Benzimidazole analogs inhibit respiratory syncytial virus G protein function.

Human respiratory syncytial virus (hRSV) is a highly contagious Paramyxovirus that infects most children by age two, generating an estimated 75,000-125,000 hospitalizations in the U.S. annually. hRSV is the most common cause of bronchiolitis and pneumonia among infants and children under 1year of age, with significant mortality among high-risk groups. A regulatory agency-approved vaccine is not available, and existing prophylaxis and therapies are limited to use in high-risk pediatric patients; thus additional therapies are sorely needed. Here, we identify a series of benzimidazole analogs that inhibit hRSV infection in vitro with high potency, using a previously-reported high-throughput screening assay. The lead compound, SRI 29365 (1-[6-(2-furyl)[1,2,4]triazolo[3,4-b][1,3,4]thiadiazol-3-yl]methyl-1H-benzimidazole), has an EC50 of 66µM and a selectivity >50. We identified additional compounds with varying potencies by testing commercially-available chemical analogs. Time-of-addition experiments indicated that SRI 29365 effectively inhibits viral replication only if present during the early stages of viral infection. We isolated a virus with resistance to SRI 29365 and identified mutations in the transmembrane domain of the viral G protein genomic sequence that suggested that the compound inhibits G-protein mediated attachment of hRSV to cells. Additional experiments with multiple cell types indicated that SRI 29365 antiviral activity correlates with the binding of cell surface heparin by full-length G protein. Lastly, SRI 29365 did not reduce hRSV titers or morbidity/mortality in efficacy studies using a cotton rat model. Although SRI 29365 and analogs inhibit hRSV replication in vitro, this work suggests that the G-protein may not be a valid drug target in vivo.

Lifting the mask: identification of new small molecule inhibitors of uropathogenic Escherichia coli group 2 capsule biogenesis.

Uropathogenic Escherichia coli (UPEC) is the leading cause of community-acquired urinary tract infections (UTIs), with over 100 million UTIs occurring annually throughout the world. Increasing antimicrobial resistance among UPEC limits ambulatory care options, delays effective treatment, and may increase overall morbidity and mortality from complications such as urosepsis. The polysaccharide capsules of UPEC are an attractive target a therapeutic, based on their importance in defense against the host immune responses; however, the large number of antigenic types has limited their incorporation into vaccine development. The objective of this study was to identify small-molecule inhibitors of UPEC capsule biogenesis. A large-scale screening effort entailing 338,740 compounds was conducted in a cell-based, phenotypic screen for inhibition of capsule biogenesis in UPEC. The primary and concentration-response assays yielded 29 putative inhibitors of capsule biogenesis, of which 6 were selected for further studies. Secondary confirmatory assays identified two highly active agents, named DU003 and DU011, with 50% inhibitory concentrations of 1.0 µM and 0.69 µM, respectively. Confirmatory assays for capsular antigen and biochemical measurement of capsular sugars verified the inhibitory action of both compounds and demonstrated minimal toxicity and off-target effects. Serum sensitivity assays demonstrated that both compounds produced significant bacterial death upon exposure to active human serum. DU011 administration in mice provided near complete protection against a lethal systemic infection with the prototypic UPEC K1 isolate UTI89. This work has provided a conceptually new class of molecules to combat UPEC infection, and future studies will establish the molecular basis for their action along with efficacy in UTI and other UPEC infections.

Global Human-Kinase Screening Identifies Therapeutic Host Targets against Influenza.

During viral infection of human cells, host kinases mediate signaling activities that are used by all viruses for replication; therefore, targeting of host kinases is of broad therapeutic interest. Here, host kinases were globally screened during human influenza virus (H1N1) infection to determine the time-dependent effects of virus infection and replication on kinase function. Desthiobiotin-labeled analogs of adenosine triphosphate and adenosine diphosphate were used to probe and covalently label host kinases in infected cell lysates, and probe affinity was determined. Using infected human A549 cells, we screened for time-dependent signal changes and identified host kinases whose probe affinities differed significantly when compared to uninfected cells. Our screen identified 10 novel host kinases that have not been previously shown to be involved with influenza virus replication, and we validated the functional importance of these novel kinases during infection using targeted small interfering RNAs (siRNAs). The effects of kinase-targeted siRNA knockdowns on replicating virus levels were measured by quantitative reverse-transcription PCR and cytoprotection assays. We identified several novel host kinases that, when knocked down, enhanced or reduced the viral load in cell culture. This preliminary work represents the first screen of the changing host kinome in influenza virus-infected human cells.

Screening methods for influenza antiviral drug discovery.

INTRODUCTION: Influenza antiviral high-throughput screens have been extensive, and yet no approved influenza antivirals have been identified through high-throughput screening. This underscores the idea that development of successful screens should focus on the exploitation of the underrepresented viral targets and novel, therapeutic host targets. AREAS COVERED: The authors review conventional screening applications and emerging technologies with the potential to enhance influenza antiviral discovery. Real-world examples from the authors' work in biocontained environments are also provided. Future innovations are discussed, including the use of targeted libraries, multiplexed assays, proximity-based endpoint methods, non-laboratory-adapted virus strains, and primary cells, for immediate physiological relevance and translational applications. EXPERT OPINION: The lack of successful anti-influenza drug discovery using high-throughput screening should not deter future efforts. Increased understanding of the functions of viral targets and host-pathogen interactions has broadened the target reservoir. Future screening efforts should focus on identifying new drugs against unexploited viral and host targets using currently developed assays, and on the development of novel, innovative assays to discover new drugs with novel mechanisms. Innovative screens must be designed to identify compounds that specifically inhibit protein-protein or protein-RNA interactions or other virus/host factor interactions that are crucial for viral replication. Finally, the use of recent viral isolates, increased biocontainment (for highly-pathogenic strains), primary cell lines, and targeted compound libraries must converge in efficient high-throughput primary screens to generate high-content, physiologically-relevant data on compounds with robust antiviral activity.

High-throughput screening identifies a bisphenol inhibitor of SV40 large T antigen ATPase activity.

The authors conducted a high-throughput screening campaign for inhibitors of SV40 large T antigen ATPase activity to identify candidate antivirals that target the replication of polyomaviruses. The primary assay was adapted to 1536-well microplates and used to screen the National Institutes of Health Molecular Libraries Probe Centers Network library of 306 015 compounds. The primary screen had an Z value of ~0.68, signal/background = 3, and a high (5%) DMSO tolerance. Two counterscreens and two secondary assays were used to prioritize hits by EC(50), cytotoxicity, target specificity, and off-target effects. Hits that inhibited ATPase activity by >44% in the primary screen were tested in dose-response efficacy and eukaryotic cytotoxicity assays. After evaluation of hit cytotoxicity, drug likeness, promiscuity, and target specificity, three compounds were chosen for chemical optimization. Chemical optimization identified a class of bisphenols as the most effective biochemical inhibitors. Bisphenol A inhibited SV40 large T antigen ATPase activity with an IC(50) of 41 µM in the primary assay and 6.2 µM in a cytoprotection assay. This compound class is suitable as probes for biochemical investigation of large T antigen ATPase activity, but because of their cytotoxicity, further optimization is necessary for their use in studying polyomavirus replication in vivo.