RESUMEN
Functional genomic approaches have facilitated the discovery of rare genetic disorders and improved efforts to decipher their underlying etiology. PPP2R5D-related disorder is an early childhood onset condition characterized by intellectual disability, hypotonia, autism-spectrum disorder, macrocephaly, and dysmorphic features. The disorder is caused by de novo single nucleotide changes in PPP2R5D, which generate heterozygous dominant missense variants. PPP2R5D is known to encode a B'-type (B'56δ) regulatory subunit of a PP2A-serine/threonine phosphatase. To help elucidate the molecular mechanisms altered in PPP2R5D-related disorder, we used a CRISPR-single-base editor to generate HEK-293 cells in which a single transition (c.1258G>A) was introduced into one allele, precisely recapitulating a clinically relevant E420K variant. Unbiased quantitative proteomic and phosphoproteomic analyses of endogenously expressed proteins revealed heterozygous-dominant changes in kinase/phosphatase signaling. These data combined with orthogonal validation studies revealed a previously unrecognized interaction of PPP2R5D with AKT in human cells, leading to constitutively active AKT-mTOR signaling, increased cell size, and uncoordinated cellular growth in E420K-variant cells. Rapamycin reduced cell size and dose-dependently reduced RPS6 phosphorylation in E420K-variant cells, suggesting that inhibition of mTOR1 can suppress both the observed RPS6 hyperphosphorylation and increased cell size. Together, our findings provide a deeper understanding of PPP2R5D and insight into how the E420K-variant alters signaling networks influenced by PPP2R5D. Our comprehensive approach, which combines precise genome editing, isobaric tandem mass tag labeling of peptides generated from endogenously expressed proteins, and concurrent liquid chromatography-mass spectrometry (LC-MS3), also provides a roadmap that can be used to rapidly explore the etiologies of additional genetic disorders.
Asunto(s)
Enfermedades Genéticas Congénitas/genética , Predisposición Genética a la Enfermedad , Proteína Fosfatasa 2/genética , Proteómica , Serina-Treonina Quinasas TOR/genética , Trastorno Autístico/genética , Trastorno Autístico/patología , Sistemas CRISPR-Cas/genética , Enfermedades Genéticas Congénitas/patología , Humanos , Discapacidad Intelectual/genética , Discapacidad Intelectual/patología , Megalencefalia/genética , Megalencefalia/patología , Mutación/genética , Polimorfismo de Nucleótido Simple/genética , Proteínas Proto-Oncogénicas c-akt/genéticaRESUMEN
Starting from a weak screening hit, potent and selective inhibitors of the MALT1 protease function were elaborated. Advanced compounds displayed high potency in biochemical and cellular assays. Compounds showed activity in a mechanistic Jurkat T cell activation assay as well as in the B-cell lymphoma line OCI-Ly3, which suggests potential use of MALT1 inhibitors in the treatment of autoimmune diseases as well as B-cell lymphomas with a dysregulated NF-κB pathway. Initially, rat pharmacokinetic properties of this compound series were dominated by very high clearance which could be linked to amide cleavage. Using a rat hepatocyte assay a good in vitro-in vivo correlation could be established which led to the identification of compounds with improved PK properties.
Asunto(s)
Antineoplásicos/farmacología , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas/antagonistas & inhibidores , Piperidinas/farmacología , Animales , Antineoplásicos/síntesis química , Antineoplásicos/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Hepatocitos/efectos de los fármacos , Humanos , Células Jurkat , Microsomas/efectos de los fármacos , Estructura Molecular , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas/metabolismo , Piperidinas/síntesis química , Piperidinas/química , Proteolisis/efectos de los fármacos , Ratas , Relación Estructura-ActividadRESUMEN
MAP-activated protein kinase 2 (MK2) plays an important role in the regulation of innate immune response as well as in cell survival upon DNA damage. Despite its potential for the treatment of inflammation and cancer, to date no MK2 low molecular weight inhibitors have reached the clinic, mainly due to inadequate absorption, distribution, metabolism, and excretion (ADME) properties. We describe here an approach based on specifically placed fluorine within a recently described pyrrole-based MK2 inhibitor scaffold for manipulation of its physicochemical and ADME properties. While preserving target potency, the novel fluoro-derivatives showed greatly improved permeability as well as enhanced solubility and reduced in vivo clearance leading to significantly increased oral exposure.
RESUMEN
Pyrrolo[2,3-f]isoquinoline based amino acids, tetracyclic lactams and cyclic ketone analogues are described as novel MK2 inhibitors with IC(50) as low as 5nM and good selectivity profiles against a number of related kinases including ERK, p38alpha and JNKs. TNFalpha release was suppressed from human peripheral blood mononuclear cells (hPBMCs), and a representative compound inhibited LPS induced TNFalpha release in mice illustrating the potential of this series to provide orally active MK2 inhibitors.
Asunto(s)
Compuestos Heterocíclicos de 4 o más Anillos/química , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/química , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Administración Oral , Aminoácidos/síntesis química , Aminoácidos/química , Aminoácidos/farmacología , Animales , Sitios de Unión , Cristalografía por Rayos X , Compuestos Heterocíclicos de 4 o más Anillos/síntesis química , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Isoquinolinas/química , Cetonas/síntesis química , Cetonas/química , Cetonas/farmacología , Lactamas/síntesis química , Lactamas/química , Lactamas/farmacología , Ratones , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/metabolismo , Pirroles/química , Relación Estructura-Actividad , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
New, selective 3-aminopyrazole based MK2-inhibitors were discovered by scaffold hopping strategy. The new derivatives proved to inhibit intracellular phosphorylation of hsp27 as well as LPS-induced TNFalpha release in cells. In addition, selected derivative 14e also inhibited LPS-induced TNFalpha release in vivo.
Asunto(s)
Descubrimiento de Drogas , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Pirazoles/química , Línea Celular Tumoral , Células Cultivadas , Cristalografía por Rayos X , Descubrimiento de Drogas/métodos , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Conformación Proteica , Proteínas Serina-Treonina Quinasas/metabolismo , Pirazoles/metabolismo , Pirazoles/farmacologíaRESUMEN
Pyrrolo-pyrimidones of the general structure 1 were synthesized and evaluated for their potential as MK2 inhibitors. Potent derivatives were discovered which inhibit MK2 in the nanomolar range and show potent inhibition of cytokine release from LPS-stimulated monocytes. These derivatives were shown to inhibit phosphorylation of hsp27, a downstream target of MK2 and are modestly selective in a panel of 28 kinases.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Pirimidinonas/síntesis química , Pirimidinonas/farmacología , Pirroles/síntesis química , Pirroles/farmacología , Artritis Reumatoide/tratamiento farmacológico , Técnicas Químicas Combinatorias , Citocinas/metabolismo , Relación Dosis-Respuesta a Droga , Diseño de Fármacos , Proteínas de Choque Térmico HSP27/antagonistas & inhibidores , Proteínas de Choque Térmico HSP27/metabolismo , Humanos , Leucocitos Mononucleares/efectos de los fármacos , Lipopolisacáridos/farmacología , Estructura Molecular , Monocitos/efectos de los fármacos , Fosforilación , Pirimidinonas/química , Pirroles/química , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidoresRESUMEN
Cinnamides as novel CCR1 antagonist chemotypes are described with high affinity to human and rodent receptors. A1B1 and A4B7 showed oral activity in the mouse collagen induced arthritis.
Asunto(s)
Antiinflamatorios no Esteroideos/química , Antiinflamatorios no Esteroideos/uso terapéutico , Artritis Experimental/tratamiento farmacológico , Receptores de Quimiocina/antagonistas & inhibidores , Administración Oral , Animales , Antiinflamatorios no Esteroideos/administración & dosificación , Humanos , Ratones , Ratas , Receptores CCR1 , Relación Estructura-ActividadRESUMEN
A class of potent, selective adenosine A(3) receptor antagonists was obtained via optimisation of the screening hit N-[4-(4-methoxyphenyl)-thiazol-2-yl]-acetamide. Structural modifications of this hit revealed very quickly that a 5-(pyridin-4-yl) substituent on the 2-aminothiazole ring was optimal for high potency at the adenosine A(3) receptor. Structure activity relationship studies led to both potent and selective A(3) receptor antagonists, including N-[5-pyridin-4-yl-4-(3,4,5-trimethoxyphenyl)-thiazol-2-yl]-acetamide, a highly potent aden-osine A(3) receptor antagonist with greater than 100- fold selectivity against the related adenosine receptors. As well as demonstrating selective in vitro binding on the human A(3) adenosine receptor, this compound was also shown to selectively block the rat A(3) receptor in vivo. This important new compound can be readily synthesised in four steps from commercially available starting materials.
Asunto(s)
Acetamidas/química , Antagonistas del Receptor de Adenosina A3 , Tiazoles/química , Acetamidas/síntesis química , Acetamidas/farmacología , Animales , Diseño de Fármacos , Humanos , Ratas , Relación Estructura-Actividad , Tiazoles/síntesis química , Tiazoles/farmacologíaRESUMEN
A series of potent p38 inhibitors based on the dihydroquinazoline scaffold was synthesized using a novel Pd-catalyzed cyclization reaction of aryl-benzyl ureas. Optimization of this compound class led to compound 20, which inhibits p38alpha in vitro with IC(50)=14 nM and is active in the mouse TNFalpha-release model.