RESUMO
The chemical diversity and known safety profiles of drugs previously tested in humans make them a valuable set of compounds to explore potential therapeutic utility in indications outside those originally targeted, especially neglected tropical diseases. This practice of "drug repurposing" has become commonplace in academic and other nonprofit drug-discovery efforts, with the appeal that significantly less time and resources are required to advance a candidate into the clinic. Here, we report a comprehensive open-access, drug repositioning screening set of 12,000 compounds (termed ReFRAME; Repurposing, Focused Rescue, and Accelerated Medchem) that was assembled by combining three widely used commercial drug competitive intelligence databases (Clarivate Integrity, GVK Excelra GoStar, and Citeline Pharmaprojects), together with extensive patent mining of small molecules that have been dosed in humans. To date, 12,000 compounds (â¼80% of compounds identified from data mining) have been purchased or synthesized and subsequently plated for screening. To exemplify its utility, this collection was screened against Cryptosporidium spp., a major cause of childhood diarrhea in the developing world, and two active compounds previously tested in humans for other therapeutic indications were identified. Both compounds, VB-201 and a structurally related analog of ASP-7962, were subsequently shown to be efficacious in animal models of Cryptosporidium infection at clinically relevant doses, based on available human doses. In addition, an open-access data portal (https://reframedb.org) has been developed to share ReFRAME screen hits to encourage additional follow-up and maximize the impact of the ReFRAME screening collection.
Assuntos
Antiprotozoários/farmacologia , Criptosporidiose/tratamento farmacológico , Cryptosporidium/efeitos dos fármacos , Bases de Dados de Produtos Farmacêuticos , Descoberta de Drogas , Reposicionamento de Medicamentos/métodos , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Criptosporidiose/parasitologia , Avaliação Pré-Clínica de Medicamentos/métodos , Feminino , Ensaios de Triagem em Larga Escala , Humanos , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Chronic pain is a highly prevalent and poorly managed human health problem. We used microarray-based expression genomics in 25 inbred mouse strains to identify dorsal root ganglion (DRG)-expressed genetic contributors to mechanical allodynia, a prominent symptom of chronic pain. We identified expression levels of Chrna6, which encodes the α6 subunit of the nicotinic acetylcholine receptor (nAChR), as highly associated with allodynia. We confirmed the importance of α6* (α6-containing) nAChRs by analyzing both gain- and loss-of-function mutants. We find that mechanical allodynia associated with neuropathic and inflammatory injuries is significantly altered in α6* mutants, and that α6* but not α4* nicotinic receptors are absolutely required for peripheral and/or spinal nicotine analgesia. Furthermore, we show that Chrna6's role in analgesia is at least partially due to direct interaction and cross-inhibition of α6* nAChRs with P2X2/3 receptors in DRG nociceptors. Finally, we establish the relevance of our results to humans by the observation of genetic association in patients suffering from chronic postsurgical and temporomandibular pain.
Assuntos
Dor Crônica/genética , Receptores Nicotínicos/genética , Receptores Purinérgicos P2X2/metabolismo , Receptores Purinérgicos P2X3/metabolismo , Animais , Regulação para Baixo , Transferência Ressonante de Energia de Fluorescência , Gânglios Espinais/metabolismo , Humanos , Camundongos , Camundongos Mutantes , Antagonistas do Receptor Purinérgico P2X/farmacologiaRESUMO
Complement-mediated bactericidal activity has long been regarded as the serological correlate of protective immunity against Neisseria meningitidis. This was affirmed in 2005 at a WHO-sponsored meningococcal serology standardization workshop. The assay currently employed by most laboratories involves determining surviving bacterial colony counts on agar as a readout which is labor-intensive, time-consuming, and not amendable to rapid data analysis for clinical trials. Consequently, there is an acute need to develop a sensitive, high-throughput bactericidal assay to enable a rapid and robust assessment of the effectiveness of vaccine candidates. To this end, we have developed an automated, kinetic assay based on the fluorescent respiration product of resazurin which reduces assay volume, shortens assay time, and facilitates automation of data analysis. We demonstrate proof of concept for applicability of this high-throughput system with multiple meningococcal strains and utilizing different lots of human complement. The assay is robust and highly reproducible. Titers obtained by the fluorescence readout method are strongly correlated with the data obtained using the conventional, agar plate-based assay. These results demonstrate that the detection of bacteria that have survived the bactericidal reaction by measuring metabolic activity using a fluorescent dye as an alternative readout is a promising approach for the development of a high-throughput bactericidal assay.
Assuntos
Anticorpos Antibacterianos/imunologia , Automação/métodos , Atividade Bactericida do Sangue , Ensaios de Triagem em Larga Escala/métodos , Viabilidade Microbiana/imunologia , Neisseria meningitidis/imunologia , Neisseria meningitidis/metabolismo , Corantes Fluorescentes/metabolismo , Humanos , Imunoensaio/métodos , Vacinas Meningocócicas/imunologia , Oxazinas/metabolismo , Reprodutibilidade dos Testes , Coloração e Rotulagem/métodos , Xantenos/metabolismoRESUMO
Online gene annotation resources are indispensable for analysis of genomics data. However, the landscape of these online resources is highly fragmented, and scientists often visit dozens of these sites for each gene in a candidate gene list. Here, we introduce BioGPS http://biogps.gnf.org, a centralized gene portal for aggregating distributed gene annotation resources. Moreover, BioGPS embraces the principle of community intelligence, enabling any user to easily and directly contribute to the BioGPS platform.
Assuntos
Biologia Computacional/métodos , Bases de Dados Genéticas , Modelos Genéticos , Computadores , Sistemas de Gerenciamento de Base de Dados , Bases de Dados de Proteínas , Genes , Genética , Genômica , Humanos , Internet , Modelos Biológicos , SoftwareRESUMO
Two decades of research identified more than a dozen clock genes and defined a biochemical feedback mechanism of circadian oscillator function. To identify additional clock genes and modifiers, we conducted a genome-wide small interfering RNA screen in a human cellular clock model. Knockdown of nearly 1000 genes reduced rhythm amplitude. Potent effects on period length or increased amplitude were less frequent; we found hundreds of these and confirmed them in secondary screens. Characterization of a subset of these genes demonstrated a dosage-dependent effect on oscillator function. Protein interaction network analysis showed that dozens of gene products directly or indirectly associate with known clock components. Pathway analysis revealed these genes are overrepresented for components of insulin and hedgehog signaling, the cell cycle, and the folate metabolism. Coupled with data showing many of these pathways are clock regulated, we conclude the clock is interconnected with many aspects of cellular function.
Assuntos
Relógios Biológicos , Ritmo Circadiano , Estudo de Associação Genômica Ampla , Linhagem Celular , Técnicas de Silenciamento de Genes , Humanos , Interferência de RNA , RNA Interferente Pequeno/metabolismoRESUMO
The growing resistance to current first-line antimalarial drugs represents a major health challenge. To facilitate the discovery of new antimalarials, we have implemented an efficient and robust high-throughput cell-based screen (1,536-well format) based on proliferation of Plasmodium falciparum (Pf) in erythrocytes. From a screen of approximately 1.7 million compounds, we identified a diverse collection of approximately 6,000 small molecules comprised of >530 distinct scaffolds, all of which show potent antimalarial activity (<1.25 microM). Most known antimalarials were identified in this screen, thus validating our approach. In addition, we identified many novel chemical scaffolds, which likely act through both known and novel pathways. We further show that in some cases the mechanism of action of these antimalarials can be determined by in silico compound activity profiling. This method uses large datasets from unrelated cellular and biochemical screens and the guilt-by-association principle to predict which cellular pathway and/or protein target is being inhibited by select compounds. In addition, the screening method has the potential to provide the malaria community with many new starting points for the development of biological probes and drugs with novel antiparasitic activities.
Assuntos
Antimaláricos/análise , Antimaláricos/farmacologia , Biologia Computacional , Animais , Antimaláricos/química , Antimaláricos/uso terapêutico , Análise por Conglomerados , Avaliação Pré-Clínica de Medicamentos , Resistência a Medicamentos/efeitos dos fármacos , Antagonistas do Ácido Fólico/análise , Antagonistas do Ácido Fólico/química , Antagonistas do Ácido Fólico/farmacologia , Malária/tratamento farmacológico , Modelos Moleculares , Parasitos/efeitos dos fármacos , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/enzimologia , Reprodutibilidade dos Testes , Relação Estrutura-Atividade , Tetra-Hidrofolato Desidrogenase/químicaRESUMO
Traditional approaches to natural product discovery involve cell-based screening of natural product extracts followed by compound isolation and characterization. Their importance notwithstanding, continued mining leads to depletion of natural resources and the reisolation of previously identified metabolites. Metagenomic strategies aimed at localizing the biosynthetic cluster genes and expressing them in surrogate hosts offers one possible alternative. A fundamental question that naturally arises when pursuing such a strategy is, how large must the genomic library be to effectively represent the genome of an organism(s) and the biosynthetic gene clusters they harbor? Such an issue is certainly augmented in the absence of expensive robotics to expedite colony picking and/or screening of clones. We have developed an algorism, named BPC (biosynthetic pathway coverage), supported by molecular simulations to deduce the number of BAC clones required to achieve proper coverage of the genome and their respective biosynthetic pathways. The strategy has been applied to the construction of a large-insert BAC library from a marine microorganism, Hon6 (isolated from Honokohau, Maui) thought to represent a new species. The genomic library is constructed with a BAC yeast shuttle vector pClasper lacZ paving the way for the culturing of libraries in both prokaryotic and eukaryotic hosts. Flow cytometric methods are utilized to estimate the genome size of the organism and BPC implemented to assess P-coverage or percent coverage. A genetic selection strategy is illustrated, applications of which could expedite screening efforts in the identification and localization of biosynthetic pathways from marine microbial consortia, offering a powerful complement to genome sequencing and degenerate probe strategies. Implementing this approach, we report on the biotin biosynthetic pathway from the marine microorganism Hon6.
Assuntos
Biotina/biossíntese , Biblioteca Genômica , Halomonadaceae/genética , Halomonadaceae/metabolismo , Biotina/genética , DNA/genética , Citometria de Fluxo , Halomonadaceae/classificação , Reação em Cadeia da Polimerase , Especificidade da EspécieRESUMO
Genome-wide gene expression profiling has been extensively used to generate biological hypotheses based on differential expression. Recently, many studies have used microarrays to measure gene expression levels across genetic mapping populations. These gene expression phenotypes have been used for genome-wide association analyses, an analysis referred to as expression QTL (eQTL) mapping. Here, eQTL analysis was performed in adipose tissue from 28 inbred strains of mice. We focused our analysis on "trans-eQTL bands", defined as instances in which the expression patterns of many genes were all associated to a common genetic locus. Genes comprising trans-eQTL bands were screened for enrichments in functional gene sets representing known biological pathways, and genes located at associated trans-eQTL band loci were considered candidate transcriptional modulators. We demonstrate that these patterns were enriched for previously characterized relationships between known upstream transcriptional regulators and their downstream target genes. Moreover, we used this strategy to identify both novel regulators and novel members of known pathways. Finally, based on a putative regulatory relationship identified in our analysis, we identified and validated a previously uncharacterized role for cyclin H in the regulation of oxidative phosphorylation. We believe that the specific molecular hypotheses generated in this study will reveal many additional pathway members and regulators, and that the analysis approaches described herein will be broadly applicable to other eQTL data sets.
Assuntos
Tecido Adiposo/metabolismo , Genes Reguladores , Genômica/métodos , Locos de Características Quantitativas , Adipócitos , Animais , Ciclina H , Ciclinas/genética , Ciclinas/metabolismo , Metabolismo Energético , Expressão Gênica , Perfilação da Expressão Gênica , Camundongos , Camundongos Endogâmicos , Análise de Sequência com Séries de Oligonucleotídeos , Polimorfismo de Nucleotídeo Único , Transcrição GênicaRESUMO
The discovery of quantitative trait loci (QTL) in model organisms has relied heavily on the ability to perform controlled breeding to generate genotypic and phenotypic diversity. Recently, we and others have demonstrated the use of an existing set of diverse inbred mice (referred to here as the mouse diversity panel, MDP) as a QTL mapping population. The use of the MDP population has many advantages relative to traditional F(2) mapping populations, including increased phenotypic diversity, a higher recombination frequency, and the ability to collect genotype and phenotype data in community databases. However, these methods are complicated by population structure inherent in the MDP and the lack of an analytical framework to assess statistical power. To address these issues, we measured gene expression levels in hypothalamus across the MDP. We then mapped these phenotypes as quantitative traits with our association algorithm, resulting in a large set of expression QTL (eQTL). We utilized these eQTL, and specifically cis-eQTL, to develop a novel nonparametric method for association analysis in structured populations like the MDP. These eQTL data confirmed that the MDP is a suitable mapping population for QTL discovery and that eQTL results can serve as a gold standard for relative measures of statistical power.
Assuntos
Técnicas Genéticas , Genoma , Endogamia , Dinâmica Populacional , Análise de Variância , Animais , Análise por Conglomerados , Expressão Gênica , Hipotálamo/metabolismo , Camundongos , Locos de Características Quantitativas/genética , Estatísticas não ParamétricasRESUMO
Rapid quantitative methods for characterizing small molecules, peptides, proteins, or RNAs in a broad array of cellular assays would allow one to discover new biological activities associated with these molecules and also provide a more comprehensive profile of drug candidates early in the drug development process. Here we describe a robotic system, termed the automated compound profiler, capable of both propagating a large number of cell lines in parallel and assaying large collections of molecules simultaneously against a matrix of cellular assays in a highly reproducible manner. To illustrate its utility, we have characterized a set of 1,400 kinase inhibitors in a panel of 35 activated tyrosine-kinase-dependent cellular assays in dose-response format in a single experiment. Analysis of the resulting multidimensional dataset revealed subclusters of both inhibitors and kinases with closely correlated activities. The approach also identified activities for the p38 inhibitor BIRB796 and the dual src/abl inhibitor BMS-354825 and exposed the expected side activities for Glivec/STI571, including cellular inhibition of c-kit and platelet-derived growth factor receptor. This methodology provides a powerful tool for unraveling the cellular biology and molecular pharmacology of both naturally occurring and synthetic chemical diversity.
Assuntos
Fosfotransferases/antagonistas & inibidores , Fosfotransferases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Robótica/métodos , Animais , Automação , Linhagem Celular , Bases de Dados Factuais , Avaliação Pré-Clínica de Medicamentos/métodos , Camundongos , Fosfotransferases/genética , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/isolamento & purificação , Reprodutibilidade dos Testes , Relação Estrutura-Atividade , Fatores de TempoRESUMO
An exhaustive ring-based algorithm, HierS, has been developed in order to provide an intuitive approach to compound clustering for analyzing high-throughput screening results. The recursive algorithm rapidly identifies all possible ring-delimited substructures within a set of compounds. Molecules are grouped by shared ring substructures (scaffolds) so that common scaffolds obtain higher membership. Once all of the scaffolds for a set of compounds are identified, the hierarchical structural relationships between the scaffold structures are established. The complex network of hierarchical relationships is then utilized to navigate compounds in a structurally directed fashion. When the scaffold hierarchy is traversed, over-represented structural features can be rapidly identified so that excess compounds that contain them can be removed without significantly impacting the structural diversity landscape of the compound set. Furthermore, the removed compounds can provide the opportunity to follow-up on active compounds that had previously been discarded because of practical limitations on follow-up capacity. A Web-based interface has been developed that incorporates this algorithm in order to allow for an interactive analysis. In addition, biological data are coupled to scaffolds by the inclusion of activity histograms, which indicate how the compounds in each scaffold class performed in previous high-throughput screening campaigns.
Assuntos
Algoritmos , Desenho de Fármacos , Relação Quantitativa Estrutura-Atividade , Compostos de Anilina/química , Antineoplásicos/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Internet , Naftalenos/química , Pirazóis/química , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidoresRESUMO
The design and evaluation of a new genetic selection system for evolving catalytic antibodies with aldolase activity are described. Through a series of model selections, we have identified selection conditions where expression of a catalytically active antibody confers a growth advantage to Escherichia coli. In addition, we provide evidence that the growth advantage is a direct result of catalytic activity.