RESUMEN
We previously piloted the concept of a Connectivity Map (CMap), whereby genes, drugs, and disease states are connected by virtue of common gene-expression signatures. Here, we report more than a 1,000-fold scale-up of the CMap as part of the NIH LINCS Consortium, made possible by a new, low-cost, high-throughput reduced representation expression profiling method that we term L1000. We show that L1000 is highly reproducible, comparable to RNA sequencing, and suitable for computational inference of the expression levels of 81% of non-measured transcripts. We further show that the expanded CMap can be used to discover mechanism of action of small molecules, functionally annotate genetic variants of disease genes, and inform clinical trials. The 1.3 million L1000 profiles described here, as well as tools for their analysis, are available at https://clue.io.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Línea Celular Tumoral , Resistencia a Antineoplásicos , Perfilación de la Expresión Génica/economía , Humanos , Neoplasias/tratamiento farmacológico , Especificidad de Órganos , Preparaciones Farmacéuticas/metabolismo , Análisis de Secuencia de ARN/economía , Análisis de Secuencia de ARN/métodos , Bibliotecas de Moléculas PequeñasRESUMEN
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 NIH 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.
Asunto(s)
Descubrimiento de Drogas , Bibliotecas de Moléculas Pequeñas , Animales , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Ensayos Analíticos de Alto Rendimiento , Humanos , National Institutes of Health (U.S.) , Estados UnidosRESUMEN
Ferroptosis is a form of nonapoptotic cell death for which key regulators remain unknown. We sought a common mediator for the lethality of 12 ferroptosis-inducing small molecules. We used targeted metabolomic profiling to discover that depletion of glutathione causes inactivation of glutathione peroxidases (GPXs) in response to one class of compounds and a chemoproteomics strategy to discover that GPX4 is directly inhibited by a second class of compounds. GPX4 overexpression and knockdown modulated the lethality of 12 ferroptosis inducers, but not of 11 compounds with other lethal mechanisms. In addition, two representative ferroptosis inducers prevented tumor growth in xenograft mouse tumor models. Sensitivity profiling in 177 cancer cell lines revealed that diffuse large B cell lymphomas and renal cell carcinomas are particularly susceptible to GPX4-regulated ferroptosis. Thus, GPX4 is an essential regulator of ferroptotic cancer cell death.
Asunto(s)
Carbolinas/farmacología , Muerte Celular/efectos de los fármacos , Glutatión Peroxidasa/antagonistas & inhibidores , Piperazinas/farmacología , Animales , Carcinoma de Células Renales/tratamiento farmacológico , Línea Celular Tumoral , Técnicas de Silenciamiento del Gen , Glutatión/metabolismo , Glutatión Peroxidasa/genética , Glutatión Peroxidasa/metabolismo , Xenoinjertos , Humanos , Linfoma de Células B/tratamiento farmacológico , Ratones , Trasplante de Neoplasias , Neoplasias/tratamiento farmacológico , Fosfolípido Hidroperóxido Glutatión PeroxidasaRESUMEN
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/genéticaRESUMEN
CARD9 is a central component of anti-fungal innate immune signaling via C-type lectin receptors, and several immune-related disorders are associated with CARD9 alterations. Here, we used a rare CARD9 variant that confers protection against inflammatory bowel disease as an entry point to investigating CARD9 regulation. We showed that the protective variant of CARD9, which is C-terminally truncated, acted in a dominant-negative manner for CARD9-mediated cytokine production, indicating an important role for the C terminus in CARD9 signaling. We identified TRIM62 as a CARD9 binding partner and showed that TRIM62 facilitated K27-linked poly-ubiquitination of CARD9. We identified K125 as the ubiquitinated residue on CARD9 and demonstrated that this ubiquitination was essential for CARD9 activity. Furthermore, we showed that similar to Card9-deficient mice, Trim62-deficient mice had increased susceptibility to fungal infection. In this study, we utilized a rare protective allele to uncover a TRIM62-mediated mechanism for regulation of CARD9 activation.
Asunto(s)
Proteínas Adaptadoras de Señalización CARD/fisiología , Candidiasis Invasiva/inmunología , Receptores de Angiotensina/fisiología , Receptores de Endotelina/fisiología , Ubiquitina-Proteína Ligasas/fisiología , Adyuvantes Inmunológicos/farmacología , Animales , Proteínas Adaptadoras de Señalización CARD/química , Proteínas Adaptadoras de Señalización CARD/deficiencia , Proteínas Adaptadoras de Señalización CARD/genética , Candidiasis Invasiva/genética , Colitis/inducido químicamente , Colitis/genética , Colitis/prevención & control , Citocinas/biosíntesis , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Genes Dominantes , Predisposición Genética a la Enfermedad , Células HEK293 , Células HeLa , Humanos , Enfermedades Inflamatorias del Intestino/genética , Ratones , Ratones de la Cepa 129 , Ratones Noqueados , Mapeo de Interacción de Proteínas , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiología , Procesamiento Proteico-Postraduccional , Estructura Terciaria de Proteína , Receptores de Angiotensina/química , Receptores de Angiotensina/deficiencia , Receptores de Endotelina/química , Receptores de Endotelina/deficiencia , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal , Organismos Libres de Patógenos Específicos , Proteínas de Motivos Tripartitos , Ubiquitina-Proteína Ligasas/química , UbiquitinaciónRESUMEN
This Letter is being retracted owing to issues with Fig. 1d and Supplementary Fig. 31b, and the unavailability of original data for these figures that raise concerns regarding the integrity of the figures. Nature published two previous corrections related to this Letter1,2. These issues in aggregate undermine the confidence in the integrity of this study. Authors Michael Foley, Monica Schenone, Nicola J. Tolliday, Todd R. Golub, Steven A. Carr, Alykhan F. Shamji, Andrew M. Stern and Stuart L. Schreiber agree with the Retraction. Authors Lakshmi Raj, Takao Ide, Aditi U. Gurkar, Anna Mandinova and Sam W. Lee disagree with the Retraction. Author Xiaoyu Li did not respond.
RESUMEN
Plasticity of the cell state has been proposed to drive resistance to multiple classes of cancer therapies, thereby limiting their effectiveness. A high-mesenchymal cell state observed in human tumours and cancer cell lines has been associated with resistance to multiple treatment modalities across diverse cancer lineages, but the mechanistic underpinning for this state has remained incompletely understood. Here we molecularly characterize this therapy-resistant high-mesenchymal cell state in human cancer cell lines and organoids and show that it depends on a druggable lipid-peroxidase pathway that protects against ferroptosis, a non-apoptotic form of cell death induced by the build-up of toxic lipid peroxides. We show that this cell state is characterized by activity of enzymes that promote the synthesis of polyunsaturated lipids. These lipids are the substrates for lipid peroxidation by lipoxygenase enzymes. This lipid metabolism creates a dependency on pathways converging on the phospholipid glutathione peroxidase (GPX4), a selenocysteine-containing enzyme that dissipates lipid peroxides and thereby prevents the iron-mediated reactions of peroxides that induce ferroptotic cell death. Dependency on GPX4 was found to exist across diverse therapy-resistant states characterized by high expression of ZEB1, including epithelial-mesenchymal transition in epithelial-derived carcinomas, TGFß-mediated therapy-resistance in melanoma, treatment-induced neuroendocrine transdifferentiation in prostate cancer, and sarcomas, which are fixed in a mesenchymal state owing to their cells of origin. We identify vulnerability to ferroptic cell death induced by inhibition of a lipid peroxidase pathway as a feature of therapy-resistant cancer cells across diverse mesenchymal cell-state contexts.
Asunto(s)
Glutatión Peroxidasa/metabolismo , Peroxidación de Lípido/efectos de los fármacos , Neoplasias/tratamiento farmacológico , Neoplasias/enzimología , Cadherinas/metabolismo , Muerte Celular , Línea Celular Tumoral , Linaje de la Célula , Transdiferenciación Celular , Resistencia a Antineoplásicos/genética , Transición Epitelial-Mesenquimal , Humanos , Hierro/metabolismo , Peróxidos Lipídicos/metabolismo , Masculino , Melanoma/tratamiento farmacológico , Melanoma/enzimología , Melanoma/metabolismo , Melanoma/patología , Mesodermo/efectos de los fármacos , Mesodermo/enzimología , Mesodermo/metabolismo , Mesodermo/patología , Neoplasias/genética , Neoplasias/patología , Fosfolípido Hidroperóxido Glutatión Peroxidasa , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/enzimología , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Proteómica , Proteínas Proto-Oncogénicas B-raf/genética , Reproducibilidad de los Resultados , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/genéticaRESUMEN
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/genéticaRESUMEN
Enhancing production of the anti-inflammatory cytokine interleukin-10 (IL-10) is a promising strategy to suppress pathogenic inflammation. To identify new mechanisms regulating IL-10 production, we conducted a phenotypic screen for small molecules that enhance IL-10 secretion from activated dendritic cells. Mechanism-of-action studies using a prioritized hit from the screen, BRD6989, identified the Mediator-associated kinase CDK8, and its paralog CDK19, as negative regulators of IL-10 production during innate immune activation. The ability of BRD6989 to upregulate IL-10 is recapitulated by multiple, structurally differentiated CDK8 and CDK19 inhibitors and requires an intact cyclin C-CDK8 complex. Using a highly parallel pathway reporter assay, we identified a role for enhanced AP-1 activity in IL-10 potentiation following CDK8 and CDK19 inhibition, an effect associated with reduced phosphorylation of a negative regulatory site on c-Jun. These findings identify a function for CDK8 and CDK19 in regulating innate immune activation and suggest that these kinases may warrant consideration as therapeutic targets for inflammatory disorders.
Asunto(s)
Quinasa 8 Dependiente de Ciclina/metabolismo , Interleucina-10/biosíntesis , Células Mieloides/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Células Cultivadas , Quinasa 8 Dependiente de Ciclina/inmunología , Relación Dosis-Respuesta a Droga , Humanos , Interleucina-10/inmunología , Ratones , Ratones Endogámicos C57BL , Estructura Molecular , Células Mieloides/inmunología , Células Mieloides/metabolismo , Bibliotecas de Moléculas Pequeñas/química , Relación Estructura-ActividadRESUMEN
High cancer death rates indicate the need for new anticancer therapeutic agents. Approaches to discovering new cancer drugs include target-based drug discovery and phenotypic screening. Here, we identified phosphodiesterase 3A modulators as cell-selective cancer cytotoxic compounds through phenotypic compound library screening and target deconvolution by predictive chemogenomics. We found that sensitivity to 6-(4-(diethylamino)-3-nitrophenyl)-5-methyl-4,5-dihydropyridazin-3(2H)-one, or DNMDP, across 766 cancer cell lines correlates with expression of the gene PDE3A, encoding phosphodiesterase 3A. Like DNMDP, a subset of known PDE3A inhibitors kill selected cancer cells, whereas others do not. Furthermore, PDE3A depletion leads to DNMDP resistance. We demonstrated that DNMDP binding to PDE3A promotes an interaction between PDE3A and Schlafen 12 (SLFN12), suggestive of a neomorphic activity. Coexpression of SLFN12 with PDE3A correlates with DNMDP sensitivity, whereas depletion of SLFN12 results in decreased DNMDP sensitivity. Our results implicate PDE3A modulators as candidate cancer therapeutic agents and demonstrate the power of predictive chemogenomics in small-molecule discovery.
Asunto(s)
Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3/metabolismo , Citotoxinas/farmacología , Neoplasias/terapia , Piridazinas/química , Piridazinas/farmacología , Línea Celular , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Citotoxinas/química , Citotoxinas/aislamiento & purificación , Sistemas de Liberación de Medicamentos , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Genómica , Humanos , ImmunoblottingRESUMEN
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 â¼19,000 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 PolimerasaRESUMEN
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/químicaRESUMEN
Malignant transformation, driven by gain-of-function mutations in oncogenes and loss-of-function mutations in tumour suppressor genes, results in cell deregulation that is frequently associated with enhanced cellular stress (for example, oxidative, replicative, metabolic and proteotoxic stress, and DNA damage). Adaptation to this stress phenotype is required for cancer cells to survive, and consequently cancer cells may become dependent upon non-oncogenes that do not ordinarily perform such a vital function in normal cells. Thus, targeting these non-oncogene dependencies in the context of a transformed genotype may result in a synthetic lethal interaction and the selective death of cancer cells. Here we used a cell-based small-molecule screening and quantitative proteomics approach that resulted in the unbiased identification of a small molecule that selectively kills cancer cells but not normal cells. Piperlongumine increases the level of reactive oxygen species (ROS) and apoptotic cell death in both cancer cells and normal cells engineered to have a cancer genotype, irrespective of p53 status, but it has little effect on either rapidly or slowly dividing primary normal cells. Significant antitumour effects are observed in piperlongumine-treated mouse xenograft tumour models, with no apparent toxicity in normal mice. Moreover, piperlongumine potently inhibits the growth of spontaneously formed malignant breast tumours and their associated metastases in mice. Our results demonstrate the ability of a small molecule to induce apoptosis selectively in cells that have a cancer genotype, by targeting a non-oncogene co-dependency acquired through the expression of the cancer genotype in response to transformation-induced oxidative stress.
Asunto(s)
Apoptosis/efectos de los fármacos , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Dioxolanos/farmacología , Estrés Oxidativo/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Línea Celular , Línea Celular Tumoral , Transformación Celular Neoplásica , Ensayo Cometa , Daño del ADN/efectos de los fármacos , Dioxolanos/efectos adversos , Dioxolanos/química , Genotipo , Ratones , Metástasis de la Neoplasia/tratamiento farmacológico , Metástasis de la Neoplasia/patología , Bibliotecas de Moléculas Pequeñas/química , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
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/metabolismoRESUMEN
A coding polymorphism (Thr300Ala) in the essential autophagy gene, autophagy related 16-like 1 (ATG16L1), confers increased risk for the development of Crohn disease, although the mechanisms by which single disease-associated polymorphisms contribute to pathogenesis have been difficult to dissect given that environmental factors likely influence disease initiation in these patients. Here we introduce a knock-in mouse model expressing the Atg16L1 T300A variant. Consistent with the human polymorphism, T300A knock-in mice do not develop spontaneous intestinal inflammation, but exhibit morphological defects in Paneth and goblet cells. Selective autophagy is reduced in multiple cell types from T300A knock-in mice compared with WT mice. The T300A polymorphism significantly increases caspase 3- and caspase 7-mediated cleavage of Atg16L1, resulting in lower levels of full-length Atg16Ll T300A protein. Moreover, Atg16L1 T300A is associated with decreased antibacterial autophagy and increased IL-1ß production in primary cells and in vivo. Quantitative proteomics for protein interactors of ATG16L1 identified previously unknown nonoverlapping sets of proteins involved in ATG16L1-dependent antibacterial autophagy or IL-1ß production. These findings demonstrate how the T300A polymorphism leads to cell type- and pathway-specific disruptions of selective autophagy and suggest a mechanism by which this polymorphism contributes to disease.
Asunto(s)
Proteínas Portadoras/genética , Enfermedad de Crohn/inmunología , Células de Paneth/patología , Polimorfismo de Nucleótido Simple/genética , Infecciones por Salmonella/inmunología , Animales , Autofagia/genética , Proteínas Relacionadas con la Autofagia , Western Blotting , Cromatografía Liquida , Enfermedad de Crohn/genética , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Técnicas de Sustitución del Gen , Células Caliciformes/patología , Ratones , Proteómica , Reacción en Cadena en Tiempo Real de la Polimerasa , Espectrometría de Masas en TándemRESUMEN
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 , HumanosRESUMEN
Lysosomes perform a critical cellular function as a site of degradation for diverse cargoes including proteins, organelles, and pathogens delivered through distinct pathways, and defects in lysosomal function have been implicated in a number of diseases. Recent studies have elucidated roles for the lysosome in the regulation of protein synthesis, metabolism, membrane integrity, and other processes involved in homeostasis. Complex small-molecule natural products have greatly contributed to the investigation of lysosomal function in cellular physiology. Here we report the discovery of a novel, small-molecule modulator of lysosomal acidification derived from diversity-oriented synthesis through high-content screening.
Asunto(s)
Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Lisosomas/efectos de los fármacos , Lisosomas/enzimología , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , ATPasas de Translocación de Protón Vacuolares/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Humanos , Lisosomas/metabolismo , Macrólidos/farmacología , ATPasas de Translocación de Protón Vacuolares/antagonistas & inhibidoresRESUMEN
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/fisiologíaRESUMEN
Piperlongumine is a naturally occurring small molecule recently identified to be toxic selectively to cancer cells in vitro and in vivo. This compound was found to elevate cellular levels of reactive oxygen species (ROS) selectively in cancer cell lines. The synthesis of 80 piperlongumine analogs has revealed structural modifications that retain, enhance, and ablate key piperlongumine-associated effects on cells, including elevation of ROS, cancer cell death, and selectivity for cancer cells over nontransformed cell types. Structure/activity relationships suggest that the electrophilicity of the C2-C3 olefin is critical for the observed effects on cells. Furthermore, we show that analogs lacking a reactive C7-C8 olefin can elevate ROS to levels observed with piperlongumine but show markedly reduced cell death, suggesting that ROS-independent mechanisms, including cellular cross-linking events, may also contribute to piperlongumine's induction of apoptosis. In particular, we have identified irreversible protein glutathionylation as a process associated with cellular toxicity. We propose a mechanism of action for piperlongumine that may be relevant to other small molecules having two sites of reactivity, one with greater and the other with lesser electrophilicity.