Targeting Chikungunya Virus Replication by Benzoannulene Inhibitors.

A benzo[6]annulene, 4-(tert-butyl)-N-(3-methoxy-5,6,7,8-tetrahydronaphthalen-2-yl) benzamide (1a), was identified as an inhibitor against Chikungunya virus (CHIKV) with antiviral activity EC90 = 1.45 µM and viral titer reduction (VTR) of 2.5 log at 10 µM with no observed cytotoxicity (CC50 = 169 µM) in normal human dermal fibroblast cells. Chemistry efforts to improve potency, efficacy, and drug-like properties of 1a resulted in a novel lead compound 8q, which possessed excellent cellular antiviral activity (EC90 = 270 nM and VTR of 4.5 log at 10 µM) and improved liver microsomal stability. CHIKV resistance to an analog of 1a, compound 1c, tracked to a mutation in the nsP3 macrodomain. Further mechanism of action studies showed compounds working through inhibition of human dihydroorotate dehydrogenase in addition to CHIKV nsP3 macrodomain. Moderate efficacy was observed in an in vivo CHIKV challenge mouse model for compound 8q as viral replication was rescued from the pyrimidine salvage pathway.

Discovery of novel inhibitors of ribosome biogenesis by innovative high throughput screening strategies.

Over the past two decades, ribosome biogenesis has emerged as an attractive target for cancer treatment. In this study, two high-throughput screens were used to identify ribosome biogenesis inhibitors. Our primary screen made use of the HaloTag selective labeling strategy to identify compounds that decreased the abundance of newly synthesized ribosomes in A375 malignant melanoma cells. This screen identified 5786 hit compounds. A subset of those initial hit compounds were tested using a secondary screen that directly measured pre-ribosomal RNA (pre-rRNA) abundance as a reporter of rRNA synthesis rate, using quantitative RT-PCR. From the secondary screen, we identified two structurally related compounds that are potent inhibitors of rRNA synthesis. These two compounds, Ribosome Biogenesis Inhibitors 1 and 2 (RBI1 and RBI2), induce a substantial decrease in the viability of A375 cells, comparable to the previously published ribosome biogenesis inhibitor CX-5461. Anchorage-independent cell growth assays further confirmed that RBI2 inhibits cell growth and proliferation. Thus, the RBI compounds have promising properties for further development as potential cancer chemotherapeutics.

Studies on Dibenzylamines as Inhibitors of Venezuelan Equine Encephalitis Virus.

Alphaviruses are arthropod-transmitted members of the Togaviridae family that can cause severe disease in humans, including debilitating arthralgia and severe neurological complications. Currently, there are no approved vaccines or antiviral therapies directed against the alphaviruses, and care is limited to treating disease symptoms. A phenotypic cell-based high-throughput screen was performed to identify small molecules that inhibit the replication of Venezuelan Equine Encephalitis Virus (VEEV). The compound, 1-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-N-(3-fluoro-4-methoxybenzyl)ethan-1-amine (1), was identified as a highly active, potent inhibitor of VEEV with an effective concentration for 90% inhibition of virus (EC90) of 0.89 µM and 7.49 log reduction in virus titers at 10 µM concentration. These data suggest that further investigation of compound 1 as an antiviral therapeutic against VEEV, and perhaps other alphaviruses, is warranted. Experiments suggested that the antiviral activity of compound 1 is directed at an early step in the VEEV replication cycle by blocking viral RNA and protein synthesis.

High-throughput screening and Bayesian machine learning for copper-dependent inhibitors of Staphylococcus aureus.

One potential source of new antibacterials is through probing existing chemical libraries for copper-dependent inhibitors (CDIs), i.e., molecules with antibiotic activity only in the presence of copper. Recently, our group demonstrated that previously unknown staphylococcal CDIs were frequently present in a small pilot screen. Here, we report the outcome of a larger industrial anti-staphylococcal screen consisting of 40 771 compounds assayed in parallel, both in standard and in copper-supplemented media. Ultimately, 483 had confirmed copper-dependent IC50 values under 50 µM. Sphere-exclusion clustering revealed that these hits were largely dominated by sulfur-containing motifs, including benzimidazole-2-thiones, thiadiazines, thiazoline formamides, triazino-benzimidazoles, and pyridinyl thieno-pyrimidines. Structure-activity relationship analysis of the pyridinyl thieno-pyrimidines generated multiple improved CDIs, with activity likely dependent on ligand/ion coordination. Molecular fingerprint-based Bayesian classification models were built using Discovery Studio and Assay Central, a new platform for sharing and distributing cheminformatic models in a portable format, based on open-source tools. Finally, we used the latter model to evaluate a library of FDA-approved drugs for copper-dependent activity in silico. Two anti-helminths, albendazole and thiabendazole, scored highly and are known to coordinate copper ions, further validating the model's applicability.

Mycobacterium tuberculosis High-Throughput Screening.

High-throughput screening is a valuable way to identify hit compounds that combined with a robust medicinal chemistry program could lead to the identification of new antibiotics. Here, we discuss our method for screening large compound libraries with virulent Mycobacterium tuberculosis, possibly one of the more difficult bacteria to use because of its slow growth and assignment to Biosafety Level-3 by the CDC and NIH. The principles illuminated here, however, are relevant to the execution of most bacteria high-throughput screens.

Adapting high-throughput screening methods and assays for biocontainment laboratories.

High-throughput screening (HTS) has been integrated into the drug discovery process, and multiple assay formats have been widely used in many different disease areas but with limited focus on infectious agents. In recent years, there has been an increase in the number of HTS campaigns using infectious wild-type pathogens rather than surrogates or biochemical pathogen-derived targets. Concurrently, enhanced emerging pathogen surveillance and increased human mobility have resulted in an increase in the emergence and dissemination of infectious human pathogens with serious public health, economic, and social implications at global levels. Adapting the HTS drug discovery process to biocontainment laboratories to develop new drugs for these previously uncharacterized and highly pathogenic agents is now feasible, but HTS at higher biosafety levels (BSL) presents a number of unique challenges. HTS has been conducted with multiple bacterial and viral pathogens at both BSL-2 and BSL-3, and pilot screens have recently been extended to BSL-4 environments for both Nipah and Ebola viruses. These recent successful efforts demonstrate that HTS can be safely conducted at the highest levels of biological containment. This review outlines the specific issues that must be considered in the execution of an HTS drug discovery program for high-containment pathogens. We present an overview of the requirements for HTS in high-level biocontainment laboratories.

Development of (E)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-5-nitro-N-phenylbenzamide, ML336: Novel 2-amidinophenylbenzamides as potent inhibitors of venezuelan equine encephalitis virus.

Venezuelan equine encephalitis virus (VEEV) is an emerging pathogenic alphavirus that can cause significant disease in humans. Given the absence of therapeutic options available and the significance of VEEV as a weaponized agent, an optimization effort was initiated around a quinazolinone screening hit 1 with promising cellular antiviral activity (EC50 = 0.8 µM), limited cytotoxic liability (CC50 > 50 µM), and modest in vitro efficacy in reducing viral progeny (63-fold at 5 µM). Scaffold optimization revealed a novel rearrangement affording amidines, specifically compound 45, which was found to potently inhibit several VEEV strains in the low nanomolar range without cytotoxicity (EC50 = 0.02-0.04 µM, CC50 > 50 µM) while limiting in vitro viral replication (EC90 = 0.17 µM). Brain exposure was observed in mice with 45. Significant protection was observed in VEEV-infected mice at 5 mg kg(-1) day(-1) and viral replication appeared to be inhibited through interference of viral nonstructural proteins.

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.

Discovery of a novel compound with anti-venezuelan equine encephalitis virus activity that targets the nonstructural protein 2.

Alphaviruses present serious health threats as emerging and re-emerging viruses. Venezuelan equine encephalitis virus (VEEV), a New World alphavirus, can cause encephalitis in humans and horses, but there are no therapeutics for treatment. To date, compounds reported as anti-VEEV or anti-alphavirus inhibitors have shown moderate activity. To discover new classes of anti-VEEV inhibitors with novel viral targets, we used a high-throughput screen based on the measurement of cell protection from live VEEV TC-83-induced cytopathic effect to screen a 340,000 compound library. Of those, we identified five novel anti-VEEV compounds and chose a quinazolinone compound, CID15997213 (IC50 = 0.84 µM), for further characterization. The antiviral effect of CID15997213 was alphavirus-specific, inhibiting VEEV and Western equine encephalitis virus, but not Eastern equine encephalitis virus. In vitro assays confirmed inhibition of viral RNA, protein, and progeny synthesis. No antiviral activity was detected against a select group of RNA viruses. We found mutations conferring the resistance to the compound in the N-terminal domain of nsP2 and confirmed the target residues using a reverse genetic approach. Time of addition studies showed that the compound inhibits the middle stage of replication when viral genome replication is most active. In mice, the compound showed complete protection from lethal VEEV disease at 50 mg/kg/day. Collectively, these results reveal a potent anti-VEEV compound that uniquely targets the viral nsP2 N-terminal domain. While the function of nsP2 has yet to be characterized, our studies suggest that the protein might play a critical role in viral replication, and further, may represent an innovative opportunity to develop therapeutic interventions for alphavirus infection.

A BSL-4 high-throughput screen identifies sulfonamide inhibitors of Nipah virus.

Nipah virus is a biosafety level 4 (BSL-4) pathogen that causes severe respiratory illness and encephalitis in humans. To identify novel small molecules that target Nipah virus replication as potential therapeutics, Southern Research Institute and Galveston National Laboratory jointly developed an automated high-throughput screening platform that is capable of testing 10,000 compounds per day within BSL-4 biocontainment. Using this platform, we screened a 10,080-compound library using a cell-based, high-throughput screen for compounds that inhibited the virus-induced cytopathic effect. From this pilot effort, 23 compounds were identified with EC50 values ranging from 3.9 to 20.0 µM and selectivities >10. Three sulfonamide compounds with EC50 values <12 µM were further characterized for their point of intervention in the viral replication cycle and for broad antiviral efficacy. Development of HTS capability under BSL-4 containment changes the paradigm for drug discovery for highly pathogenic agents because this platform can be readily modified to identify prophylactic and postexposure therapeutic candidates against other BSL-4 pathogens, particularly Ebola, Marburg, and Lassa viruses.

(S)-N-(2,5-Dimethylphenyl)-1-(quinoline-8-ylsulfonyl)pyrrolidine-2-carboxamide as a small molecule inhibitor probe for the study of respiratory syncytial virus infection.

A high-throughput, cell-based screen was used to identify chemotypes as inhibitors for human respiratory syncytial virus (hRSV). Optimization of a sulfonylpyrrolidine scaffold resulted in compound 5o that inhibited a virus-induced cytopathic effect in the entry stage of infection (EC50 = 2.3 ± 0.8 µM) with marginal cytotoxicity (CC50 = 30.9 ± 1.1 µM) and reduced viral titer by 100-fold. Compared to ribavirin, sulfonylpyrrolidine 5o demonstrated an improved in vitro potency and selectivity index.

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.