RESUMO
A series of furano[3,2-d]pyrimidine Syk inhibitors were synthesized and optimized for their enzyme potency and selectivity versus other kinases. In addition, ADME properties were assessed and compounds were prepared with optimized profiles for in vivo experiments. Compound 23 was identified as having acceptable pharmacokinetic properties and demonstrated efficacy in a rat collagen induced arthritis model.
Assuntos
Artrite Experimental/tratamento farmacológico , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/uso terapêutico , Pirimidinas/química , Pirimidinas/uso terapêutico , Quinase Syk/antagonistas & inibidores , Animais , Artrite Experimental/enzimologia , Cães , Furanos/síntese química , Furanos/química , Furanos/farmacologia , Furanos/uso terapêutico , Humanos , Simulação de Acoplamento Molecular , Inibidores de Proteínas Quinases/farmacocinética , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacocinética , Pirimidinas/farmacologia , Ratos , Quinase Syk/metabolismoRESUMO
BACKGROUND: Structure-based drug design (SBDD) can accelerate inhibitor lead design and optimization, and efficient methods including protein purification, characterization, crystallization, and high-resolution diffraction are all needed for rapid, iterative structure determination. Janus kinases are important targets that are amenable to structure-based drug design. Here we present the first mouse Tyk2 crystal structures, which are complexed to 3-aminoindazole compounds. RESULTS: A comprehensive construct design effort included N- and C-terminal variations, kinase-inactive mutations, and multiple species orthologs. High-throughput cloning and expression methods were coupled with an abbreviated purification protocol to optimize protein solubility and stability. In total, 50 Tyk2 constructs were generated. Many displayed poor expression, inadequate solubility, or incomplete affinity tag processing. One kinase-inactive murine Tyk2 construct, complexed with an ATP-competitive 3-aminoindazole inhibitor, provided crystals that diffracted to 2.5-2.6 Å resolution. This structure revealed initial "hot-spot" regions for SBDD, and provided a robust platform for ligand soaking experiments. Compared to previously reported human Tyk2 inhibitor crystal structures (Chrencik et al. (2010) J Mol Biol 400:413), our structures revealed a key difference in the glycine-rich loop conformation that is induced by the inhibitor. Ligand binding also conferred resistance to proteolytic degradation by thermolysin. As crystals could not be obtained with the unliganded enzyme, this enhanced stability is likely important for successful crystallization and inhibitor soaking methods. CONCLUSIONS: Practical criteria for construct performance and prioritization, the optimization of purification protocols to enhance protein yields and stability, and use of high-throughput construct exploration enable structure determination methods early in the drug discovery process. Additionally, specific ligands stabilize Tyk2 protein and may thereby enable crystallization.
Assuntos
Desenho de Fármacos , Indazóis/química , Indazóis/farmacologia , TYK2 Quinase/antagonistas & inibidores , TYK2 Quinase/química , Sequência de Aminoácidos , Animais , Cristalização , Cristalografia por Raios X , Estabilidade Enzimática/efeitos dos fármacos , Humanos , Janus Quinases/antagonistas & inibidores , Janus Quinases/metabolismo , Camundongos , Dados de Sequência Molecular , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Estrutura Secundária de Proteína , Proteólise/efeitos dos fármacos , Relação Estrutura-Atividade , TYK2 Quinase/isolamento & purificaçãoRESUMO
The bioisosteric replacement of the indole core of CRTH2 antagonists using thienopyrroles was investigated, resulting in potent antagonists with good selectivity over DP1. Early ADME/PK assessment of this chemotype demonstrated bioavailability in mice.
Assuntos
Acetatos/farmacologia , Pirróis/química , Receptores Imunológicos/antagonistas & inibidores , Receptores de Prostaglandina/antagonistas & inibidores , Acetatos/química , Acetatos/farmacocinética , Animais , Disponibilidade Biológica , Camundongos , Microssomos Hepáticos/metabolismo , RatosRESUMO
We describe structure-based optimization of a series of novel 2,4-diaminopyrimidine MK2 inhibitors. Co-crystal structures (see accompanying Letter) demonstrated a unique inhibitor binding mode. Resulting inhibitors had IC(50) values as low as 19nM and moderate selectivity against a kinase panel. Compounds 15, 31a, and 31b inhibit TNFalpha production in peripheral human monocytes.
Assuntos
Anti-Inflamatórios/química , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Inibidores de Proteínas Quinases/química , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Pirimidinas/química , Administração Oral , Animais , Anti-Inflamatórios/síntese química , Anti-Inflamatórios/farmacocinética , Sítios de Ligação , Simulação por Computador , Cristalografia por Raios X , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacocinética , Proteínas Serina-Treonina Quinases/metabolismo , Pirimidinas/síntese química , Pirimidinas/farmacocinética , Ratos , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Microsomal prostaglandin E2 synthase-1 (mPGES-1) catalyzes the formation of prostaglandin E2 (PGE2) from the endoperoxide prostaglandin H2 (PGH2). Expression of this enzyme is induced during the inflammatory response, and mouse knockout experiments suggest it may be an attractive target for antiarthritic therapies. Assaying the activity of this enzyme in vitro is challenging because of the unstable nature of the PGH2 substrate. Here, the authors present an mPGES-1 activity assay suitable for characterization of enzyme preparations and for determining the potency of inhibitor compounds. This plate-based competition assay uses homogenous time-resolved fluorescence to measure PGE2 produced by the enzyme. The assay is insensitive to DMSO concentration up to 10% and does not require extensive washes after the initial enzyme reaction is concluded, making it a simple and convenient way to assess mPGES-1 inhibition.
Assuntos
Oxirredutases Intramoleculares/metabolismo , Microssomos/metabolismo , Espectrometria de Fluorescência/métodos , Animais , Artrite/tratamento farmacológico , Baculoviridae/metabolismo , Ligação Competitiva , Regulação Enzimológica da Expressão Gênica , Humanos , Inflamação , Concentração Inibidora 50 , Insetos , Microssomos/enzimologia , Peróxidos/metabolismo , Prostaglandina H2/metabolismo , Prostaglandina-E Sintases , Fatores de TempoRESUMO
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.
Assuntos
Proteína Tirosina Quinase p56(lck) Linfócito-Específica/antagonistas & inibidores , Piridinas/química , Sítios de Ligação , Desenho de Fármacos , Humanos , Concentração Inibidora 50 , Piridinas/farmacologia , Relação Estrutura-Atividade , Quinases da Família src/antagonistas & inibidoresRESUMO
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.
Assuntos
Rejeição de Enxerto/prevenção & controle , Imunossupressores/farmacologia , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/antagonistas & inibidores , Pirazóis/síntese química , Pirazóis/farmacologia , Pirimidinas/síntese química , Pirimidinas/farmacologia , Quinases da Família src/antagonistas & inibidores , Animais , Química Farmacêutica/métodos , Cristalografia por Raios X , Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Transplante de Coração/métodos , Humanos , Imunofenotipagem , Concentração Inibidora 50 , Cinética , Complexo Principal de Histocompatibilidade , Modelos Químicos , Modelos Moleculares , Preparações Farmacêuticas , Ratos , Fatores de Tempo , Transplante Homólogo/métodosRESUMO
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.