RESUMEN

Inhibition of mixed lineage kinase 3 (MLK3) is a potential strategy for treatment of Parkinson's disease and HIV-1 associated neurocognitive disorders (HAND), requiring an inhibitor that can achieve significant brain concentration levels. We report here URMC-099 (1) an orally bioavailable (F = 41%), potent (IC50 = 14 nM) MLK3 inhibitor with excellent brain exposure in mouse PK models and minimal interference with key human CYP450 enzymes or hERG channels. The compound inhibits LPS-induced TNFα release in microglial cells, HIV-1 Tat-induced release of cytokines in human monocytes and up-regulation of phospho-JNK in Tat-injected brains of mice. Compound 1 likely functions in HAND preclinical models by inhibiting multiple kinase pathways, including MLK3 and LRRK2 (IC50 = 11 nM). We compare the kinase specificity and BBB penetration of 1 with CEP-1347 (2). Compound 1 is well tolerated, with excellent in vivo activity in HAND models, and is under investigation for further development.

Asunto(s)

Descubrimiento de Drogas/métodos , Quinasas Quinasa Quinasa PAM/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Piridinas/farmacología , Pirroles/farmacología , Administración Oral , Animales , Área Bajo la Curva , Disponibilidad Biológica , Barrera Hematoencefálica/metabolismo , Encéfalo/metabolismo , Carbazoles/síntesis química , Carbazoles/farmacocinética , Carbazoles/farmacología , Células Cultivadas , Trastornos del Conocimiento/complicaciones , Trastornos del Conocimiento/prevención & control , Infecciones por VIH/complicaciones , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Quinasas Quinasa Quinasa PAM/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Modelos Químicos , Estructura Molecular , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/farmacocinética , Piridinas/síntesis química , Piridinas/farmacocinética , Pirroles/síntesis química , Pirroles/farmacocinética , Factor de Necrosis Tumoral alfa/metabolismo , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/farmacología , Proteina Quinasa Quinasa Quinasa 11 Activada por Mitógeno

RESUMEN

Human immunodeficiency virus (HIV)-associated neurocognitive disorders (HAND) is a significant source of disability in the HIV-infected population. Even with stringent adherence to anti-retroviral therapy, >50% of patients living with HIV-1 will develop HAND (Heaton et al., 2010). Because suppression of viral replication alone is not enough to stop HAND progression, there is a need for an adjunctive neuroprotective therapy in this population. To this end, we have developed a small-molecule brain-penetrant inhibitor with activity against mixed-lineage kinase 3 (MLK3), named URMC-099. MLK3 activation is associated with many of the pathologic hallmarks of HAND (Bodner et al., 2002, 2004; Sui et al., 2006) and therefore represents a prime target for adjunctive therapy based on small-molecule kinase inhibition. Here we demonstrate the anti-inflammatory and neuroprotective effects of URMC-099 in multiple murine and rodent models of HAND. In vitro, URMC-099 treatment reduced inflammatory cytokine production by HIV-1 Tat-exposed microglia and prevented destruction and phagocytosis of cultured neuronal axons by these cells. In vivo, URMC-099 treatment reduced inflammatory cytokine production, protected neuronal architecture, and altered the morphologic and ultrastructural response of microglia to HIV-1 Tat exposure. In conclusion, these data provide compelling in vitro and in vivo evidence to investigate the utility of URMC-099 in other models of HAND with the goal of advancement to an adjunctive therapeutic agent.

Asunto(s)

Infecciones por VIH/complicaciones , Infecciones por VIH/tratamiento farmacológico , Inflamación/prevención & control , Quinasas Quinasa Quinasa PAM/antagonistas & inhibidores , Fármacos Neuroprotectores/uso terapéutico , Animales , Trasplante de Médula Ósea , Receptor 1 de Quimiocinas CX3C , Línea Celular Transformada/efectos de los fármacos , Línea Celular Transformada/virología , Células Cultivadas , Citocinas , Modelos Animales de Enfermedad , Embrión de Mamíferos , Productos del Gen tat/inmunología , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Infecciones por VIH/virología , VIH-1/fisiología , Hipocampo/patología , Humanos , Inflamación/genética , Inflamación/patología , Inflamación/virología , Ratones , Ratones Transgénicos , Microscopía Inmunoelectrónica , Fagocitosis/efectos de los fármacos , Fagocitosis/genética , Fosforilación/efectos de los fármacos , Piridinas/farmacología , Piridinas/uso terapéutico , Pirroles/farmacología , Pirroles/uso terapéutico , Ratas , Receptores de Quimiocina/genética , Receptores de Quimiocina/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Estadísticas no Paramétricas , Factores de Tiempo , Transfección , Productos del Gen tat del Virus de la Inmunodeficiencia Humana , Proteina Quinasa Quinasa Quinasa 11 Activada por Mitógeno

RESUMEN

The present article describes a selection of a new class of small molecule antagonists for the h-GnRH receptor, their preparation, and evaluation in vitro. Three computational methods were combined into a consensus score, to rank order virtual templates. The top 5% of templates were further evaluated in silico and assessed for novelty and synthetic accessibility. The tetrahydropyrido[4,3-d]pyrimidine-2,4-dione core was selected for synthesis and evaluated in vitro. Using an array approach for analog design and synthesis, we were able to drive the binding below 10nM for the h-GnRH receptor after two rounds of optimization.

Asunto(s)

Hidrógeno/química , Pirimidinas/química , Pirimidinas/farmacología , Receptores LHRH/antagonistas & inhibidores , Bases de Datos Factuales , Etilaminas/química , Humanos , Modelos Moleculares , Estructura Molecular , Pirimidinas/síntesis química , Receptores LHRH/metabolismo , Relación Estructura-Actividad

RESUMEN

A novel series of 2-(4,5,6,7-tetrahydro-1H-pyrrolo[3,2-c]pyridin-3-yl)-ethylamine derivatives were designed and synthesized as GnRH receptor antagonists. SAR studies led to a series of highly active molecules against both the rat and human receptors. Furthermore, one potent compound, 17j, demonstrated dose-dependent LH suppression in castrated rats.

Asunto(s)

Piridinas/farmacología , Receptores LHRH/antagonistas & inhibidores , Animales , Células Cultivadas , Humanos , Piridinas/química , Ratas , Relación Estructura-Actividad

RESUMEN

Nonpeptide antagonists of the human gonadotropin-releasing hormone receptor (GnRH-R) have been the subject of considerable interest because of their potential as a new class of oral therapeutics for the treatment of sex hormone-dependent diseases and infertility. While many classes of competitive GnRH-R antagonists have been described, we present here the first characterization of an allosteric nonpeptide GnRH-R antagonist. Previously, 5-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthalen-2-ylmethyl)furan-2-carboxylic acid (2,4,6-trimethoxyphenyl)amide (here called Furan-1) had been demonstrated to be a potent GnRH-R antagonist both in vitro and in vivo. Using mutagenesis, the binding sites for Furan-1 and another potent nonpeptide antagonist (NBI-42902) have been mapped and are shown to be adjacent but nonoverlapping. Furan-1 is shown to affect the binding kinetics of radiolabeled peptide agonists as well as radiolabeled NBI-42902, and the kinetic data fit the allosteric ternary complex model. Furan-1 is considerably negatively cooperative with the nonpeptide antagonist and extremely negatively cooperative with the peptide agonist [125I-His5,d-Tyr6]GnRH so that it is nearly indistinguishable from an orthosteric competitive compound. Taken together, these data were used to develop a model of the nonpeptides bound to the GnRH-R binding site consistent with the current data.

Asunto(s)

Furanos/metabolismo , Antagonistas de Hormonas/metabolismo , Receptores LHRH/antagonistas & inhibidores , Receptores LHRH/metabolismo , Timina/análogos & derivados , Regulación Alostérica/genética , Animales , Sitios de Unión/genética , Unión Competitiva/genética , Células COS , Línea Celular Tumoral , Chlorocebus aethiops , Furanos/farmacología , Humanos , Concentración 50 Inhibidora , Mutagénesis Sitio-Dirigida , Ensayo de Unión Radioligante , Ratas , Receptores LHRH/agonistas , Receptores LHRH/genética , Estereoisomerismo , Timina/metabolismo , Timina/farmacología

RESUMEN

Here we characterize the biological activity of a hairpin polyamide 1 that inhibits binding of the minor-groove transcription factor LEF-1, constitutively expressed in colon cancers. Genome-wide analysis of mRNA expression in DLD1 colon cancer cells treated with 1 reveals that a limited number of genes are affected; the most significant changes correspond to genes related to cell cycle, signaling, and proteolysis rather than the anticipated WNT signaling pathway. Treated cells display increased doubling time and hypersensitivity to DNA damage that most likely results from downregulation of DNA-damage checkpoint genes, including YWAE (14-3-3epsilon protein) and DDIT3. Promoter analyses on a genomic level revealed numerous potential polyamide binding sites and multiple possible mechanisms for transcriptional antagonism, underscoring the utility of gene expression profiling in understanding the effects of polyamides on transcription at the cellular level.

Asunto(s)

ADN/metabolismo , Nylons/metabolismo , ARN Mensajero/biosíntesis , Sitios de Unión , Calreticulina/genética , Neoplasias del Colon , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/metabolismo , Bases de Datos Genéticas , Regulación hacia Abajo/genética , Regulación hacia Abajo/fisiología , Ensayo de Cambio de Movilidad Electroforética , Genes bcl-1/genética , Humanos , Factor de Unión 1 al Potenciador Linfoide , Microscopía Fluorescente , Conformación de Ácido Nucleico , Nylons/química , Análisis de Secuencia por Matrices de Oligonucleótidos , Regiones Promotoras Genéticas , Factores de Transcripción/análisis , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción Genética/fisiología , Células Tumorales Cultivadas

RESUMEN

Specific, designed, nonperiodic arrangements of gold nanocrystals that are 5 and 10 nm in diameter can be prepared with double-stranded DNA serving as a template (see drawing; A' and B' denote oligonucleotide sequences complementary to sequences A and B). The methods described should be applicable to nanocrystals composed of various materials.