Peter R. McDonald

Open Access High Throughput Drug Discovery in the Public Domain: A Mount Everest in the Making

High throughput screening (HTS) facilitates screening large numbers of compounds against a biochemical target of interest using validated biological or biophysical assays. In recent years, a significant number of drugs in clinical trails originated from HTS campaigns, validating HTS as a bona fide mechanism for hit finding. In the current drug discovery landscape, the pharmaceutical industry is embracing open innovation strategies with academia to maximize their research capabilities and to feed their drug discovery pipeline. The goals of academic research have therefore expanded from target identification and validation to probe discovery, chemical genomics, and compound library screening. This trend is reflected in the emergence of HTS centers in the public domain over the past decade, ranging in size from modestly equipped academic screening centers to well endowed Molecular Libraries Probe Centers Network (MLPCN) centers funded by the NIH Roadmap initiative. These centers facilitate a comprehensive approach to probe discovery in academia and utilize both classical and cutting-edge assay technologies for executing primary and secondary screening campaigns. The various facets of academic HTS centers as well as their implications on technology transfer and drug discovery are discussed, and a roadmap for successful drug discovery in the public domain is presented. New lead discovery against therapeutic targets, especially those involving the rare and neglected diseases, is indeed a Mount Everestonian size task, and requires diligent implementation of pharmaceutical industrys best practices for a successful outcome.

Screening of natural compounds as activators of the keap1-nrf2 pathway.

Nuclear factor erythroid 2-related factor 2 is a master regulator that promotes transcription of cytoprotective genes in response to oxidative/electrophilic stress. A large number of natural dietary compounds are thought to protect against oxidative stress, and a few have been reported to induce genes involved in antioxidant defense through activating nuclear factor erythroid 2-related factor 2. Therefore, a library of 54 natural compounds were collected to determine whether they are nuclear factor erythroid 2-related factor 2 activators and to compare their efficacy and potency to activate nuclear factor erythroid 2-related factor 2. The assay utilized AREc32 cells that contain a luciferase gene under the control of antioxidant response element promoters. Each natural compound was tested at 13 concentrations between 0.02 and 30 µM. Known nuclear factor erythroid 2-related factor 2 activators tert-butylhydroquinone and 2-cyano-3,12-dioxooleana-1,9-diene-28-imidazolide were used as positive controls in parallel with the natural compounds. Among the 54 tested natural compounds, andrographolide had the highest efficacy, followed by trans-chalcone, sulforaphane, curcumin, flavone, kahweol, and carnosol, all of which had better efficacy than tert-butylhydroquinone. Among the compounds tested, 2-cyano-3,12-dioxooleana-1,9-diene-28-imidazolide was the most potent, having an EC50 of 0.41 µM. Seven of the natural compounds, namely andrographolide, trans-chalcone, sulforaphane, curcumin, flavone, kahweol, and cafestol had lower EC50 values than tert-butylhydroquinone but higher than 2-cyano-3,12-dioxooleana-1,9-diene-28-imidazolide. The present study provides insights into which natural compounds activate the Keap1-nuclear factor erythroid 2-related factor 2 pathway and thus might be useful for detoxifying oxidative/electrophilic stress.

Implementation of a High-Throughput Screen for Identifying Small Molecules to Activate the Keap1-Nrf2-ARE Pathway

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that induces a battery of cytoprotective genes involved in antioxidant defense through binding to Antioxidant Response Elements (ARE) located in the promoter regions of these genes. To identify Nrf2 activators for the treatment of oxidative/electrophilic stress-induced diseases, the present study developed a high-throughput assay to evaluate Nrf2 activation using AREc32 cells that contain a luciferase gene under the control of ARE promoters. Of the 47,000 compounds screened, 238 (top 0.5% hits) of the chemicals increased the luminescent signal more than 14.4-fold and were re-tested at eleven concentrations in a range of 0.01–30 µM. Of these 238 compounds, 231 (96%) increased the luminescence signal in a concentration-dependent manner. Chemical structure relationship analysis of these 231 compounds indicated enrichment of four chemical scaffolds (diaryl amides and diaryl ureas, oxazoles and thiazoles, pyranones and thiapyranones, and pyridinones and pyridazinones). In addition, 30 of these 231 compounds were highly effective and/or potent in activating Nrf2, with a greater than 80-fold increase in luminescence, or an EC50 lower than 1.6 µM. These top 30 compounds were also screened in Hepa1c1c7 cells for an increase in Nqo1 mRNA, the prototypical Nrf2-target gene. Of these 30 compounds, 17 increased Nqo1 mRNA in a concentration-dependent manner. In conclusion, the present study documents the development, implementation, and validation of a high-throughput screen to identify activators of the Keap1-Nrf2-ARE pathway. Results from this screening identified Nrf2 activators, and provide novel insights into chemical scaffolds that might prevent oxidative/electrophilic stress-induced toxicity and carcinogenesis.

Targeting Epithelial-Mesenchymal Transition for Identification of Inhibitors for Pancreatic Cancer Cell Invasion and Tumor Spheres Formation

Background Pancreatic cancer has an enrichment of stem-like cancer cells (CSCs) that contribute to chemoresistant tumors prone to metastasis and recurrence. Drug screening assays based on cytotoxicity cannot identify specific CSC inhibitors, because CSCs comprise only a small portion of cancer cell population, and it is difficult to propagate stable CSC populations in vitro for high-throughput screening (HTS) assays. Based on the important role of cancer cell epithelial-to-mesenchymal transition (EMT) in promoting CSCs, we hypothesized that inhibition of EMT can be a useful strategy for inhibiting CSCs, and therefore a feasible approach for HTS can be built for identification of CSC inhibitors, based on assays detecting EMT inhibition. Methods An immunofluorescent assay was established and optimized for HTS to identify compounds that enhance E-cadherin expression, as a hallmark of inhibition of EMT. Four chemical libraries containing 41,472 compounds were screened in PANC-1 pancreatic cancer cell line. Positive hits were validated for EMT and CSC inhibition in vitro using sphere formation assay, western blotting, immune fluorescence, and scratch assay. Results Initial hits were refined to 73 compounds with a secondary screening, among which 17 exhibited concentration dependent induction of E-cadherin expression. Six compounds were selected for further study which belonged to 2 different chemical structural clusters. A novel compound 1-(benzylsulfonyl) indoline (BSI, Compound #38) significantly inhibited pancreatic cancer cell migration and invasion. BSI inhibited histone deacetylase, increased histone 4 acetylation preferably, resulting in E-cadherin up-regulation. BSI effectively inhibited tumor spheres formation. Six more analogues of BSI were tested for anti-migration and anti-CSC activities. Conclusion This study demonstrated a feasible approach for discovery of agents targeting EMT and CSCs using HTS, and identified a class of novel chemicals that could be developed as anti-EMT and anti-CSC drug leads.

Recent Trends in Collaborative, Open Source Drug Discovery

The pharmaceutical sector has traditionally played a predominant role in screening compounds against a molecular target and optimizing chemistry to develop drugs. The basic research studies in the academic sector have provided the pharmaceutical companies with many biological targets with potential impact on therapeutics. These well- defined contributions of the pharmaceutical and academic sectors in drug discovery field are being redefined to meet the fiscal and innovation challenges in the current drug discovery landscape. There is an increased capital and personnel investment in academia in the areas of highthroughput screening and technology transfer. The pharma has adopted an open innovation paradigm which seeks to complement internal intellect with external global talent and expertise, with the overall goal of expediting complex data interpretation and introduction of more effective and safe drugs. The pharma- academia sectors are collaborating on several mutually beneficial alliances and the changing landscape at pharma- academiainterface necessitates an urgent need for a better understanding of the role of academia in translational research and suitable technology transfer terms to sustain collaborative opportunities.The collaborative partnership between pharma and academia is expected to provide the required boost to identify novel targets and develop new and effective drugs.

The University of Kansas High-Throughput Screening Laboratory. Part II: enabling collaborative drug-discovery partnerships through cutting-edge screening technology

The University of Kansas High-Throughput Screening (KU HTS) core is a state-of-the-art drug-discovery facility with an entrepreneurial open-service policy, which provides centralized resources supporting public- and private-sector research initiatives. The KU HTS core was established in 2002 at the University of Kansas with support from an NIH grant and the state of Kansas. It collaborates with investigators from national and international academic, nonprofit and pharmaceutical organizations in executing HTS-ready assay development and screening of chemical libraries for target validation, probe selection, hit identification and lead optimization. This is part two of a contribution from the KU HTS laboratory.

The University of Kansas High-Throughput Screening laboratory. Part I: meeting drug-discovery needs in the heartland of America with entrepreneurial flair.

The University of Kansas High-Throughput Screening (KU HTS) core is a state-of-the-art drug-discovery facility with an entrepreneurial open-service policy, which provides centralized resources supporting public- and private-sector research initiatives. The KU HTS core applies pharmaceutical industry project-management principles in an academic setting by bringing together multidisciplinary teams to fill critical scientific and technology gaps, using an experienced team of industry-trained researchers and project managers. The KU HTS proactively engages in supporting grant applications for extramural funding, intellectual-property management and technology transfer. The KU HTS staff further provides educational opportunities for the KU faculty and students to learn cutting-edge technologies in drug-discovery platforms through seminars, workshops, internships and course teaching. This is the first instalment of a two-part contribution from the KU HTS laboratory.

Measuring and Statistically Testing the Size of the Effect of a Chemical Compound on a Continuous In-Vitro Pharmacological Response Through a New Statistical Model of Response Detection Limit

Biomolecular screening research frequently searches for the chemical compounds that are most likely to make a biochemical or cell-based assay system produce a strong continuous response. Several doses are tested with each compound and it is assumed that, if there is a dose–response relationship, the relationship follows a monotonic curve, usually a version of the median-effect equation. However, the null hypothesis of no relationship cannot be statistically tested using this equation. We used a linearized version of this equation to define a measure of pharmacological effect size, and use this measure to rank the investigated compounds in order of their overall capability to produce strong responses. The null hypothesis that none of the examined doses of a particular compound produced a strong response can be tested with this approach. The proposed approach is based on a new statistical model of the important concept of response detection limit, a concept that is usually neglected in the analysis of dose–response data with continuous responses. The methodology is illustrated with data from a study searching for compounds that neutralize the infection by a human immunodeficiency virus of brain glioblastoma cells.

Compound Ranking Based on a New Mathematical Measure of Effectiveness Using Time Course Data from Cell-Based Assays

The half maximal inhibitory concentration (IC₅₀) has several limitations that make it unsuitable for examining a large number of compounds in cytotoxicity studies, particularly when multiple exposure periods are tested. This article proposes a new approach to measure drug effectiveness, which allows ranking compounds according to their toxic effects on live cells. This effectiveness measure, which combines all exposure times tested, compares the growth rates of a particular cell line in the presence of the compound with its growth rate in the presence of DMSO alone. Our approach allows measuring a wider spectrum of toxicity than the IC₅₀ approach, and allows automatic analyses of a large number of compounds. It can be easily implemented in linear regression software, provides a comparable measure of effectiveness for each investigated compound (both toxic and non-toxic), and allows statistically testing the null hypothesis that a compound is non-toxic versus the alternative that it is toxic. Importantly, our approach allows defining an automated decision rule for deciding whether a compound is significantly toxic. As an illustration, we describe the results of a cellbased study of the cytotoxicity of 24 analogs of novobiocin, a C-terminal inhibitor of heat shock protein 90 (Hsp90); the compounds were ranked in order of cytotoxicity to a panel of 18 cancer cell lines and 1 normal cell line. Our approach may also be a good alternative to computing the half maximal effective concentration (EC₅₀) in studies searching for compounds that promote cell growth.

Comparative oncology approach to drug repurposing in osteosarcoma

Background Osteosarcoma is an orphan disease for which little improvement in survival has been made since the late 1980s. New drug discovery for orphan diseases is limited by the cost and time it takes to develop new drugs. Repurposing already approved FDA-drugs can help overcome this limitation. Another limitation of cancer drug discovery is the lack of preclinical models that accurately recapitulate what occurs in humans. For OS using dogs as a model can minimize this limitation as OS in canines develops spontaneously, is locally invasive and metastasizes to the lungs as it does in humans. Methods In our present work we used high-throughput screens to identify drugs from a library of 2,286 FDA-approved drugs that demonstrated selective growth inhibition against both human and canine OS cell lines. The identified lead compound was then tested for synergy with 7 other drugs that have demonstrated activity against OS. These results were confirmed with in vitro assays and an in vivo murine model of OS. Results We identified 13 drugs that demonstrated selective growth inhibition against both human and canine OS cell lines. Auranofin was selected for further in vitro combination drug screens. Auranofin showed synergistic effects with vorinostat and rapamycin on OS viability and apoptosis induction. Auranofin demonstrated single-agent growth inhibition in both human and canine OS xenografts, and cooperative growth inhibition was observed in combination with rapamycin or vorinostat. There was a significant decrease in Ki67-positive cells and an increase in cleaved caspase-3 levels in tumor tissues treated with a combination of auranofin and vorinostat or rapamycin. Conclusions Auranofin, alone or in combination with rapamycin or vorinostat, may be useful new treatment strategies for OS. Future studies may evaluate the efficacy of auranofin in dogs with OS as a prelude to human clinical evaluation.