RESUMO
A method is presented for the detection of circulating tumor cells (CTC) using mass spectrometry (MS), through reporter-ion amplification. Particles functionalized with short-chain peptides are bound to cells through antibody-antigen interactions. Selective release and MS detection of peptides is shown to detect as few as 690 cells isolated from a 10 mL blood sample. Here we present proof-of-concept results that pave the way for further investigations.
Assuntos
Separação Celular/métodos , Células Neoplásicas Circulantes , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Anticorpos/química , Anticorpos/imunologia , Contagem de Células , Linhagem Celular Tumoral , Humanos , Técnicas Imunológicas/métodos , Limite de Detecção , Camundongos , Peso Molecular , Nanopartículas/química , Células Neoplásicas Circulantes/imunologia , Peptídeos/química , Dióxido de Silício/químicaRESUMO
Matrix metalloproteinase-13 (MMP-13) is a zinc-dependent protease responsible for the cleavage of type II collagen, the major structural protein of articular cartilage. Degradation of this cartilage matrix leads to the development of osteoarthritis. We previously have described highly potent and selective carboxylic acid containing MMP-13 inhibitors; however, nephrotoxicity in preclinical toxicology species precluded development. The accumulation of compound in the kidneys mediated by human organic anion transporter 3 (hOAT3) was hypothesized as a contributing factor for the finding. Herein we report our efforts to optimize the MMP-13 potency and pharmacokinetic properties of non-carboxylic acid leads resulting in the identification of compound 43a lacking the previously observed preclinical toxicology at comparable exposures.
Assuntos
Metaloproteinase 13 da Matriz/efeitos dos fármacos , Inibidores de Metaloproteinases de Matriz/síntese química , Inibidores de Metaloproteinases de Matriz/farmacologia , Osteoartrite/tratamento farmacológico , Pirimidinas/síntese química , Pirimidinas/farmacologia , Tetrazóis/síntese química , Tetrazóis/farmacologia , Animais , Cartilagem Articular/efeitos dos fármacos , Cartilagem Articular/patologia , Colagenases/efeitos dos fármacos , Cães , Desenho de Fármacos , Humanos , Rim/metabolismo , Macaca fascicularis , Masculino , Inibidores de Metaloproteinases de Matriz/toxicidade , Modelos Moleculares , Transportadores de Ânions Orgânicos Sódio-Independentes/metabolismo , Ligação Proteica , Ratos , Ratos Sprague-Dawley , Relação Estrutura-AtividadeRESUMO
Microsomal prostaglandin E(2) synthase-1 (mPGES-1) is a novel therapeutic target for the treatment of inflammation and pain. In the preceding letter, we detailed the discovery of clinical candidate PF-04693627, a potent mPGES-1 inhibitor possessing a novel benzoxazole structure. While PF-04693627 was undergoing further preclinical profiling, we sought to identify a back-up mPGES-1 inhibitor that differentiated itself from PF-04693627. The design, synthesis, mPGES-1 activity and in vivo PK of a novel set of substituted benzoxazoles are described herein. Also described is a conformation-based hypothesis for mPGES-1 activity based on the preferred conformation of the cyclohexane ring within this class of inhibitors.
Assuntos
Benzoxazóis/química , Benzoxazóis/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Oxirredutases Intramoleculares/antagonistas & inibidores , Benzoxazóis/síntese química , Desenho de Fármacos , Inibidores Enzimáticos/síntese química , Humanos , Oxirredutases Intramoleculares/química , Oxirredutases Intramoleculares/metabolismo , Modelos Moleculares , Conformação Molecular , Prostaglandina-E Sintases , Relação Estrutura-AtividadeRESUMO
Potent, highly selective and orally-bioavailable MMP-13 inhibitors have been identified based upon a (pyridin-4-yl)-2H-tetrazole scaffold. Co-crystal structure analysis revealed that the inhibitors bind at the S(1)(') active site pocket and are not ligands for the catalytic zinc atom. Compound 29b demonstrated reduction of cartilage degradation biomarker (TIINE) levels associated with cartilage protection in a preclinical rat osteoarthritis model.
Assuntos
Inibidores de Metaloproteinases de Matriz , Osteoartrite/tratamento farmacológico , Ácidos Picolínicos/química , Inibidores de Proteases/química , Tetrazóis/química , Administração Oral , Animais , Sítios de Ligação , Cartilagem/efeitos dos fármacos , Cartilagem/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Modelos Animais de Doenças , Descoberta de Drogas , Metaloproteinase 13 da Matriz/metabolismo , Ácidos Picolínicos/síntese química , Ácidos Picolínicos/farmacologia , Inibidores de Proteases/síntese química , Inibidores de Proteases/farmacologia , Ratos , Tetrazóis/síntese química , Tetrazóis/farmacologia , Zinco/químicaRESUMO
NF-kappa B-induced gene expression contributes significantly to the pathogenesis of inflammatory diseases such as arthritis. I kappa B kinase (IKK) is the converging point for the activation of NF-kappa B by a broad spectrum of inflammatory agonists and is thus a novel target for therapeutic intervention. We describe a small molecule, selective inhibitor of IKK-2, SC-514, which does not inhibit other IKK isoforms or other serine-threonine and tyrosine kinases. SC-514 inhibits the native IKK complex or recombinant human IKK-1/IKK-2 heterodimer and IKK-2 homodimer similarly. IKK-2 inhibition by SC-514 is selective, reversible, and competitive with ATP. SC-514 inhibits transcription of NF-kappa B-dependent genes in IL-1 beta-induced rheumatoid arthritis-derived synovial fibroblasts in a dose-dependent manner. When the mechanism of NF-kappa B activation was evaluated in the presence of this inhibitor, several interesting observations were found. First, SC-514 did not inhibit the phosphorylation and activation of the IKK complex. Second, there was a delay but not a complete blockade in I kappa B alpha phosphorylation and degradation; likewise there was a slightly slowed, decreased import of p65 into the nucleus and a faster export of p65 from the nucleus. Finally, both I kappa B alpha and p65 were comparable substrates for IKK-2, with similar Km and Kcat values, and SC-514 inhibited the phosphorylation of either substrate similarly. Thus, the effect of SC-514 on cytokine gene expression may be a combination of inhibiting I kappa B alpha phosphorylation/degradation, affecting NF-kappa B nuclear import/export as well as the phosphorylation and transactivation of p65.