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1.
Cell ; 177(4): 1067-1079.e19, 2019 05 02.
Artículo en Inglés | MEDLINE | ID: mdl-31051099

RESUMEN

The precise control of CRISPR-Cas9 activity is required for a number of genome engineering technologies. Here, we report a generalizable platform that provided the first synthetic small-molecule inhibitors of Streptococcus pyogenes Cas9 (SpCas9) that weigh <500 Da and are cell permeable, reversible, and stable under physiological conditions. We developed a suite of high-throughput assays for SpCas9 functions, including a primary screening assay for SpCas9 binding to the protospacer adjacent motif, and used these assays to screen a structurally diverse collection of natural-product-like small molecules to ultimately identify compounds that disrupt the SpCas9-DNA interaction. Using these synthetic anti-CRISPR small molecules, we demonstrated dose and temporal control of SpCas9 and catalytically impaired SpCas9 technologies, including transcription activation, and identified a pharmacophore for SpCas9 inhibition using structure-activity relationships. These studies establish a platform for rapidly identifying synthetic, miniature, cell-permeable, and reversible inhibitors against both SpCas9 and next-generation CRISPR-associated nucleases.


Asunto(s)
Proteína 9 Asociada a CRISPR/antagonistas & inhibidores , Sistemas CRISPR-Cas/fisiología , Ensayos Analíticos de Alto Rendimiento/métodos , Proteína 9 Asociada a CRISPR/metabolismo , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/fisiología , ADN/metabolismo , Endonucleasas/metabolismo , Edición Génica/métodos , Genoma , Bibliotecas de Moléculas Pequeñas , Streptococcus pyogenes/genética , Especificidad por Sustrato
2.
Cell ; 154(5): 1151-1161, 2013 Aug 29.
Artículo en Inglés | MEDLINE | ID: mdl-23993102

RESUMEN

The high rate of clinical response to protein-kinase-targeting drugs matched to cancer patients with specific genomic alterations has prompted efforts to use cancer cell line (CCL) profiling to identify additional biomarkers of small-molecule sensitivities. We have quantitatively measured the sensitivity of 242 genomically characterized CCLs to an Informer Set of 354 small molecules that target many nodes in cell circuitry, uncovering protein dependencies that: (1) associate with specific cancer-genomic alterations and (2) can be targeted by small molecules. We have created the Cancer Therapeutics Response Portal (http://www.broadinstitute.org/ctrp) to enable users to correlate genetic features to sensitivity in individual lineages and control for confounding factors of CCL profiling. We report a candidate dependency, associating activating mutations in the oncogene ß-catenin with sensitivity to the Bcl-2 family antagonist, navitoclax. The resource can be used to develop novel therapeutic hypotheses and to accelerate discovery of drugs matched to patients by their cancer genotype and lineage.


Asunto(s)
Bases de Datos Farmacéuticas , Descubrimiento de Drogas , Neoplasias/tratamiento farmacológico , Antineoplásicos/química , Línea Celular Tumoral , Humanos , Neoplasias/genética
3.
Cell ; 150(3): 575-89, 2012 Aug 03.
Artículo en Inglés | MEDLINE | ID: mdl-22863010

RESUMEN

The mechanism by which cells decide to skip mitosis to become polyploid is largely undefined. Here we used a high-content image-based screen to identify small-molecule probes that induce polyploidization of megakaryocytic leukemia cells and serve as perturbagens to help understand this process. Our study implicates five networks of kinases that regulate the switch to polyploidy. Moreover, we find that dimethylfasudil (diMF, H-1152P) selectively increased polyploidization, mature cell-surface marker expression, and apoptosis of malignant megakaryocytes. An integrated target identification approach employing proteomic and shRNA screening revealed that a major target of diMF is Aurora kinase A (AURKA). We further find that MLN8237 (Alisertib), a selective inhibitor of AURKA, induced polyploidization and expression of mature megakaryocyte markers in acute megakaryocytic leukemia (AMKL) blasts and displayed potent anti-AMKL activity in vivo. Our findings provide a rationale to support clinical trials of MLN8237 and other inducers of polyploidization and differentiation in AMKL.


Asunto(s)
Azepinas/farmacología , Descubrimiento de Drogas , Leucemia Megacarioblástica Aguda/tratamiento farmacológico , Megacariocitos/metabolismo , Poliploidía , Pirimidinas/farmacología , Bibliotecas de Moléculas Pequeñas , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/farmacología , Animales , Aurora Quinasa A , Aurora Quinasas , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Humanos , Leucemia Megacarioblástica Aguda/genética , Megacariocitos/citología , Megacariocitos/patología , Ratones , Ratones Endogámicos C57BL , Mapas de Interacción de Proteínas , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/metabolismo , Quinasas Asociadas a rho/metabolismo
4.
Nature ; 585(7826): 603-608, 2020 09.
Artículo en Inglés | MEDLINE | ID: mdl-32939090

RESUMEN

Ferroptosis-an iron-dependent, non-apoptotic cell death process-is involved in various degenerative diseases and represents a targetable susceptibility in certain cancers1. The ferroptosis-susceptible cell state can either pre-exist in cells that arise from certain lineages or be acquired during cell-state transitions2-5. However, precisely how susceptibility to ferroptosis is dynamically regulated remains poorly understood. Here we use genome-wide CRISPR-Cas9 suppressor screens to identify the oxidative organelles peroxisomes as critical contributors to ferroptosis sensitivity in human renal and ovarian carcinoma cells. Using lipidomic profiling we show that peroxisomes contribute to ferroptosis by synthesizing polyunsaturated ether phospholipids (PUFA-ePLs), which act as substrates for lipid peroxidation that, in turn, results in the induction of ferroptosis. Carcinoma cells that are initially sensitive to ferroptosis can switch to a ferroptosis-resistant state in vivo in mice, which is associated with extensive downregulation of PUFA-ePLs. We further find that the pro-ferroptotic role of PUFA-ePLs can be extended beyond neoplastic cells to other cell types, including neurons and cardiomyocytes. Together, our work reveals roles for the peroxisome-ether-phospholipid axis in driving susceptibility to and evasion from ferroptosis, highlights PUFA-ePL as a distinct functional lipid class that is dynamically regulated during cell-state transitions, and suggests multiple regulatory nodes for therapeutic interventions in diseases that involve ferroptosis.


Asunto(s)
Éteres/metabolismo , Ferroptosis , Peroxisomas/metabolismo , Fosfolípidos/química , Fosfolípidos/metabolismo , Animales , Sistemas CRISPR-Cas/genética , Diferenciación Celular , Línea Celular , Éteres/química , Femenino , Edición Génica , Humanos , Neoplasias Renales/metabolismo , Neoplasias Renales/patología , Peroxidación de Lípido , Masculino , Ratones , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Neuronas/citología , Neuronas/metabolismo , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Peroxisomas/genética
5.
Proc Natl Acad Sci U S A ; 114(43): 11392-11397, 2017 10 24.
Artículo en Inglés | MEDLINE | ID: mdl-29073062

RESUMEN

Advances in human genetics have dramatically expanded our understanding of complex heritable diseases. Genome-wide association studies have identified an allelic series of CARD9 variants associated with increased risk of or protection from inflammatory bowel disease (IBD). The predisposing variant of CARD9 is associated with increased NF-κB-mediated cytokine production. Conversely, the protective variant lacks a functional C-terminal domain and is unable to recruit the E3 ubiquitin ligase TRIM62. Here, we used biochemical insights into CARD9 variant proteins to create a blueprint for IBD therapeutics and recapitulated the mechanism of the CARD9 protective variant using small molecules. We developed a multiplexed bead-based technology to screen compounds for disruption of the CARD9-TRIM62 interaction. We identified compounds that directly and selectively bind CARD9, disrupt TRIM62 recruitment, inhibit TRIM62-mediated ubiquitinylation of CARD9, and demonstrate cellular activity and selectivity in CARD9-dependent pathways. Taken together, small molecules targeting CARD9 illustrate a path toward improved IBD therapeutics.


Asunto(s)
Proteínas Adaptadoras de Señalización CARD/antagonistas & inhibidores , Proteínas Adaptadoras de Señalización CARD/genética , Ensayo de Inmunoadsorción Enzimática/métodos , Variación Genética , Enfermedades Inflamatorias del Intestino/genética , Evaluación Preclínica de Medicamentos , Marcadores Genéticos , Ensayos Analíticos de Alto Rendimiento , Humanos , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , Unión Proteica , Sensibilidad y Especificidad , Proteínas de Motivos Tripartitos/antagonistas & inhibidores , Proteínas de Motivos Tripartitos/genética , Ubiquitina-Proteína Ligasas/antagonistas & inhibidores , Ubiquitina-Proteína Ligasas/genética
6.
Nat Chem Biol ; 12(2): 109-16, 2016 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-26656090

RESUMEN

Changes in cellular gene expression in response to small-molecule or genetic perturbations have yielded signatures that can connect unknown mechanisms of action (MoA) to ones previously established. We hypothesized that differential basal gene expression could be correlated with patterns of small-molecule sensitivity across many cell lines to illuminate the actions of compounds whose MoA are unknown. To test this idea, we correlated the sensitivity patterns of 481 compounds with ∼19,000 basal transcript levels across 823 different human cancer cell lines and identified selective outlier transcripts. This process yielded many novel mechanistic insights, including the identification of activation mechanisms, cellular transporters and direct protein targets. We found that ML239, originally identified in a phenotypic screen for selective cytotoxicity in breast cancer stem-like cells, most likely acts through activation of fatty acid desaturase 2 (FADS2). These data and analytical tools are available to the research community through the Cancer Therapeutics Response Portal.


Asunto(s)
Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología , Aflatoxinas/química , Aflatoxinas/farmacología , Western Blotting , Neoplasias de la Mama/tratamiento farmacológico , Línea Celular Tumoral , Simulación por Computador , Sistemas de Liberación de Medicamentos , Femenino , Humanos , Estructura Molecular , Análisis de Componente Principal , Reacción en Cadena en Tiempo Real de la Polimerasa
7.
Proc Natl Acad Sci U S A ; 112(31): E4281-7, 2015 Aug 04.
Artículo en Inglés | MEDLINE | ID: mdl-26195741

RESUMEN

Studies of human genetics and pathophysiology have implicated the regulation of autophagy in inflammation, neurodegeneration, infection, and autoimmunity. These findings have motivated the use of small-molecule probes to study how modulation of autophagy affects disease-associated phenotypes. Here, we describe the discovery of the small-molecule probe BRD5631 that is derived from diversity-oriented synthesis and enhances autophagy through an mTOR-independent pathway. We demonstrate that BRD5631 affects several cellular disease phenotypes previously linked to autophagy, including protein aggregation, cell survival, bacterial replication, and inflammatory cytokine production. BRD5631 can serve as a valuable tool for studying the role of autophagy in the context of cellular homeostasis and disease.


Asunto(s)
Autofagia/efectos de los fármacos , Genética Médica , Enfermedad de Niemann-Pick Tipo C/genética , Enfermedad de Niemann-Pick Tipo C/patología , Bibliotecas de Moléculas Pequeñas/farmacología , Bacterias/efectos de los fármacos , Proteínas Portadoras/metabolismo , Agregación Celular/efectos de los fármacos , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Ensayos Analíticos de Alto Rendimiento , Humanos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/metabolismo , Interleucina-1beta/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Glicoproteínas de Membrana/metabolismo , Modelos Biológicos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/patología , Proteína Niemann-Pick C1 , Enfermedad de Niemann-Pick Tipo C/metabolismo , Péptidos/metabolismo , Fenotipo , Bibliotecas de Moléculas Pequeñas/química
8.
Proc Natl Acad Sci U S A ; 111(34): 12468-73, 2014 Aug 26.
Artículo en Inglés | MEDLINE | ID: mdl-25114223

RESUMEN

Genetic alterations that reduce the function of the immunoregulatory cytokine IL-10 contribute to colitis in mouse and man. Myeloid cells such as macrophages (MΦs) and dendritic cells (DCs) play an essential role in determining the relative abundance of IL-10 versus inflammatory cytokines in the gut. As such, using small molecules to boost IL-10 production by DCs-MΦs represents a promising approach to increase levels of this cytokine specifically in gut tissues. Toward this end, we screened a library of well-annotated kinase inhibitors for compounds that enhance production of IL-10 by murine bone-marrow-derived DCs stimulated with the yeast cell wall preparation zymosan. This approach identified a number of kinase inhibitors that robustly up-regulate IL-10 production including the Food and Drug Administration (FDA)-approved drugs dasatinib, bosutinib, and saracatinib that target ABL, SRC-family, and numerous other kinases. Correlating the kinase selectivity profiles of the active compounds with their effect on IL-10 production suggests that inhibition of salt-inducible kinases (SIKs) mediates the observed IL-10 increase. This was confirmed using the SIK-targeting inhibitor HG-9-91-01 and a series of structural analogs. The stimulatory effect of SIK inhibition on IL-10 is also associated with decreased production of the proinflammatory cytokines IL-1ß, IL-6, IL-12, and TNF-α, and these coordinated effects are observed in human DCs-MΦs and anti-inflammatory CD11c(+) CX3CR1(hi) cells isolated from murine gut tissue. Collectively, these studies demonstrate that SIK inhibition promotes an anti-inflammatory phenotype in activated myeloid cells marked by robust IL-10 production and establish these effects as a previously unidentified activity associated with several FDA-approved multikinase inhibitors.


Asunto(s)
Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Interleucina-10/biosíntesis , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Compuestos de Anilina/farmacología , Animales , Células Cultivadas , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Citocinas/biosíntesis , Dasatinib , Células Dendríticas/enzimología , Evaluación Preclínica de Medicamentos , Humanos , Mediadores de Inflamación/metabolismo , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , Enfermedades Inflamatorias del Intestino/enzimología , Enfermedades Inflamatorias del Intestino/inmunología , Intestino Delgado/efectos de los fármacos , Intestino Delgado/enzimología , Intestino Delgado/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Células Mieloides/efectos de los fármacos , Células Mieloides/enzimología , Células Mieloides/inmunología , Nitrilos/farmacología , Compuestos de Fenilurea/química , Compuestos de Fenilurea/farmacología , Inhibidores de Proteínas Quinasas/química , Pirimidinas/química , Pirimidinas/farmacología , Quinolinas/farmacología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunología , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/enzimología , Linfocitos T Reguladores/inmunología , Tiazoles/farmacología , Factores de Transcripción/metabolismo
9.
Proc Natl Acad Sci U S A ; 111(30): 10911-6, 2014 Jul 29.
Artículo en Inglés | MEDLINE | ID: mdl-25024206

RESUMEN

High-throughput screening has become a mainstay of small-molecule probe and early drug discovery. The question of how to build and evolve efficient screening collections systematically for cell-based and biochemical screening is still unresolved. It is often assumed that chemical structure diversity leads to diverse biological performance of a library. Here, we confirm earlier results showing that this inference is not always valid and suggest instead using biological measurement diversity derived from multiplexed profiling in the construction of libraries with diverse assay performance patterns for cell-based screens. Rather than using results from tens or hundreds of completed assays, which is resource intensive and not easily extensible, we use high-dimensional image-based cell morphology and gene expression profiles. We piloted this approach using over 30,000 compounds. We show that small-molecule profiling can be used to select compound sets with high rates of activity and diverse biological performance.


Asunto(s)
Evaluación Preclínica de Medicamentos/métodos , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Línea Celular Tumoral , Humanos
10.
PLoS Genet ; 9(5): e1003487, 2013 May.
Artículo en Inglés | MEDLINE | ID: mdl-23696745

RESUMEN

Although genetic and non-genetic studies in mouse and human implicate the CD40 pathway in rheumatoid arthritis (RA), there are no approved drugs that inhibit CD40 signaling for clinical care in RA or any other disease. Here, we sought to understand the biological consequences of a CD40 risk variant in RA discovered by a previous genome-wide association study (GWAS) and to perform a high-throughput drug screen for modulators of CD40 signaling based on human genetic findings. First, we fine-map the CD40 risk locus in 7,222 seropositive RA patients and 15,870 controls, together with deep sequencing of CD40 coding exons in 500 RA cases and 650 controls, to identify a single SNP that explains the entire signal of association (rs4810485, P = 1.4×10(-9)). Second, we demonstrate that subjects homozygous for the RA risk allele have ∼33% more CD40 on the surface of primary human CD19+ B lymphocytes than subjects homozygous for the non-risk allele (P = 10(-9)), a finding corroborated by expression quantitative trait loci (eQTL) analysis in peripheral blood mononuclear cells from 1,469 healthy control individuals. Third, we use retroviral shRNA infection to perturb the amount of CD40 on the surface of a human B lymphocyte cell line (BL2) and observe a direct correlation between amount of CD40 protein and phosphorylation of RelA (p65), a subunit of the NF-κB transcription factor. Finally, we develop a high-throughput NF-κB luciferase reporter assay in BL2 cells activated with trimerized CD40 ligand (tCD40L) and conduct an HTS of 1,982 chemical compounds and FDA-approved drugs. After a series of counter-screens and testing in primary human CD19+ B cells, we identify 2 novel chemical inhibitors not previously implicated in inflammation or CD40-mediated NF-κB signaling. Our study demonstrates proof-of-concept that human genetics can be used to guide the development of phenotype-based, high-throughput small-molecule screens to identify potential novel therapies in complex traits such as RA.


Asunto(s)
Artritis Reumatoide/tratamiento farmacológico , Artritis Reumatoide/genética , Antígenos CD40/antagonistas & inhibidores , Antígenos CD40/genética , Evaluación Preclínica de Medicamentos , Alelos , Animales , Antígenos CD19/genética , Artritis Reumatoide/patología , Linfocitos B/citología , Linfocitos B/metabolismo , Antígenos CD40/metabolismo , Predisposición Genética a la Enfermedad , Estudio de Asociación del Genoma Completo , Ensayos Analíticos de Alto Rendimiento , Humanos , Ratones , FN-kappa B/genética , FN-kappa B/metabolismo , Sitios de Carácter Cuantitativo/genética , Transducción de Señal , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología
11.
J Am Chem Soc ; 137(24): 7929-34, 2015 Jun 24.
Artículo en Inglés | MEDLINE | ID: mdl-26042473

RESUMEN

Phenotypic cell-based screening is a powerful approach to small-molecule discovery, but a major challenge of this strategy lies in determining the intracellular target and mechanism of action (MoA) for validated hits. Here, we show that the small-molecule BRD0476, a novel suppressor of pancreatic ß-cell apoptosis, inhibits interferon-gamma (IFN-γ)-induced Janus kinase 2 (JAK2) and signal transducer and activation of transcription 1 (STAT1) signaling to promote ß-cell survival. However, unlike common JAK-STAT pathway inhibitors, BRD0476 inhibits JAK-STAT signaling without suppressing the kinase activity of any JAK. Rather, we identified the deubiquitinase ubiquitin-specific peptidase 9X (USP9X) as an intracellular target, using a quantitative proteomic analysis in rat ß cells. RNAi-mediated and CRISPR/Cas9 knockdown mimicked the effects of BRD0476, and reverse chemical genetics using a known inhibitor of USP9X blocked JAK-STAT signaling without suppressing JAK activity. Site-directed mutagenesis of a putative ubiquitination site on JAK2 mitigated BRD0476 activity, suggesting a competition between phosphorylation and ubiquitination to explain small-molecule MoA. These results demonstrate that phenotypic screening, followed by comprehensive MoA efforts, can provide novel mechanistic insights into ostensibly well-understood cell signaling pathways. Furthermore, these results uncover USP9X as a potential target for regulating JAK2 activity in cellular inflammation.


Asunto(s)
Células Secretoras de Insulina/efectos de los fármacos , Interferón gamma/inmunología , Janus Quinasa 2/inmunología , Sustancias Protectoras/química , Sustancias Protectoras/farmacología , Factor de Transcripción STAT1/inmunología , Animales , Apoptosis/efectos de los fármacos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Humanos , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/inmunología , Fosforilación/efectos de los fármacos , Ratas , Transducción de Señal/efectos de los fármacos , Ubiquitina Tiolesterasa/inmunología , Ubiquitinación/efectos de los fármacos
12.
Nat Chem Biol ; 9(4): 232-40, 2013 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-23508189

RESUMEN

Target-identification and mechanism-of-action studies have important roles in small-molecule probe and drug discovery. Biological and technological advances have resulted in the increasing use of cell-based assays to discover new biologically active small molecules. Such studies allow small-molecule action to be tested in a more disease-relevant setting at the outset, but they require follow-up studies to determine the precise protein target or targets responsible for the observed phenotype. Target identification can be approached by direct biochemical methods, genetic interactions or computational inference. In many cases, however, combinations of approaches may be required to fully characterize on-target and off-target effects and to understand mechanisms of small-molecule action.


Asunto(s)
Biomarcadores Farmacológicos/metabolismo , Descubrimiento de Drogas , Evaluación Preclínica de Medicamentos , Ensayos Analíticos de Alto Rendimiento , Bibliotecas de Moléculas Pequeñas/metabolismo , Animales , Biomarcadores Farmacológicos/química , Humanos , Marcaje Isotópico , Espectrometría de Masas , Terapia Molecular Dirigida , Fenotipo , Interferencia de ARN , Genética Inversa , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Estudios de Validación como Asunto
13.
Nat Chem Biol ; 9(12): 840-848, 2013 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-24161946

RESUMEN

Efforts to develop more effective therapies for acute leukemia may benefit from high-throughput screening systems that reflect the complex physiology of the disease, including leukemia stem cells (LSCs) and supportive interactions with the bone marrow microenvironment. The therapeutic targeting of LSCs is challenging because LSCs are highly similar to normal hematopoietic stem and progenitor cells (HSPCs) and are protected by stromal cells in vivo. We screened 14,718 compounds in a leukemia-stroma co-culture system for inhibition of cobblestone formation, a cellular behavior associated with stem-cell function. Among those compounds that inhibited malignant cells but spared HSPCs was the cholesterol-lowering drug lovastatin. Lovastatin showed anti-LSC activity in vitro and in an in vivo bone marrow transplantation model. Mechanistic studies demonstrated that the effect was on target, via inhibition of HMG-CoA reductase. These results illustrate the power of merging physiologically relevant models with high-throughput screening.


Asunto(s)
Antineoplásicos/farmacología , Ensayos de Selección de Medicamentos Antitumorales/métodos , Leucemia , Células Madre Neoplásicas/efectos de los fármacos , Línea Celular Tumoral , Células Madre Hematopoyéticas , Humanos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Lovastatina/farmacología , Células Madre Neoplásicas/citología , Células Madre Neoplásicas/fisiología
14.
Proc Natl Acad Sci U S A ; 108(17): 6817-22, 2011 Apr 26.
Artículo en Inglés | MEDLINE | ID: mdl-21482810

RESUMEN

Using a diverse collection of small molecules we recently found that compound sets from different sources (commercial; academic; natural) have different protein-binding behaviors, and these behaviors correlate with trends in stereochemical complexity for these compound sets. These results lend insight into structural features that synthetic chemists might target when synthesizing screening collections for biological discovery. We report extensive characterization of structural properties and diversity of biological performance for these compounds and expand comparative analyses to include physicochemical properties and three-dimensional shapes of predicted conformers. The results highlight additional similarities and differences between the sets, but also the dependence of such comparisons on the choice of molecular descriptors. Using a protein-binding dataset, we introduce an information-theoretic measure to assess diversity of performance with a constraint on specificity. Rather than relying on finding individual active compounds, this measure allows rational judgment of compound subsets as groups. We also apply this measure to publicly available data from ChemBank for the same compound sets across a diverse group of functional assays. We find that performance diversity of compound sets is relatively stable across a range of property values as judged by this measure, both in protein-binding studies and functional assays. Because building screening collections with improved performance depends on efficient use of synthetic organic chemistry resources, these studies illustrate an important quantitative framework to help prioritize choices made in building such collections.


Asunto(s)
Bases de Datos Factuales , Evaluación Preclínica de Medicamentos/métodos , Modelos Químicos , Estructura Molecular , Relación Estructura-Actividad
15.
Proc Natl Acad Sci U S A ; 107(34): 15099-104, 2010 Aug 24.
Artículo en Inglés | MEDLINE | ID: mdl-20696901

RESUMEN

High-content screening for small-molecule inducers of insulin expression identified the compound BRD7389, which caused alpha-cells to adopt several morphological and gene expression features of a beta-cell state. Assay-performance profile analysis suggests kinase inhibition as a mechanism of action, and we show that biochemical and cellular inhibition of the RSK kinase family by BRD7389 is likely related to its ability induce a beta-cell-like state. BRD7389 also increases the endocrine cell content and function of donor human pancreatic islets in culture.


Asunto(s)
Células Secretoras de Glucagón/efectos de los fármacos , Células Secretoras de Glucagón/metabolismo , Insulina/biosíntesis , Inhibidores de Proteínas Quinasas/farmacología , Quinolonas/farmacología , Animales , Diferenciación Celular/efectos de los fármacos , Línea Celular , Evaluación Preclínica de Medicamentos , Expresión Génica/efectos de los fármacos , Células Secretoras de Glucagón/citología , Humanos , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/metabolismo , Ratones , Estructura Molecular , Inhibidores de Proteínas Quinasas/química , Quinolonas/química , Interferencia de ARN , Proteínas Quinasas S6 Ribosómicas/antagonistas & inhibidores , Proteínas Quinasas S6 Ribosómicas/genética , Técnicas de Cultivo de Tejidos
16.
Nat Commun ; 14(1): 1967, 2023 04 08.
Artículo en Inglés | MEDLINE | ID: mdl-37031208

RESUMEN

Predicting assay results for compounds virtually using chemical structures and phenotypic profiles has the potential to reduce the time and resources of screens for drug discovery. Here, we evaluate the relative strength of three high-throughput data sources-chemical structures, imaging (Cell Painting), and gene-expression profiles (L1000)-to predict compound bioactivity using a historical collection of 16,170 compounds tested in 270 assays for a total of 585,439 readouts. All three data modalities can predict compound activity for 6-10% of assays, and in combination they predict 21% of assays with high accuracy, which is a 2 to 3 times higher success rate than using a single modality alone. In practice, the accuracy of predictors could be lower and still be useful, increasing the assays that can be predicted from 37% with chemical structures alone up to 64% when combined with phenotypic data. Our study shows that unbiased phenotypic profiling can be leveraged to enhance compound bioactivity prediction to accelerate the early stages of the drug-discovery process.


Asunto(s)
Descubrimiento de Drogas , Transcriptoma , Descubrimiento de Drogas/métodos , Bioensayo , Ensayos Analíticos de Alto Rendimiento/métodos
17.
ACS Chem Biol ; 17(5): 1131-1142, 2022 05 20.
Artículo en Inglés | MEDLINE | ID: mdl-35439415

RESUMEN

Type 2 diabetes is marked by progressive ß-cell failure, leading to loss of ß-cell mass. Increased levels of circulating glucose and free fatty acids associated with obesity lead to ß-cell glucolipotoxicity. There are currently no therapeutic options to address this facet of ß-cell loss in obese type 2 diabetes patients. To identify small molecules capable of protecting ß-cells, we performed a high-throughput screen of 20,876 compounds in the rat insulinoma cell line INS-1E in the presence of elevated glucose and palmitate. We found 312 glucolipotoxicity-protective small molecules (1.49% hit rate) capable of restoring INS-1E viability, and we focused on 17 with known biological targets. 16 of the 17 compounds were kinase inhibitors with activity against specific families including but not limited to cyclin-dependent kinases (CDK), PI-3 kinase (PI3K), Janus kinase (JAK), and Rho-associated kinase 2 (ROCK2). 7 of the 16 kinase inhibitors were PI3K inhibitors. Validation studies in dissociated human islets identified 10 of the 17 compounds, namely, KD025, ETP-45658, BMS-536924, AT-9283, PF-03814735, torin-2, AZD5438, CP-640186, ETP-46464, and GSK2126458 that reduced glucolipotoxicity-induced ß-cell death. These 10 compounds decreased markers of glucolipotoxicity including caspase activation, mitochondrial depolarization, and increased calcium flux. Together, these results provide a path forward toward identifying novel treatments to preserve ß-cell viability in the face of glucolipotoxicity.


Asunto(s)
Diabetes Mellitus Tipo 2 , Células Secretoras de Insulina , Animales , Apoptosis , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , Glucosa/metabolismo , Humanos , Células Secretoras de Insulina/metabolismo , Palmitatos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Ratas
18.
Nat Cell Biol ; 24(12): 1766-1775, 2022 12.
Artículo en Inglés | MEDLINE | ID: mdl-36396978

RESUMEN

The need to control the activity and fidelity of CRISPR-associated nucleases has resulted in a demand for inhibitory anti-CRISPR molecules. The small-molecule inhibitor discovery platforms available at present are not generalizable to multiple nuclease classes, only target the initial step in the catalytic activity and require high concentrations of nuclease, resulting in inhibitors with suboptimal attributes, including poor potency. Here we report a high-throughput discovery pipeline consisting of a fluorescence resonance energy transfer-based assay that is generalizable to contemporary and emerging nucleases, operates at low nuclease concentrations and targets all catalytic steps. We applied this pipeline to identify BRD7586, a cell-permeable small-molecule inhibitor of SpCas9 that is twofold more potent than other inhibitors identified to date. Furthermore, unlike the reported inhibitors, BRD7586 enhanced SpCas9 specificity and its activity was independent of the genomic loci, DNA-repair pathway or mode of nuclease delivery. Overall, these studies describe a general pipeline to identify inhibitors of contemporary and emerging CRISPR-associated nucleases.


Asunto(s)
Genómica
19.
Clin Transl Sci ; 15(8): 1848-1855, 2022 08.
Artículo en Inglés | MEDLINE | ID: mdl-36125173

RESUMEN

Within clinical, biomedical, and translational science, an increasing number of projects are adopting graphs for knowledge representation. Graph-based data models elucidate the interconnectedness among core biomedical concepts, enable data structures to be easily updated, and support intuitive queries, visualizations, and inference algorithms. However, knowledge discovery across these "knowledge graphs" (KGs) has remained difficult. Data set heterogeneity and complexity; the proliferation of ad hoc data formats; poor compliance with guidelines on findability, accessibility, interoperability, and reusability; and, in particular, the lack of a universally accepted, open-access model for standardization across biomedical KGs has left the task of reconciling data sources to downstream consumers. Biolink Model is an open-source data model that can be used to formalize the relationships between data structures in translational science. It incorporates object-oriented classification and graph-oriented features. The core of the model is a set of hierarchical, interconnected classes (or categories) and relationships between them (or predicates) representing biomedical entities such as gene, disease, chemical, anatomic structure, and phenotype. The model provides class and edge attributes and associations that guide how entities should relate to one another. Here, we highlight the need for a standardized data model for KGs, describe Biolink Model, and compare it with other models. We demonstrate the utility of Biolink Model in various initiatives, including the Biomedical Data Translator Consortium and the Monarch Initiative, and show how it has supported easier integration and interoperability of biomedical KGs, bringing together knowledge from multiple sources and helping to realize the goals of translational science.


Asunto(s)
Reconocimiento de Normas Patrones Automatizadas , Ciencia Traslacional Biomédica , Conocimiento
20.
Clin Transl Sci ; 2022 May 25.
Artículo en Inglés | MEDLINE | ID: mdl-35611543

RESUMEN

Clinical, biomedical, and translational science has reached an inflection point in the breadth and diversity of available data and the potential impact of such data to improve human health and well-being. However, the data are often siloed, disorganized, and not broadly accessible due to discipline-specific differences in terminology and representation. To address these challenges, the Biomedical Data Translator Consortium has developed and tested a pilot knowledge graph-based "Translator" system capable of integrating existing biomedical data sets and "translating" those data into insights intended to augment human reasoning and accelerate translational science. Having demonstrated feasibility of the Translator system, the Translator program has since moved into development, and the Translator Consortium has made significant progress in the research, design, and implementation of an operational system. Herein, we describe the current system's architecture, performance, and quality of results. We apply Translator to several real-world use cases developed in collaboration with subject-matter experts. Finally, we discuss the scientific and technical features of Translator and compare those features to other state-of-the-art, biomedical graph-based question-answering systems.

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