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1.
Cell ; 173(4): 864-878.e29, 2018 05 03.
Artículo en Inglés | MEDLINE | ID: mdl-29681454

RESUMEN

Diversity in the genetic lesions that cause cancer is extreme. In consequence, a pressing challenge is the development of drugs that target patient-specific disease mechanisms. To address this challenge, we employed a chemistry-first discovery paradigm for de novo identification of druggable targets linked to robust patient selection hypotheses. In particular, a 200,000 compound diversity-oriented chemical library was profiled across a heavily annotated test-bed of >100 cellular models representative of the diverse and characteristic somatic lesions for lung cancer. This approach led to the delineation of 171 chemical-genetic associations, shedding light on the targetability of mechanistic vulnerabilities corresponding to a range of oncogenotypes present in patient populations lacking effective therapy. Chemically addressable addictions to ciliogenesis in TTC21B mutants and GLUT8-dependent serine biosynthesis in KRAS/KEAP1 double mutants are prominent examples. These observations indicate a wealth of actionable opportunities within the complex molecular etiology of cancer.


Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/patología , Proliferación Celular/efectos de los fármacos , Neoplasias Pulmonares/patología , Bibliotecas de Moléculas Pequeñas/farmacología , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Línea Celular Tumoral , Familia 4 del Citocromo P450/deficiencia , Familia 4 del Citocromo P450/genética , Descubrimiento de Drogas , Puntos de Control de la Fase G1 del Ciclo Celular/efectos de los fármacos , Glucocorticoides/farmacología , Proteínas Facilitadoras del Transporte de la Glucosa/antagonistas & inhibidores , Proteínas Facilitadoras del Transporte de la Glucosa/genética , Proteínas Facilitadoras del Transporte de la Glucosa/metabolismo , Humanos , Proteína 1 Asociada A ECH Tipo Kelch/genética , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Mutación , Factor 2 Relacionado con NF-E2/antagonistas & inhibidores , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Receptor Notch2/genética , Receptor Notch2/metabolismo , Receptores de Glucocorticoides/antagonistas & inhibidores , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/metabolismo
2.
Mol Cell ; 84(2): 202-220.e15, 2024 Jan 18.
Artículo en Inglés | MEDLINE | ID: mdl-38103559

RESUMEN

Compounds binding to the bromodomains of bromodomain and extra-terminal (BET) family proteins, particularly BRD4, are promising anticancer agents. Nevertheless, side effects and drug resistance pose significant obstacles in BET-based therapeutics development. Using high-throughput screening of a 200,000-compound library, we identified small molecules targeting a phosphorylated intrinsically disordered region (IDR) of BRD4 that inhibit phospho-BRD4 (pBRD4)-dependent human papillomavirus (HPV) genome replication in HPV-containing keratinocytes. Proteomic profiling identified two DNA damage response factors-53BP1 and BARD1-crucial for differentiation-associated HPV genome amplification. pBRD4-mediated recruitment of 53BP1 and BARD1 to the HPV origin of replication occurs in a spatiotemporal and BRD4 long (BRD4-L) and short (BRD4-S) isoform-specific manner. This recruitment is disrupted by phospho-IDR-targeting compounds with little perturbation of the global transcriptome and BRD4 chromatin landscape. The discovery of these protein-protein interaction inhibitors (PPIi) not only demonstrates the feasibility of developing PPIi against phospho-IDRs but also uncovers antiviral agents targeting an epigenetic regulator essential for virus-host interaction and cancer development.


Asunto(s)
Infecciones por Papillomavirus , Factores de Transcripción , Humanos , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Virus del Papiloma Humano , Infecciones por Papillomavirus/tratamiento farmacológico , Infecciones por Papillomavirus/genética , Proteómica , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Papillomaviridae/genética , Papillomaviridae/metabolismo , Proteínas Virales/genética , Replicación Viral/fisiología , Reparación del ADN , Proteínas que Contienen Bromodominio
3.
Cell ; 155(3): 552-66, 2013 Oct 24.
Artículo en Inglés | MEDLINE | ID: mdl-24243015

RESUMEN

Context-specific molecular vulnerabilities that arise during tumor evolution represent an attractive intervention target class. However, the frequency and diversity of somatic lesions detected among lung tumors can confound efforts to identify these targets. To confront this challenge, we have applied parallel screening of chemical and genetic perturbations within a panel of molecularly annotated NSCLC lines to identify intervention opportunities tightly linked to molecular response indicators predictive of target sensitivity. Anchoring this analysis on a matched tumor/normal cell model from a lung adenocarcinoma patient identified three distinct target/response-indicator pairings that are represented with significant frequencies (6%-16%) in the patient population. These include NLRP3 mutation/inflammasome activation-dependent FLIP addiction, co-occurring KRAS and LKB1 mutation-driven COPI addiction, and selective sensitivity to a synthetic indolotriazine that is specified by a seven-gene expression signature. Target efficacies were validated in vivo, and mechanism-of-action studies informed generalizable principles underpinning cancer cell biology.


Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Ensayos de Selección de Medicamentos Antitumorales , Indoles/farmacología , Neoplasias Pulmonares/metabolismo , Triazinas/farmacología , Animales , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Proteínas Portadoras , Línea Celular Tumoral , Proteína Coatómero/metabolismo , Femenino , Genes ras , Xenoinjertos , Humanos , Neoplasias Pulmonares/patología , Lisosomas/metabolismo , Ratones , Terapia Molecular Dirigida , Proteína con Dominio Pirina 3 de la Familia NLR , Trasplante de Neoplasias , Fosforilación Oxidativa
4.
Proc Natl Acad Sci U S A ; 121(7): e2318024121, 2024 Feb 13.
Artículo en Inglés | MEDLINE | ID: mdl-38330014

RESUMEN

Lipid synthesis is regulated by the actions of Scap, a polytopic membrane protein that binds cholesterol in membranes of the endoplasmic reticulum (ER). When ER cholesterol levels are low, Scap activates SREBPs, transcription factors that upregulate genes for synthesis of cholesterol, fatty acids, and triglycerides. When ER cholesterol levels rise, the sterol binds to Scap, triggering conformational changes that prevent activation of SREBPs and halting synthesis of lipids. To achieve a molecular understanding of how cholesterol regulates the Scap/SREBP machine and to identify therapeutics for dysregulated lipid metabolism, cholesterol-mimetic compounds that specifically bind and inhibit Scap are needed. To accomplish this goal, we focused on Anthrolysin O (ALO), a pore-forming bacterial toxin that binds cholesterol with a specificity and sensitivity that is uncannily similar to Scap. We reasoned that a small molecule that would bind and inhibit ALO might also inhibit Scap. High-throughput screening of a ~300,000-compound library for ALO-binding unearthed one molecule, termed UT-59, which binds to Scap's cholesterol-binding site. Upon binding, UT-59 triggers the same conformation changes in Scap as those induced by cholesterol and blocks activation of SREBPs and lipogenesis in cultured cells. UT-59 also inhibits SREBP activation in the mouse liver. Unlike five previously reported inhibitors of SREBP activation, UT-59 is the only one that acts specifically by binding to Scap's cholesterol-binding site. Our approach to identify specific Scap inhibitors such as UT-59 holds great promise in developing therapeutic leads for human diseases stemming from elevated SREBP activation, such as fatty liver and certain cancers.


Asunto(s)
Toxinas Bacterianas , Lipogénesis , Animales , Ratones , Humanos , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Colesterol/metabolismo , Toxinas Bacterianas/metabolismo
5.
Proc Natl Acad Sci U S A ; 119(51): e2213116119, 2022 12 20.
Artículo en Inglés | MEDLINE | ID: mdl-36512492

RESUMEN

New antimicrobials are needed for the treatment of extensively drug-resistant Acinetobacter baumannii. The de novo pyrimidine biosynthetic enzyme dihydroorotate dehydrogenase (DHODH) is a validated drug target for malaria and human autoimmune diseases. We provide genetic evidence that A. baumannii DHODH (AbDHODH) is essential for bacterial survival in rodent infection models. We chemically validate the target by repurposing a unique library of ~450 triazolopyrimidine/imidazopyrimidine analogs developed for our malaria DHODH program to identify 21 compounds with submicromolar activity on AbDHODH. The most potent (DSM186, DHODH IC50 28 nM) had a minimal inhibitory concentration of ≤1 µg/ml against geographically diverse A. baumannii strains, including meropenem-resistant isolates. A structurally related analog (DSM161) with a long in vivo half-life conferred significant protection in the neutropenic mouse thigh infection model. Encouragingly, the development of resistance to these compounds was not identified in vitro or in vivo. Lastly, the X-ray structure of AbDHODH bound to DSM186 was solved to 1.4 Å resolution. These data support the potential of AbDHODH as a drug target for the development of antimicrobials for the treatment of A. baumannii and potentially other high-risk bacterial infections.


Asunto(s)
Acinetobacter baumannii , Humanos , Ratones , Animales , Dihidroorotato Deshidrogenasa , Pruebas de Sensibilidad Microbiana , Meropenem , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/química , Antibacterianos/farmacología , Antibacterianos/uso terapéutico
6.
Genes Dev ; 31(17): 1770-1783, 2017 09 01.
Artículo en Inglés | MEDLINE | ID: mdl-28982760

RESUMEN

Direct reprogramming of fibroblasts to cardiomyocytes represents a potential means of restoring cardiac function following myocardial injury. AKT1 in the presence of four cardiogenic transcription factors, GATA4, HAND2, MEF2C, and TBX5 (AGHMT), efficiently induces the cardiac gene program in mouse embryonic fibroblasts but not adult fibroblasts. To identify additional regulators of adult cardiac reprogramming, we performed an unbiased screen of transcription factors and cytokines for those that might enhance or suppress the cardiogenic activity of AGHMT in adult mouse fibroblasts. Among a collection of inducers and repressors of cardiac reprogramming, we discovered that the zinc finger transcription factor 281 (ZNF281) potently stimulates cardiac reprogramming by genome-wide association with GATA4 on cardiac enhancers. Concomitantly, ZNF281 suppresses expression of genes associated with inflammatory signaling, suggesting the antagonistic convergence of cardiac and inflammatory transcriptional programs. Consistent with an inhibitory influence of inflammatory pathways on cardiac reprogramming, blockade of these pathways with anti-inflammatory drugs or components of the nucleosome remodeling deacetylase (NuRD) complex, which associate with ZNF281, stimulates cardiac gene expression. We conclude that ZNF281 acts at a nexus of cardiac and inflammatory gene programs, which exert opposing influences on fibroblast to cardiac reprogramming.


Asunto(s)
Reprogramación Celular/genética , Regulación de la Expresión Génica/genética , Factores de Transcripción/metabolismo , Antiinflamatorios/farmacología , Reprogramación Celular/efectos de los fármacos , Fibroblastos/fisiología , Factor de Transcripción GATA4/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Estudio de Asociación del Genoma Completo , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/metabolismo , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/fisiología , Proteínas Represoras , Transcriptoma
7.
J Pharmacol Exp Ther ; 384(3): 393-405, 2023 03.
Artículo en Inglés | MEDLINE | ID: mdl-36635085

RESUMEN

Metabolic flux augmentation via glucose transport activation may be desirable in glucose transporter 1 (Glut1) deficiency syndrome (G1D) and dementia, whereas suppression might prove useful in cancer. Using lung adenocarcinoma cells that predominantly express Glut1 relative to other glucose transporters, we screened 9,646 compounds for effects on the accumulation of an extracellularly applied fluorescent glucose analog. Five drugs currently prescribed for unrelated indications or preclinically characterized robustly enhanced intracellular fluorescence. Additionally identified were 37 novel activating and nine inhibitory compounds lacking previous biologic characterization. Because few glucose-related mechanistic or pharmacological studies were available for these compounds, we developed a method to quantify G1D mouse behavior to infer potential therapeutic value. To this end, we designed a five-track apparatus to record and evaluate spontaneous locomotion videos. We applied this to a G1D mouse model that replicates the ataxia and other manifestations cardinal to the human disorder. Because the first two drugs that we examined in this manner (baclofen and acetazolamide) exerted various impacts on several gait aspects, we used deep learning neural networks to more comprehensively assess drug effects. Using this method, 49 locomotor parameters differentiated G1D from control mice. Thus, we used parameter modifiability to quantify efficacy on gait. We tested this by measuring the effects of saline as control and glucose as G1D therapy. The results indicate that this in vivo approach can estimate preclinical suitability from the perspective of G1D locomotion. This justifies the use of this method to evaluate our drugs or other interventions and sort candidates for further investigation. SIGNIFICANCE STATEMENT: There are few or no activators and few clinical inhibitors of glucose transport. Using Glut1-rich cells exposed to a glucose analog, we identified, in highthroughput fashion, a series of novel modulators. Some were drugs used to modify unrelated processes and some represented large but little studied chemical compound families. To facilitate their preclinical efficacy characterization regardless of potential mechanism of action, we developed a gait testing platform for deep learning neural network analysis of drug impact on Glut1-deficient mouse locomotion.


Asunto(s)
Errores Innatos del Metabolismo de los Carbohidratos , Aprendizaje Profundo , Animales , Humanos , Ratones , Errores Innatos del Metabolismo de los Carbohidratos/metabolismo , Glucosa/metabolismo , Transportador de Glucosa de Tipo 1
8.
Cancer Immunol Immunother ; 71(7): 1671-1680, 2022 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-34816323

RESUMEN

Natural killer (NK) cells are cytotoxic lymphocytes that play a major role in the innate immune system. NK cells exhibit potent cytotoxic activity against cancer cells and virally infected cells without antigen priming. These unique cytotoxic properties make NK cells a promising therapeutic against cancer. Limitations of NK cell therapy include deficiencies in high clinical efficacy often due to a need for a high NK cell to target cell ratio to achieve effective killing. In order to address the suboptimal efficacy of current adoptive NK cell therapy, a high throughput screen (HTS) was designed and performed to identify drug-like compounds that increase NK cytotoxic activity against tumor cells without affecting the normal cells. This screen was performed in a 384-well plate format utilizing an expanded primary NK cell product and ovarian cancer cells as a target cell (TC) line. Of the 8000 diverse small molecules screened, 16 hits were identified (0.2% hit rate) based on both a robust Z (RZ) score < -3 and a greater than 10% increase in NK cell killing. A validation screen had a confirmation rate of 70%. Select compounds were further validated and characterized by additional cytotoxicity assays including activity against multiple blood cancer and solid tumor cell lines, with no effect on primary human T cells. This work demonstrates that high-throughput screening can be reliably used to identify compounds that increase NK tumoricidal activity in vitro that can be further investigated and translated for potential clinical application. Précis: Our work led to the identification of promising compound that potently increases NK cell-mediated killing of a variety of different cancer cells, but no impact on the killing of normal cells. This compound demonstrates the utility of this assay.


Asunto(s)
Detección Precoz del Cáncer , Neoplasias , Línea Celular Tumoral , Citotoxicidad Inmunológica , Humanos , Inmunoterapia Adoptiva , Células Asesinas Naturales , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Linfocitos T
9.
PLoS Pathog ; 16(4): e1008407, 2020 04.
Artículo en Inglés | MEDLINE | ID: mdl-32240278

RESUMEN

Influenza A viruses are human pathogens with limited therapeutic options. Therefore, it is crucial to devise strategies for the identification of new classes of antiviral medications. The influenza A virus genome is constituted of 8 RNA segments. Two of these viral RNAs are transcribed into mRNAs that are alternatively spliced. The M1 mRNA encodes the M1 protein but is also alternatively spliced to yield the M2 mRNA during infection. M1 to M2 mRNA splicing occurs at nuclear speckles, and M1 and M2 mRNAs are exported to the cytoplasm for translation. M1 and M2 proteins are critical for viral trafficking, assembly, and budding. Here we show that gene knockout of the cellular protein NS1-BP, a constituent of the M mRNA speckle-export pathway and a binding partner of the virulence factor NS1 protein, inhibits M mRNA nuclear export without altering bulk cellular mRNA export, providing an avenue to preferentially target influenza virus. We performed a high-content, image-based chemical screen using single-molecule RNA-FISH to label viral M mRNAs followed by multistep quantitative approaches to assess cellular mRNA and cell toxicity. We identified inhibitors of viral mRNA biogenesis and nuclear export that exhibited no significant activity towards bulk cellular mRNA at non-cytotoxic concentrations. Among the hits is a small molecule that preferentially inhibits nuclear export of a subset of viral and cellular mRNAs without altering bulk cellular mRNA export. These findings underscore specific nuclear export requirements for viral mRNAs and phenocopy down-regulation of the mRNA export factor UAP56. This RNA export inhibitor impaired replication of diverse influenza A virus strains at non-toxic concentrations. Thus, this screening strategy yielded compounds that alone or in combination may serve as leads to new ways of treating influenza virus infection and are novel tools for studying viral RNA trafficking in the nucleus.


Asunto(s)
Transporte Activo de Núcleo Celular/efectos de los fármacos , Antivirales/farmacología , Núcleo Celular/virología , Virus de la Influenza A/metabolismo , Gripe Humana/virología , ARN Mensajero/metabolismo , ARN Viral/metabolismo , Evaluación Preclínica de Medicamentos , Humanos , Virus de la Influenza A/genética , ARN Mensajero/genética , ARN Viral/genética , Replicación Viral/efectos de los fármacos
10.
Nature ; 538(7623): 114-117, 2016 Oct 06.
Artículo en Inglés | MEDLINE | ID: mdl-27680702

RESUMEN

The common participation of oncogenic KRAS proteins in many of the most lethal human cancers, together with the ease of detecting somatic KRAS mutant alleles in patient samples, has spurred persistent and intensive efforts to develop drugs that inhibit KRAS activity. However, advances have been hindered by the pervasive inter- and intra-lineage diversity in the targetable mechanisms that underlie KRAS-driven cancers, limited pharmacological accessibility of many candidate synthetic-lethal interactions and the swift emergence of unanticipated resistance mechanisms to otherwise effective targeted therapies. Here we demonstrate the acute and specific cell-autonomous addiction of KRAS-mutant non-small-cell lung cancer cells to receptor-dependent nuclear export. A multi-genomic, data-driven approach, utilizing 106 human non-small-cell lung cancer cell lines, was used to interrogate 4,725 biological processes with 39,760 short interfering RNA pools for those selectively required for the survival of KRAS-mutant cells that harbour a broad spectrum of phenotypic variation. Nuclear transport machinery was the sole process-level discriminator of statistical significance. Chemical perturbation of the nuclear export receptor XPO1 (also known as CRM1), with a clinically available drug, revealed a robust synthetic-lethal interaction with native or engineered oncogenic KRAS both in vitro and in vivo. The primary mechanism underpinning XPO1 inhibitor sensitivity was intolerance to the accumulation of nuclear IκBα (also known as NFKBIA), with consequent inhibition of NFκB transcription factor activity. Intrinsic resistance associated with concurrent FSTL5 mutations was detected and determined to be a consequence of YAP1 activation via a previously unappreciated FSTL5-Hippo pathway regulatory axis. This occurs in approximately 17% of KRAS-mutant lung cancers, and can be overcome with the co-administration of a YAP1-TEAD inhibitor. These findings indicate that clinically available XPO1 inhibitors are a promising therapeutic strategy for a considerable cohort of patients with lung cancer when coupled to genomics-guided patient selection and observation.


Asunto(s)
Transporte Activo de Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Carioferinas/antagonistas & inhibidores , Carioferinas/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/genética , Receptores Citoplasmáticos y Nucleares/antagonistas & inhibidores , Receptores Citoplasmáticos y Nucleares/metabolismo , Proteínas Adaptadoras Transductoras de Señales/antagonistas & inhibidores , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Núcleo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/metabolismo , Femenino , Proteínas Relacionadas con la Folistatina/genética , Genes Letales/genética , Vía de Señalización Hippo , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Ratones , Mutación , Inhibidor NF-kappaB alfa/metabolismo , FN-kappa B/antagonistas & inhibidores , FN-kappa B/metabolismo , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/metabolismo , Fosfoproteínas/antagonistas & inhibidores , Fosfoproteínas/metabolismo , Porfirinas/farmacología , Proteínas Serina-Treonina Quinasas/metabolismo , Interferencia de ARN , ARN Interferente Pequeño , Transducción de Señal , Factores de Transcripción de Dominio TEA , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/metabolismo , Verteporfina , Ensayos Antitumor por Modelo de Xenoinjerto , Proteínas Señalizadoras YAP , Proteína Exportina 1
11.
Biochem J ; 477(17): 3253-3269, 2020 09 18.
Artículo en Inglés | MEDLINE | ID: mdl-32776146

RESUMEN

The carbohydrate response element binding protein (ChREBP) is a glucose-responsive transcription factor that plays a critical role in glucose-mediated induction of genes involved in hepatic glycolysis and lipogenesis. In response to fluctuating blood glucose levels ChREBP activity is regulated mainly by nucleocytoplasmic shuttling of ChREBP. Under high glucose ChREBP binds to importin α and importin ß and translocates into the nucleus to initiate transcription. We have previously shown that the nuclear localization signal site (NLS) for ChREBP is bipartite with the NLS extending from Arg158 to Lys190. Here, we report the 2.5 Šcrystal structure of the ChREBP-NLS peptide bound to importin α. The structure revealed that the NLS binding is monopartite, with the amino acid residues K171RRI174 from the ChREBP-NLS interacting with ARM2-ARM5 on importin α. We discovered that importin α also binds to the primary binding site of the 14-3-3 proteins with high affinity, which suggests that both importin α and 14-3-3 are each competing with the other for this broad-binding region (residues 117-196) on ChREBP. We screened a small compound library and identified two novel compounds that inhibit the ChREBP-NLS/importin α interaction, nuclear localization, and transcription activities of ChREBP. These candidate molecules support developing inhibitors of ChREBP that may be useful in treatment of obesity and the associated diseases.


Asunto(s)
Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/química , Señales de Localización Nuclear/química , alfa Carioferinas/química , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Cristalografía por Rayos X , Células Hep G2 , Humanos , Señales de Localización Nuclear/genética , Señales de Localización Nuclear/metabolismo , alfa Carioferinas/genética , alfa Carioferinas/metabolismo
12.
Molecules ; 26(19)2021 Sep 29.
Artículo en Inglés | MEDLINE | ID: mdl-34641449

RESUMEN

We discovered SW033291 in a high throughput chemical screen aimed at identifying 15-prostaglandin dehydrogenase (15-PGDH) modulators. The compound exhibited inhibitory activity in in vitro biochemical and cell-based assays of 15-PGDH activity. We subsequently demonstrated that this compound, and several analogs thereof, are effective in in vivo mouse models of bone marrow transplant, colitis, and liver regeneration, where increased levels of PGE2 positively potentiate tissue regeneration. To better understand the binding of SW033291, we carried out docking studies for both the substrate, PGE2, and an inhibitor, SW033291, to 15-PGDH. Our models suggest similarities in the ways that PGE2 and SW033291 interact with key residues in the 15-PGDH-NAD+ complex. We carried out molecular dynamics simulations (MD) of SW033291 bound to this complex, in order to understand the dynamics of the binding interactions for this compound. The butyl side chain (including the sulfoxide) of SW033291 participates in crucial binding interactions that are similar to those observed for the C15-OH and the C16-C20 alkyl chain of PGE2. In addition, interactions with residues Ser138, Tyr151, and Gln148 play key roles in orienting and stabilizing SW033291 in the binding site and lead to enantioselectivity for the R-enantiomer. Finally, we compare the binding mode of (R)-S(O)-SW033291 with the binding interactions of published 15-PGDH inhibitors.


Asunto(s)
Hidroxiprostaglandina Deshidrogenasas/antagonistas & inhibidores , Piridinas/química , Piridinas/farmacología , Tiofenos/química , Tiofenos/farmacología , Sitios de Unión , Humanos , Simulación de Dinámica Molecular
13.
Proc Natl Acad Sci U S A ; 114(31): E6427-E6436, 2017 08 01.
Artículo en Inglés | MEDLINE | ID: mdl-28716915

RESUMEN

The cervix represents a formidable structural barrier for successful induction of labor. Approximately 10% of pregnancies undergo induction of cervical ripening and labor with prostaglandin (PG) E2 or PGE analogs, often requiring many hours of hospitalization and monitoring. On the other hand, preterm cervical ripening in the second trimester predicts preterm birth. The regulatory mechanisms of this paradoxical function of the cervix are unknown. Here, we show that PGE2 uses cell-specific EP2 receptor-mediated increases in Ca2+ to dephosphorylate and translocate histone deacetylase 4 (HDAC4) to the nucleus for repression of 15-hydroxy prostaglandin dehydrogenase (15-PGDH). The crucial role of 15-PGDH in cervical ripening was confirmed in vivo. Although PGE2 or 15-PGDH inhibitor alone did not alter gestational length, treatment with 15-PGDH inhibitor + PGE2 or metabolism-resistant dimethyl-PGE2 resulted in preterm cervical ripening and delivery in mice. The ability of PGE2 to selectively autoamplify its own synthesis in stromal cells by signaling transcriptional repression of 15-PGDH elucidates long sought-after molecular mechanisms that govern PG action in the cervix. This report details unique mechanisms of action in the cervix and serves as a catalyst for (i) the use of 15-PGDH inhibitors to initiate or amplify low-dose PGE2-mediated cervical ripening or (ii) EP2 receptor antagonists, HDAC4 inhibitors, and 15-PGDH activators to prevent preterm cervical ripening and preterm birth.


Asunto(s)
Maduración Cervical/metabolismo , Dinoprostona/metabolismo , Histona Desacetilasas/metabolismo , Hidroxiprostaglandina Deshidrogenasas/metabolismo , Nacimiento Prematuro/fisiopatología , Subtipo EP2 de Receptores de Prostaglandina E/metabolismo , Proteínas Represoras/metabolismo , Animales , Calcio/metabolismo , Línea Celular , Cuello del Útero/citología , Cuello del Útero/fisiología , Ciclooxigenasa 2/metabolismo , Regulación hacia Abajo , Femenino , Histona Desacetilasa 2/genética , Histona Desacetilasas/genética , Humanos , Ratones , Ratones Endogámicos C57BL , Embarazo , Prostaglandina-E Sintasas/antagonistas & inhibidores , Prostaglandina-E Sintasas/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/genética , Proteínas Represoras/antagonistas & inhibidores , Proteínas Represoras/genética
14.
Nat Chem Biol ; 12(4): 218-25, 2016 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-26829472

RESUMEN

A hallmark of targeted cancer therapies is selective toxicity among cancer cell lines. We evaluated results from a viability screen of over 200,000 small molecules to identify two chemical series, oxalamides and benzothiazoles, that were selectively toxic at low nanomolar concentrations to the same 4 of 12 human lung cancer cell lines. Sensitive cell lines expressed cytochrome P450 (CYP) 4F11, which metabolized the compounds into irreversible inhibitors of stearoyl CoA desaturase (SCD). SCD is recognized as a promising biological target in cancer and metabolic disease. However, SCD is essential to sebocytes, and accordingly SCD inhibitors cause skin toxicity. Mouse sebocytes did not activate the benzothiazoles or oxalamides into SCD inhibitors, providing a therapeutic window for inhibiting SCD in vivo. We thus offer a strategy to target SCD in cancer by taking advantage of high CYP expression in a subset of tumors.


Asunto(s)
Antineoplásicos/farmacología , Benzotiazoles/farmacología , Descubrimiento de Drogas/métodos , Neoplasias Pulmonares/enzimología , Ácido Oxámico/análogos & derivados , Estearoil-CoA Desaturasa/antagonistas & inhibidores , Animales , Antineoplásicos/farmacocinética , Antineoplásicos/uso terapéutico , Antineoplásicos/toxicidad , Benzotiazoles/farmacocinética , Benzotiazoles/uso terapéutico , Benzotiazoles/toxicidad , Línea Celular Tumoral , Sistema Enzimático del Citocromo P-450/metabolismo , Familia 4 del Citocromo P450 , Femenino , Humanos , Neoplasias Pulmonares/patología , Masculino , Ratones , Ratones SCID , Estructura Molecular , Terapia Molecular Dirigida , Ácido Oxámico/farmacocinética , Ácido Oxámico/farmacología , Ácido Oxámico/uso terapéutico , Ácido Oxámico/toxicidad , Unión Proteica , Glándulas Sebáceas/efectos de los fármacos , Glándulas Sebáceas/enzimología , Glándulas Sebáceas/patología , Ensayos Antitumor por Modelo de Xenoinjerto
15.
Nat Chem Biol ; 11(1): 58-63, 2015 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-25402767

RESUMEN

A fundamental challenge in treating disease is identifying molecular states that affect cellular responses to drugs. Here, we focus on glycogen synthase kinase 3 (GSK-3), a key regulator for many of the hallmark behaviors of cancer cells. We alter GSK-3 activity in colon epithelial cells to test its role in modulating drug response. We find that GSK-3 activity broadly affects the cellular sensitivities to a panel of oncology drugs and kinase inhibitors. Specifically, inhibition of GSK-3 activity can strongly desensitize or sensitize cells to kinase inhibitors (for example, mTOR or PLK1 inhibitors, respectively). Additionally, colorectal cancer cell lines, in which GSK-3 function is commonly suppressed, are resistant to mTOR inhibitors and yet highly sensitive to PLK1 inhibitors, and this is further exacerbated by additional GSK-3 inhibition. Finally, by conducting a kinome-wide RNAi screen, we find that GSK-3 modulates the cell proliferative phenotype of a large fraction (∼35%) of the kinome, which includes ∼50% of current, clinically relevant kinase-targeted drugs. Our results highlight an underappreciated interplay of GSK-3 with therapeutically important kinases and suggest strategies for identifying disease-specific molecular profiles that can guide optimal selection of drug treatment.


Asunto(s)
Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Glucógeno Sintasa Quinasa 3/fisiología , Inhibidores de Proteínas Quinasas/farmacología , Antineoplásicos/farmacología , Proteínas de Ciclo Celular/antagonistas & inhibidores , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Quinasa Tipo Polo 1
16.
J Am Chem Soc ; 138(33): 10561-70, 2016 08 24.
Artículo en Inglés | MEDLINE | ID: mdl-27459345

RESUMEN

Poylcyclic tetrahydroxanthones comprise a large class of cytototoxic natural products. No mechanism of action has been described for any member of the family. We report the synthesis of kibdelone C and several simplified analogs. Both enantiomers of kibdeleone C show low nanomolar cytotoxicity toward multiple human cancer cell lines. Moreover, several simplified derivatives with improved chemical stability display higher activity than the natural product itself. In vitro studies rule out interaction with DNA or inhibition of topoisomerase, both of which are common modes of action for polycyclic aromatic compounds. However, celluar studies reveal that kibdelone C and its simplified derivatives disrupt the actin cytoseketon without directly binding actin or affecting its polymerization in vitro.


Asunto(s)
Xantonas/síntesis química , Xantonas/farmacología , Línea Celular Tumoral , Técnicas de Química Sintética , Citoesqueleto/efectos de los fármacos , Citoesqueleto/metabolismo , Humanos , Xantonas/química
17.
Bioorg Med Chem Lett ; 26(16): 3923-7, 2016 08 15.
Artículo en Inglés | MEDLINE | ID: mdl-27426302

RESUMEN

The MAP3K (Mitogen Activated Protein Kinase Kinase Kinase) TAOK2 (Thousand-And-One Kinase 2) is an activator of p38 MAP kinase cascade that is up-regulated in response to environmental stresses. A synthetic lethal screen performed using a NSCLC (non-small cell lung cancer) cell line, and a second screen identifying potential modulators of autophagy have implicated TAOK2 as a potential cancer therapeutic target. Using a 200,000 compound high throughput screen, we identified three specific small molecule compounds that inhibit the kinase activity of TAOK2. These compounds also showed inhibition of autophagy. Based on SAR (structure-activity relationship) studies, we have predicted the modifications on the reactive groups for the three compounds.


Asunto(s)
Inhibidores de Proteínas Quinasas/química , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/química , Autofagia/efectos de los fármacos , Línea Celular Tumoral , Evaluación Preclínica de Medicamentos , Ensayos Analíticos de Alto Rendimiento , Humanos , Unión Proteica , Inhibidores de Proteínas Quinasas/metabolismo , Inhibidores de Proteínas Quinasas/toxicidad , Proteínas Serina-Treonina Quinasas/metabolismo , Bibliotecas de Moléculas Pequeñas/metabolismo , Bibliotecas de Moléculas Pequeñas/toxicidad , Relación Estructura-Actividad , Temperatura de Transición , Proteínas Quinasas p38 Activadas por Mitógenos/química , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo
18.
Proc Natl Acad Sci U S A ; 110(51): 20364-71, 2013 Dec 17.
Artículo en Inglés | MEDLINE | ID: mdl-24277826

RESUMEN

A long-standing controversy is whether autophagy is a bona fide cause of mammalian cell death. We used a cell-penetrating autophagy-inducing peptide, Tat-Beclin 1, derived from the autophagy protein Beclin 1, to investigate whether high levels of autophagy result in cell death by autophagy. Here we show that Tat-Beclin 1 induces dose-dependent death that is blocked by pharmacological or genetic inhibition of autophagy, but not of apoptosis or necroptosis. This death, termed "autosis," has unique morphological features, including increased autophagosomes/autolysosomes and nuclear convolution at early stages, and focal swelling of the perinuclear space at late stages. We also observed autotic death in cells during stress conditions, including in a subpopulation of nutrient-starved cells in vitro and in hippocampal neurons of neonatal rats subjected to cerebral hypoxia-ischemia in vivo. A chemical screen of ~5,000 known bioactive compounds revealed that cardiac glycosides, antagonists of Na(+),K(+)-ATPase, inhibit autotic cell death in vitro and in vivo. Furthermore, genetic knockdown of the Na(+),K(+)-ATPase α1 subunit blocks peptide and starvation-induced autosis in vitro. Thus, we have identified a unique form of autophagy-dependent cell death, a Food and Drug Administration-approved class of compounds that inhibit such death, and a crucial role for Na(+),K(+)-ATPase in its regulation. These findings have implications for understanding how cells die during certain stress conditions and how such cell death might be prevented.


Asunto(s)
Autofagia/efectos de los fármacos , Isquemia Encefálica/metabolismo , Péptidos de Penetración Celular/farmacología , Proteínas del Tejido Nervioso/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Animales , Isquemia Encefálica/patología , Glicósidos Cardíacos/farmacología , Células HeLa , Humanos , Ratas , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores
19.
J Biol Chem ; 288(27): 19673-84, 2013 Jul 05.
Artículo en Inglés | MEDLINE | ID: mdl-23671287

RESUMEN

A novel scintillation proximity high throughput assay (SPA) to identify inhibitors of DNA methyltransferases was developed and used to screen over 180,000 compounds. The majority of the validated hits shared a quinone core and several were found to generate the reactive oxygen species, H2O2. Inhibition of the production of H2O2 by the addition of catalase blocked the ability of this group of compounds to inhibit DNA methyltransferase (DNMT) activity. However, a related compound, SW155246, was identified that existed in an already reduced form of the quinone. This compound did not generate H2O2, and catalase did not block its ability to inhibit DNA methyltransferase. SW155246 showed a 30-fold preference for inhibition of human DNMT1 versus human or murine DNMT3A or -3B, inhibited global methylation in HeLa cells, and reactivated expression of the tumor suppressor gene RASSF1A in A549 cells. To our knowledge, this work represents the first description of selective chemical inhibitors of the DNMT1 enzyme.


Asunto(s)
Bioensayo , ADN (Citosina-5-)-Metiltransferasas/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Animales , ADN (Citosina-5-)-Metiltransferasa 1 , ADN (Citosina-5-)-Metiltransferasas/genética , ADN (Citosina-5-)-Metiltransferasas/metabolismo , ADN Metiltransferasa 3A , Inhibidores Enzimáticos/química , Células HeLa , Humanos , Peróxido de Hidrógeno/farmacología , Ratones , Oxidantes/farmacología , Células Sf9 , Spodoptera , Proteínas Supresoras de Tumor/biosíntesis , Proteínas Supresoras de Tumor/genética , ADN Metiltransferasa 3B
20.
J Nat Prod ; 77(5): 1245-8, 2014 May 23.
Artículo en Inglés | MEDLINE | ID: mdl-24754815

RESUMEN

Three new acylated arylamine derivatives (1-3), carpatamides A-C, were isolated from a marine-derived Streptomyces sp. based on activity screening against non-small-cell lung cancer (NSCLC). The structures of 1-3 were established on the basis of comprehensive spectroscopic analyses and chemical methods. Compounds 1 and 3 showed moderate cytotoxicity against NSCLC cell lines HCC366, A549, and HCC44 with IC50 values ranging from 2.2 to 8.4 µM.


Asunto(s)
Aminas/aislamiento & purificación , Aminas/farmacología , Antineoplásicos/aislamiento & purificación , Antineoplásicos/farmacología , Streptomyces/química , Amidas , Aminas/química , Antineoplásicos/química , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Concentración 50 Inhibidora , Biología Marina , Estructura Molecular , Resonancia Magnética Nuclear Biomolecular
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