RESUMEN
To identify Janus kinase (JAK) inhibitors that selectively target gastrointestinal tissues with limited systemic exposures, a class of imidazopyrrolopyridines with a range of physical properties was prepared and evaluated. We identified compounds with low intrinsic permeability and determined a correlation between permeability and physicochemical properties, clogP and tPSA, for a subset of compounds. This low intrinsic permeability translated into compounds displaying high colonic exposure and low systemic exposure after oral dosing at 25 mg/kg in mouse. In a mouse PK/PD model, oral dosing of lead compound 2 demonstrated dose-dependent inhibition of pSTAT phosphorylation in colonic explants post-oral dose but low systemic exposure and no measurable systemic pharmacodynamic activity. We thus demonstrate the utility of JAK inhibitors with low intrinsic permeability as a feasible approach to develop gut-restricted, pharmacologically active molecules with a potential advantage over systemically available compounds that are limited by systemic on-target adverse events.
Asunto(s)
Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , Inhibidores de las Cinasas Janus/farmacología , Inhibidores de las Cinasas Janus/farmacocinética , Piridinas/farmacología , Piridinas/farmacocinética , Administración Oral , Animales , Perros , Descubrimiento de Drogas , Femenino , Humanos , Enfermedades Inflamatorias del Intestino/metabolismo , Inhibidores de las Cinasas Janus/administración & dosificación , Inhibidores de las Cinasas Janus/química , Quinasas Janus/antagonistas & inhibidores , Quinasas Janus/metabolismo , Células de Riñón Canino Madin Darby , Ratones , Ratones Endogámicos C57BL , Permeabilidad , Fosforilación/efectos de los fármacos , Piridinas/administración & dosificación , Piridinas/químicaRESUMEN
The increasing dissemination of carbapenemases in Gram-negative bacteria has threatened the clinical usefulness of the ß-lactam class of antimicrobials. A program was initiated to discover a new series of serine ß-lactamase inhibitors containing a boronic acid pharmacophore, with the goal of finding a potent inhibitor of serine carbapenemase enzymes that are currently compromising the utility of the carbapenem class of antibacterials. Potential lead structures were screened in silico by modeling into the active sites of key serine ß-lactamases. Promising candidate molecules were synthesized and evaluated in biochemical and whole-cell assays. Inhibitors were identified with potent inhibition of serine carbapenemases, particularly the Klebsiella pneumoniae carbapenemase (KPC), with no inhibition of mammalian serine proteases. Studies in vitro and in vivo show that RPX7009 (9f) is a broad-spectrum inhibitor, notably restoring the activity of carbapenems against KPC-producing strains. Combined with a carbapenem, 9f is a promising product for the treatment of multidrug resistant Gram-negative bacteria.
Asunto(s)
Antibacterianos/química , Proteínas Bacterianas/antagonistas & inhibidores , Ácidos Borónicos/química , Compuestos Heterocíclicos con 1 Anillo/química , Inhibidores de beta-Lactamasas/química , Animales , Antibacterianos/farmacocinética , Antibacterianos/farmacología , Ácidos Borónicos/farmacocinética , Ácidos Borónicos/farmacología , Carbapenémicos/farmacología , Cristalografía por Rayos X , Farmacorresistencia Bacteriana , Sinergismo Farmacológico , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Gramnegativas/enzimología , Bacterias Gramnegativas/aislamiento & purificación , Compuestos Heterocíclicos con 1 Anillo/farmacocinética , Compuestos Heterocíclicos con 1 Anillo/farmacología , Ratones , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Ratas , Estereoisomerismo , Relación Estructura-Actividad , Inhibidores de beta-Lactamasas/farmacocinética , Inhibidores de beta-Lactamasas/farmacología , beta-LactamasasRESUMEN
We describe the identification, SAR, and in vivo pharmacology of a new series of Src-family selective Lck inhibitors. These thienopyridines were designed based on a desire to access the unique residues in the extended hinge region of Lck.
Asunto(s)
Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/antagonistas & inhibidores , Piridinas/química , Sitios de Unión , Diseño de Fármacos , Humanos , Concentración 50 Inhibidora , Piridinas/farmacología , Relación Estructura-Actividad , Familia-src Quinasas/antagonistas & inhibidoresRESUMEN
We describe the identification, SAR, and pharmacology of the src-family selective lck inhibitor A-770041 that prolongs the survival of major histocompatibility mismatched allografts in models of solid organ transplant rejection for greater than 65 days.
Asunto(s)
Rechazo de Injerto/prevención & control , Inmunosupresores/farmacología , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/antagonistas & inhibidores , Pirazoles/síntesis química , Pirazoles/farmacología , Pirimidinas/síntesis química , Pirimidinas/farmacología , Familia-src Quinasas/antagonistas & inhibidores , Animales , Química Farmacéutica/métodos , Cristalografía por Rayos X , Diseño de Fármacos , Inhibidores Enzimáticos/farmacología , Trasplante de Corazón/métodos , Humanos , Inmunofenotipificación , Concentración 50 Inhibidora , Cinética , Complejo Mayor de Histocompatibilidad , Modelos Químicos , Modelos Moleculares , Preparaciones Farmacéuticas , Ratas , Factores de Tiempo , Trasplante Homólogo/métodosRESUMEN
Lck, one of eight members of the Src family of tyrosine kinases, is activated after T cell stimulation and is required for T-cell proliferation and interleukin (IL)-2 production. Inhibition of Lck has been a target to prevent lymphocyte activation and acute rejection. Here, we report the pharmacologic characterization of 1-methyl-1H-indole-2-carboxylic acid (4-{1-[4-(4-acetyl-piperazin-l-yl)-cyclohexyl]-4-amino-1H-pyrazolo[3,4-d]pyrimidin-3-yl}-2-methoxy-phenyl)-amide (A-770041), an orally bioavailable pyrazolo[3,4-d]pyrimidine with increased selectivity for Lck compared with previously reported compounds. A-770041 is a 147 nM inhibitor of Lck (1 mM ATP) and is 300-fold selective against Fyn, the other Src family kinase involved in T-cell signaling. Concanavalin A-stimulated IL-2 production in whole blood is inhibited by A-770041 with an EC50 of approximately 80 nM. A-770041 is orally bioavailable (F = 34.1 +/- 7.2% at 10 mg/kg) and has a t(1/2) of 4.1 +/- 0.1 h. Concanavalin A-induced IL-2 production in vivo is inhibited by oral administration of A-770041 (in vivo EC50 = 78 +/- 28 nM). Doses of A-770041 at or above 10 mg/kg/day prevent rejection of hearts transplanted heterotopically in rats from Brown Norway donors to Lewis recipients across a major histocompatibility barrier for least 65 days. Grafts from animals treated with 20 mg/kg/day A-770041 or 10 mg/day Cyclosporin A had minimal microvascular changes or multifocal mononuclear infiltrates. However, mineralization in myocytes from the grafts from A-770041-treated animals was less than animals treated with Cyclosporin A. Lck inhibition is an attractive target to prevent acute rejection.
Asunto(s)
Rechazo de Injerto/prevención & control , Trasplante de Corazón/efectos adversos , Péptidos y Proteínas de Señalización Intracelular/farmacología , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/antagonistas & inhibidores , Pirazoles/farmacología , Pirimidinas/farmacología , Animales , Interleucina-2/metabolismo , Péptidos y Proteínas de Señalización Intracelular/farmacocinética , Activación de Linfocitos/efectos de los fármacos , Masculino , Ratas , Ratas Endogámicas BN , Ratas Endogámicas Lew , Linfocitos T/inmunología , Trasplante HomólogoRESUMEN
The Src family kinases Lck and Fyn play an important role in T cell development and function. We have synthesized a novel small molecule, A-420983, which inhibits Lck and Fyn, as well as other Src family kinases, but has selectivity with respect to non-Src family kinases. A-420983 completely inhibited antigen-stimulated production of IFN-gamma and IL-4 by mouse Th1 and Th2 cells, respectively. Antigen-induced T cell proliferation was also blocked by treatment with A-420983. In contrast, IL-15-induced proliferation was unaffected by A-420983, suggesting that TCR-independent pathways of T cell activation were not impaired. When mice were dosed orally, A-420983 inhibited TCR-mediated c-jun and ZAP-70 phosphorylation in CD4+ T cells and suppressed the disease course of established EAE. Treatment with A-420983 for 7 days resulted in a block in thymocyte development at the CD4- CD8- stage, consistent with inhibition of Lck and Fyn in vivo. These results demonstrate that a small molecule inhibitor of Lck and Fyn can block TCR-induced T cell activation in vitro and in vivo. Furthermore, CNS demyelination mediated by activated encephalitogenic CD4+ T cells is dependent upon the kinase activity of these Src family members. We conclude that inhibition of Src family kinases may represent a promising strategy for the treatment of T cell-mediated disorders.
Asunto(s)
Linfocitos T CD4-Positivos/efectos de los fármacos , Activación de Linfocitos/efectos de los fármacos , Pirazoles/farmacología , Pirimidinas/farmacología , Familia-src Quinasas/antagonistas & inhibidores , Animales , Línea Celular , Proliferación Celular/efectos de los fármacos , Femenino , Ratones , Ratones Endogámicos , Timo/citología , Timo/efectos de los fármacosRESUMEN
We have identified the pyrazolo[3,4-d]pyrimidine A-420983 (compound 7) as a potent inhibitor of lck. A-420983 exhibits oral efficacy in animal models of delayed-type hypersensitivity and organ transplant rejection.
Asunto(s)
Rechazo de Injerto/tratamiento farmacológico , Inmunosupresores/síntesis química , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/antagonistas & inhibidores , Pirazoles/uso terapéutico , Pirimidinas/uso terapéutico , Administración Oral , Animales , Perros , Trasplante de Corazón/efectos adversos , Humanos , Hipersensibilidad Tardía/tratamiento farmacológico , Inmunosupresores/farmacocinética , Inmunosupresores/uso terapéutico , Concentración 50 Inhibidora , Interleucina-2/biosíntesis , Ratones , Ratones Endogámicos C57BL , Microsomas Hepáticos , Modelos Animales , Modelos Moleculares , Estructura Molecular , Pirazoles/administración & dosificación , Pirazoles/farmacocinética , Pirimidinas/administración & dosificación , Pirimidinas/farmacocinética , Relación Estructura-ActividadRESUMEN
Protein tyrosine kinases play critical roles in cell signaling and are considered attractive targets for drug discovery. The authors have applied microARCS (microarrayed compound screening) technology to develop a high-throughput screen for finding inhibitors of the p56lck tyrosine kinase. Initial assay development was performed in a homogeneous time-resolved (LANCE) format in 96-well microplates and then converted into the gel-based microARCS format. The microARCS methodology is a well-less screening format in which 8640 compounds are arrayed on a microplate-sized piece of polystyene and subsequently assayed by placing reagents cast in agarose gels in contact with these compound sheets. A blotting paper soaked with adenosine triphosphate is applied on the gel to initiate the kinase reaction in the gel. Using this screening methodology, 300,000 compounds were screened in less than 40 h. Substantial reagent reduction was achieved by converting this tyrosine kinase assay from a 96-well plate assay to microARCS, resulting in significant cost savings.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/antagonistas & inhibidores , Análisis de Secuencia por Matrices de Oligonucleótidos , Electroforesis en Gel de Agar , Especificidad por SustratoRESUMEN
A series of para-substituted 3-phenyl pyrazolopyrimidines was synthesized and evaluated as inhibitors of lck. The nature of the substitution affected enzyme selectivity and potency for lck, src, kdr, and tie-2. The para-phenoxyphenyl analogue 2 is an orally active lck inhibitor with a bioavailability of 69% and exhibits an extended duration of action in animal models of T cell inhibition.