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1.  Fostering collaborative research for rare genetic disease: the example of niemann-pick type C disease 
Rare disease represents one of the most significant issues facing the medical community and health care providers worldwide, yet the majority of these disorders never emerge from their obscurity, drawing little attention from the medical community or the pharmaceutical industry. The challenge therefore is how best to mobilize rare disease stakeholders to enhance basic, translational and clinical research to advance understanding of pathogenesis and accelerate therapy development. Here we describe a rare, fatal brain disorder known as Niemann-Pick type C (NPC) and an innovative research collaborative known as Support of Accelerated Research for NPC (SOAR-NPC) which illustrates one pathway through which knowledge of a rare disease and its possible treatments are being successfully advanced. Use of the “SOAR” mechanism, we believe, offers a blueprint for similar advancement for many other rare disorders.
doi:10.1186/s13023-016-0540-x
PMCID: PMC5131440  PMID: 27903269
Cyclodextrin; Collaborative science; Drug pipeline; Lysosomal disease; Miglustat; Niemann-Pick C; Patient advocacy; Rare disease; Therapy development; Translational medicine
4.  Advancing Biological Understanding and Therapeutics Discovery with Small Molecule Probes 
Cell  2015;161(6):1252-1265.
Small-molecule probes can illuminate biological processes and aid in the assessment of emerging therapeutic targets by perturbing biological systems in a manner distinct from other experimental approaches. Despite the tremendous promise of chemical tools for investigating biology and disease, small-molecule probes were unavailable for most targets and pathways as recently as a decade ago. In 2005, the U.S. National Institutes of Health launched the decade-long Molecular Libraries Program with the intent of innovating in and broadening access to small-molecule science. This Perspective describes how novel small-molecule probes identified through the program are enabling the exploration of biological pathways and therapeutic hypotheses not otherwise testable. These experiences illustrate how small-molecule probes can help bridge the chasm between biological research and the development of medicines, but also highlight the need to innovate the science of therapeutic discovery.
doi:10.1016/j.cell.2015.05.023
PMCID: PMC4564295  PMID: 26046436
5.  Structure Activity Relationship of Imidazo-pyridinium Analogs as Antagonists of Neuropeptide S Receptor 
Journal of medicinal chemistry  2013;56(22):9045-9056.
The discovery and characterization of a novel chemical series of phosphorothioyl-containing imidazopyridines as potent Neuropeptide S Receptor antagonists is presented. The synthesis of analogs and their SAR with respect to the Gq, Gs, and ERK pathways is detailed. Pharmacokinetics and in vivo efficacy of a potent analog in a food intake rodent model are also included underscoring its potential therapeutic value for the treatment of sleep, anxiety, and addiction disorders.
Graphical Abstract
doi:10.1021/jm400904m
PMCID: PMC4877059  PMID: 24171469
Neuropeptide S Receptor antagonist; sleep disorders; addiction disorders; imidazo-pyridinium
6.  Gene Therapy: The View from NCATS 
Human Gene Therapy  2016;27(1):7-13.
doi:10.1089/hum.2016.29018.pjb
PMCID: PMC4743673  PMID: 26784641
7.  Niemann-Pick disease type C: induced pluripotent stem cell-derived neuronal cells for modeling neural disease and evaluating drug efficacy 
Journal of biomolecular screening  2014;19(8):1164-1173.
Niemann-Pick disease type C (NPC) is a rare neurodegenerative disorder caused by recessive mutations in NPC1 or NPC2 gene that result in lysosomal accumulation of unesterified cholesterol in patient cells. Patient fibroblasts have been used for evaluation of compound efficacy although neuronal degeneration is the hallmark of NPC disease. Here we report the application of human NPC1 neural stem cells as a cell-based disease model to evaluate nine compounds that have been reported to be efficacious in the NPC1 fibroblasts and mouse models. These cells are differentiated from NPC1 induced pluripotent stem cells and exhibit a phenotype of lysosomal cholesterol accumulation. Treatment of these cells with hydroxypropyl-β-cyclodextrin, methyl-β-cyclodextrin and δ-tocopherol significantly ameliorated the lysosomal cholesterol accumulation. Combined treatment with cyclodextrin and δ-tocopherol shows an additive/synergistic effect that otherwise requires 10-fold higher concentration of cyclodextrin alone. Additionally, we found that hydroxypropyl-β-cyclodextrin is much more potent and efficacious in the NPC1 neural stem cells, compared to the NPC1 fibroblasts. However, miglustat, SAHA, curcumin, lovastatin, pravastatin and rapamycin did not have significant effect in these cells. The results demonstrate that patient derived NPC1 neural stem cells can be used as a model system for evaluation of drug efficacy and study of disease pathogenesis.
doi:10.1177/1087057114537378
PMCID: PMC4529815  PMID: 24907126
Niemann-Pick disease type C; NPC1; induced pluripotent stem cells; differentiated neurons; cyclodextrin; δ-tocopherol; drug combination therapy
8.  Lessons from Toxicology: Developing a 21st-Century Paradigm for Medical Research 
Environmental Health Perspectives  2015;123(11):A268-A272.
Summary
Biomedical developments in the 21st century provide an unprecedented opportunity to gain a dynamic systems-level and human-specific understanding of the causes and pathophysiologies of disease. This understanding is a vital need, in view of continuing failures in health research, drug discovery, and clinical translation. The full potential of advanced approaches may not be achieved within a 20th-century conceptual framework dominated by animal models. Novel technologies are being integrated into environmental health research and are also applicable to disease research, but these advances need a new medical research and drug discovery paradigm to gain maximal benefits. We suggest a new conceptual framework that repurposes the 21st-century transition underway in toxicology. Human disease should be conceived as resulting from integrated extrinsic and intrinsic causes, with research focused on modern human-specific models to understand disease pathways at multiple biological levels that are analogous to adverse outcome pathways in toxicology. Systems biology tools should be used to integrate and interpret data about disease causation and pathophysiology. Such an approach promises progress in overcoming the current roadblocks to understanding human disease and successful drug discovery and translation. A discourse should begin now to identify and consider the many challenges and questions that need to be solved.
doi:10.1289/ehp.1510345
PMCID: PMC4629751  PMID: 26523530
9.  Identification of Small Molecule Modulators of Gene Transcription with Anticancer Activity 
ACS Chemical Biology  2014;9(11):2603-2611.
Epigenetic regulation of gene expression is essential in many biological processes, and its deregulation contributes to pathology including tumor formation. We used an image-based cell assay that measures the induction of a silenced GFP-estrogen receptor reporter to identify novel classes of small molecules involved in the regulation of gene expression. Using this Locus Derepression assay, we queried 283,122 compounds by quantitative high-throughput screening evaluating compounds at multiple concentrations. After confirmation and independent validation, the Locus Derepression assay identified 19 small molecules as new actives that induce the GFP message over 2-fold. Viability assays demonstrated that 17 of these actives have anti-proliferative activity, and two of them show selectivity for cancer versus patient-matched normal cells and cause unique changes in gene expression patterns in cancer cells by altering histone marks. Hence, these compounds represent chemical tools for understanding the molecular mechanisms of epigenetic control of transcription and for modulating cell growth pathways.
doi:10.1021/cb500532x
PMCID: PMC4245161  PMID: 25188650
10.  Detection of Phospholipidosis Induction: A Cell-Based Assay in High-Throughput and High–Content Format 
Drug-induced phospholipidosis is characterized by the accumulation of intracellular phospholipids in cells exposed to cationic amphiphilic drugs. The appearance of unicentric or multicentric multi-lamellar bodies viewed under electron microscope (EM) is the morphological hallmark of phospholipidosis. Although the EM method is the gold standard for detecting cellular phospholipidosis, this method has its drawbacks, including low throughput, high cost, and unsuitability for screening a large chemical library. In this study, a cell-based phospholipidosis assay has been developed using the LipidTOX Red reagent in HepG2 cells and miniaturized into a 1536-well plate format. In order to validate this assay for high throughput screening, the LOPAC library of 1280 compounds was screened on a quantitative high throughput screening platform. A group of known phospholipidosis inducers, such as amiodarone, propranolol, chlorpromazine, desipramine, promazine, clomipramine, and amitriptyline, was identified by the screen, consistent with previous reports. Several novel phospholipidosis inducers including NAN-190, ebastine, GR127935 and cis-(Z)-flupenthixol were identified in this study and confirmed using the EM method. These results demonstrate that this assay can be used to evaluate and profile large numbers of chemicals for drug-induced phospholipidosis.
doi:10.1177/1087057113502851
PMCID: PMC4550094  PMID: 24003057
LipidTOX; phospholipidosis; qHTS
11.  Genome-Wide Interactions of Mouse-Plasmodium yoelii Parasite and Identification of Regulators of Type I Interferon Response 
Cell reports  2015;12(4):661-672.
SUMMARY
Invading pathogens trigger specific host responses, an understanding of which might identify genes that function in pathogen recognition and elimination. In this study, we performed trans-species expression quantitative trait locus (ts-eQTL) analysis using genotypes of the Plasmodium yoelii malaria parasite and phenotypes of mouse gene expression. We significantly linked 1,054 host genes to parasite genetic loci (LOD score ≥ 3.0). Using LOD score patterns, which produced results that differed from direct expression level clustering, we grouped host genes that function in related pathways, allowing functional prediction of unknown genes. As a proof of principle, 14 of 15 randomly selected genes predicted to function in type I interferon (IFN-I) responses were experimentally validated using overexpression, shRNA knockdown, viral infection, and/or infection of KO mice. This study demonstrates an effective strategy for studying gene function, establishes a functional gene database, and identifies regulators in IFN-I pathways.
Graphical Abstract
doi:10.1016/j.celrep.2015.06.058
PMCID: PMC4520759  PMID: 26190101
Plasmodium; pathogen-host interaction; innate signaling; microarray; linkage
12.  Novel Phenotypic Outcomes Identified for a Public Collection of Approved Drugs from a Publicly Accessible Panel of Assays 
PLoS ONE  2015;10(7):e0130796.
Phenotypic assays have a proven track record for generating leads that become first-in-class therapies. Whole cell assays that inform on a phenotype or mechanism also possess great potential in drug repositioning studies by illuminating new activities for the existing pharmacopeia. The National Center for Advancing Translational Sciences (NCATS) pharmaceutical collection (NPC) is the largest reported collection of approved small molecule therapeutics that is available for screening in a high-throughput setting. Via a wide-ranging collaborative effort, this library was analyzed in the Open Innovation Drug Discovery (OIDD) phenotypic assay modules publicly offered by Lilly. The results of these tests are publically available online at www.ncats.nih.gov/expertise/preclinical/pd2 and via the PubChem Database (https://pubchem.ncbi.nlm.nih.gov/) (AID 1117321). Phenotypic outcomes for numerous drugs were confirmed, including sulfonylureas as insulin secretagogues and the anti-angiogenesis actions of multikinase inhibitors sorafenib, axitinib and pazopanib. Several novel outcomes were also noted including the Wnt potentiating activities of rotenone and the antifolate class of drugs, and the anti-angiogenic activity of cetaben.
doi:10.1371/journal.pone.0130796
PMCID: PMC4503722  PMID: 26177200
13.  Auranofin Induces Lethal Oxidative and Endoplasmic Reticulum Stress and Exerts Potent Preclinical Activity against Chronic Lymphocytic Leukemia 
Cancer research  2014;74(9):2520-2532.
Chronic lymphocytic leukemia (CLL) exhibits high remission rates after initial chemoimmunotherapy, but with relapses with treatment, refractory disease is the most common outcome, especially in CLL with the deletion of chromosome 11q or 17p. In addressing the need of treatments for relapsed disease, we report the identification of an existing U.S. Food and Drug Administration-approved small-molecule drug to repurpose for CLL treatment. Auranofin (Ridaura) is approved for use in treating rheumatoid arthritis, but it exhibited preclinical efficacy in CLL cells. By inhibiting thioredoxin reductase activity and increasing intracellular reactive oxygen species levels, auranofin induced a lethal endoplasmic reticulum stress response in cultured and primary CLL cells. In addition, auranofin displayed synergistic lethality with heme oxygenase-1 and glutamate-cysteine ligase inhibitors against CLL cells. Auranofin overcame apoptosis resistance mediated by protective stromal cells, and it also killed primary CLL cells with deletion of chromosome 11q or 17p. In TCL-1 transgenic mice, an in vivo model of CLL, auranofin treatment markedly reduced tumor cell burden and improved mouse survival. Our results provide a rationale to reposition the approved drug auranofin for clinical evaluation in the therapy of CLL.
doi:10.1158/0008-5472.CAN-13-2033
PMCID: PMC4172421  PMID: 24599128
14.  Population-Based in Vitro Hazard and Concentration–Response Assessment of Chemicals: The 1000 Genomes High-Throughput Screening Study 
Environmental Health Perspectives  2015;123(5):458-466.
Background: Understanding of human variation in toxicity to environmental chemicals remains limited, so human health risk assessments still largely rely on a generic 10-fold factor (10½ each for toxicokinetics and toxicodynamics) to account for sensitive individuals or subpopulations.
Objectives: We tested a hypothesis that population-wide in vitro cytotoxicity screening can rapidly inform both the magnitude of and molecular causes for interindividual toxicodynamic variability.
Methods: We used 1,086 lymphoblastoid cell lines from the 1000 Genomes Project, representing nine populations from five continents, to assess variation in cytotoxic response to 179 chemicals. Analysis included assessments of population variation and heritability, and genome-wide association mapping, with attention to phenotypic relevance to human exposures.
Results: For about half the tested compounds, cytotoxic response in the 1% most “sensitive” individual occurred at concentrations within a factor of 10½ (i.e., approximately 3) of that in the median individual; however, for some compounds, this factor was > 10. Genetic mapping suggested important roles for variation in membrane and transmembrane genes, with a number of chemicals showing association with SNP rs13120371 in the solute carrier SLC7A11, previously implicated in chemoresistance.
Conclusions: This experimental approach fills critical gaps unaddressed by recent large-scale toxicity testing programs, providing quantitative, experimentally based estimates of human toxicodynamic variability, and also testable hypotheses about mechanisms contributing to interindividual variation.
Citation: Abdo N, Xia M, Brown CC, Kosyk O, Huang R, Sakamuru S, Zhou YH, Jack JR, Gallins P, Xia K, Li Y, Chiu WA, Motsinger-Reif AA, Austin CP, Tice RR, Rusyn I, Wright FA. 2015. Population-based in vitro hazard and concentration–response assessment of chemicals: the 1000 Genomes high-throughput screening study. Environ Health Perspect 123:458–466; http://dx.doi.org/10.1289/ehp.1408775
doi:10.1289/ehp.1408775
PMCID: PMC4421772  PMID: 25622337
15.  Innovation in Therapeutics Development at the NCATS 
Neuropsychopharmacology  2013;39(1):230-232.
doi:10.1038/npp.2013.247
PMCID: PMC3857672  PMID: 24317308
16.  Profiling of the Tox21 Chemical Collection for Mitochondrial Function to Identify Compounds that Acutely Decrease Mitochondrial Membrane Potential 
Background: Mitochondrial dysfunction has been implicated in the pathogenesis of a variety of disorders including cancer, diabetes, and neurodegenerative and cardiovascular diseases. Understanding whether different environmental chemicals and druglike molecules impact mitochondrial function represents an initial step in predicting exposure-related toxicity and defining a possible role for such compounds in the onset of various diseases.
Objectives: We sought to identify individual chemicals and general structural features associated with changes in mitochondrial membrane potential (MMP).
Methods: We used a multiplexed [two end points in one screen; MMP and adenosine triphosphate (ATP) content] quantitative high throughput screening (qHTS) approach combined with informatics tools to screen the Tox21 library of 10,000 compounds (~ 8,300 unique chemicals) at 15 concentrations each in triplicate to identify chemicals and structural features that are associated with changes in MMP in HepG2 cells.
Results: Approximately 11% of the compounds (913 unique compounds) decreased MMP after 1 hr of treatment without affecting cell viability (ATP content). In addition, 309 compounds decreased MMP over a concentration range that also produced measurable cytotoxicity [half maximal inhibitory concentration (IC50) in MMP assay/IC50 in viability assay ≤ 3; p < 0.05]. More than 11% of the structural clusters that constitute the Tox21 library (76 of 651 clusters) were significantly enriched for compounds that decreased the MMP.
Conclusions: Our multiplexed qHTS approach allowed us to generate a robust and reliable data set to evaluate the ability of thousands of drugs and environmental compounds to decrease MMP. The use of structure-based clustering analysis allowed us to identify molecular features that are likely responsible for the observed activity.
Citation: Attene-Ramos MS, Huang R, Michael S, Witt KL, Richard A, Tice RR, Simeonov A, Austin CP, Xia M. 2015. Profiling of the Tox21 chemical collection for mitochondrial function to identify compounds that acutely decrease mitochondrial membrane potential. Environ Health Perspect 123:49–56; http://dx.doi.org/10.1289/ehp.1408642
doi:10.1289/ehp.1408642
PMCID: PMC4286281  PMID: 25302578
17.  The NCGC Pharmaceutical Collection: A comprehensive resource of clinically approved drugs enabling repurposing and chemical genomics 
Science translational medicine  2011;3(80):80ps16.
Small-molecule compounds approved for use as drugs may be “repurposed” for new indications and studied to determine the mechanisms of their beneficial and adverse effects. A comprehensive collection of all small-molecule drugs approved for human use would be invaluable for systematic repurposing across human diseases, particularly for rare and neglected diseases, for which the cost and time required for development of a new chemical entity are often prohibitive. Previous efforts to build such a comprehensive collection have been limited by the complexities, redundancies, and semantic inconsistencies of drug naming within and among regulatory agencies worldwide; a lack of clear conceptualization of what constitutes a drug; and a lack of access to physical samples. We report here the creation of a definitive, complete, and nonredundant list of all approved molecular entities as a freely available electronic resource and a physical collection of small molecules amenable to high-throughput screening.
doi:10.1126/scitranslmed.3001862
PMCID: PMC3098042  PMID: 21525397
18.  A systematic study of mitochondrial toxicity of environmental chemicals using quantitative high throughput screening 
Chemical research in toxicology  2013;26(9):1323-1332.
A goal of the Tox21 program is to transit toxicity testing from traditional in vivo models to in vitro assays that assess how chemicals affect cellular responses and toxicity pathways. A critical contribution of the NIH Chemical Genomics center (NCGC) to the Tox21 program is the implementation of a quantitative high throughput screening (qHTS) approach, using cell- and biochemical-based assays to generate toxicological profiles for thousands of environmental compounds. Here, we evaluated the effect of chemical compounds on mitochondrial membrane potential in HepG2 cells by screening a library of 1,408 compounds provided by the National Toxicology Program (NTP) in a qHTS platform. Compounds were screened over 14 concentrations, and results showed that 91 and 88 compounds disrupted mitochondrial membrane potential after treatment for one or five h, respectively. Seventy-six compounds active at both time points were clustered by structural similarity, producing 11 clusters and 23 singletons. Thirty-eight compounds covering most of the active chemical space were more extensively evaluated. Thirty-six of the 38 compounds were confirmed to disrupt mitochondrial membrane potential using a fluorescence plate reader and 35 were confirmed using a high content imaging approach. Among the 38 compounds, 4 and 6 induced LDH release, a measure of cytotoxicity, at 1 or 5 h, respectively. Compounds were further assessed for mechanism of action (MOA) by measuring changes in oxygen consumption rate, which enabled identification of 20 compounds as uncouplers. This comprehensive approach allows for evaluation of thousands of environmental chemicals for mitochondrial toxicity and identification of possible MOAs.
doi:10.1021/tx4001754
PMCID: PMC4154066  PMID: 23895456
mitochondrial membrane potential assay; mitochondrial toxicity; NTP 1408 compound library; oxygen consumption rate; qHTS; Tox21 collaboration
19.  A High-throughput Screening Assay for Determining Cellular Levels of Total Tau Protein 
Current Alzheimer research  2013;10(7):679-687.
The microtubule-associated protein (MAP) tau has been implicated in the pathology of numerous neurodegenerative diseases. In the past decade, the hyperphosphorylated and aggregated states of tau protein have been important targets in the drug discovery field for the potential treatment of Alzheimer’s disease. Although several compounds have been reported to reduce the hyperphosphorylated state of tau or impact the stabilization of tau, their therapeutic activities are still to be validated. Recently, reduction of total cellular tau protein has emerged as an alternate intervention point for drug development and a potential treatment of tauopathies. We have developed and optimized a homogenous assay, using the AlphaLISA and HTRF assay technologies, for the quantification of total cellular tau protein levels in the SH-SY5Y neuroblastoma cell line. The signal-to-basal ratios were 375 and 5.3, and the Z’ factors were 0.67 and 0.60 for the AlphaLISA and HTRF tau assays, respectively. The clear advantages of this homogeneous tau assay over conventional total tau assays, such as ELISA and Western blot, are the elimination of plate wash steps and miniaturization of the assay into 1536-well plate format for the ultra–high-throughput screening of large compound libraries.
PMCID: PMC4010324  PMID: 23905996
Alzheimer’s disease; FRET-based assay; high throughput screening assay; prions; neurodegenerative diseases; Tau protein
20.  Are hERG Channel Blockers Also Phospholipidosis Inducers? 
Both pharmacophore models of the human ether-à-go-go-related gene (hERG) channel blockers and phospholipidosis (PLD) inducers contain a hydrophobic moiety and a hydrophilic motif / positively charged center, so it is interesting to investigate the overlap between the ligand chemical spaces of both targets. We have assayed over 4,000 non-redundant drug-like compounds for both their hERG inhibitory activity and PLD inducing potential in a quantitative high throughput screening (qHTS) format. Seventy-seven percent of PLD inducing compounds identified from the screening were also found to be hERG channel blockers, and 96.9% of the dually active compounds were positively charged. Among the 48 compounds that induced PLD without inhibiting hERG channel, 24 compounds (50.0%) carried steroidal structures. According to our results, hERG channel blockers and PLD inducers share a large chemical space. In addition, a positively charged hERG channel blocker will most likely induce PLD, while a steroid PLD inducer is less likely a hERG channel blocker.
doi:10.1016/j.bmcl.2013.06.034
PMCID: PMC3736554  PMID: 23856051
hERG; phospholipidosis; qHTS
21.  The Tox21 robotic platform for assessment of environmental chemicals - from vision to reality 
Drug discovery today  2013;18(0):716-723.
Since its establishment in 2008, the US Tox21 inter-agency collaboration has made great progress in developing and evaluating cellular models for the evaluation of environmental chemicals as a proof of principle. Currently, the program has entered its production phase (Tox21 Phase II) focusing initially on the areas of modulation of nuclear receptors and stress response pathways. During Tox21 Phase II, the set of chemicals to be tested has been expanded to nearly 10,000 (10K) compounds and a fully automated screening platform has been implemented. The Tox21 robotic system combined with informatics efforts is capable of screening and profiling the collection of 10K environmental chemicals in triplicate in a week. In this article, we describe the Tox21 screening process, compound library preparation, data processing, and robotic system validation.
doi:10.1016/j.drudis.2013.05.015
PMCID: PMC3771082  PMID: 23732176
10K compound library; in vitro assays; quantitative high-throughput screening; robotic platform; Tox21 collaboration; toxicity testing
22.  Profiling of the Tox21 10K compound library for agonists and antagonists of the estrogen receptor alpha signaling pathway 
Scientific Reports  2014;4:5664.
The U.S. Tox21 program has screened a library of approximately 10,000 (10K) environmental chemicals and drugs in three independent runs for estrogen receptor alpha (ERα) agonist and antagonist activity using two types of ER reporter gene cell lines, one with an endogenous full length ERα (ER-luc; BG1 cell line) and the other with a transfected partial receptor consisting of the ligand binding domain (ER-bla; ERα β-lactamase cell line), in a quantitative high-throughput screening (qHTS) format. The ability of the two assays to correctly identify ERα agonists and antagonists was evaluated using a set of 39 reference compounds with known ERα activity. Although both assays demonstrated adequate (i.e. >80%) predictivity, the ER-luc assay was more sensitive and the ER-bla assay more specific. The qHTS assay results were compared with results from previously published ERα binding assay data and showed >80% consistency. Actives identified from both the ER-bla and ER-luc assays were analyzed for structure-activity relationships (SARs) revealing known and potentially novel ERα active structure classes. The results demonstrate the feasibility of qHTS to identify environmental chemicals with the potential to interact with the ERα signaling pathway and the two different assay formats improve the confidence in correctly identifying these chemicals.
doi:10.1038/srep05664
PMCID: PMC4092345  PMID: 25012808
23.  Collaborative Development of 2-Hydroxypropyl-β-Cyclodextrin for the Treatment of Niemann-Pick Type C1 Disease 
In 2010, the National Institutes of Health (NIH) established the Therapeutics for Rare and Neglected Diseases (TRND) program within the National Center for Advancing Translational Science (NCATS), which was created to stimulate drug discovery and development for rare and neglected tropical diseases through a collaborative model between the NIH, academic scientists, nonprofit organizations, and pharmaceutical and biotechnology companies. This paper describes one of the first TRND programs, the development of 2-hydroxypropyl-β-cyclodextrin (HP-β-CD) for the treatment of Niemann-Pick disease type C1 (NPC1). NPC is a neurodegenerative, autosomal recessive rare disease caused by a mutation in either the NPC1 (about 95% of cases) or the NPC2 gene (about 5% of cases). These mutations affect the intracellular trafficking of cholesterol and other lipids, which leads to a progressive accumulation of unesterified cholesterol and glycosphingolipids in the CNS and visceral organs. Affected individuals typically exhibit ataxia, swallowing problems, seizures, and progressive impairment of motor and intellectual function in early childhood, and usually die in adolescence. There is no disease modifying therapy currently approved for NPC1 in the US. A collaborative drug development program has been established between TRND, public and private partners that has completed the pre-clinical development of HP-β-CD through IND filing for the current Phase I clinical trial that is underway. Here we discuss how this collaborative effort helped to overcome scientific, clinical and financial challenges facing the development of new drug treatments for rare and neglected diseases, and how it will incentivize the commercialization of HP-β-CD for the benefit of the NPC patient community.
PMCID: PMC4048128  PMID: 24283970
2-hydroxypropyl-β-cyclodextrin; Niemann-Pick disease type C1; neurodegenerative rare disease; translational research
24.  Bisphenol A affects androgen receptor function via multiple mechanisms 
Chemico-biological interactions  2013;203(3):556-564.
Bisphenol A (BPA), is a well-known endocrine disruptor compound (EDC) that affects the normal development and function of the female and male reproductive system, however the mechanisms of action remain unclear. To investigate the molecular mechanisms of how BPA may affect ten different nuclear receptors, stable cell lines containing individual nuclear receptor ligand binding domain (LBD)-linked to the β-Gal reporter were examined by a quantitative high throughput screening (qHTS) format in the Tox21 Screening Program of the NIH. The results showed that two receptors, estrogen receptor alpha (ERα) and androgen receptor (AR), are affected by BPA in opposite direction. To confirm the observed effects of BPA on ERα and AR, we performed transient transfection experiments with full-length receptors and their corresponding response elements linked to luciferase reporters. We also included in this study two BPA analogs, bisphenol AF (BPAF) and bisphenol S (BPS). As seen in African green monkey kidney CV1 cells, the present study confirmed that BPA and BPAF act as ERα agonists (half maximal effective concentration EC50 of 10-100 nM) and as AR antagonists (half maximal inhibitory concentration IC50 of 1-2 μM). Both BPA and BPAF antagonized AR function via competitive inhibition of the action of synthetic androgen R1881. BPS with lower estrogenic activity (EC50 of 2.2 μM), did not compete with R1881 for AR binding, when tested at 30 μM. Finally, the effects of BPA were also evaluated in a nuclear translocation assays using EGPF-tagged receptors. Similar to 17β-estradiol (E2) which was used as control, BPA was able to enhance ERα nuclear foci formation but at a 100-fold higher concentration. Although BPA was able to bind AR, the nuclear translocation was reduced. Furthermore, BPA was unable to induce functional foci in the nuclei and is consistent with the transient transfection study that BPA is unable to activate AR.
doi:10.1016/j.cbi.2013.03.013
PMCID: PMC3722857  PMID: 23562765
Bisphenol A and related compounds; androgen receptor; qHTS; transfection; imaging analysis
25.  Lomofungin and dilomofungin: inhibitors of MBNL1-CUG RNA binding with distinct cellular effects 
Nucleic Acids Research  2014;42(10):6591-6602.
Myotonic dystrophy type 1 (DM1) is a dominantly inherited neuromuscular disorder resulting from expression of RNA containing an expanded CUG repeat (CUGexp). The pathogenic RNA is retained in nuclear foci. Poly-(CUG) binding proteins in the Muscleblind-like (MBNL) family are sequestered in foci, causing misregulated alternative splicing of specific pre-mRNAs. Inhibitors of MBNL1-CUGexp binding have been shown to restore splicing regulation and correct phenotypes in DM1 models. We therefore conducted a high-throughput screen to identify novel inhibitors of MBNL1-(CUG)12 binding. The most active compound was lomofungin, a natural antimicrobial agent. We found that lomofungin undergoes spontaneous dimerization in DMSO, producing dilomofungin, whose inhibition of MBNL1–(CUG)12 binding was 17-fold more potent than lomofungin itself. However, while dilomofungin displayed the desired binding characteristics in vitro, when applied to cells it produced a large increase of CUGexp RNA in nuclear foci, owing to reduced turnover of the CUGexp transcript. By comparison, the monomer did not induce CUGexp accumulation in cells and was more effective at rescuing a CUGexp-induced splicing defect. These results support the feasibility of high-throughput screens to identify compounds targeting toxic RNA, but also demonstrate that ligands for repetitive sequences may have unexpected effects on RNA decay.
doi:10.1093/nar/gku275
PMCID: PMC4041448  PMID: 24799433

Results 1-25 (133)