Clinton Maddox

Antituberculosis Activity of the Molecular Libraries Screening Center Network Library

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.

Assay development and high throughput antiviral drug screening against Bluetongue virus

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 5000cells/well in medium containing 1% FBS and a multiplicity of infection at 0.01 in 384-well plate, with Z′-values ≥0.70, Coefficient of Variations ≥5.68 and signal-to-background ratio ≥7.10. This assay was further validated using a 9532 compound library. The fully validated assay was then used to screen the 194950 compound collection, which identified 693 compounds with >30% CPE inhibition. The 10-concentration dose response assay identified 185 structures with IC50 ≤100μM, 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.

Adapting Cell-Based Assays to the High Throughput Screening Platform: Problems Encountered and Lessons Learned

In recent years, cell-based phenotypic assays have emerged as an effective and robust addition to the array of assay technologies available for drug discovery in the high-throughput screening (HTS) arena. Previously, biochemical target-based assays have been the technology of choice. With the emergence of stem cells as a basis for a new screening technology, it is important to keep in mind the lessons that have been learned from the adaptation of existing stable cell lines onto the HTS drug discovery platform, with special consideration being given to assay miniaturization, liquid-handling complications, and instrument-introduced artifacts. We present an overview of the problems encountered with the implementation of multiple cell-based assays at the High Throughput Screening Center at Southern Research Institute as well as empirically defined effective solutions to these problems. These include examples of artifacts induced by temperature differences throughout the screening campaign, cell-plating conditions including the effect of room temperature incubation on assay consistency, DMSO carry over, and incubator-induced artifacts.

Discovery of Novel Benzoquinazolinones and Thiazoloimidazoles, Inhibitors of Influenza H5N1 and H1N1 Viruses, from a Cell-Based High-Throughput Screen

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 SI50 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.

Identification of novel small molecule activators of nuclear factor-κb with neuroprotective action via high-throughput screening

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.

A cell based high-throughput screening approach for the discovery of new inhibitors of respiratory syncytial virus.

BackgroundHuman 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® from MedImmune) that is limited to use in high risk pediatric patients. Thus, the discovery of new inhibitors for hRSV would be clinically beneficial.ResultsWe 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®). 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 μM. 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.ConclusionsA 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.

(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.

HTS-Driven Discovery of New Chemotypes with West Nile Virus Inhibitory Activity

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 μM 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.

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.

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.

A High-Throughput Screening Strategy to Overcome Virus Instability

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.