RESUMO
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.
Assuntos
Doenças Genéticas Inatas/genética , Predisposição Genética para Doença , Proteína Fosfatase 2/genética , Proteômica , Serina-Treonina Quinases TOR/genética , Transtorno Autístico/genética , Transtorno Autístico/patologia , Sistemas CRISPR-Cas/genética , Doenças Genéticas Inatas/patologia , Humanos , Deficiência Intelectual/genética , Deficiência Intelectual/patologia , Megalencefalia/genética , Megalencefalia/patologia , Mutação/genética , Polimorfismo de Nucleotídeo Único/genética , Proteínas Proto-Oncogênicas c-akt/genéticaRESUMO
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.
Assuntos
Antineoplásicos/farmacologia , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/antagonistas & inibidores , Piperidinas/farmacologia , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Hepatócitos/efeitos dos fármacos , Humanos , Células Jurkat , Microssomos/efeitos dos fármacos , Estrutura Molecular , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/metabolismo , Piperidinas/síntese química , Piperidinas/química , Proteólise/efeitos dos fármacos , Ratos , Relação Estrutura-AtividadeRESUMO
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.
RESUMO
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.
Assuntos
Compostos Heterocíclicos de 4 ou mais Anéis/química , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Inibidores de Proteínas Quinases/química , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Administração Oral , Aminoácidos/síntese química , Aminoácidos/química , Aminoácidos/farmacologia , Animais , Sítios de Ligação , Cristalografia por Raios X , Compostos Heterocíclicos de 4 ou mais Anéis/síntese química , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Isoquinolinas/química , Cetonas/síntese química , Cetonas/química , Cetonas/farmacologia , Lactamas/síntese química , Lactamas/química , Lactamas/farmacologia , Camundongos , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Pirróis/química , Relação Estrutura-Atividade , Fator de Necrose Tumoral alfa/metabolismoRESUMO
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.
Assuntos
Descoberta de Drogas , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Pirazóis/química , Linhagem Celular Tumoral , Células Cultivadas , Cristalografia por Raios X , Descoberta de Drogas/métodos , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Conformação Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Pirazóis/metabolismo , Pirazóis/farmacologiaRESUMO
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.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Pirimidinonas/síntese química , Pirimidinonas/farmacologia , Pirróis/síntese química , Pirróis/farmacologia , Artrite Reumatoide/tratamento farmacológico , Técnicas de Química Combinatória , Citocinas/metabolismo , Relação Dose-Resposta a Droga , Desenho de Fármacos , Proteínas de Choque Térmico HSP27/antagonistas & inibidores , Proteínas de Choque Térmico HSP27/metabolismo , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Estrutura Molecular , Monócitos/efeitos dos fármacos , Fosforilação , Pirimidinonas/química , Pirróis/química , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidoresRESUMO
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.
Assuntos
Anti-Inflamatórios não Esteroides/química , Anti-Inflamatórios não Esteroides/uso terapêutico , Artrite Experimental/tratamento farmacológico , Receptores de Quimiocinas/antagonistas & inibidores , Administração Oral , Animais , Anti-Inflamatórios não Esteroides/administração & dosagem , Humanos , Camundongos , Ratos , Receptores CCR1 , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
Acetamidas/química , Antagonistas do Receptor A3 de Adenosina , Tiazóis/química , Acetamidas/síntese química , Acetamidas/farmacologia , Animais , Desenho de Fármacos , Humanos , Ratos , Relação Estrutura-Atividade , Tiazóis/síntese química , Tiazóis/farmacologiaRESUMO
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.