High-throughput Chemical Screening Research Articles

Novel Chemical Suppressors of Long QT Syndrome Identified by an In Vivo Functional Screen

Genetic long QT (LQT) syndrome is a life-threatening disorder caused by mutations that result in prolongation of cardiac repolarization. Recent work has demonstrated that a zebrafish model of LQT syndrome faithfully recapitulates several features of human disease, including prolongation of ventricular action potential duration, spontaneous early afterdepolarizations, and 2:1 atrioventricular block in early stages of development. Because of their transparency, small size, and absorption of small molecules from their environment, zebrafish are amenable to high-throughput chemical screens. We describe a small-molecule screen using the zebrafish KCNH2 mutant breakdance to identify compounds that can rescue the LQT type 2 phenotype. Zebrafish breakdance embryos were exposed to test compounds at 48 hours of development and scored for rescue of 2:1 atrioventricular block at 72 hours in a 96-well format. Only compounds that suppressed the LQT phenotype in 3 of 3 fish were considered hits. Screen compounds were obtained from commercially available small-molecule libraries (Prestwick and Chembridge). Initial hits were confirmed with dose-response testing and time-course studies. Optical mapping with the voltage-sensitive dye di-4 ANEPPS was performed to measure compound effects on cardiac action potential durations. Screening of 1200 small molecules resulted in the identification of flurandrenolide and 2-methoxy-N-(4-methylphenyl) benzamide (2-MMB) as compounds that reproducibly suppressed the LQT phenotype. Optical mapping confirmed that treatment with each compound caused shortening of ventricular action potential durations. Structure activity studies and steroid receptor knockdown suggest that flurandrenolide functions via the glucocorticoid signaling pathway. Using a zebrafish model of LQT type 2 syndrome in a high-throughput chemical screen, we have identified 2 compounds, flurandrenolide and the novel compound 2-MMB, as small molecules that rescue the zebrafish LQT type 2 syndrome by shortening the ventricular action potential duration. We provide evidence that flurandrenolide functions via the glucocorticoid receptor-mediated pathway. These 2 molecules and future discoveries from this screen should yield novel tools for the study of cardiac electrophysiology and may lead to novel therapeutics for human LQT patients.

Yeast DEL Assay Detects Protection against Radiation-Induced Cytotoxicity and Genotoxicity: Adaptation of a Microtiter Plate Version

The DEL assay in yeast detects DNA deletions that are inducible by many carcinogens. Here we use the colorimetric agent MTS to adapt the yeast DEL assay for microwell plate measurement of ionizing radiation-induced cell killing and DNA deletions. Using the microwell-based DEL assay, cell killing and genotoxic DNA deletions both increased with radiation dose between 0 and 2000 Gy. We used the microwell-based DEL assay to assess the effectiveness of varying concentrations of five different radioprotectors, N-acetyl-l-cysteine, l-ascorbic acid, DMSO, Tempol and Amifostine, and one radiosensitizer, 5-bromo-2-deoxyuridine. The microwell format of the DEL assay was able to successfully detect protection against and sensitization to both radiation-induced cytotoxicity and genotoxicity. Such radioprotection and sensitization detected by the microwell-based DEL assay was validated and compared with similar measurements made using the traditional agar-based assay format. The yeast DEL assay in microwell format is an effective tool for rapidly detecting chemical protectors and sensitizers to ionizing radiation and is automatable for chemical high-throughput screening purposes.

Stem Cell Expansion

The ability to expand hematopoietic stem cells (HSCs) during ex-vivo culture has been an important goal for over 20 years. Using a high-throughput chemical screen, Boitano et al. (p. [1345][1], published online 5 August; see the Perspective by [Sauvageau and Humphries][2] ) found that a purine derivative, StemRegenin1 (SR1), promoted the expansion of human HSCs. Treatment of HSCs with SR1 (which blocked the activity of the aryl hydrocarbon receptor) led to the expansion of CD34+ cells and a 12 to 17-fold increase in the number of HSCs that engraft immune deficient mice. [1]: /lookup/doi/10.1126/science.1191536 [2]: /lookup/doi/10.1126/science.1195173

Zebrafish: A tool for chemical screens

The zebrafish has proven to be an invaluable vertebrate animal model for developmental biology. Recent technological advances have added an arsenal of tools to expand its use into the realm of drug discovery. This includes methodology to generate transgenic reporter lines that allow for the direct visualization of fluorescent markers in live embryos. With the addition of automated imaging and analysis of embryos treated with small molecules, these innovations have expanded its utility into high throughput chemical screens. This review will highlight some of these advances that have propelled zebrafish as a tool for drug discovery.

Aryl Hydrocarbon Receptor Antagonists Promote the Expansion of Human Hematopoietic Stem Cells

Although practiced clinically for more than 40 years, the use of hematopoietic stem cell (HSC) transplants remains limited by the ability to expand these cells ex vivo. An unbiased screen with primary human HSCs identified a purine derivative, StemRegenin 1 (SR1), that promotes the ex vivo expansion of CD34+ cells. Culture of HSCs with SR1 led to a 50-fold increase in cells expressing CD34 and a 17-fold increase in cells that retain the ability to engraft immunodeficient mice. Mechanistic studies show that SR1 acts by antagonizing the aryl hydrocarbon receptor (AHR). The identification of SR1 and AHR modulation as a means to induce ex vivo HSC expansion should facilitate the clinical use of HSC therapy.

Emergence of Chemical Biology Approaches to the RNAi/miRNA Pathway

RNA interference (RNAi) is a well-conserved mechanism that uses small noncoding RNAs to silence gene expression posttranscriptionally. Gene regulation by RNAi is now recognized as one of the major regulatory pathways in eukaryotic cells. Although the main components of the RNAi/miRNA pathway have been identified, the molecular mechanisms regulating the activity of the RNAi/miRNA pathway have only begun to emerge within the last couple of years. Recently, high-throughput reporter assays to monitor the activity of the RNAi/miRNA pathway have been developed and used for proof-of-concept pilot screens. Both inhibitors and activators of the RNAi/miRNA pathway have been found. Although still in its infancy, a chemical biology approach using high-throughput chemical screens should open up a new avenue for dissecting the RNAi/miRNA pathway, as well as developing novel RNAi- or miRNA-based therapeutic interventions.

Identification of the ENT1 Antagonists Dipyridamole and Dilazep as Amplifiers of Oncolytic Herpes Simplex Virus-1 Replication

Oncolytic herpes simplex virus-1 (oHSV) vectors selectively replicate in tumor cells, where they kill through oncolysis while sparing normal cells. One of the drawbacks of oHSV vectors is their limited replication and spread to neighboring cancer cells. Here, we report the outcome of a high-throughput chemical library screen to identify small-molecule compounds that augment the replication of oHSV G47Delta. Of the 2,640-screened bioactives, 6 compounds were identified and subsequently validated for enhanced G47Delta replication. Two of these compounds, dipyridamole and dilazep, interfered with nucleotide metabolism by potently and directly inhibiting the equilibrative nucleoside transporter-1 (ENT1). Replicative amplification promoted by dipyridamole and dilazep were dependent on HSV mutations in ICP6, the large subunit of ribonucleotide reductase. Our results indicate that ENT1 antagonists augment oHSV replication in tumor cells by increasing cellular ribonucleoside activity.

Inhibition of Hsp70 by Methylene Blue Affects Signaling Protein Function and Ubiquitination and Modulates Polyglutamine Protein Degradation

The Hsp90/Hsp70-based chaperone machinery regulates the activity and degradation of many signaling proteins. Cycling with Hsp90 stabilizes client proteins, whereas Hsp70 interacts with chaperone-dependent E3 ubiquitin ligases to promote protein degradation. To probe these actions, small molecule inhibitors of Hsp70 would be extremely useful; however, few have been identified. Here we test the effects of methylene blue, a recently described inhibitor of Hsp70 ATPase activity, in three well established systems of increasing complexity. First, we demonstrate that methylene blue inhibits the ability of the purified Hsp90/Hsp70-based chaperone machinery to enable ligand binding by the glucocorticoid receptor and show that this effect is due to specific inhibition of Hsp70. Next, we establish that ubiquitination of neuronal nitric-oxide synthase by the native ubiquitinating system of reticulocyte lysate is dependent upon both Hsp70 and the E3 ubiquitin ligase CHIP and is blocked by methylene blue. Finally, we demonstrate that methylene blue impairs degradation of the polyglutamine expanded androgen receptor, an Hsp90 client mutated in spinal and bulbar muscular atrophy. In contrast, degradation of an amino-terminal fragment of the receptor, which lacks the ligand binding domain and, therefore, is not a client of the Hsp90/Hsp70-based chaperone machinery, is enhanced through homeostatic induction of autophagy that occurs when Hsp70-dependent proteasomal degradation is inhibited by methylene blue. Our data demonstrate the utility of methylene blue in defining Hsp70-dependent functions and reveal divergent effects on polyglutamine protein degradation depending on whether the substrate is an Hsp90 client.

Revealing a core signaling regulatory mechanism for pluripotent stem cell survival and self-renewal by small molecules

Using a high-throughput chemical screen, we identified two small molecules that enhance the survival of human embryonic stem cells (hESCs). By characterizing their mechanisms of action, we discovered an essential role of E-cadherin signaling for ESC survival. Specifically, we showed that the primary cause of hESC death following enzymatic dissociation comes from an irreparable disruption of E-cadherin signaling, which then leads to a fatal perturbation of integrin signaling. Furthermore, we found that stability of E-cadherin and the resulting survival of ESCs were controlled by specific growth factor signaling. Finally, we generated mESC-like hESCs by culturing them in mESC conditions. And these converted hESCs rely more on E-cadherin signaling and significantly less on integrin signaling. Our data suggest that differential usage of cell adhesion systems by ESCs to maintain self-renewal may explain their profound differences in terms of morphology, growth factor requirement, and sensitivity to enzymatic cell dissociation.

Opening the TRPML Gates

The intracellular TRPML channels have multiple biological roles, but the physiological stimuli that open them remained unknown. In a previous issue of Chemistry & Biology, Grimm et al. report a high-throughput chemical screen that identified a plethora of selective activators of TRPML3 that should open the way to fully characterize these channels and their physiological roles.

The Neurofibromatosis Type 1 Tumor Suppressor Controls Cell Growth by Regulating Signal Transducer and Activator of Transcription-3 Activity In vitro and In vivo

Neurofibromatosis type 1 (NF1) is a common cancer predisposition syndrome in which affected individuals develop benign and malignant nerve tumors. The NF1 gene product neurofibromin negatively regulates Ras and mammalian target of rapamycin (mTOR) signaling, prompting clinical trials to evaluate the ability of Ras and mTOR pathway inhibitors to arrest NF1-associated tumor growth. To discover other downstream targets of neurofibromin, we performed an unbiased cell-based high-throughput chemical library screen using NF1-deficient malignant peripheral nerve sheath tumor (MPNST) cells. We identified the natural product, cucurbitacin-I (JSI-124), which inhibited NF1-deficient cell growth by inducing apoptosis. We further showed that signal transducer and activator of transcription-3 (STAT3), the target of cucurbitacin-I inhibition, was hyperactivated in NF1-deficient primary astrocytes and neural stem cells, mouse glioma cells, and human MPNST cells through Ser(727) phosphorylation, leading to increased cyclin D1 expression. STAT3 was regulated in NF1-deficient cells of murine and human origin in a TORC1- and Rac1-dependent manner. Finally, cucurbitacin-I inhibited the growth of NF1-deficient MPNST cells in vivo. In summary, we used a chemical genetics approach to reveal STAT3 as a novel neurofibromin/mTOR pathway signaling molecule, define its action and regulation, and establish STAT3 as a tractable target for future NF1-associated cancer therapy studies.

Cladribine inhibits a diltiazem-induced increase in red blood cell purine nucleotide concentrations in a zebrafish model

Minimizing drug interactions is paramount to improving the efficacy and tolerability of cancer therapy. The zebrafish represents an innovative cancer model due to highly conserved genetics and inherent capacity for high-throughput chemical screening. This pilot study extends the utility of the zebrafish to a preclinical model for pharmacodynamics by examining the interaction of the nucleoside analogue, cladribine with the calcium channel blocker, diltiazem. Cladribine (0.7–3.5 mM) and/or diltiazem (2.4 mM), was injected intraperitoneally into adult zebrafish and red blood cell (RBC) lysates were assayed by HPLC for levels of purine nucleotides (e.g. ATP), potential biomarkers of cardiovascular health. Diltiazem increased RBC ATP concentrations, which were inhibited by co-injection of cladribine. These results suggest a novel drug interaction and highlight the feasibility of the zebrafish as an in vivo model for pharmacodynamic studies.

High Content Screening Identifies Decaprenyl-Phosphoribose 2′ Epimerase as a Target for Intracellular Antimycobacterial Inhibitors

A critical feature of Mycobacterium tuberculosis, the causative agent of human tuberculosis (TB), is its ability to survive and multiply within macrophages, making these host cells an ideal niche for persisting microbes. Killing the intracellular tubercle bacilli is a key requirement for efficient tuberculosis treatment, yet identifying potent inhibitors has been hampered by labor-intensive techniques and lack of validated targets. Here, we present the development of a phenotypic cell-based assay that uses automated confocal fluorescence microscopy for high throughput screening of chemicals that interfere with the replication of M. tuberculosis within macrophages. Screening a library of 57,000 small molecules led to the identification of 135 active compounds with potent intracellular anti-mycobacterial efficacy and no host cell toxicity. Among these, the dinitrobenzamide derivatives (DNB) showed high activity against M. tuberculosis, including extensively drug resistant (XDR) strains. More importantly, we demonstrate that incubation of M. tuberculosis with DNB inhibited the formation of both lipoarabinomannan and arabinogalactan, attributable to the inhibition of decaprenyl-phospho-arabinose synthesis catalyzed by the decaprenyl-phosphoribose 2′ epimerase DprE1/DprE2. Inhibition of this new target will likely contribute to new therapeutic solutions against emerging XDR-TB. Beyond validating the high throughput/content screening approach, our results open new avenues for finding the next generation of antimicrobials.

Application of quantitative structure–activity relationships for modeling drug and chemical transport across the human placenta barrier: a multivariate data analysis approach

Pharmacological agents and environmental pollutants can transfer from mother to fetus across the placental barrier, leading to reproductive toxic effects. Ex vivo human placental perfusion constitutes the most widely used method to study placental transfer and metabolism of drugs and chemicals. The aim of the present study was to evaluate whether quantitative structure-activity relationship (QSAR) methodology could serve as an effective alternative tool to estimate drugs and chemicals transport across the human placental barrier on the basis of easily interpretable molecular, physicochemical and structural properties. Multivariate data analysis (MVDA) was applied to a set of 88 structurally diverse drugs and chemicals to model placental transfer expressed by clearance index values compiled from literature sources. An adequate and robust QSAR model (r(2) = 0.73, Q(2) = 0.71, RMSEE = 0.15) was established, providing an informative illustration of the contributing physicochemical, molecular and structural properties of the compounds in placental transfer process. Descriptors reflecting the polarity of compounds proved to be the most important with a negative sign. Lipophilicity and, at a lower extent, molecular size parameters exerted positive contribution in the model. Thus, QSAR analysis may be considered as a promising alternative tool to support high-throughput screening of drugs and chemicals in respect to their transport across placental barrier.

A plasmacytoid dendritic cell (CD123+/CD11c−) based assay system to predict contact allergenicity of chemicals

A predictive allergenicity test system for assessing the contact allergenicity of chemicals is needed by the cosmetic and pharmaceutical industry to monitor product safety in the marketplace. Development of such non-animal alternative assay systems for skin sensitization and hazard identification has been pursued by policy makers and regulatory agencies. We investigated whether phenotypic and functional changes to a subset of dendritic cells (DC), plasmacytoid DC (pDC), could be used to identify contact allergens. To achieve this goal, normal human DC were generated from CD34+ progenitor cells and cryopreserved. Frozen DC were thawed and the pDC fraction (CD123+/CD11c−) was harvested using FACS sorting. The pDC were cultured, expanded, and exposed to chemical allergens ( N = 26) or non-allergens ( N = 22). Concentrations of each chemical that resulted in >50% viability was determined using FACS analysis of propidium iodide stained cells using pDC from 2 to 5 donors. Expression of the surface marker, CD86, which has been implicated in dendritic cell maturation, was used as a marker of allergenicity. CD86 expression increased (≥1.5-fold) for 25 of 26 allergens (sensitivity = 96%) but did not increase for 19 of 22 non-allergens (specificity = 86%). In a direct comparison to historical data for the regulatory approved, mouse local lymph node assay (LLNA) for 23 allergens and 22 non-allergens, the pDC method had sensitivity and specificity of 96% and 86%, respectively, while the sensitivity and specificity of the LLNA assay was 83% and 82%, respectively. In conclusion, CD86 expression in pDC appears to be a sensitive and specific indicator to identify contact allergenicity. Such an assay method utilizing normal human cells will be useful for high throughput screening of chemicals for allergenicity.

Inter-laboratory evaluation of the bioluminescent Salmonella reverse mutation assay using 10 model chemicals

We have developed the bioluminescent Salmonella reverse mutation assay as a tool for detecting mutagenicity applicable for high-throughput screening of new chemicals. In this study, we report the inter-laboratory evaluation of the assay using 10 model chemicals in five independent laboratories located in the USA (Groton, CT; Cambridge, MA and La Jolla, CA), Europe (Sandwich, Kent, UK) and Asia (Nagoya, Japan). The studies were performed in blinded fashion in all sites except for Groton and Cambridge laboratories. The chemicals were tested in at least three independent experiments using strains TA98-lux and TA100-lux in the presence and absence of metabolic activation. The results were statistically evaluated and compared to published results. Seven of the 10 compounds were positive in either TA98-lux and/or TA100-lux in the presence or absence of metabolic activation. The positive compound set included: nitrofurazone, 3-3'-dimethoxybenzidine, benzo[a]pyrene, 1,4-benzoquinone dioxime, 2-amino-5-nitrophenol, 2-bromo-4,6-dinitroaniline and busulfan. The remaining three compounds, namely, anthracene, crystal violet and benzyl chloride were negative in both Salmonella strains. Final results for individual compounds yielded 100% agreement among the laboratories and published data. Detailed comparison of all 40 individual test conditions yielded 93% (37 of 40) agreement among participating laboratories. We conclude that the bioluminescent Salmonella reverse mutation assay is a robust, accurate and economical higher throughput assay applicable for the mutagenicity screening of chemicals.

Careers in Basic Cardiovascular Research

Received August 2, 2008; accepted January 28, 2009. The opportunities in basic science for graduating PhD students and for aspiring physician-scientists have never been so extraordinary. The sequencing of the human genome and the development of the human haplotype map have enabled scientists to begin to unravel the basis of complex multigenic disorders. Rapid developments in stem cell biology are affecting many areas of cardiovascular biology and may enable repair of injured myocardium. High-throughput chemical genetic screening, proteomics, and metabolomics are changing the approaches with which investigators characterize novel signal transduction pathways and develop new therapeutic paradigms. Rapidly evolving molecular imaging technologies are noninvasively illuminating the fundamental processes contributing to cardiovascular diseases. New insights into the mechanisms involved in cardiac myocyte hypertrophy and dysfunction have important therapeutic implications for an expanding population of patients with congestive heart failure. For the young scientist looking to the future, there are major benefits in pursuing a basic science career including the excitement of discovery, the opportunity for life-long learning, and the potential to broadly affect cardiovascular science and medicine. On the other hand, the young investigator considering a basic science career is confronted by many challenges. The budget of the National Institutes of Health (NIH) has remained essentially unchanged for 5 years (when the impact of inflation is considered). The success rate of R01 grant applications has consistently declined since 2000, and the average age when an investigator receives a first R01 grant (or equivalent) increased to 42.6 in 2007 (NIH Extramural Data Book at www.report.nih.gov/index.aspx). At the same time, the increased costs of undergraduate and medical school educations have burdened potential young investigators with ever-increasing financial commitments. Many academic leaders have suggested that an entire generation of young scientists is “at risk” (www.BrokenPipeline. org). Long hours at the laboratory bench are compensated …

Identification of a potent natural triterpenoid inhibitor of proteosome chymotrypsin-like activity and NF-κB with antimyeloma activity in vitro and in vivo

AbstractAs multiple myeloma tumors universally dysregulate cyclin D genes we conducted high-throughput chemical library screens for compounds that induce suppression of cyclin D2 promoter transcription. The top-ranked compound was a natural triterpenoid, pristimerin. Strikingly, the early transcriptional response of cells treated with pristimerin closely resembles cellular responses elicited by proteosome inhibitors, with rapid induction of heat shock proteins, activating transcription factor 3 (ATF3), and CHOP. Enzymatic assays and immunoblotting confirm that pristimerin rapidly (< 90 minutes) and specifically inhibits chymotrypsin-like proteosome activity at low concentrations (< 100 nM) and causes accumulation of cellular ubiquitinated proteins. Notably, cytotoxic triterpenoids including pristimerin inhibit NF-κB activation via inhibition of IKKα or IKKβ, whereas proteosome inhibitors instead suppress NF-κB function by impairing degradation of ubiquitinated IκB. By inhibiting both IKK and the proteosome, pristimerin causes overt suppression of constitutive NF-κB activity in myeloma cells that may mediate its suppression of cyclin D. Multiple myeloma is exquisitely sensitive to proteosome or NF-κB pathway inhibition. Consistent with this, pristimerin is potently and selectively lethal to primary myeloma cells (IC50 < 100 nM), inhibits xenografted plasmacytoma tumors in mice, and is synergistically cytotoxic with bortezomib—providing the rationale for pharmaceutical development of triterpenoid dual-function proteosome/NF-κB inhibitors as therapeutics for human multiple myeloma and related malignancies.

Automated image‐based phenotypic analysis in zebrafish embryos

Presently, the zebrafish is the only vertebrate model compatible with contemporary paradigms of drug discovery. Zebrafish embryos are amenable to automation necessary for high-throughput chemical screens, and optical transparency makes them potentially suited for image-based screening. However, the lack of tools for automated analysis of complex images presents an obstacle to using the zebrafish as a high-throughput screening model. We have developed an automated system for imaging and analyzing zebrafish embryos in multi-well plates regardless of embryo orientation and without user intervention. Images of fluorescent embryos were acquired on a high-content reader and analyzed using an artificial intelligence-based image analysis method termed Cognition Network Technology (CNT). CNT reliably detected transgenic fluorescent embryos (Tg(fli1:EGFP)(y1)) arrayed in 96-well plates and quantified intersegmental blood vessel development in embryos treated with small molecule inhibitors of anigiogenesis. The results demonstrate it is feasible to adapt image-based high-content screening methodology to measure complex whole organism phenotypes.

Use of high-density tiling microarrays to identify mutations globally and elucidate mechanisms of drug resistance in Plasmodium falciparum

Using tiling microarrays, a mechanism by which Plasmodium falciparum parasites acquire resistance to the antimalarial fosmidomycin has been elucidated