RESUMEN
We report here structure-guided optimization of a novel series of NF-κB inducing kinase (NIK) inhibitors. Starting from a modestly potent, low molecular weight lead, activity was improved by designing a type 11/2 binding mode that accessed a back pocket past the methionine-471 gatekeeper. Divergent binding modes in NIK and PI3K were exploited to dampen PI3K inhibition while maintaining NIK inhibition within these series. Potent compounds were discovered that selectively inhibit the nuclear translocation of NF-κB2 (p52/REL-B) but not canonical NF-κB1 (REL-A/p50).
Asunto(s)
Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Compuestos Heterocíclicos de Anillo en Puente/farmacología , Isoxazoles/farmacología , Oxazepinas/farmacología , Oxazoles/farmacología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Transporte Activo de Núcleo Celular , Animales , Sitios de Unión , Núcleo Celular/metabolismo , Perros , Células HEK293 , Células HeLa , Compuestos Heterocíclicos de 4 o más Anillos/síntesis química , Compuestos Heterocíclicos de 4 o más Anillos/química , Compuestos Heterocíclicos de Anillo en Puente/síntesis química , Compuestos Heterocíclicos de Anillo en Puente/química , Humanos , Imidazoles/farmacología , Isoxazoles/síntesis química , Isoxazoles/química , Ratones , Subunidad p50 de NF-kappa B/metabolismo , Subunidad p52 de NF-kappa B/metabolismo , Oxazepinas/síntesis química , Oxazepinas/química , Oxazoles/síntesis química , Oxazoles/química , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Transducción de Señal/efectos de los fármacos , Quinasa de Factor Nuclear kappa BRESUMEN
Inhibition of the non-receptor tyrosine kinase ITK, a component of the T-cell receptor signalling cascade, may represent a novel treatment for allergic asthma. Here we report the structure-based optimization of a series of benzothiazole amides that demonstrate sub-nanomolar inhibitory potency against ITK with good cellular activity and kinase selectivity. We also elucidate the binding mode of these inhibitors by solving the X-ray crystal structures of several inhibitor-ITK complexes.
Asunto(s)
Benzotiazoles/química , Benzotiazoles/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Animales , Benzotiazoles/síntesis química , Cristalografía por Rayos X , Diseño de Fármacos , Humanos , Ratones , Modelos Moleculares , Proteínas Tirosina Quinasas/química , Transducción de Señal , Relación Estructura-ActividadRESUMEN
Keap1 binds to the Nrf2 transcription factor to promote its degradation, resulting in the loss of gene products that protect against oxidative stress. While cell-active small molecules have been identified that modify cysteines in Keap1 and effect the Nrf2 dependent pathway, few act through a non-covalent mechanism. We have identified and characterized several small molecule compounds that specifically bind to the Keap1 Kelch-DC domain as measured by NMR, native mass spectrometry and X-ray crystallography. One compound upregulates Nrf2 response genes measured by a luciferase cell reporter assay. The non-covalent inhibition strategy presents a reasonable course of action to avoid toxic side-effects due to non-specific cysteine modification.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Factor 2 Relacionado con NF-E2/antagonistas & inhibidores , Factor 2 Relacionado con NF-E2/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Proteínas Portadoras , Cristalografía por Rayos X , Péptidos y Proteínas de Señalización Intracelular/química , Proteína 1 Asociada A ECH Tipo Kelch , Factor 2 Relacionado con NF-E2/química , Unión Proteica/efectos de los fármacos , Estructura Terciaria de Proteína , Espectrometría de Masa por Ionización de Electrospray , Relación Estructura-Actividad , TermodinámicaRESUMEN
A novel series of 2-aminobenzimidazole inhibitors of BACE1 has been discovered using fragment-based drug discovery (FBDD) techniques. The rapid optimization of these inhibitors using structure-guided medicinal chemistry is discussed.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Inhibidores de Proteasas/farmacología , Humanos , Modelos Moleculares , Inhibidores de Proteasas/química , Relación Estructura-ActividadRESUMEN
Inhibitors of MAPKAP kinase 2 (MK2) are expected to attenuate the p38alpha signal transduction pathway in macrophages in a similar way to p38alpha inhibitors and to have a lower propensity for toxic side effects that have slowed the clinical development of the latter. Therefore, novel MK2 inhibitors may find therapeutic application in acute and chronic, TNFalpha-mediated inflammatory conditions like rheumatoid arthritis and others. Herein we have applied fragment screening, using physiologically relevant bioassays and fragment binding mode mapping by protein-observed NMR spectroscopy to the discovery of novel efficient chemical starting points for MK2.
Asunto(s)
Bioensayo/métodos , Inhibidores Enzimáticos/farmacología , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Fragmentos de Péptidos , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Driven by growing corporate compound files, the demands of target biology, and attempts to cut cost, the number of solutions to HTS has spiralled. In quick succession new assay technologies and screening platforms are appearing on the market, with the promise of screening faster than ever in low volume high density formats whilst providing high quality data. Within this world of rapid change, Pfizer has applied cutting edge technology to HTS by introducing screening in 1 microl formats utilising single molecule detection technology. Instead of resource intensive in-house development, Pfizer entered into a collaboration with Evotec OAI / Evotec Technologies and introduced their Mark-II EVOscreen platform. In this article we will outline the benefits of the approach taken at Pfizer, Sandwich, and introduce the Mark-II EVOscreen platform, illustrating the potential but also possible pitfalls of HTS miniaturisation.
Asunto(s)
Evaluación Preclínica de Medicamentos/métodos , Tecnología Farmacéutica , Clonación Molecular , Interpretación Estadística de Datos , Evaluación Preclínica de Medicamentos/economía , Polarización de Fluorescencia , Colorantes Fluorescentes , Indicadores y Reactivos , Nanotecnología , Fosfotransferasas/análisis , Fosfotransferasas/metabolismoRESUMEN
This paper describes the development of a robust, miniaturizable, and quantitative fluorescence-based assay for mitogen-activated protein kinase kinase 7 (MKK7). As a first step, the basic steady-state kinetics of the MKK7-catalyzed phosphorylation of c-Jun N-terminal kinases (JNKs) 1, 2, and 3 were defined using standard radiometric methods. Subsequently, the authors found that in addition to the holo JNKs, a series of novel small peptides (based on the region around the JNK phosphorylation site) are also substrates, provided that these were prephosphorylated on the Y residue of the TPY motif. One of these peptide substrates was used in the development of a fluorescence polarization-based assay using an antibody as a sensor. The assay was successfully miniaturized for use with conventional fluorescence polarization (FP) reader technology in 8.5 microl and on the single microl scale using Evotec proprietary 2-dimensional fluorescence intensity distribution analysis (2D-FIDA) anisotropy and liquid handling technology. The steady-state kinetic parameters derived using the FP or 2D-FIDA anisotropy format assays correlated well with those generated using a radiometric assay. Moreover, the quantitative sensitivity to known inhibitors was maintained independent of the format and assay volume. In addition, the authors found that the 2D-FIDA anisotropy assay exhibited superior performance statistics (typical Z' = approximately 0.5) relative to conventional FP (typical Z' = 0.3) and yielded the additional benefit of order-of-magnitude savings in terms of reagent costs. The 2D-FIDA anisotropy assay was used to carry out a successful high-throughput screening in 1-microl final volume against company file compounds.