RESUMEN
Severe asthma patients with low type 2 inflammation derive less clinical benefit from therapies targeting type 2 cytokines and represent an unmet need. We show that mast cell tryptase is elevated in severe asthma patients independent of type 2 biomarker status. Active ß-tryptase allele count correlates with blood tryptase levels, and asthma patients carrying more active alleles benefit less from anti-IgE treatment. We generated a noncompetitive inhibitory antibody against human ß-tryptase, which dissociates active tetramers into inactive monomers. A 2.15 Å crystal structure of a ß-tryptase/antibody complex coupled with biochemical studies reveal the molecular basis for allosteric destabilization of small and large interfaces required for tetramerization. This anti-tryptase antibody potently blocks tryptase enzymatic activity in a humanized mouse model, reducing IgE-mediated systemic anaphylaxis, and inhibits airway tryptase in Ascaris-sensitized cynomolgus monkeys with favorable pharmacokinetics. These data provide a foundation for developing anti-tryptase as a clinical therapy for severe asthma.
Asunto(s)
Anticuerpos Monoclonales Humanizados/uso terapéutico , Asma/terapia , Mastocitos/enzimología , Mastocitos/inmunología , Triptasas/antagonistas & inhibidores , Triptasas/inmunología , Adolescente , Regulación Alostérica/inmunología , Animales , Línea Celular , Femenino , Humanos , Macaca fascicularis , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos NOD , Ratones SCID , ConejosRESUMEN
Classical homocystinuria (HCU) is a rare inborn error of amino acid metabolism characterized by accumulation of homocysteine, an intermediate product of methionine metabolism, leading to significant systemic toxicities, particularly within the vascular, skeletal, and ocular systems. Most patients require lifelong dietary therapy with severe restriction of natural protein to minimize methionine intake, and many patients still struggle to maintain healthy homocysteine levels. Since eliminating methionine from the diet reduces homocysteine levels, we hypothesized that an enzyme that can degrade methionine within the gastrointestinal (GI) tract could help HCU patients maintain healthy levels while easing natural protein restrictions. We describe the preclinical development of CDX-6512, a methionine gamma lyase (MGL) enzyme that was engineered for stability and activity within the GI tract for oral administration to locally degrade methionine. CDX-6512 is stable to low pH and intestinal proteases, enabling it to survive the harsh GI environment without enteric coating and to degrade methionine freed from dietary protein within the small intestine. Administering CDX-6512 to healthy non-human primates following a high protein meal led to a dose-dependent suppression of plasma methionine. In Tg-I278T Cbs-/- mice, an animal model that recapitulates aspects of HCU disease including highly elevated serum homocysteine levels, oral dosing of CDX-6512 after a high protein meal led to suppression in serum levels of both methionine and homocysteine. When animals received a daily dose of CDX-6512 with a high protein meal for two weeks, the Tg-I278T Cbs-/- mice maintained baseline homocysteine levels, whereas homocysteine levels in untreated animals increased by 39%. These preclinical data demonstrate the potential of CDX-6512 as an oral enzyme therapy for HCU.
Asunto(s)
Homocistinuria , Humanos , Ratones , Animales , Homocistinuria/tratamiento farmacológico , Homocistinuria/genética , Metionina/metabolismo , Homocisteína , Cistationina betasintasa/genética , Cistationina betasintasa/metabolismo , Racemetionina , Tracto Gastrointestinal/metabolismoRESUMEN
Prevailing dogma holds that cell-cell communication through Notch ligands and receptors determines binary cell fate decisions during progenitor cell divisions, with differentiated lineages remaining fixed. Mucociliary clearance in mammalian respiratory airways depends on secretory cells (club and goblet) and ciliated cells to produce and transport mucus. During development or repair, the closely related Jagged ligands (JAG1 and JAG2) induce Notch signalling to determine the fate of these lineages as they descend from a common proliferating progenitor. In contrast to such situations in which cell fate decisions are made in rapidly dividing populations, cells of the homeostatic adult airway epithelium are long-lived, and little is known about the role of active Notch signalling under such conditions. To disrupt Jagged signalling acutely in adult mammals, here we generate antibody antagonists that selectively target each Jagged paralogue, and determine a crystal structure that explains selectivity. We show that acute Jagged blockade induces a rapid and near-complete loss of club cells, with a concomitant gain in ciliated cells, under homeostatic conditions without increased cell death or division. Fate analyses demonstrate a direct conversion of club cells to ciliated cells without proliferation, meeting a conservative definition of direct transdifferentiation. Jagged inhibition also reversed goblet cell metaplasia in a preclinical asthma model, providing a therapeutic foundation. Our discovery that Jagged antagonism relieves a blockade of cell-to-cell conversion unveils unexpected plasticity, and establishes a model for Notch regulation of transdifferentiation.
Asunto(s)
Anticuerpos/uso terapéutico , Transdiferenciación Celular , Pulmón/citología , Pulmón/metabolismo , Receptores Notch/metabolismo , Animales , Anticuerpos/inmunología , Anticuerpos/farmacología , Asma/tratamiento farmacológico , Asma/metabolismo , Asma/patología , Proteínas de Unión al Calcio/antagonistas & inhibidores , Proteínas de Unión al Calcio/inmunología , Proteínas de Unión al Calcio/metabolismo , Muerte Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Linaje de la Célula/efectos de los fármacos , Rastreo Celular , Transdiferenciación Celular/efectos de los fármacos , Cilios/metabolismo , Modelos Animales de Enfermedad , Femenino , Células Caliciformes/citología , Células Caliciformes/efectos de los fármacos , Células Caliciformes/patología , Homeostasis/efectos de los fármacos , Humanos , Péptidos y Proteínas de Señalización Intercelular/inmunología , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteína Jagged-1 , Proteína Jagged-2 , Ligandos , Pulmón/efectos de los fármacos , Masculino , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/inmunología , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Proteínas Serrate-Jagged , Transducción de Señal/efectos de los fármacosRESUMEN
High temperature requirement A1 (HtrA1) is a trypsin-fold serine protease implicated in the progression of age-related macular degeneration (AMD). Our interest in an antibody therapy to neutralize HtrA1 faces the complication that the target adopts a trimeric arrangement, with three active sites in close proximity. In the present study, we describe antibody 94, obtained from a human antibody phage display library, which forms a distinct macromolecular complex with HtrA1 and inhibits the enzymatic activity of recombinant and native HtrA1 forms. Using biochemical methods and negative-staining EM we were able to elucidate the molecular composition of the IgG94 and Fab94 complexes and the associated inhibition mechanism. The 246-kDa complex between the HtrA1 catalytic domain trimer (HtrA1_Cat) and Fab94 had a propeller-like organization with one Fab bound peripherally to each protomer. Low-resolution EM structures and epitope mapping indicated that the antibody binds to the surface-exposed loops B and C of the catalytic domain, suggesting an allosteric inhibition mechanism. The HtrA1_Cat-IgG94 complex (636 kDa) is a cage-like structure with three centrally located IgG94 molecules co-ordinating two HtrA1_Cat trimers and the six active sites pointing into the cavity of the cage. In both complexes, all antigen-recognition regions (paratopes) are found to bind one HtrA1 protomer and all protomers are bound by a paratope, consistent with the complete inhibition of enzyme activity. Therefore, in addition to its potential therapeutic usefulness, antibody 94 establishes a new paradigm of multimeric serine protease inhibition.
Asunto(s)
Anticuerpos Neutralizantes/farmacología , Complejo Antígeno-Anticuerpo/química , Antineoplásicos/farmacología , Melanoma/tratamiento farmacológico , Proteínas de Neoplasias/antagonistas & inhibidores , Inhibidores de Proteasas/farmacología , Serina Endopeptidasas/metabolismo , Regulación Alostérica , Sustitución de Aminoácidos , Animales , Anticuerpos Neutralizantes/química , Anticuerpos Neutralizantes/genética , Anticuerpos Neutralizantes/metabolismo , Especificidad de Anticuerpos , Antineoplásicos/química , Antineoplásicos/metabolismo , Sitios de Unión de Anticuerpos , Dominio Catalítico , Línea Celular Tumoral , Mapeo Epitopo , Serina Peptidasa A1 que Requiere Temperaturas Altas , Humanos , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/genética , Fragmentos Fab de Inmunoglobulinas/metabolismo , Fragmentos Fab de Inmunoglobulinas/farmacología , Inmunoglobulina G/química , Inmunoglobulina G/genética , Inmunoglobulina G/metabolismo , Inmunoglobulina G/farmacología , Melanoma/enzimología , Melanoma/metabolismo , Ratones , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Proteínas Mutantes/farmacología , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/farmacología , Inhibidores de Proteasas/química , Inhibidores de Proteasas/metabolismo , Subunidades de Proteína/antagonistas & inhibidores , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Serina Endopeptidasas/química , Serina Endopeptidasas/genéticaRESUMEN
BACKGROUND: Cutaneous squamous-cell carcinomas and keratoacanthomas are common findings in patients treated with BRAF inhibitors. METHODS: We performed a molecular analysis to identify oncogenic mutations (HRAS, KRAS, NRAS, CDKN2A, and TP53) in the lesions from patients treated with the BRAF inhibitor vemurafenib. An analysis of an independent validation set and functional studies with BRAF inhibitors in the presence of the prevalent RAS mutation was also performed. RESULTS: Among 21 tumor samples, 13 had RAS mutations (12 in HRAS). In a validation set of 14 samples, 8 had RAS mutations (4 in HRAS). Thus, 60% (21 of 35) of the specimens harbored RAS mutations, the most prevalent being HRAS Q61L. Increased proliferation of HRAS Q61L-mutant cell lines exposed to vemurafenib was associated with mitogen-activated protein kinase (MAPK)-pathway signaling and activation of ERK-mediated transcription. In a mouse model of HRAS Q61L-mediated skin carcinogenesis, the vemurafenib analogue PLX4720 was not an initiator or a promoter of carcinogenesis but accelerated growth of the lesions harboring HRAS mutations, and this growth was blocked by concomitant treatment with a MEK inhibitor. CONCLUSIONS: Mutations in RAS, particularly HRAS, are frequent in cutaneous squamous-cell carcinomas and keratoacanthomas that develop in patients treated with vemurafenib. The molecular mechanism is consistent with the paradoxical activation of MAPK signaling and leads to accelerated growth of these lesions. (Funded by Hoffmann-La Roche and others; ClinicalTrials.gov numbers, NCT00405587, NCT00949702, NCT01001299, and NCT01006980.).
Asunto(s)
Carcinoma de Células Escamosas/genética , Genes ras , Indoles/uso terapéutico , Mutación , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Neoplasias Cutáneas/genética , Sulfonamidas/uso terapéutico , Anciano , Anciano de 80 o más Años , Animales , Carcinoma de Células Escamosas/tratamiento farmacológico , Femenino , Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Indoles/administración & dosificación , Masculino , Ratones , Persona de Mediana Edad , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Inhibidores de Proteínas Quinasas/administración & dosificación , Neoplasias Cutáneas/tratamiento farmacológico , Neoplasias Cutáneas/patología , Sulfonamidas/administración & dosificación , VemurafenibRESUMEN
OBJECTIVE: Bruton's tyrosine kinase (BTK) plays a critical role in B cell development and function. We recently described a selective BTK inhibitor, RN486, that blocks B cell receptor (BCR) and Fcγ receptor signaling and is efficacious in animal models of arthritis. The aim of this study was to examine the potential efficacy of BTK in systemic lupus erythematosus (SLE), using an NZB × NZW mouse model of spontaneous SLE. METHODS: Mice received RN486 or its vehicle (administered in chow) at a final concentration of 30 mg/kg for 8 weeks, starting at 32 weeks of age. RESULTS: The administration of RN486 completely stopped disease progression, as determined by histologic and functional analyses of glomerular nephritis. The efficacy was associated with striking inhibition of B cell activation, as demonstrated by a significant reduction in CD69 expression in response to BCR crosslinking. RN486 markedly reduced the secretion of IgG anti-double-stranded DNA (anti-dsDNA) secretion, as determined by enzyme-linked immunosorbent and enzyme-linked immunospot assays. Flow cytometric analysis demonstrated depletion of CD138(high) B220(low) plasma cells in the spleen. RN486 inhibited secretion of IgG anti-dsDNA but not IgM anti-dsDNA, suggesting that pharmacologic blockade of BTK resembles the reported transgenic expression of low levels of endogenous BTK in B cells. In addition, RN486 may also impact the effector function of autoantibodies, as evidenced by a significant reduction in immune complex-mediated activation of human monocytes in vitro and down-regulation of the expression of macrophage-related and interferon-inducible genes in both the kidneys and spleens of treated mice. CONCLUSION: Collectively, our data suggest that BTK inhibitors may simultaneously target autoantibody-producing and effector cells in SLE, thus constituting a promising therapeutic alternative for this disease.
Asunto(s)
Linfocitos B/patología , Glomerulonefritis/tratamiento farmacológico , Glomérulos Renales/patología , Lupus Eritematoso Sistémico/tratamiento farmacológico , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Agammaglobulinemia Tirosina Quinasa , Animales , Complejo Antígeno-Anticuerpo/metabolismo , Antígenos CD/metabolismo , Antígenos de Diferenciación de Linfocitos T/metabolismo , Linfocitos B/efectos de los fármacos , Linfocitos B/metabolismo , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Regulación hacia Abajo , Glomerulonefritis/metabolismo , Glomerulonefritis/patología , Glomérulos Renales/efectos de los fármacos , Glomérulos Renales/metabolismo , Lectinas Tipo C/metabolismo , Lupus Eritematoso Sistémico/metabolismo , Lupus Eritematoso Sistémico/patología , Activación de Linfocitos/efectos de los fármacos , Ratones , Ratones Endogámicos NZB , Receptores de IgG/metabolismoRESUMEN
BACKGROUND: Thymic stromal lymphopoietin (TSLP) pathway blockade is a potential strategy for asthma treatment because the main activities of TSLP are activation of myeloid dendritic cells (mDCs) and modulation of cytokine production by mast cells. TSLP-activated mDCs prime the differentiation of naive T cells into inflammatory TH2 cells. OBJECTIVE: We sought to investigate mechanisms underlying the development of allergic lung inflammation in cynomolgus monkeys using gene expression profiling and to assess the effect of thymic stromal lymphopoietin receptor (TSLPR) blockade in this model. METHODS: An mAb against human TSLPR was generated and confirmed to be cross-reactive to cynomolgus monkey. Animals were dosed weekly with either vehicle or anti-TSLPR mAb for 6 weeks, and their responses to allergen challenge at baseline, week 2, and week 6 were assessed. RESULTS: After 6 weeks of treatment, anti-TSLPR mAb-treated animals showed reduced bronchoalveolar lavage (BAL) fluid eosinophil counts, reduced airway resistance in response to allergen challenge, and reduced IL-13 cytokine levels in BAL fluid compared with values seen in vehicle-treated animals. Expression profiling of BAL fluid cells collected before and after challenge showed a group of genes upregulated by allergen challenge that strongly overlapped with 11 genes upregulated in dendritic cells (DCs) when in vitro stimulated by TSLP (TSLP-DC gene signature). The number of genes differentially expressed in response to challenge was reduced in antibody-treated animals after 6 weeks relative to vehicle-treated animals. Expression of the TSLP-DC gene signature was also significantly reduced in antibody-treated animals. CONCLUSION: These results demonstrate promising efficacy for TSLPR blockade in an allergic lung inflammation model in which TSLP activation of mDCs might play a key role.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Asma/terapia , Modelos Animales de Enfermedad , Hipersensibilidad/terapia , Inflamación/terapia , Receptores de Citocinas/antagonistas & inhibidores , Animales , Anticuerpos Monoclonales/inmunología , Asma/inmunología , Cricetinae , Citocinas/inmunología , Citocinas/metabolismo , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Femenino , Humanos , Hipersensibilidad/inmunología , Inflamación/inmunología , Macaca fascicularis/inmunología , Receptores de Citocinas/inmunología , Células Th2/inmunología , Células Th2/metabolismo , Linfopoyetina del Estroma TímicoRESUMEN
Purpose: Diabetic macular edema (DME) is the leading cause of vision loss and blindness among working-age adults. Although current intravitreal anti-vascular endothelial growth factor (VEGF) therapies improve vision for many patients with DME, approximately half do not achieve the visual acuity required to drive. We therefore sought additional approaches to resolve edema and improve vision for these patients. Methods: We explored direct agonists of Tie2, a receptor known to stabilize vasculature and prevent leakage. We identified a multivalent PEG-Fab conjugate, Tie2.1-hexamer, that oligomerizes Tie2 and drives receptor activation and characterized its activities in vitro and in vivo. Results: Tie2.1-hexamer normalized and stabilized intercellular junctions of stressed endothelial cell monolayers in vitro, suppressed vascular leak in mice under conditions where anti-VEGF alone was ineffective, and demonstrated extended ocular exposure and robust pharmacodynamic responses in non-human primates. Conclusions: Tie2.1-hexamer directly activates the Tie2 pathway, reduces vascular leak, and is persistent within the vitreal humor. Translational Relevance: Our study presents a promising potential therapeutic for the treatment of DME.
Asunto(s)
Diabetes Mellitus , Retinopatía Diabética , Edema Macular , Ratones , Animales , Edema Macular/tratamiento farmacológico , Edema Macular/etiología , Retinopatía Diabética/tratamiento farmacológico , Factores de Crecimiento Endotelial/uso terapéutico , Agudeza Visual , Trastornos de la Visión/complicaciones , Trastornos de la Visión/tratamiento farmacológico , Ceguera/complicacionesRESUMEN
Meiotic maturation and ovulation rates in Caenorhabditis elegans are regulated by a sperm-released gradient of major sperm protein (MSP). Recent work has provided insights into the modulation of the MSP signal by the trafficking of its receptor in oocytes.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Ciclo Celular/metabolismo , Endocitosis/fisiología , Proteínas del Helminto/metabolismo , Meiosis/fisiología , Oocitos/fisiología , Proteínas Tirosina Quinasas Receptoras/metabolismo , Animales , Caenorhabditis elegans , Femenino , Masculino , Oocitos/metabolismoRESUMEN
Fertilization triggers egg activation and converts the egg into a developing embryo. The events of this egg-to-embryo transition typically include the resumption of meiosis, the reorganization of the cortical actin cytoskeleton, and the remodeling of the oocyte surface. The factors that regulate sperm-dependent egg-activation events are not well understood. Caenorhabditis elegans EGG-3, a member of the protein tyrosine phosphatase-like (PTPL) family, is essential for regulating cell-surface and cortex rearrangements during egg activation in response to sperm entry. Although fertilization occurred normally in egg-3 mutants, the polarized dispersal of F-actin is altered, a chitin eggshell is not formed, and no polar bodies are produced. EGG-3 is associated with the oocyte plasma membrane in a pattern that is similar to CHS-1 and MBK-2. CHS-1 is required for eggshell deposition, whereas MBK-2 is required for the degradation of maternal proteins during the egg-to-embryo transition. The localization of CHS-1 and EGG-3 are interdependent and both genes were required for the proper localization of MBK-2 in oocytes. Therefore, EGG-3 plays a central role in egg activation by influencing polarized F-actin dynamics and the localization or activity of molecules that are directly involved in executing the egg-to-embryo transition.
Asunto(s)
Proteínas de Caenorhabditis elegans/fisiología , Caenorhabditis elegans/embriología , Óvulo/crecimiento & desarrollo , Proteínas Tirosina Fosfatasas/fisiología , Actinas/análisis , Actinas/metabolismo , Secuencias de Aminoácidos , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/análisis , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/metabolismo , Membrana Celular/metabolismo , Embrión no Mamífero/citología , Embrión no Mamífero/metabolismo , Fertilización , Proteínas Fluorescentes Verdes/análisis , Datos de Secuencia Molecular , Óvulo/citología , Óvulo/metabolismo , Proteínas Tirosina Fosfatasas/química , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas Tirosina Quinasas/análisis , Proteínas Tirosina Quinasas/metabolismoRESUMEN
CRTH2 is one of the prostaglandin D2 receptors and plays a proinflammatory role in allergic diseases. Gene expression markers in whole blood induced by CRTH2 activation have not previously been reported. Using microarray analyses of 54 675 genes, we revealed modest gene expression changes in human whole blood stimulated in vitro by a selective CRTH2 agonist, DK-PGD2. Five genes were found to exhibit 1.5- to 2.6-fold changes in expression. The expression of Charcot-Leyden crystal protein/galectin-10 (CLC/Gal-10) in particular was consistently enhanced in human whole blood stimulated by DK-PGD2, as confirmed by quantitative real-time polymerase chain reaction analyses. DK-PGD(2)-induced increases in blood CLC/Gal-10 mRNA levels were largely attenuated by the CRTH2 antagonist CAY10471.Thus, the DK-PGD2-induced CLC/Gal-10 mRNA level can serve as a potential marker for monitoring pharmacodynamic effects of blood exposure to CRTH2 modulating agents.
Asunto(s)
Biomarcadores/metabolismo , Glicoproteínas/genética , Lisofosfolipasa/genética , ARN Mensajero/genética , Receptores Inmunológicos/sangre , Receptores de Prostaglandina/sangre , Secuencia de Bases , Cartilla de ADN , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la PolimerasaRESUMEN
An analysis of the binding motifs of known HIV-1 non-nucleoside reverse transcriptase inhibitors has led to discovery of novel piperidine-linked aminopyrimidine derivatives with broad activity against wild-type as well as drug-resistant mutant viruses. Notably, the series retains potency against the K103N/Y181C and Y188L mutants, among others. Thus, the N-benzyl compound 5k has a particularly attractive profile. Synthesis and SAR are presented and discussed, as well as crystal structures relating to the binding motifs.
Asunto(s)
Transcriptasa Inversa del VIH/antagonistas & inhibidores , VIH-1/efectos de los fármacos , Mutación , Pirimidinas/farmacología , Inhibidores de la Transcriptasa Inversa/farmacología , Descubrimiento de Drogas , Farmacorresistencia Viral/genética , VIH-1/genética , Modelos Moleculares , Pirimidinas/química , Relación Estructura-ActividadRESUMEN
Conformational modeling has been successfully applied to the design of cyclic bioisosteres used to replace a conformationally rigid amide bond in a series of thiophene carboxylate inhibitors of HCV NS5B polymerase. Select compounds were equipotent with the original amide series. Single-point mutant binding studies, in combination with inhibition structure-activity relationships, suggest this new series interacts at the Thumb-II domain of NS5B. Inhibitor binding at the Thumb-II site was ultimately confirmed by solving a crystal structure of 8b complexed with NS5B.
Asunto(s)
Amidas/química , Antivirales/síntesis química , Inhibidores Enzimáticos/síntesis química , Hepacivirus/efectos de los fármacos , Tiofenos/síntesis química , Proteínas no Estructurales Virales/antagonistas & inhibidores , Amidas/síntesis química , Amidas/farmacología , Antivirales/química , Antivirales/farmacología , Sitios de Unión , Cristalografía por Rayos X , Diseño de Fármacos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Estructura Terciaria de Proteína , Relación Estructura-Actividad , Tiofenos/química , Tiofenos/farmacología , Proteínas no Estructurales Virales/metabolismoRESUMEN
Further investigation of the recently reported piperidine-4-yl-aminopyrimidine class of non-nucleoside reverse transcriptase inhibitors (NNRTIs) has been carried out. Thus, preparation of a series of N-phenyl piperidine analogs resulted in the identification of 3-carboxamides as a particularly active series. Analogs such as 28 and 40 are very potent versus wild-type HIV-1 and a broad range of NNRTI-resistant mutant viruses. Synthesis, structure-activity relationship (SAR), clearance data, and crystallographic evidence for the binding motif are discussed.
Asunto(s)
Fármacos Anti-VIH/química , Fármacos Anti-VIH/farmacología , Transcriptasa Inversa del VIH/antagonistas & inhibidores , VIH-1/efectos de los fármacos , VIH-1/enzimología , Pirimidinas/química , Pirimidinas/farmacología , Fármacos Anti-VIH/síntesis química , Farmacorresistencia Viral , Infecciones por VIH/tratamiento farmacológico , Transcriptasa Inversa del VIH/metabolismo , VIH-1/genética , Humanos , Modelos Moleculares , Mutación , Piperidinas/síntesis química , Piperidinas/química , Piperidinas/farmacología , Pirimidinas/síntesis química , Relación Estructura-ActividadRESUMEN
Fertilization involves multiple layers of sperm-egg interactions that lead to gamete fusion and egg activation. There must be specific molecules required for these interactions. The challenge is to determine the identity of the genes encoding these molecules and how their protein products function. The nematode worm Caenorhabditis elegans has emerged as an efficient model system for gene discovery and understanding the molecular mechanisms of fertilization. The primary advantage of the C. elegans system is the ability to isolate and maintain mutants that affect sperm or eggs and no other cells. In this review we describe progress and challenges in the analysis of genes required for gamete interactions and egg activation in the worm.
Asunto(s)
Caenorhabditis elegans/genética , Caenorhabditis elegans/fisiología , Fertilización/genética , Animales , Proteínas de Caenorhabditis elegans/fisiología , Membrana Celular/metabolismo , Femenino , Genes de Helminto/genética , Masculino , Modelos Biológicos , Modelos Genéticos , Mutación , Óvulo/metabolismo , Interacciones Espermatozoide-Óvulo/genética , Espermatozoides/citologíaRESUMEN
Agonism of members of the tumor necrosis factor receptor superfamily (TNFRSF) with monoclonal antibodies is of high therapeutic interest due to their role in immune regulation and cell proliferation. A major hurdle for pharmacologic activation of this receptor class is the requirement for high-order clustering, a mechanism that imposes a reliance in vivo on Fc receptor-mediated crosslinking. This extrinsic dependence represents a potential limitation of virtually the entire pipeline of agonist TNFRSF antibody drugs, of which none have thus far been approved or reached late-stage clinical trials. We show that tetravalent biepitopic targeting enables robust intrinsic antibody agonism for two members of this family, OX40 and DR5, that is superior to extrinsically crosslinked native parental antibodies. Tetravalent biepitopic anti-OX40 engagement co-stimulated OX40low cells, obviated the requirement for CD28 co-signal for T cell activation, and enabled superior pharmacodynamic activity relative to native IgG in a murine vaccination model. This work establishes a proof of concept for an engineering approach that addresses a major gap for the therapeutic activation of this important receptor class.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Recubrimiento Inmunológico , Ligando OX40/agonistas , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/agonistas , Transducción de Señal/inmunología , Linfocitos T/inmunología , Animales , Antígenos CD28/inmunología , Células CHO , Cricetulus , Humanos , Células Jurkat , Ratones , Ratones SCID , Ratones Transgénicos , Ligando OX40/inmunología , Receptores Fc/inmunología , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/inmunología , Linfocitos T/citologíaRESUMEN
Novel non-nucleoside inhibitors of HIV-RT that contain pyridazinone isosteres were prepared, and a series of triazolinones were found to be potent inhibitors of HIV replication. These compounds were active against several NNRTI-resistant virus strains. Pharmacokinetic studies indicated that inhibitor 7e has good bioavailability in rats. Several fragments of inhibitor 7c were prepared, and the binding of these compounds to HIV-RT was analyzed by surface plasmon resonance spectroscopy.
Asunto(s)
Fármacos Anti-VIH , Piridazinas , Inhibidores de la Transcriptasa Inversa , Triazoles , Animales , Fármacos Anti-VIH/síntesis química , Fármacos Anti-VIH/química , Fármacos Anti-VIH/farmacocinética , Fármacos Anti-VIH/farmacología , Técnicas Químicas Combinatorias , Farmacorresistencia Viral/efectos de los fármacos , Estructura Molecular , Piridazinas/síntesis química , Piridazinas/química , Piridazinas/farmacocinética , Piridazinas/farmacología , Ratas , Inhibidores de la Transcriptasa Inversa/síntesis química , Inhibidores de la Transcriptasa Inversa/química , Inhibidores de la Transcriptasa Inversa/farmacología , Relación Estructura-Actividad , Resonancia por Plasmón de Superficie , Triazoles/síntesis química , Triazoles/química , Triazoles/farmacocinética , Triazoles/farmacologíaRESUMEN
Human immunodeficiency virus (HIV) RNase H activity is essential for the synthesis of viral DNA by HIV reverse transcriptase (HIV-RT). RNA cleavage by RNase H requires the presence of divalent metal ions, but the role of metal ions in the mechanism of RNA cleavage has not been resolved. We measured HIV RNase H activity associated with HIV-RT protein in the presence of different concentrations of either Mg2+, Mn2+, Co2+ or a combination of these divalent metal ions. Polymerase-independent HIV RNase H was similar to or more active with Mn2+ and Co2+ compared with Mg2+. Activation of RNase H by these metal ions followed sigmoidal dose-response curves suggesting cooperative metal ion binding. Titration of Mg2+-bound HIV RNase H with Mn2+ or Co2+ ions generated bell-shaped activity dose-response curves. Higher activity could be achieved through simultaneous binding of more than one divalent metal ion at intermediate Mn2+ and Co2+ concentrations, and complete replacement of Mg2+ occurred at higher Mn2+ or Co2+ concentrations. These results are consistent with a two-metal ion mechanism of RNA cleavage as previously suggested for a number of polymerase-associated nucleases. In contrast, the structurally highly homologous RNase HI from Escherichia coli is most strongly activated by Mg2+, is significantly inhibited by submillimolar concentrations of Mn2+ and most probably cleaves RNA via a one-metal ion mechanism. Based on this difference in active site structure, a series of small molecule N-hydroxyimides was identified with significant enzyme inhibitory potency and selectivity for HIV RNase H.
Asunto(s)
Cationes Bivalentes/metabolismo , Diseño de Fármacos , Inhibidores Enzimáticos/farmacología , VIH/enzimología , Metales/metabolismo , Ribonucleasa H/antagonistas & inhibidores , Ribonucleasa H/metabolismo , Sitios de Unión , Cationes Bivalentes/farmacología , Activación Enzimática/efectos de los fármacos , Escherichia coli/enzimología , Metales/farmacología , Especificidad por SustratoRESUMEN
The hepatitis C virus (HCV) non-structural (NS) 5A protein plays an essential role in the replication of the viral RNA by the membrane-associated replication complex (RC). Recently, a putative NS5A inhibitor, BMS-790052, exhibited the highest potency of any known anti-HCV compound in inhibiting HCV replication in vitro and showed a promising clinical effect in HCV-infected patients. The precise mechanism of action for this new class of potential anti-HCV therapeutics, however, is still unclear. In order to gain further insight into its mode of action, we sought to test the hypothesis that the antiviral effect of BMS-790052 might be mediated by interfering with the functional assembly of the HCV RC. We observed that BMS-790052 indeed altered the subcellular localization and biochemical fractionation of NS5A. Taken together, our data suggest that NS5A inhibitors such as BMS-790052 can suppress viral genome replication by altering the proper localization of NS5A into functional RCs.