Publications (13)26.78 Total impact
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Article: A cell based high-throughput screening approach for the discovery of new inhibitors of respiratory syncytial virus.
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ABSTRACT: BACKGROUND: Human respiratory syncytial virus (hRSV) is a highly contagious pathogen and is the most common cause of bronchiolitis and pneumonia for infants and children under one year of age. Worldwide, greater than 33 million children under five years of age are affected by hRSV resulting in three million hospitalizations and 200,000 deaths. However, severe lower respiratory tract disease may occur at any age, especially among the elderly or those with compromised cardiac, pulmonary, or immune systems. There is no vaccine commercially available. Existing therapies for the acute infection are ribavirin and the prophylactic humanized monoclonal antibody (Synagis(R) from MedImmune) that is limited to use in high risk pediatric patients. Thus, the discovery of new inhibitors for hRSV would be clinically beneficial. RESULTS: We have developed and validated a 384-well cell-based, high-throughput assay that measures the cytopathic effect of hRSV (strain Long) in HEp-2 cells using a luminescent-based detection system for signal endpoint (Cell Titer Glo(R)). The assay is sensitive and robust, with Z factors greater than 0.8, signal to background greater than 35, and signal to noise greater than 24. Utilizing this assay, 313,816 compounds from the Molecular Libraries Small Molecule Repository were screened at 10 muM. We identified 7,583 compounds that showed greater than 22% CPE inhibition in the primary screen. The top 2,500 compounds were selected for confirmation screening and 409 compounds showed at least 50% inhibition of CPE and were considered active. We selected fifty-one compounds, based on potency, selectivity and chemical tractability, for further evaluation in dose response and secondary assays Several compounds had SI50 values greater than 3, while the most active compound displayed an SI50 value of 58.9. CONCLUSIONS: A robust automated luminescent-based high throughput screen that measures the inhibition of hRSV-induced cytopathic effect in HEp-2 cells for the rapid identification of potential inhibitors from large compound libraries has been developed, optimized and validated. The active compounds identified in the screen represent different classes of molecules, including aryl sulfonylpyrrolidines which have not been previously identified as having anti-hRSV activity.Virology Journal 01/2013; 10(1):19. · 2.34 Impact Factor -
Article: (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.
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ABSTRACT: 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 (EC(50) = 2.3 ± 0.8 μM) with marginal cytotoxicity (CC(50) = 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.Journal of Medicinal Chemistry 10/2012; 55(20):8582-7. · 4.80 Impact Factor -
Article: High throughput screening of a library based on kinase inhibitor scaffolds against Mycobacterium tuberculosis H37Rv.
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ABSTRACT: Kinase targets are being pursued in a variety of diseases beyond cancer, including immune and metabolic as well as viral, parasitic, fungal and bacterial. In particular, there is a relatively recent interest in kinase and ATP-binding targets in Mycobacterium tuberculosis in order to identify inhibitors and potential drugs for essential proteins that are not targeted by current drug regimens. Herein, we report the high throughput screening results for a targeted library of approximately 26,000 compounds that was designed based on current kinase inhibitor scaffolds and known kinase binding sites. The phenotypic data presented herein may form the basis for selecting scaffolds/compounds for further enzymatic screens against specific kinase or other ATP-binding targets in Mycobacterium tuberculosis based on the apparent activity against the whole bacteria in vitro.Tuberculosis (Edinburgh, Scotland) 06/2011; 92(1):72-83. · 2.54 Impact Factor -
Article: A high-throughput screen with isogenic PTEN+/+ and PTEN-/- cells identifies CID1340132 as a novel compound that induces apoptosis in PTEN and PIK3CA mutant human cancer cells.
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ABSTRACT: The PTEN tumor suppressor gene is one of the most commonly mutated genes in human cancer. Because inactivation of PTEN is a somatic event, PTEN mutations represent an important genetic difference between cancer cells and normal cells and therefore a potential anticancer drug target. However, it remains a substantial challenge to identify compounds that target loss-of-function events such as mutations of tumor suppressors. In an effort to identify small molecules that preferentially kill cells with mutations of PTEN, the authors developed and implemented a high-throughput, paired cell-based screen composed of parental HCT116 cells and their PTEN gene-targeted derivatives. From 138 758 compounds tested, two hits were identified, and one, N'-[(1-benzyl-1H-indol-3-yl)methylene]benzenesulfonohydrazide (CID1340132), was further studied using a variety of cell-based models, including HCT116, MCF10A, and HEC1A cells with targeted deletion of either their PTEN or PIK3CA genes. Preferential killing of PTEN and PIK3CA mutant cells was accompanied by DNA damage, inhibition of DNA synthesis, and apoptosis. Taken together, these data validate a cell-based screening approach for identifying lead compounds that target cells with specific tumor suppressor gene mutations and describe a novel compound with preferential killing activity toward PTEN and PIK3CA mutant cells.Journal of Biomolecular Screening 02/2011; 16(4):383-93. · 2.05 Impact Factor -
Article: Identification of novel small molecule activators of nuclear factor-κB with neuroprotective action via high-throughput screening.
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ABSTRACT: Neuronal noncytokine-dependent p50/p65 nuclear factor-κB (the primary NF-κB complex in the brain) activation has been shown to exert neuroprotective actions. Thus neuronal activation of NF-κB could represent a viable neuroprotective target. We have developed a cell-based assay able to detect NF-κB expression enhancement, and through its use we have identified small molecules able to up-regulate NF-κB expression and hence trigger its activation in neurons. We have successfully screened approximately 300,000 compounds and identified 1,647 active compounds. Cluster analysis of the structures within the hit population yielded 14 enriched chemical scaffolds. One high-potency and chemically attractive representative of each of these 14 scaffolds and four singleton structures were selected for follow-up. The experiments described here highlighted that seven compounds caused noncanonical long-lasting NF-κB activation in primary astrocytes. Molecular NF-κB docking experiments indicate that compounds could be modulating NF-κB-induced NF-κB expression via enhancement of NF-κB binding to its own promoter. Prototype compounds increased p65 expression in neurons and caused its nuclear translocation without affecting the inhibitor of NF-κB (I-κB). One of the prototypical compounds caused a large reduction of glutamate-induced neuronal death. In conclusion, we have provided evidence that we can use small molecules to activate p65 NF-κB expression in neurons in a cytokine receptor-independent manner, which results in both long-lasting p65 NF-κB translocation/activation and decreased glutamate neurotoxicity.Journal of Neuroscience Research 11/2010; 89(1):58-72. · 2.74 Impact Factor -
Article: A high-throughput screening strategy to overcome virus instability.
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ABSTRACT: Respiratory syncytial virus (RSV) is a widely distributed pathogen that causes severe disease in children, the elderly, and immunocompromised individuals. Both vaccine development and drug discovery have been hampered by the inherent instability of the virus itself. Drug discovery efforts have had limited success due, at least in part, to the lack of an antiviral assay robust enough for high-throughput screening. Instability of the purified virus has long been recognized as a problem in RSV research and has been a major hurdle to producing a virus-based screening assay. Using frozen RSV-infected cells as the source of infectious material, we have overcome the problem of virus instability and validated a cell-based high-throughput screening assay to screen for inhibitors of RSV-induced cytopathic effect. The assay was validated with 1,280 compounds identified as potentially active against RSV (Long strain) in a virus-based screen. To date over 300,000 compounds have been screened over several months with minimal variability in cell or virus controls. Long-term assay stability studies are still in progress.Assay and Drug Development Technologies 11/2010; 9(2):184-90. · 1.73 Impact Factor -
Article: Discovery of novel benzoquinazolinones and thiazoloimidazoles, inhibitors of influenza H5N1 and H1N1 viruses, from a cell-based high-throughput screen.
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ABSTRACT: A highly reproducible and robust cell-based high-throughput screening (HTS) assay was adapted for screening of small molecules for antiviral activity against influenza virus strain A/Vietnam/1203/2004 (H5N1). The NIH Molecular Libraries Small Molecule Repository (MLSMR) Molecular Libraries Screening Centers Network (MLSCN) 100,000-compound library was screened at 50 µM. The "hit" rate (>25% inhibition of the viral cytopathic effect) from the single-dose screen was 0.32%. The hits were evaluated for their antiviral activity, cell toxicity, and selectivity in dose-response experiments. The screen yielded 5 active compounds (SI value >3). One compound showed an SI(50) value of greater than 3, 3 compounds had SI values ranging from greater than 14 to 34, and the most active compound displayed an SI value of 94. The active compounds represent 2 different classes of molecules, benzoquinazolinones and thiazoloimidazoles, which have not been previously identified as having antiviral/anti-influenza activity. These molecules were also effective against influenza A/California/04/2009 virus (H1N1) and other H1N1 and H5N1 virus strains in vitro but not H3N2 strains. Real-time qRT-PCR results reveal that these chemotypes significantly reduced M1 RNA levels as compared to the no-drug influenza-infected Madin Darby canine kidney cells.Journal of Biomolecular Screening 11/2010; 16(1):73-81. · 2.05 Impact Factor -
Chapter: A Cell Based Assay for the Identification of Lead Compounds with Anti-Viral Activity Against West Nile Virus
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ABSTRACT: West Nile Virus (WNV) is a mosquito-borne pathogen that causes febrile illness and, occasionally, encephalitis when transmitted to humans. Infection can cause significant health problems such as West Nile fever and neuroinvasive disease. The goal of the assay was to identify and develop chemical probes that inhibit in vitro replication of WNV. An unbiased phenotypic whole virus cell-based assay was utilized to select chemical probes that inhibit WNV propagation without adversely affecting the host cell. The two probe candidates described in this report, ML143 (CID-2557019) and ML142 (CID-928277), provide two small molecules that demonstrate modest yet useful potency against the West Nile Virus, but with a robust margin of selectivity against cell cytotoxicity, providing potentially useful evaluation tools for small animal toxicity and efficacy studies.01/2010; -
Article: HTS-driven discovery of new chemotypes with West Nile Virus inhibitory activity.
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ABSTRACT: West Nile virus (WNV) is a positive sense, single-stranded RNA virus that can cause illness in humans when transmitted via mosquito vectors. Unfortunately, no antivirals or vaccines are currently available, and therefore efficient and safe antivirals are urgently needed. We developed a high throughput screen to discover small molecule probes that inhibit virus infection of Vero E6 cells. A primary screen of a 13,001 compound library at a 10 microM final concentration was conducted using the 384-well format. Z' values ranged from 0.54-0.83 with a median of 0.74. Average S/B was 17 and S/N for each plate ranged from 10.8 to 23.9. Twenty-six compounds showed a dose response in the HT screen and were further evaluated in a time of addition assay and in a titer reduction assay. Seven compounds showed potential as small molecule probes directed at WNV. The hit rate from the primary screen was 0.185% (24 compounds out of 13,001 compounds) and from the secondary screens was 0.053% (7 out of 13,001 compounds) respectively.Molecules 01/2010; 15(3):1690-704. · 2.39 Impact Factor -
Article: Antituberculosis activity of the molecular libraries screening center network library.
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ABSTRACT: There is an urgent need for the discovery and development of new antitubercular agents that target novel biochemical pathways and treat drug-resistant forms of the disease. One approach to addressing this need is through high-throughput screening of drug-like small molecule libraries against the whole bacterium in order to identify a variety of new, active scaffolds that will stimulate additional biological research and drug discovery. Through the Molecular Libraries Screening Center Network, the NIAID Tuberculosis Antimicrobial Acquisition and Coordinating Facility tested a 215,110-compound library against Mycobacterium tuberculosis strain H37Rv. A medicinal chemistry survey of the results from the screening campaign is reported herein.Tuberculosis (Edinburgh, Scotland) 09/2009; 89(5):354-63. · 2.54 Impact Factor -
Article: Assay development and high-throughput antiviral drug screening against Bluetongue virus.
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ABSTRACT: Bluetongue virus (BTV) infection is one of the most important diseases of domestic livestock. There are no antivirals available against BTV disease. In this paper, we present the development, optimization and validation of an in vitro cell-based high-throughput screening (HTS) assay using the luminescent-based CellTiter-Glo reagent to identify novel antivirals against BTV. Conditions of the cytopathic effect (CPE)-based assay were optimized at cell density of 5000 cells/well in medium containing 1% FBS and a multiplicity of infection at 0.01 in 384-well plate, with Z'-values > or = 0.70, Coefficient of Variations > or = 5.68 and signal-to-background ratio > or = 7.10. This assay was further validated using a 9532 compound library. The fully validated assay was then used to screen the 194,950 compound collection, which identified 693 compounds with >30% CPE inhibition. The 10-concentration dose response assay identified 185 structures with IC(50) < or =100 microM, out of which 42 compounds were grouped into six analog series corresponding to six scaffolds enriched within the active set compared to their distribution in the library. The CPE-based assay development demonstrated its robustness and reliability, and its application in the HTS campaign will make significant contribution to the antiviral drug discovery against BTV disease.Antiviral research 06/2009; 83(3):267-73. · 3.61 Impact Factor -
Chapter: Identification of a Series of Quinazolinediones as Potent, Selective, Post-Entry Inhibitors of Human Respiratory Syncytial Virus (hRSV) via a Cell-Based High Throughput Screen and Chemical Optimization
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ABSTRACT: Respiratory Syncytial Virus (RSV) is the most common cause of bronchiolitis and pneumonia among infants under one year of age. Most children will be infected with RSV prior to their second birthday, leading to 75,000-125,000 hospitalizations and medical costs exceeding $650 million annually. The virus is highly contagious and is associated with substantial morbidity and mortality. Nevertheless, severe lower respiratory tract disease may occur at any age, especially among the elderly or those with compromised cardiac, pulmonary, or immune systems. FDA-approved drugs for the acute infection are ribavirin and the prophylactic humanized monoclonal antibody, Synagis(®), which is limited to use in high risk pediatric patients. Due to the lack of a vaccine and the presence of toxicological limitations in existing therapies, there is substantial need for effective treatments with an improved profile. Of the 313,816 Molecular Libraries Small Molecule Repository (MLSMR) compounds screened in a cell-based, RSV inhibition assay, 51 compounds were selected based on potency, selectivity and chemical tractability for further evaluation in dose response and secondary assays. Collaboration between the assay provider at the University of Louisville, the screening center at Southern Research Institute and the University of Kansas Specialized Chemistry Center narrowed the structure activity relationship (SAR) focus to three scaffolds. The probe, ML275, resulted from structural modification and optimization of a quinazolinedione chemical series, generating a compound with an antiviral EC50 value of 0.81 ± 0.75 μM and a 247-fold selectivity index (SI) for antiviral activity over HEp-2 cell cytotoxicity. Additionally, ML275 demonstrated a 6.7 log reduction of in vitro viral titer, or reduction by approximately 5,000,000-fold. ML275, determined to be a post-entry inhibitor of viral replication, has been broadly profiled for off-target liabilities and assessed for PAMPA permeability and hepatocyte toxicity. -
Chapter: A Cell Based HTS Approach for the Discovery of New Inhibitors of RSV
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ABSTRACT: Respiratory Syncitial Virus (RSV) is a highly contagious member of the family Paramyxoviridae. It has been estimated that most children will be infected with RSV prior to their second birthday generating an estimated 75,000 – 125,000 hospitalizations in children. RSV is associated with substantial morbidity and mortality and is the most common cause of bronchiolitis and pneumonia among infants and children under one year of age. Nevertheless, severe lower respiratory tract disease may occur at any age, especially among the elderly or among those with compromised cardiac, pulmonary, or immune systems. There are no vaccines commercially available. Existing therapies for the acute infection are ribavirin and the prophylactic humanized monoclonal antibody (Synagis(®) from MedImmune) that is limited to use in high risk pediatric patients. The economic impact of RSV infections due to hospitalizations and indirect medical costs is greater than $650 million annually; thus, finding inhibitors for RSV would be extensively valuable. The cell-based RSV inhibition screen produced novel compounds that may be developed further into potential prophylactic therapies. Of the 313,816 Molecular Libraries Small Molecule Repository (MLSMR) compounds screened, 51 compounds were selected, based on potency, selectivity and chemical tractability, for further evaluation in dose response and secondary assays. Collaboration between the assay provider, the screening center at Southern Research Institute and the University of Kansas Specialized Chemistry Center narrowed the SAR focus to three scaffolds. The probe, ML232, had an antiviral EC50 value of 2.25 μM and a 13.7-fold Selectivity Index (SI) for antiviral activity over mammalian cell cytotoxicity.
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2012
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Southern Research Institute
Birmingham, AL, USA
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