RESUMEN
BACKGROUND: In a stable, inotrope-dependent pediatric patient with dilated cardiomyopathy, we evaluated the cost-effectiveness of continuous-flow VAD implantation compared to a watchful waiting approach using chronic inotropic therapy. METHODS: We used a state-transition model to estimate the costs and outcomes of 14-year-old (INTERMACS profile 3) patients receiving either VAD or watchful waiting. We measured benefits in terms of lifetime QALYs gained. Model inputs were taken from the literature. We calculated the ICER, or the cost per additional QALY gained, of VADs and performed multiple sensitivity analyses to test how our assumptions influenced the results. RESULTS: Compared to watchful waiting, VADs produce 0.97 more QALYs for an additional $156 639, leading to an ICER of $162 123 per QALY gained from a healthcare perspective. VADs have 17% chance of being cost-effective given a cost-effectiveness threshold of $100 000 per QALY gained. Sensitivity analyses suggest that VADs can be cost-effective if the costs of implantation decrease or if hospitalization costs or mortality among watchful waiting patients is higher. CONCLUSIONS: As a bridge to transplant, VADs provide a health benefit to children who develop stable, inotrope-dependent heart failure, but immediate implantation is not yet a cost-effective strategy compared to watchful waiting based on commonly used cost-effectiveness thresholds. Early VAD support can be cost-effective in sicker patients and if device implantation is cheaper. In complex conditions such as pediatric heart failure, cost-effectiveness should be just one of many factors that inform clinical decision-making.
Asunto(s)
Cardiomiopatía Dilatada/economía , Cardiomiopatía Dilatada/cirugía , Análisis Costo-Beneficio , Costos de la Atención en Salud/estadística & datos numéricos , Insuficiencia Cardíaca/economía , Insuficiencia Cardíaca/cirugía , Corazón Auxiliar/economía , Adolescente , Cardiomiopatía Dilatada/tratamiento farmacológico , Cardiotónicos/economía , Cardiotónicos/uso terapéutico , Simulación por Computador , Insuficiencia Cardíaca/tratamiento farmacológico , Humanos , Cadenas de Markov , Modelos Económicos , Años de Vida Ajustados por Calidad de Vida , Estados Unidos , Espera Vigilante/economíaRESUMEN
Identification of voltage-gated sodium channel NaV1.7 inhibitors for chronic pain therapeutic development is an area of vigorous pursuit. In an effort to identify more potent leads compared to our previously reported GpTx-1 peptide series, electrophysiology screening of fractionated tarantula venom discovered the NaV1.7 inhibitory peptide JzTx-V from the Chinese earth tiger tarantula Chilobrachys jingzhao. The parent peptide displayed nominal selectivity over the skeletal muscle NaV1.4 channel. Attribute-based positional scan analoging identified a key Ile28Glu mutation that improved NaV1.4 selectivity over 100-fold, and further optimization yielded the potent and selective peptide leads AM-8145 and AM-0422. NMR analyses revealed that the Ile28Glu substitution changed peptide conformation, pointing to a structural rationale for the selectivity gains. AM-8145 and AM-0422 as well as GpTx-1 and HwTx-IV competed for ProTx-II binding in HEK293 cells expressing human NaV1.7, suggesting that these NaV1.7 inhibitory peptides interact with a similar binding site. AM-8145 potently blocked native tetrodotoxin-sensitive (TTX-S) channels in mouse dorsal root ganglia (DRG) neurons, exhibited 30- to 120-fold selectivity over other human TTX-S channels and exhibited over 1,000-fold selectivity over other human tetrodotoxin-resistant (TTX-R) channels. Leveraging NaV1.7-NaV1.5 chimeras containing various voltage-sensor and pore regions, AM-8145 mapped to the second voltage-sensor domain of NaV1.7. AM-0422, but not the inactive peptide analog AM-8374, dose-dependently blocked capsaicin-induced DRG neuron action potential firing using a multi-electrode array readout and mechanically-induced C-fiber spiking in a saphenous skin-nerve preparation. Collectively, AM-8145 and AM-0422 represent potent, new engineered NaV1.7 inhibitory peptides derived from the JzTx-V scaffold with improved NaV selectivity and biological activity in blocking action potential firing in both DRG neurons and C-fibers.
Asunto(s)
Analgésicos/aislamiento & purificación , Canal de Sodio Activado por Voltaje NAV1.7/efectos de los fármacos , Péptidos/química , Bloqueadores de los Canales de Sodio/aislamiento & purificación , Venenos de Araña/química , Potenciales de Acción/efectos de los fármacos , Sustitución de Aminoácidos , Analgésicos/farmacología , Animales , Capsaicina/farmacología , Línea Celular , Evaluación Preclínica de Medicamentos , Ganglios Espinales/efectos de los fármacos , Humanos , Masculino , Ratones Endogámicos C57BL , Mutagénesis Sitio-Dirigida , Fibras Nerviosas Amielínicas/efectos de los fármacos , Resonancia Magnética Nuclear Biomolecular , Técnicas de Placa-Clamp , Estimulación Física , Ingeniería de Proteínas , Proteínas Recombinantes/efectos de los fármacos , Bloqueadores de los Canales de Sodio/farmacología , Relación Estructura-Actividad , Tetrodotoxina/farmacologíaRESUMEN
Potent and selective antagonists of the voltage-gated sodium channel NaV1.7 represent a promising avenue for the development of new chronic pain therapies. We generated a small molecule atropisomer quinolone sulfonamide antagonist AMG8379 and a less active enantiomer AMG8380. Here we show that AMG8379 potently blocks human NaV1.7 channels with an IC50 of 8.5 nM and endogenous tetrodotoxin (TTX)-sensitive sodium channels in dorsal root ganglion (DRG) neurons with an IC50 of 3.1 nM in whole-cell patch clamp electrophysiology assays using a voltage protocol that interrogates channels in a partially inactivated state. AMG8379 was 100- to 1000-fold selective over other NaV family members, including NaV1.4 expressed in muscle and NaV1.5 expressed in the heart, as well as TTX-resistant NaV channels in DRG neurons. Using an ex vivo mouse skin-nerve preparation, AMG8379 blocked mechanically induced action potential firing in C-fibers in both a time-dependent and dose-dependent manner. AMG8379 similarly reduced the frequency of thermally induced C-fiber spiking, whereas AMG8380 affected neither mechanical nor thermal responses. In vivo target engagement of AMG8379 in mice was evaluated in multiple NaV1.7-dependent behavioral endpoints. AMG8379 dose-dependently inhibited intradermal histamine-induced scratching and intraplantar capsaicin-induced licking, and reversed UVB radiation skin burn-induced thermal hyperalgesia; notably, behavioral effects were not observed with AMG8380 at similar plasma exposure levels. AMG8379 is a potent and selective NaV1.7 inhibitor that blocks sodium current in heterologous cells as well as DRG neurons, inhibits action potential firing in peripheral nerve fibers, and exhibits pharmacodynamic effects in translatable models of both itch and pain.
Asunto(s)
Canal de Sodio Activado por Voltaje NAV1.7/efectos de los fármacos , Bloqueadores de los Canales de Sodio/farmacología , Potenciales de Acción/efectos de los fármacos , Animales , Conducta Animal/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ganglios Espinales/citología , Ganglios Espinales/efectos de los fármacos , Humanos , Ratones , Ratones Endogámicos C57BL , Músculo Esquelético/metabolismo , Miocardio/metabolismo , Neuronas/efectos de los fármacos , Dolor/prevención & control , Dolor/psicología , Técnicas de Placa-Clamp , Prurito/prevención & control , Prurito/psicología , Quinolonas/farmacología , Bibliotecas de Moléculas Pequeñas , Estereoisomerismo , Sulfonamidas/farmacologíaRESUMEN
The majority of potent and selective hNaV1.7 inhibitors possess common pharmacophoric features that include a heteroaryl sulfonamide headgroup and a lipophilic aromatic tail group. Recently, reports of similar aromatic tail groups in combination with an acyl sulfonamide headgroup have emerged, with the acyl sulfonamide bestowing levels of selectivity over hNaV1.5 comparable to the heteroaryl sulfonamide. Beginning with commercially available carboxylic acids that met selected pharmacophoric requirements in the lipophilic tail, a parallel synthetic approach was applied to rapidly generate the derived acyl sulfonamides. A biaryl acyl sulfonamide hit from this library was elaborated, optimizing for potency and selectivity with attention to physicochemical properties. The resulting novel leads are potent, ligand and lipophilic efficient, and selective over hNaV1.5. Representative lead 36 demonstrates selectivity over other human NaV isoforms and good pharmacokinetics in rodents. The biaryl acyl sulfonamides reported herein may also offer ADME advantages over known heteroaryl sulfonamide inhibitors.
Asunto(s)
Benzamidas/química , Canal de Sodio Activado por Voltaje NAV1.7/metabolismo , Sulfonamidas/química , Bloqueadores del Canal de Sodio Activado por Voltaje/química , Animales , Benzamidas/síntesis química , Benzamidas/farmacocinética , Benzamidas/farmacología , Línea Celular , Femenino , Histamina , Humanos , Masculino , Ratones Endogámicos C57BL , Microsomas Hepáticos/metabolismo , Simulación del Acoplamiento Molecular , Prurito/inducido químicamente , Prurito/tratamiento farmacológico , Ensayo de Unión Radioligante , Ratas , Relación Estructura-Actividad , Sulfonamidas/síntesis química , Sulfonamidas/farmacocinética , Sulfonamidas/farmacología , Bloqueadores del Canal de Sodio Activado por Voltaje/síntesis química , Bloqueadores del Canal de Sodio Activado por Voltaje/farmacocinética , Bloqueadores del Canal de Sodio Activado por Voltaje/farmacologíaRESUMEN
There has been significant interest in developing a transient receptor potential A1 (TRPA1) antagonist for the treatment of pain due to a wealth of data implicating its role in pain pathways. Despite this, identification of a potent small molecule tool possessing pharmacokinetic properties allowing for robust in vivo target coverage has been challenging. Here we describe the optimization of a potent, selective series of quinazolinone-based TRPA1 antagonists. High-throughput screening identified 4, which possessed promising potency and selectivity. A strategy focused on optimizing potency while increasing polarity in order to improve intrinsic clearance culminated with the discovery of purinone 27 (AM-0902), which is a potent, selective antagonist of TRPA1 with pharmacokinetic properties allowing for >30-fold coverage of the rat TRPA1 IC50 in vivo. Compound 27 demonstrated dose-dependent inhibition of AITC-induced flinching in rats, validating its utility as a tool for interrogating the role of TRPA1 in in vivo pain models.
Asunto(s)
Proteínas del Tejido Nervioso/antagonistas & inhibidores , Oxadiazoles/síntesis química , Oxadiazoles/farmacología , Purinas/síntesis química , Purinas/farmacología , Quinazolinas/síntesis química , Quinazolinas/farmacología , Canales de Potencial de Receptor Transitorio/antagonistas & inhibidores , Animales , Transporte Biológico Activo , Células CHO , Canales de Calcio , Cricetulus , Perros , Relación Dosis-Respuesta a Droga , Descubrimiento de Drogas , Ensayos Analíticos de Alto Rendimiento , Humanos , Técnicas In Vitro , Células de Riñón Canino Madin Darby , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/metabolismo , Modelos Moleculares , Dimensión del Dolor/efectos de los fármacos , Ratas , Relación Estructura-Actividad , Canal Catiónico TRPA1RESUMEN
The transient receptor potential ankyrin 1 (TRPA1) channel is activated by noxious stimuli including chemical irritants and endogenous inflammatory mediators. Antagonists of this channel are currently being investigated for use as therapeutic agents for treating pain, airway disorders, and itch. A novel azabenzofuran series was developed that demonstrated in vitro inhibition of allyl isothiocyanate (AITC)-induced (45)Ca(2+) uptake with nanomolar potencies against both human and rat TRPA1. From this series, compound 10 demonstrated in vivo target coverage in an AITC-induced flinching model in rats while providing unbound plasma concentrations up to 16-fold higher than the TRPA1 rat IC50.
Asunto(s)
Bloqueadores de los Canales de Calcio/farmacología , Diseño de Fármacos , Compuestos Heterocíclicos con 3 Anillos/farmacología , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Canales Catiónicos TRPC/antagonistas & inhibidores , Canales de Potencial de Receptor Transitorio/antagonistas & inhibidores , Animales , Bloqueadores de los Canales de Calcio/síntesis química , Bloqueadores de los Canales de Calcio/química , Canales de Calcio/metabolismo , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Compuestos Heterocíclicos con 3 Anillos/síntesis química , Compuestos Heterocíclicos con 3 Anillos/química , Humanos , Isotiocianatos/antagonistas & inhibidores , Estructura Molecular , Proteínas del Tejido Nervioso/metabolismo , Ratas , Relación Estructura-Actividad , Canal Catiónico TRPA1 , Canales Catiónicos TRPC/metabolismo , Canales de Potencial de Receptor Transitorio/metabolismoRESUMEN
Small molecule inhibitors of Janus kinase (JAK) family members (JAK1, JAK2, JAK3, and Tyk2) are currently being pursued as potential new modes of therapy for a variety of diseases, including the inhibition of JAK2 for the treatment of myeloproliferative disorders. Selective inhibition within the JAK family can be beneficial in avoiding undesirable side effects (e.g., immunosuppression) caused by parallel inhibition of other JAK members. In an effort to design an assay paradigm for the development of JAK2 selective inhibitors, we investigated whether compound selectivity differed between cellular and purified enzyme environments. A set of JAK2 inhibitors was tested in a high-throughput JAK family cell assay suite and in corresponding purified enzyme assays. The high-throughput JAK cell assay suite comprises Ba/F3 cells individually expressing translocated ETS leukemia (TEL) fusions of each JAK family member (TEL-JAK Ba/F3) and an AlphaScreen phosphorylated-STAT5 (pSTAT5) immunoassay. Compound potencies from the TEL-JAK Ba/F3 pSTAT5 assays were similar to those determined in downstream cell proliferation measurements and more physiologically relevant cytokine-induced pSTAT5 PBMC assays. However, compound selectivity data between cell and purified enzyme assays were discrepant because of different potency shifts between cell and purified enzyme values for each JAK family member. For any JAK small molecule development program, our results suggest that relying solely on enzyme potency and selectivity data may be misleading. Adopting the high-throughput TEL-JAK Ba/F3 pSTAT5 cell assay suite in lead development paradigms should provide a more meaningful understanding of selectivity and facilitate the development of more selective JAK inhibitors.
Asunto(s)
Janus Quinasa 2/antagonistas & inhibidores , Proteínas de Fusión Oncogénica/antagonistas & inhibidores , Células Precursoras de Linfocitos B/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Factor de Transcripción STAT5/metabolismo , Animales , Línea Celular , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Ensayos Analíticos de Alto Rendimiento/métodos , Humanos , Janus Quinasa 2/genética , Janus Quinasa 2/metabolismo , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , Fosforilación/efectos de los fármacos , Células Precursoras de Linfocitos B/metabolismo , Reproducibilidad de los ResultadosRESUMEN
Lysine demethylase 1 (LSD1) and Jumonji C domain-containing oxygenase D2C (JMJD2C) participate in regulating the methylation status of histone H3 lysine residues. In some contexts, LSD1 and JMJD2C activity causes enhanced cellular proliferation, which may lead to tumorigenesis. The authors explored the utility of time-resolved fluorescence resonance energy transfer (TR-FRET) immunoassays, which employed peptides consisting of the first 21 amino acids of histone H3 in which lysine 4 (H3K4) or lysine 9 (H3K9) was methylated (me) to quantify LSD1 and JMJD2C activity. The LSD1 assay monitored demethylation of the H3K4me1 peptide using an antibody that recognizes H3K4me1 but not the unmethylated peptide product. The JMJD2C assay measured demethylation of H3K9me3 with an antibody that selectively recognizes H3K9me2. The optimized conditions resulted in robust assays (Z' > 0.7) that required only 3 to 6 nM of enzyme in a reaction volume of 6 to 10 µL. These assays were used to compare the activity of different LSD1 constructs and to determine the apparent K(m) of each JMJD2C substrate. Finally, both assays were used in a high-throughput setting for identifying demethylase inhibitors. Compounds discovered by these TR-FRET methods may lead to powerful tools for ascertaining the roles of demethylases in a cellular context and ultimately for potential cancer treatments.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Transferencia Resonante de Energía de Fluorescencia/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Histona Demetilasas/antagonistas & inhibidores , Histona Demetilasas con Dominio de Jumonji/antagonistas & inhibidores , Secuencia de Aminoácidos , Inhibidores Enzimáticos/metabolismo , Inmunoensayo/métodos , Lisina/metabolismo , Metilación , Datos de Secuencia Molecular , Péptidos/metabolismoRESUMEN
Protein kinases are recognized as important drug targets due to the pivotal roles they play in human disease. Many kinase inhibitors are ATP competitive, leading to potential problems with poor selectivity and significant loss of potency in vivo due to cellular ATP concentrations being much higher than K(m). Consequently, there has been growing interest in the development of ATP-noncompetitive inhibitors to overcome these problems. There are challenges to identifying ATP-noncompetitive inhibitors from compound library screens because ATP-noncompetitive inhibitors are often weaker and commonly excluded by potency-based hit selection criteria in favor of abundant and highly potent ATP-competitive inhibitors in screening libraries. Here we report the development of a time-resolved fluorescence resonance energy transfer (TR-FRET) assay for protein kinase cyclin-dependent kinase 4 (CDK4) and the identification of ATP-noncompetitive inhibitors by high-throughput screening after employing a strategy to favor this type of inhibitors. We also present kinetic characterization that is consistent with the proposed mode of inhibition.
Asunto(s)
Adenosina Trifosfato/metabolismo , Quinasa 4 Dependiente de la Ciclina/antagonistas & inhibidores , Transferencia Resonante de Energía de Fluorescencia/métodos , Inhibidores de Proteínas Quinasas/farmacología , Línea Celular Tumoral , Cromatografía Líquida de Alta Presión , Quinasa 4 Dependiente de la Ciclina/metabolismo , Humanos , Cinética , Espectrometría de Masas , Modelos MolecularesRESUMEN
Developing Janus kinase 2 (Jak2) inhibitors has become a significant focus for small molecule drug discovery programs in recent years due to the identification of a Jak2 gain-of-function mutation in the majority of patients with myeloproliferative disorders (MPD). Here, we describe the discovery of a thienopyridine series of Jak2 inhibitors that culminates with compounds showing 100- to >500-fold selectivity over the related Jak family kinases in enzyme assays. Selectivity for Jak2 was also observed in TEL-Jak cellular assays, as well as in cytokine-stimulated peripheral blood mononuclear cell (PBMC) and whole blood assays. X-ray cocrystal structures of 8 and 19 bound to the Jak2 kinase domain aided structure-activity relationship efforts and, along with a previously reported small molecule X-ray cocrystal structure of the Jak1 kinase domain, provided structural rationale for the observed high levels of Jak2 selectivity.
Asunto(s)
Janus Quinasa 2/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/síntesis química , Tienopiridinas/síntesis química , Animales , Línea Celular Tumoral , Permeabilidad de la Membrana Celular , Cristalografía por Rayos X , Humanos , Janus Quinasa 1/química , Janus Quinasa 2/química , Leucocitos Mononucleares/efectos de los fármacos , Modelos Moleculares , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Estructura Terciaria de Proteína , Relación Estructura-Actividad , Porcinos , Tienopiridinas/química , Tienopiridinas/farmacologíaRESUMEN
A series of alpha-amidosulfones were found to be potent and selective agonists of CB(2). The discovery, synthesis, and structure-activity relationships of this series of agonists are reported. In addition, the pharmacokinetic properties of the most promising compounds are profiled.
Asunto(s)
Receptor Cannabinoide CB2/agonistas , Sulfonas/química , Amidas/química , Amidas/farmacología , Animales , Humanos , Microsomas Hepáticos , Farmacocinética , Ratas , Relación Estructura-Actividad , Sulfonas/farmacologíaRESUMEN
The CB2 receptor is an attractive therapeutic target for analgesic and anti-inflammatory agents. Herein we describe the discovery of a novel class of oxadiazole derivatives from which potent and selective CB2 agonist leads were developed. Initial hit 7 was identified from a cannabinoid target-biased library generated by virtual screening of sample collections using a pharmacophore model in combination with a series of physicochemical filters. 7 was demonstrated to be a selective CB2 agonist (CB2 EC50 = 93 nM, Emax = 98%, CB1 EC50 > 10 microM). However, this compound exhibited poor solubility and relatively high clearance in rat, resulting in low oral bioavailability. In this paper, we report detailed SAR studies on 7 en route toward improving potency, physicochemical properties, and solubility. This effort resulted in identification of 63 that is a potent and selective agonist at CB2 (EC50 = 2 nM, Emax = 110%) with excellent pharmacokinetic properties.
Asunto(s)
Aminoquinolinas/síntesis química , Oxadiazoles/síntesis química , Receptor Cannabinoide CB2/agonistas , Administración Oral , Aminoquinolinas/administración & dosificación , Aminoquinolinas/farmacocinética , Animales , Disponibilidad Biológica , Células CHO , Cricetinae , Cricetulus , Evaluación Preclínica de Medicamentos , Humanos , Modelos Moleculares , Oxadiazoles/administración & dosificación , Oxadiazoles/farmacocinética , Ratas , Relación Estructura-ActividadRESUMEN
Structural modifications to the central portion of the N-arylamide oxadiazole scaffold led to the identification of N-arylpiperidine oxadiazoles as conformationally constrained analogs that offered improved stability and comparable potency and selectivity. The simple, modular scaffold allowed for the use of expeditious and divergent synthetic routes, which provided two-directional SAR in parallel. Several potent and selective agonists from this novel ligand class are described.
Asunto(s)
Microsomas Hepáticos/metabolismo , Oxadiazoles , Receptor Cannabinoide CB2/agonistas , Animales , Técnicas Químicas Combinatorias , Humanos , Conformación Molecular , Estructura Molecular , Oxadiazoles/síntesis química , Oxadiazoles/química , Oxadiazoles/farmacocinética , Oxadiazoles/farmacología , Ratas , Relación Estructura-ActividadRESUMEN
The lymphocyte-specific kinase (Lck), a member of the Src family of cytoplasmic tyrosine kinases, is expressed in T cells and natural killer (NK) cells. Genetic evidence, including knockout mice and human mutations, demonstrates that Lck kinase activity is critical for normal T cell development, activation, and signaling. Selective inhibition of Lck is expected to offer a new therapy for the treatment of T-cell-mediated autoimmune and inflammatory disease. With the aid of X-ray structure-based analysis, aminopyrimidine amides 2 and 3 were designed from aminoquinazolines 1, which had previously been demonstrated to exhibit potent inhibition of Lck and T cell proliferation. In this report, we describe the synthesis and structure-activity relationships of a series of novel aminopyrimidine amides 3 possessing improved cellular potency and selectivity profiles relative to their aminoquinazoline predecessors 1. Orally bioavailable compound 13b inhibited the anti-CD3-induced production of interleukin-2 (IL-2) in mice in a dose-dependent manner (ED 50 = 9.4 mg/kg).
Asunto(s)
Amidas/farmacología , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas/farmacología , Linfocitos T/efectos de los fármacos , Administración Oral , Amidas/síntesis química , Amidas/química , Animales , Proliferación Celular/efectos de los fármacos , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Diseño de Fármacos , Activación Enzimática/efectos de los fármacos , Femenino , Humanos , Interleucina-2/antagonistas & inhibidores , Interleucina-2/metabolismo , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/metabolismo , Lipopolisacáridos/farmacología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Modelos Moleculares , Estructura Molecular , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Pirimidinas/síntesis química , Pirimidinas/química , Ratas , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Estereoisomerismo , Relación Estructura-Actividad , Linfocitos T/metabolismoRESUMEN
N-3-(Phenylcarbamoyl)arylpyrimidine-5-carboxamides are a novel class of selective Lck inhibitors. This series of compounds derives its selectivity from a hydrogen bond with the gatekeeper Thr316 of the enzyme. X-ray co-crystal structural data, structure-activity relationships, and the synthesis of these inhibitors are reported herein.
Asunto(s)
Amidas/síntesis química , Amidas/farmacología , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/antagonistas & inhibidores , Pirimidinas/síntesis química , Pirimidinas/farmacología , Amidas/química , Técnicas Químicas Combinatorias , Cristalografía por Rayos X , Diseño de Fármacos , Humanos , Enlace de Hidrógeno , Conformación Molecular , Estructura Molecular , Pirimidinas/química , Relación Estructura-Actividad , Treonina/químicaRESUMEN
2,3-Diarylfuro[2,3-b]pyridine-4-amines are a novel class of potent and selective inhibitors of Lck. The discovery, synthesis, and structure activity relationships of this series of inhibitors are reported. The most promising compounds were also profiled to deduce their pharmacokinetic properties.
Asunto(s)
Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/antagonistas & inhibidores , Piridinas/síntesis química , Piridinas/farmacocinética , Aminas/síntesis química , Aminas/farmacocinética , Animales , Humanos , Concentración 50 Inhibidora , Farmacocinética , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/farmacocinética , Ratas , Ratas Sprague-Dawley , Relación Estructura-ActividadRESUMEN
4-Amino-5,6-biaryl-furo[2,3-d]pyrimidines were identified as potent non-selective inhibitors of Lck. A novel, divergent, and practical synthetic route was developed to access derivatives from bifunctional intermediates. Lead optimization was guided by X-ray crystallographic data, and preliminary SAR led to the identification of compounds with improved cellular potency and selectivity.
Asunto(s)
Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/antagonistas & inhibidores , Pirimidinas/síntesis química , Pirimidinas/farmacocinética , Relación Estructura-Actividad Cuantitativa , Animales , Antiinflamatorios/síntesis química , Humanos , Concentración 50 Inhibidora , Interleucina-2/metabolismo , Prueba de Cultivo Mixto de Linfocitos , Linfocitos/efectos de los fármacos , Farmacocinética , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/farmacocinética , Ratas , Ratas Sprague-DawleyRESUMEN
The lymphocyte-specific kinase (Lck) is a cytoplasmic tyrosine kinase of the Src family expressed in T cells and natural killer (NK) cells. Genetic evidence in both mice and humans demonstrates that Lck kinase activity is critical for signaling mediated by the T cell receptor (TCR), which leads to normal T cell development and activation. Selective inhibition of Lck is expected to offer a new therapy for the treatment of T-cell-mediated autoimmune and inflammatory disease. Screening of our kinase-preferred collection identified aminoquinazoline 1 as a potent, nonselective inhibitor of Lck and T cell proliferation. In this report, we describe the synthesis and structure-activity relationships of a series of novel aminoquinazolines possessing in vitro mechanism-based potency. Optimized, orally bioavailable compounds 32 and 47 exhibit anti-inflammatory activity (ED(50) of 22 and 11 mg/kg, respectively) in the anti-CD3-induced production of interleukin-2 (IL-2) in mice.
Asunto(s)
Antiinflamatorios no Esteroideos/síntesis química , Benzamidas/síntesis química , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/antagonistas & inhibidores , Quinazolinas/síntesis química , Administración Oral , Animales , Antiinflamatorios no Esteroideos/química , Antiinflamatorios no Esteroideos/farmacología , Benzamidas/química , Benzamidas/farmacología , Disponibilidad Biológica , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Femenino , Humanos , Técnicas In Vitro , Interleucina-2/biosíntesis , Masculino , Ratones , Ratones Endogámicos BALB C , Modelos Moleculares , Quinazolinas/química , Quinazolinas/farmacología , Ratas , Ratas Sprague-Dawley , Relación Estructura-Actividad , Linfocitos T/citología , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
The lymphocyte-specific kinase (Lck) is a cytoplasmic tyrosine kinase of the Src family expressed in T cells and NK cells. Genetic evidence in both mice and humans demonstrates that Lck kinase activity is critical for signaling mediated by the T cell receptor (TCR), which leads to normal T cell development and activation. A small molecule inhibitor of Lck is expected to be useful in the treatment of T cell-mediated autoimmune and inflammatory disorders and/or organ transplant rejection. In this paper, we describe the synthesis, structure-activity relationships, and pharmacological characterization of 2-aminopyrimidine carbamates, a new class of compounds with potent and selective inhibition of Lck. The most promising compound of this series, 2,6-dimethylphenyl 2-((3,5-bis(methyloxy)-4-((3-(4-methyl-1-piperazinyl)propyl)oxy)phenyl)amino)-4-pyrimidinyl(2,4-bis(methyloxy)phenyl)carbamate (43) exhibits good activity when evaluated in in vitro assays and in an in vivo model of T cell activation.
Asunto(s)
Aminopiridinas/síntesis química , Antiinflamatorios/síntesis química , Carbamatos/síntesis química , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/antagonistas & inhibidores , Pirimidinas/síntesis química , Administración Oral , Aminopiridinas/química , Aminopiridinas/farmacología , Animales , Antiinflamatorios/química , Antiinflamatorios/farmacología , Disponibilidad Biológica , Carbamatos/química , Carbamatos/farmacología , Cristalografía por Rayos X , Humanos , Técnicas In Vitro , Células Jurkat , Activación de Linfocitos , Prueba de Cultivo Mixto de Linfocitos , Ratones , Ratones Endogámicos BALB C , Modelos Moleculares , Estructura Molecular , Pirimidinas/química , Pirimidinas/farmacología , Ratas , Ratas Sprague-Dawley , Relación Estructura-Actividad , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunologíaRESUMEN
BACKGROUND: CD56+ human natural killer (NK) cells are the principal anti-pig cytotoxic effectors in vitro. Expression of certain human leukocyte antigen (HLA) class I molecules in porcine cells can inhibit NK cell-mediated natural cytotoxicity in serum-free medium, but had not been shown to inhibit antibody-dependent cellular cytotoxicity (ADCC) by CD16+ NK cells in the presence of human xenoreactive immunoglobulin G. Moreover, expression of HLA molecules might amplify the previously weak CD8+ cytotoxic T-lymphocyte (CTL) response against porcine cells. METHODS: A novel porcine B-lymphoblastoid cell line (13271) was stably transfected with HLA-Cw*0304 gene constructs encoding wild-type (wt) Cw3 or genetically modified Cw3 unable to interact with CD8 (Cw3-D227K). The Cw3 transfectants were used in limiting dilution assays to estimate the CTL precursor frequency in CD56-depleted human peripheral blood mononuclear cells (PBMC) obtained from eight unrelated donors. The 13271 transfectants were also used as targets for clonal and polyclonal NK cells in the presence and absence of human serum, to measure inhibition of ADCC. RESULTS: Expression of Cw3-wt in 13271 cells significantly increased the human CTL response compared with the empty-vector control transfectant, whereas no significant increase resulted from expression of CD8-interaction mutant Cw3-D227K molecules. The Cw3-D227K mutant was indistinguishable from Cw3-wt in its ability to inhibit both natural cytotoxicity and ADCC mediated by human NK clones that have the appropriate CD158b inhibitory receptor. CONCLUSIONS: Transgenic expression of HLA molecules in pig cells will likely amplify the CD8+ CTL response against the xenograft. Disruption of HLA-CD8 interaction could minimize this amplification without compromising NK-cell inhibition.