RESUMEN
Plants have evolved mechanisms to abscise organs as they develop or when exposed to unfavorable conditions.1 Uncontrolled abscission of petals, fruits, or leaves can impair agricultural productivity.2,3,4,5 Despite its importance for abscission progression, our understanding of the IDA signaling pathway and its regulation remains incomplete. IDA is secreted to the apoplast, where it is perceived by the receptors HAESA (HAE) and HAESA-LIKE2 (HSL2) and somatic embryogenesis receptor kinase (SERK) co-receptors.6,7,8,9 These plasma membrane receptors activate an intracellular cascade of mitogen-activated protein kinases (MAPKs) by an unknown mechanism.10,11,12 Here, we characterize brassinosteroid signaling kinases (BSKs) as regulators of floral organ abscission in Arabidopsis. BSK1 localizes to the plasma membrane of abscission zone cells, where it interacts with HAESA receptors to regulate abscission. Furthermore, we demonstrate that YODA (YDA) has a leading role among other MAPKKKs in controlling abscission downstream of the HAESA/BSK complex. This kinase axis, comprising a leucine-rich repeat receptor kinase, a BSK, and an MAPKKK, is known to regulate stomatal patterning, early embryo development, and immunity.10,13,14,15,16 How specific cellular responses are obtained despite signaling through common effectors is not well understood. We show that the identified abscission-promoting allele of BSK1 also enhances receptor signaling in other BSK-mediated pathways, suggesting conservation of signaling mechanisms. Furthermore, we provide genetic evidence supporting independence of BSK1 function from its kinase activity in several developmental processes. Together, our findings suggest that BSK1 facilitates signaling between plasma membrane receptor kinases and MAPKKKs via conserved mechanisms across multiple facets of plant development.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Flores , Transducción de Señal , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Arabidopsis/fisiología , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Flores/crecimiento & desarrollo , Flores/genética , Regulación de la Expresión Génica de las Plantas , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Quinasas/metabolismo , Proteínas Quinasas/genéticaRESUMEN
Stomata regulate photosynthesis and water loss. They have been an active subject of research for centuries, but our knowledge of the genetic components that regulate stomatal development in crops remains very limited in comparison to the model plant Arabidopsis thaliana. Leaf stomatal density was found to vary by over 2.5-fold across a panel of 235 rice accessions. Using GWAS, we successfully identified five different QTLs associated with stomatal density on chromosomes 2, 3, 9, and 12. Forty-two genes were identified within the haplotype blocks corresponding to these QTLs. Of these, nine genes contained haplotypes that were associated with different stomatal densities. These include a gene encoding a trehalose-6-phosphate synthase, an enzyme that has previously been associated with altered stomatal density in Arabidopsis, and genes encoding a B-BOX zinc finger family protein, a leucine-rich repeat family protein, and the 40 S ribosomal protein S3a, none of which have previously been linked to stomatal traits. We investigated further and show that a closely related B-BOX protein regulates stomatal development in Arabidopsis. The results of this study provide information on genetic associations with stomatal density in rice. The QTLs and candidate genes may be useful in future breeding programs for low or high stomatal density and, consequently, improved photosynthetic capacity, water use efficiency, or drought tolerance.
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Arabidopsis , Oryza , Oryza/metabolismo , Estudio de Asociación del Genoma Completo , Arabidopsis/genética , Arabidopsis/metabolismo , Fitomejoramiento , Agua/metabolismoRESUMEN
Colorectal cancer (CRC) remains one of the most lethal human malignancies, and pursuit of new therapeutic targets for treatment has been a major research focus. Cyclin-dependent kinase 9 (CDK9), which plays a crucial role in transcription, has emerged as a target for cancer treatment. CDKI-73, one of the most potent and pharmacologically superior CDK9 inhibitors, has demonstrated excellent anti-tumour efficacy against several types of cancers. In this study, we evaluated its therapeutic potential against CRC. CDKI-73 elicited high cytotoxicity against all colon cancer cell lines tested. Cell cycle and apoptosis analysis in HCT 116 and HT29 cells revealed that CDKI-73 induced cell death without accumulation of DNA at any phase of the cell cycle. Moreover, it caused depolarisation of mitochondrial membrane, leading to caspase-independent apoptosis. Knockdown by shRNA demonstrated the CDK9-targeted mechanism of CDKI-73, which also affected the Mnk/eIF4E signalling axis. In addition, RT-qPCR analysis showed that CDKI-73 down-regulated multiple pro-survival factors at the mRNA level. Its in vivo anti-tumour efficacy was further evaluated in Balb/c nude mice bearing HCT 116 xenograft tumours. CDKI-73 significantly inhibited tumour growth (***P < 0.001) without overt toxicity. Analysis of the tumour tissues collected from the xenografted animals confirmed that the in vivo anti-tumour efficacy was associated with CDK9 targeting of CDKI-73. Overall, this study provides compelling evidence that CDKI-73 is a promising drug candidate for treating colorectal cancer.
Asunto(s)
Antineoplásicos/uso terapéutico , Neoplasias Colorrectales/tratamiento farmacológico , Quinasa 9 Dependiente de la Ciclina/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/uso terapéutico , Pirimidinas/uso terapéutico , Sulfonamidas/uso terapéutico , Animales , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Neoplasias Colorrectales/metabolismo , Quinasa 9 Dependiente de la Ciclina/metabolismo , Femenino , Células HCT116 , Células HT29 , Humanos , Ratones Endogámicos BALB C , Ratones Desnudos , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas/farmacología , Sulfonamidas/farmacologíaRESUMEN
Acute myeloid leukemia (AML) is the most common form of acute leukemia with dismal long-term prognosis with age. The most aggressive subtype of AML is MLL-AML that is characterized by translocations of the mixed-lineage leukemia gene (MLL) and resistance to conventional chemotherapy. Cyclin dependent kinase 9 (CDK9) plays a crucial role in the MLL-driven oncogenic transcription, and hence, inhibiting activity of CDK9 has been proposed as a promising strategy for treatment of AML. We investigated the therapeutic potential of CDKI-73, one of the most potent CDK9 inhibitors, against a panel of AML cell lines and samples derived from 97 patients. CDKI-73 induced cancer cells undergoing apoptosis through transcriptional downregulation of anti-apoptotic proteins Bcl-2, Mcl-1 and XIAP by majorly targeting CDK9. Contrastively, it was relatively low toxic to the bone marrow cells of healthy donors. In MV4-11 xenograft mouse models, oral administration of CDKI-73 resulted in a marked inhibition of tumor growth (p < 0.0001) and prolongation of animal life span (P < 0.001) without causing body weight loss and other overt toxicities. The study suggests that CDKI-73 can be developed as a highly efficacious and orally deliverable therapeutic agent for treatment of AML.
Asunto(s)
Antineoplásicos/uso terapéutico , Quinasa 9 Dependiente de la Ciclina/antagonistas & inhibidores , Leucemia Mieloide Aguda/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/uso terapéutico , Pirimidinas/uso terapéutico , Sulfonamidas/uso terapéutico , Administración Oral , Adulto , Animales , Antineoplásicos/farmacocinética , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Disponibilidad Biológica , Células de la Médula Ósea/efectos de los fármacos , Línea Celular Tumoral , Femenino , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Masculino , Ratones Endogámicos BALB C , Ratones Desnudos , Persona de Mediana Edad , Inhibidores de Proteínas Quinasas/farmacocinética , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas/farmacocinética , Pirimidinas/farmacología , Sulfonamidas/farmacocinética , Sulfonamidas/farmacología , Carga Tumoral/efectos de los fármacosRESUMEN
A hallmark of prostate cancer progression is dysregulation of lipid metabolism via overexpression of fatty acid synthase (FASN), a key enzyme in de novo fatty acid synthesis. Metastatic castration-resistant prostate cancer (mCRPC) develops resistance to inhibitors of androgen receptor (AR) signaling through a variety of mechanisms, including the emergence of the constitutively active AR variant V7 (AR-V7). Here, we developed an FASN inhibitor (IPI-9119) and demonstrated that selective FASN inhibition antagonizes CRPC growth through metabolic reprogramming and results in reduced protein expression and transcriptional activity of both full-length AR (AR-FL) and AR-V7. Activation of the reticulum endoplasmic stress response resulting in reduced protein synthesis was involved in IPI-9119-mediated inhibition of the AR pathway. In vivo, IPI-9119 reduced growth of AR-V7-driven CRPC xenografts and human mCRPC-derived organoids and enhanced the efficacy of enzalutamide in CRPC cells. In human mCRPC, both FASN and AR-FL were detected in 87% of metastases. AR-V7 was found in 39% of bone metastases and consistently coexpressed with FASN. In patients treated with enzalutamide and/or abiraterone FASN/AR-V7 double-positive metastases were found in 77% of cases. These findings provide a compelling rationale for the use of FASN inhibitors in mCRPCs, including those overexpressing AR-V7.
Asunto(s)
Lipogénesis , Proteínas de Neoplasias/metabolismo , Neoplasias de la Próstata Resistentes a la Castración/metabolismo , Receptores Androgénicos/metabolismo , Transducción de Señal , Animales , Línea Celular Tumoral , Inhibidores Enzimáticos/farmacología , Acido Graso Sintasa Tipo I/antagonistas & inhibidores , Acido Graso Sintasa Tipo I/genética , Acido Graso Sintasa Tipo I/metabolismo , Humanos , Masculino , Ratones , Metástasis de la Neoplasia , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/genética , Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , Neoplasias de la Próstata Resistentes a la Castración/genética , Neoplasias de la Próstata Resistentes a la Castración/patología , Receptores Androgénicos/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Latent transforming growth factor-ß-1 binding protein-2 (LTBP-2) belongs to the LTBP-fibrillin superfamily of extracellular proteins. Unlike other LTBPs, LTBP-2 does not covalently bind transforming growth factor-ß1 (TGF-ß1) but appears to be implicated in the regulation of TGF-ß1 bioactivity, although the mechanisms are largely unknown. In experiments originally designed to study the displacement of latent TGF-ß1 complexes from matrix storage, we found that the addition of exogenous LTBP-2 to cultured human MSU-1.1 fibroblasts caused an increase in TGF-ß1 levels in the medium. However, the TGF-ß1 increase was due to an upregulation of TGF-ß1 expression and secretion rather than a displacement of matrix-stored TGF-ß1. The secreted TGF-ß1 was mainly in an inactive form, and its concentration peaked around 15 h after addition of LTBP-2. Using a series of recombinant LTBP-2 fragments, the bioactivity was identified to a small region of LTBP-2 consisting of an 8-Cys motif flanked by four epidermal growth factor (EGF)-like repeats. The LTBP-2 stimulation of TGF-ß expression involved the phosphorylation of both Akt and p38 mitogen-activated protein kinase (MAPK) signalling proteins, and specific inactivation of each protein individually blocked TGF-ß1 increase. The search for the cell surface receptor mediating this LTBP-2 activity proved inconclusive. Inhibitory antibodies to integrins ß1 and αVß5 showed no reduction of LTBP-2 stimulation of TGF-ß1. However, TGF-ß1 upregulation was partially inhibited by anti-αVß3 integrin antibodies, suggestive of a direct or indirect role for this integrin. Overall, the study indicates that LTBP-2 can directly upregulate cellular TGF-ß1 expression and secretion by interaction with cells via a short central bioactive region. This may be significant in connective tissue disorders involving aberrant TGF-ß1 signalling.
Asunto(s)
Fibroblastos/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Línea Celular , Fibrosis/metabolismo , Humanos , Fosforilación , Reacción en Cadena de la PolimerasaRESUMEN
Recent clinical trials using immunotherapy have demonstrated its potential to control cancer by disinhibiting the immune system. Immune checkpoint blocking (ICB) antibodies against cytotoxic-T-lymphocyte-associated protein 4 or programmed cell death protein 1/programmed death-ligand 1 have displayed durable clinical responses in various cancers. Although these new immunotherapies have had a notable effect on cancer treatment, multiple mechanisms of immune resistance exist in tumours. Among the key mechanisms, myeloid cells have a major role in limiting effective tumour immunity. Growing evidence suggests that high infiltration of immune-suppressive myeloid cells correlates with poor prognosis and ICB resistance. These observations suggest a need for a precision medicine approach in which the design of the immunotherapeutic combination is modified on the basis of the tumour immune landscape to overcome such resistance mechanisms. Here we employ a pre-clinical mouse model system and show that resistance to ICB is directly mediated by the suppressive activity of infiltrating myeloid cells in various tumours. Furthermore, selective pharmacologic targeting of the gamma isoform of phosphoinositide 3-kinase (PI3Kγ), highly expressed in myeloid cells, restores sensitivity to ICB. We demonstrate that targeting PI3Kγ with a selective inhibitor, currently being evaluated in a phase 1 clinical trial (NCT02637531), can reshape the tumour immune microenvironment and promote cytotoxic-T-cell-mediated tumour regression without targeting cancer cells directly. Our results introduce opportunities for new combination strategies using a selective small molecule PI3Kγ inhibitor, such as IPI-549, to overcome resistance to ICB in patients with high levels of suppressive myeloid cell infiltration in tumours.
Asunto(s)
Puntos de Control del Ciclo Celular/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Melanoma/tratamiento farmacológico , Melanoma/inmunología , Células Mieloides/efectos de los fármacos , Células Mieloides/inmunología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Inhibidores de Proteínas Quinasas/farmacología , Animales , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Resistencia a Antineoplásicos/inmunología , Femenino , Humanos , Tolerancia Inmunológica/efectos de los fármacos , Masculino , Melanoma/patología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Células Mieloides/enzimología , Metástasis de la Neoplasia/tratamiento farmacológico , Fenotipo , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de Proteínas Quinasas/uso terapéutico , Linfocitos T Citotóxicos/efectos de los fármacos , Linfocitos T Citotóxicos/inmunología , Microambiente Tumoral/efectos de los fármacos , Microambiente Tumoral/inmunologíaRESUMEN
Cytarabine (Ara-C) is a first line clinical therapeutic agent for treatment of acute myeloid leukemia (AML). However, this therapy is limited due to high rate of resistance and relapse. Recent research has revealed that the poor prognosis and resistance to Ara-C in AML were associated with its abnormally activated MAPK pathways. In this study, we showed a strong synergistic effect of Ara-C with either our Mnk inhibitor (MNKI-8e) or short hairpin RNA (shRNA) mediated knockdown of Mnks in MV4-11 AML cells. We investigated the underlying mechanisms for this synergism. We showed that both MNKI-8e and Mnk shRNAs enhanced the ability of Ara-C to induce apoptosis. We found that Ara-C increased the phosphorylation of Erk1/2, p38 and eIF4E, which correlated with an enhanced level of anti-apoptotic Mcl-1 protein. Inhibition of Mnk activity suppressed the Ara-C-induced MAPK activity, and thus enhanced apoptosis in MV4-11 cells. Taken together, our study suggests that MAPK-Mnk-eIF4E pathway plays a critical role in Ara-C-treated MV4-11 cells and targeting Mnk may be a promising therapeutic strategy for sensitizing leukemic cells to Ara-C therapy.
Asunto(s)
Apoptosis , ATPasas Transportadoras de Cobre/antagonistas & inhibidores , Citarabina/farmacología , Leucemia Mieloide Aguda/patología , Antimetabolitos Antineoplásicos/farmacología , Caspasa 3/metabolismo , Caspasa 7/metabolismo , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular , Resistencia a Antineoplásicos/efectos de los fármacos , Activación Enzimática , Regulación Leucémica de la Expresión Génica , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Sistema de Señalización de MAP Quinasas , Fosforilación , ARN Interferente Pequeño/metabolismo , Transducción de SeñalRESUMEN
Latent transforming growth factor-beta-1 binding protein-2 (LTBP-2) belongs to the fibrillin-LTBP superfamily of extracellular matrix proteins. LTBPs and fibrillins are involved in the sequestration and storage of latent growth factors, particularly transforming growth factor ß (TGF-ß), in tissues. Unlike other LTBPs, LTBP-2 does not covalently bind TGF-ß, and its molecular functions remain unclear. We are screening LTBP-2 for binding to other growth factors and have found very strong saturable binding to fibroblast growth factor-2 (FGF-2) (Kd = 1.1 nM). Using a series of recombinant LTBP-2 fragments a single binding site for FGF-2 was identified in a central region of LTBP-2 consisting of six tandem epidermal growth factor-like (EGF-like) motifs (EGFs 9-14). This region was also shown to contain a heparin/heparan sulphate-binding site. FGF-2 stimulation of fibroblast proliferation was completely negated by the addition of 5-fold molar excess of LTBP-2 to the assay. Confocal microscopy showed strong co-localisation of LTBP-2 and FGF-2 in fibrotic keloid tissue suggesting that the two proteins may interact in vivo. Overall the study indicates that LTBP-2 is a potent inhibitor of FGF-2 that may influence FGF-2 bioactivity during wound repair particularly in fibrotic tissues.
Asunto(s)
Sitios de Unión , Factor 2 de Crecimiento de Fibroblastos/antagonistas & inhibidores , Factor 2 de Crecimiento de Fibroblastos/química , Proteínas de Unión a TGF-beta Latente/química , Proteínas de Unión a TGF-beta Latente/metabolismo , Línea Celular , Proliferación Celular/efectos de los fármacos , Fibrilinas , Factor 2 de Crecimiento de Fibroblastos/farmacología , Fibroblastos/metabolismo , Heparina/metabolismo , Humanos , Queloide/metabolismo , Proteínas de Unión a TGF-beta Latente/farmacología , Proteínas de Microfilamentos/metabolismo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Transporte de Proteínas , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/agonistas , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/metabolismo , Proteínas Recombinantes , Piel/metabolismoRESUMEN
Pim oncogenes are highly expressed in many types of hematological and solid cancers. Pim kinases regulate the network of signaling pathways that are critical for tumorigenesis and development, making Pim kinases the attractive drug targets. Currently, two approaches have been employed in designing Pim kinase inhibitors: ATP-mimetics and non-ATP mimetics; but all target the ATP-binding pocket and are ATP-competitive. In this review, we summarize the current progress in understanding the Pim-related structure and biology, and provide insights into the binding modes of some prototypical Pim-1 inhibitors. The challenges as well as opportunities are highlighted for development of Pim kinase inhibitors as potential anticancer agents.
Asunto(s)
Neoplasias/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas c-pim-1/antagonistas & inhibidores , Unión Competitiva , Evaluación Preclínica de Medicamentos , Humanos , Simulación del Acoplamiento Molecular , Unión Proteica , Inhibidores de Proteínas Quinasas/química , Proteínas Proto-Oncogénicas c-pim-1/metabolismo , Factores de Transcripción STAT/metabolismoRESUMEN
We chronicle and dissect the history of the field of Experimental Microbial Evolution, beginning with work by Monod. Early research was largely carried out by microbiologists and biochemists, who used experimental evolutionary change as a tool to understand structure-function relationships. These studies attracted the interest of evolutionary biologists who recognized the power of the approach to address issues such as the tempo of adaptive change, the costs and benefits of sex, parallelism, and the role which contingency plays in the evolutionary process. In the 1980s and 1990s, an ever-expanding body of microbial, physiological and biochemical data, together with new technologies for manipulating microbial genomes, allowed such questions to be addressed in ever-increasing detail. Since then, technological advances leading to low-cost, high-throughput DNA sequencing have made it possible for these and other fundamental questions in evolutionary biology to be addressed at the molecular level.
Asunto(s)
Evolución Molecular , Genoma Microbiano , Bases de Datos Genéticas/historia , Evolución Molecular Dirigida/historia , Secuenciación de Nucleótidos de Alto Rendimiento/historia , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Historia del Siglo XX , Historia del Siglo XXIRESUMEN
Dysregulation of cellular transcription and translation is a fundamental hallmark of cancer. As CDK9 and Mnks play pivotal roles in the regulation of RNA transcription and protein synthesis, respectively, they are important targets for drug development. We herein report the cellular mechanism of a novel CDK9 inhibitor CDKI-73 in an ovarian cancer cell line (A2780). We also used shRNA-mediated CDK9 knockdown to investigate the importance of CDK9 in the maintenance of A2780 cells. This study revealed that CDKI-73 rapidly inhibited cellular CDK9 kinase activity and down-regulated the RNAPII phosphorylation. This subsequently caused a decrease in the eIF4E phosphorylation by blocking Mnk1 kinase activity. Consistently, CDK9 shRNA was also found to down-regulate the Mnk1 expression. Both CDKI-73 and CDK9 shRNA decreased anti-apoptotic proteins Mcl-1 and Bcl-2 and induced apoptosis. The study confirmed that CDK9 is required for cell survival and that ovarian cancer may be susceptible to CDK9 inhibition strategy. The data also implied a role of CDK9 in eIF4E-mediated translational control, suggesting that CDK9 may have important implication in the Mnk-eIF4E axis, the key determinants of PI3K/Akt/mTOR- and Ras/Raf/MAPK-mediated tumorigenic activity. As such, CDK9 inhibitor drug candidate CDKI-73 should have a major impact on these pathways in human cancers.
Asunto(s)
Antineoplásicos/farmacología , Quinasa 9 Dependiente de la Ciclina/antagonistas & inhibidores , Neoplasias Ováricas/enzimología , Pirimidinas/farmacología , Sulfonamidas/farmacología , Apoptosis/efectos de los fármacos , Western Blotting , Línea Celular Tumoral , Femenino , Humanos , Biosíntesis de Proteínas/efectos de los fármacos , ARN , Reacción en Cadena en Tiempo Real de la Polimerasa , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Transcripción Genética/efectos de los fármacosRESUMEN
A major goal of genetics is to define the relationship between phenotype and genotype, while a major goal of ecology is to identify the rules that govern community assembly. Achieving these goals by analyzing natural systems can be difficult, as selective pressures create dynamic fitness landscapes that vary in both space and time. Laboratory experimental evolution offers the benefit of controlling variables that shape fitness landscapes, helping to achieve both goals. We previously showed that a clonal population of E. coli experimentally evolved under continuous glucose limitation gives rise to a genetically diverse community consisting of one clone, CV103, that best scavenges but incompletely utilizes the limiting resource, and others, CV101 and CV116, that consume its overflow metabolites. Because this community can be disassembled and reassembled, and involves cooperative interactions that are stable over time, its genetic diversity is sustained by clonal reinforcement rather than by clonal interference. To understand the genetic factors that produce this outcome, and to illuminate the community's underlying physiology, we sequenced the genomes of ancestral and evolved clones. We identified ancestral mutations in intermediary metabolism that may have predisposed the evolution of metabolic interdependence. Phylogenetic reconstruction indicates that the lineages that gave rise to this community diverged early, as CV103 shares only one Single Nucleotide Polymorphism with the other evolved clones. Underlying CV103's phenotype we identified a set of mutations that likely enhance glucose scavenging and maintain redox balance, but may do so at the expense of carbon excreted in overflow metabolites. Because these overflow metabolites serve as growth substrates that are differentially accessible to the other community members, and because the scavenging lineage shares only one SNP with these other clones, we conclude that this lineage likely served as an "engine" generating diversity by creating new metabolic niches, but not the occupants themselves.
Asunto(s)
Evolución Molecular Dirigida , Escherichia coli/genética , Glucosa/metabolismo , Selección Genética , Sustitución de Aminoácidos/genética , Codón sin Sentido/genética , Variación Genética , Genoma Bacteriano , Glucosa/genética , Glicerol/metabolismo , Filogenia , Polimorfismo de Nucleótido SimpleRESUMEN
ON01910.Na is a highly effective anticancer agent that induces mitotic arrest and apoptosis. Clinical studies with ON01910 in cancer patients have shown efficacy along with an impressive safety profile. While ON01910 is highly active against cancer cells, it has a low oral availability and requires continuous intravenous infusion or multiple gram doses to ensure sufficient drug exposure for biological activity in patients. We have identified two novel series of styrylsulfonyl-methylpyridines. Lead compounds 8, 9a, 18 and 19a are highly potent mitotic inhibitors and selectively cytotoxic to cancer cells. Impressively, these compounds possess excellent pharmaceutical properties and two lead drug candidates 9a and 18 demonstrated antitumor activities in animal models.
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Aminopiridinas/síntesis química , Aminopiridinas/farmacología , Antineoplásicos/síntesis química , Antineoplásicos/farmacología , Glicina/análogos & derivados , Estirenos/síntesis química , Estirenos/farmacología , Sulfonamidas/síntesis química , Sulfonamidas/farmacología , Sulfonas/farmacología , Animales , Anexina A5 , Antineoplásicos/farmacocinética , Apoptosis/efectos de los fármacos , Área Bajo la Curva , Disponibilidad Biológica , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cristalografía por Rayos X , Diseño de Fármacos , Descubrimiento de Drogas , Glicina/farmacología , Semivida , Indicadores y Reactivos , Estimación de Kaplan-Meier , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Ratones , Microsomas Hepáticos/metabolismo , Modelos Moleculares , Ratas , Relación Estructura-Actividad , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
1. IPI-926 is a novel semisynthetic cyclopamine derivative that is a potent and selective Smoothened inhibitor that blocks the hedgehog signal transduction pathway. 2. The in vivo clearance of IPI-926 is low in mouse and dog and moderate in monkey. The volume of distribution is high across species. Oral bioavailability ranges from moderate in monkey to high in mouse and dog. Predicted human clearance using simple allometry is low (24 L h(-1)), predicted volume of distribution is high (469 L) and predicted half-life is long (20 h). 3. IPI-926 is highly bound to plasma proteins and has minimal interaction with human α-1-acid glycoprotein. 4. In vitro metabolic stability ranges from stable to moderately stable. Twelve oxidative metabolites were detected in mouse, rat, dog, monkey and human liver microsome incubations and none were unique to human. 5. IPI-926 is not a potent reversible inhibitor of CYP1A2, 2C8, 2C9 or 3A4 (testosterone). IPI-926 is a moderate inhibitor of CYP2C19, 2D6 and 3A4 (midazolam) with KI values of 19, 16 and 4.5 µM, respectively. IPI-926 is both a substrate and inhibitor (IC50 = 1.9 µM) of P-glycoprotein. 6. In summary, IPI-926 has desirable pre-clinical absorption, distribution, metabolism and excretion properties.
Asunto(s)
Alcaloides de Veratrum/farmacocinética , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Administración Oral , Animales , Hidrocarburo de Aril Hidroxilasas/antagonistas & inhibidores , Hidrocarburo de Aril Hidroxilasas/metabolismo , Disponibilidad Biológica , Citocromo P-450 CYP2C19 , Perros , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacocinética , Femenino , Semivida , Proteínas Hedgehog/antagonistas & inhibidores , Proteínas Hedgehog/metabolismo , Hepatocitos/metabolismo , Humanos , Macaca fascicularis , Masculino , Ratones , Ratones Endogámicos , Microsomas Hepáticos/metabolismo , Orosomucoide/metabolismo , Ratas Sprague-Dawley , Distribución Tisular , Alcaloides de Veratrum/administración & dosificación , Alcaloides de Veratrum/metabolismoRESUMEN
Abstract A phase 1 study of IPI-504 (retaspimycin hydrochloride) administered intravenously twice weekly for 2 weeks at 22.5, 45, 90, 150, 225, 300 or 400 mg/m(2) followed by 10 days off-treatment was conducted to determine the safety and maximum tolerated dose (MTD) of IPI-504 in patients with relapsed or relapsed/refractory multiple myeloma (MM). Anti-tumor activity and pharmacokinetics were also evaluated. Eighteen patients (mean age 60.5 years; median 9 prior therapies) were enrolled. No dose-limiting toxicities (DLTs) were reported for IPI-504 doses up to 400 mg/m(2). The most common treatment-related adverse event was grade 1 infusion site pain (four patients). All other treatment-related events were assessed as grade 1 or 2 in severity. The area under the curve (AUC) increased with increasing dose, and the mean half-life was approximately 2-4 h for IPI-504 and its metabolites. Four patients had stable disease, demonstrating modest single-agent activity in relapsed or relapsed/refractory MM.
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Antineoplásicos/uso terapéutico , Benzoquinonas/uso terapéutico , Lactamas Macrocíclicas/uso terapéutico , Mieloma Múltiple/tratamiento farmacológico , Anciano , Antineoplásicos/efectos adversos , Antineoplásicos/farmacocinética , Benzoquinonas/efectos adversos , Benzoquinonas/farmacocinética , Femenino , Humanos , Lactamas Macrocíclicas/efectos adversos , Lactamas Macrocíclicas/farmacocinética , Masculino , Persona de Mediana Edad , Recurrencia , Resultado del TratamientoRESUMEN
Several Hsp90 (heat shock protein 90) inhibitors are currently under clinical evaluation as anticancer agents. However, the correlation between the duration and magnitude of Hsp90 inhibition and the downstream effects on client protein degradation and cancer cell growth inhibition has not been thoroughly investigated. To investigate the relationship between Hsp90 inhibition and cellular effects, we developed a method that measures drug occupancy on Hsp90 after treatment with the Hsp90 inhibitor IPI-504 in living cells and in tumor xenografts. In cells, we find the level of Hsp90 occupancy to be directly correlated with cell growth inhibition. At the molecular level, the relationship between Hsp90 occupancy and Hsp90 client protein degradation was examined for different client proteins. For sensitive Hsp90 clients (e.g. HER2 (human epidermal growth factor receptor 2), client protein levels directly mirror Hsp90 occupancy at all time points after IPI-504 administration. For insensitive client proteins, we find that protein abundance matches Hsp90 occupancy only after prolonged incubation with drug. Additionally, we investigate the correlation between plasma pharmacokinetics (PK), tumor PK, pharmacodynamics (PD) (client protein degradation), tumor growth inhibition, and Hsp90 occupancy in a xenograft model of human cancer. Our results indicate Hsp90 occupancy to be a better predictor of PD than either plasma PK or tumor PK. In the nonsmall cell lung cancer xenograft model studied, a linear correlation between Hsp90 occupancy and tumor growth inhibition was found. This novel binding assay was evaluated both in vitro and in vivo and could be used as a pharmacodynamic readout in the clinic.
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Antineoplásicos/farmacocinética , Benzoquinonas/farmacocinética , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Lactamas Macrocíclicas/farmacocinética , Neoplasias Pulmonares/tratamiento farmacológico , Proteínas de Neoplasias/antagonistas & inhibidores , Ensayos Antitumor por Modelo de Xenoinjerto , Animales , Antineoplásicos/farmacología , Benzoquinonas/farmacología , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Células HeLa , Humanos , Lactamas Macrocíclicas/farmacología , Neoplasias Pulmonares/metabolismo , Ratones , Ratones DesnudosRESUMEN
Latent transforming growth factor-beta-1 binding protein-2 (LTBP-2) is a protein of poorly understood function associated with fibrillin-1-containing microfibrils during elastinogenesis. In this study we investigated the molecular interactions of LTBP-2 with heparin and heparan sulfate proteoglycans (HSPGs) since unidentified cell surface HSPGs are critical for normal fiber assembly. In solid phase assays, heparin conjugated to albumin (HAC) bound strongly to recombinant full-length human LTBP-2. This interaction was completely blocked by addition of excess heparin, but not chondroitin sulfate, confirming specificity. Analysis of binding to LTBP-2 fragments showed that HAC bound strongly to N-terminal fragment LTBP-2 NT(H) and more weakly to central fragment LTBP-2 C(H). No binding was detected to C-terminal fragment LTBP-2 CT(H). Kds for heparin binding were calculated for full-length LTBP-2, LTBP-2 NT(H) and LTBP-2 C(H) as 0.9 nM, 0.7 nM and 80 nM respectively. HAC interaction with fragment LTBP-2 NT(H) was not sensitive to EDTA or EGTA indicating that binding had no requirement for Ca(2+) ions whereas HAC binding to fragment LTBP-2 C(H) was markedly reduced by these chelating agents indicating a degree of Ca(2+) dependence. Inhibition studies with synthetic peptides identified three major heparin binding sequences in fragment LTBP-2 NT(H), including sequence LTEKIKKIKIV in the first large cysteine-free domain of LTBP-2, adjacent to the previously identified fibulin-5 binding site. LTBP-2 was found to interact strongly in a heparin-inhibitable manner with cell surface HSPG syndecan-4, but showed no interaction with recombinant syndecan-2. LTBP-2 also showed strong interaction with the heparan sulfate chains of basement membrane HSPG, perlecan. The potential importance of HSPG-LTBP-2 interactions in elastic fiber assembly and microfibril attachment to basement membranes is discussed.
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Membrana Basal/fisiología , Proteoglicanos de Heparán Sulfato/fisiología , Heparina/fisiología , Proteínas de Unión a TGF-beta Latente/fisiología , Microfibrillas/fisiología , Sindecano-4/fisiología , Secuencia de Aminoácidos , Sitios de Unión/fisiología , Quelantes/farmacología , Ácido Edético/farmacología , Ácido Egtácico/farmacología , Ensayo de Inmunoadsorción Enzimática , Cinética , Datos de Secuencia Molecular , Fragmentos de Péptidos/fisiología , Proteínas Recombinantes/metabolismoRESUMEN
The structure of aldehyde reductase (ALR1) in ternary complex with the coenzyme NADPH and [5-(3-carboxymethoxy-4-methoxybenzylidene)-2,4-dioxothiazolidin-3-yl]acetic acid (CMD), a potent inhibitor of aldose reductase (ALR2), was determined at 1.99A resolution. The partially disordered inhibitor formed a tight network of hydrogen bonds with the active site residues (Tyr50 and His113) and coenzyme. Molecular modelling calculations and inhibitory activity measurements of CMD and [5-(3-hydroxy-4-methoxybenzylidene)-2,4-dioxothiazolidin-3-yl]acetic acid (HMD) indicated that pi-stacking interactions with several conserved active site tryptophan residues and hydrogen-bonding interactions with the non-conserved C-terminal residue Leu300 in ALR2 (Pro301 in ALR1) contributed to inhibitor selectivity. In particular for the potent inhibitor CMD, the rotameric state of the conserved residue Trp219 (Trp220 in ALR1) is important in forming a pi-stacking interaction with the inhibitor in ALR2 and contributes to the difference in the binding of the inhibitor to the enzymes.