal models of traumatic brain injury and improved solubility and pharmacokinetic profiles. Pharmacodynamic analysis reveals that a nonprogesterone steroidal analogue may be primarily responsible for the observed activity.

Discovery of a Fluorinated Enigmol Analog with Enhanced in Vivo Pharmacokinetic and Anti-Tumor Properties

The orally bioavailable 1-deoxy-sphingosine analog, Enigmol, has demonstrated anticancer activity in numerous in vivo settings. However, as no Enigmol analog with enhanced potency in vitro has been identified, a new strategy to improve efficacy in vivo by increasing tumor uptake was adopted. Herein, synthesis and biological evaluation of two novel fluorinated Enigmol analogs, CF3-Enigmol and CF2-Enigmol, are reported. Each analog was equipotent to Enigmol in vitro, but achieved higher plasma and tissue levels than Enigmol in vivo. Although plasma and tissue exposures were anticipated to trend with fluorine content, CF2-Enigmol absorbed into tissue at strikingly higher concentrations than CF3-Enigmol. Using mouse xenograft models of prostate cancer, we also show that CF3-Enigmol underperformed Enigmol-mediated inhibition of tumor growth and elicited systemic toxicity. By contrast, CF2-Enigmol was not systemically toxic and demonstrated significantly enhanced antitumor activity as compared to Enigmol.

Identification and Characterization of Influenza Virus Entry Inhibitors through Dual Myxovirus High-Throughput Screening

ABSTRACT Influenza A virus (IAV) infections cause major morbidity and mortality, generating an urgent need for novel antiviral therapeutics. We recently established a dual myxovirus high-throughput screening protocol that combines a fully replication-competent IAV-WSN strain and a respiratory syncytial virus reporter strain for the simultaneous identification of IAV-specific, paramyxovirus-specific, and broad-spectrum inhibitors. In the present study, this protocol was applied to a screening campaign to assess a diverse chemical library with over 142,000 entries. Focusing on IAV-specific hits, we obtained a hit rate of 0.03% after cytotoxicity testing and counterscreening. Three chemically distinct hit classes with nanomolar potency and favorable cytotoxicity profiles were selected. Time-of-addition, minigenome, and viral entry studies demonstrated that these classes block hemagglutinin (HA)-mediated membrane fusion. Antiviral activity extends to an isolate from the 2009 pandemic and, in one case, another group 1 subtype. Target identification through biolayer interferometry confirmed binding of all hit compounds to HA. Resistance profiling revealed two distinct escape mechanisms: primary resistance, associated with reduced compound binding, and secondary resistance, associated with unaltered binding. Secondary resistance was mediated, unusually, through two different pairs of cooperative mutations, each combining a mutation eliminating the membrane-proximal stalk N-glycan with a membrane-distal change in HA1 or HA2. Chemical synthesis of an analog library combined with in silico docking extracted a docking pose for the hit classes. Chemical interrogation spotlights IAV HA as a major druggable target for small-molecule inhibition. Our study identifies novel chemical scaffolds with high developmental potential, outlines diverse routes of IAV escape from entry inhibition, and establishes a path toward structure-aided lead development. IMPORTANCE This study is one of the first to apply a fully replication-competent third-generation IAV reporter strain to a large-scale high-throughput screen (HTS) drug discovery campaign, allowing multicycle infection and screening in physiologically relevant human respiratory cells. A large number of potential druggable targets was thus chemically interrogated, but mechanistic characterization, positive target identification, and resistance profiling demonstrated that three chemically promising and structurally distinct hit classes selected for further analysis all block HA-mediated membrane fusion. Viral escape from inhibition could be achieved through primary and secondary resistance mechanisms. In silico docking predicted compound binding to a microdomain located at the membrane-distal site of the prefusion HA stalk that was also previously suggested as a target site for chemically unrelated HA inhibitors. This study identifies an unexpected chemodominance of the HA stalk microdomain for small-molecule inhibitors in IAV inhibitor screening campaigns and highlights a novel mechanism of cooperative resistance to IAV entry blockers.

β-d-N4-Hydroxycytidine Is a Potent Anti-alphavirus Compound That Induces a High Level of Mutations in the Viral Genome

ABSTRACT Venezuelan equine encephalitis virus (VEEV) is a representative member of the New World alphaviruses. It is transmitted by mosquito vectors and causes highly debilitating disease in humans, equids, and other vertebrate hosts. Despite a continuous public health threat, very few compounds with anti-VEEV activity in cell culture and in mouse models have been identified to date, and rapid development of virus resistance to some of them has been recorded. In this study, we investigated the possibility of using a modified nucleoside analog, β-d-N4-hydroxycytidine (NHC), as an anti-VEEV agent and defined the mechanism of its anti-VEEV activity. The results demonstrate that NHC is a very potent antiviral agent. It affects both the release of genome RNA-containing VEE virions and their infectivity. Both of these antiviral activities are determined by the NHC-induced accumulation of mutations in virus-specific RNAs. The antiviral effect is most prominent when NHC is applied early in the infectious process, during the amplification of negative- and positive-strand RNAs in infected cells. Most importantly, only a low-level resistance of VEEV to NHC can be developed, and it requires acquisition and cooperative function of more than one mutation in nsP4. These adaptive mutations are closely located in the same segment of nsP4. Our data suggest that NHC is more potent than ribavirin as an anti-VEEV agent and likely can be used to treat other alphavirus infections. IMPORTANCE Venezuelan equine encephalitis virus (VEEV) can cause widespread epidemics among humans and domestic animals. VEEV infections result in severe meningoencephalitis and long-term sequelae. No approved therapeutics exist for treatment of VEEV infections. Our study demonstrates that β-d-N4-hydroxycytidine (NHC) is a very potent anti-VEEV compound, with the 50% effective concentration being below 1 μM. The mechanism of NHC antiviral activity is based on induction of high mutation rates in the viral genome. Accordingly, NHC treatment affects both the rates of particle release and the particle infectivity. Most importantly, in contrast to most of the anti-alphavirus drugs that are under development, resistance of VEEV to NHC develops very inefficiently. Even low levels of resistance require acquisition of multiple mutations in the gene of the VEEV-specific RNA-dependent RNA polymerase nsP4.