RESUMO
Structure-activity relationships of a lead hydroxamic acid inhibitor of recombinant human stromelysin were systematically defined by taking advantage of a concise synthesis that allowed diverse functionality to be explored at each position in a template. An ex vivo rat model and an in vivo rabbit model of stromelysin-induced cartilage degradation were used to further optimize these analogs for oral activity and duration of action. The culmination of these modifications resulted in CGS 27023A, a potent, orally active stromelysin inhibitor that blocks the erosion of cartilage matrix.
Assuntos
Cartilagem/metabolismo , Ácidos Hidroxâmicos , Inibidores de Metaloproteinases de Matriz , Inibidores de Proteases/farmacologia , Pirazinas , Administração Oral , Animais , Sítios de Ligação , Cartilagem/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Ensaio de Imunoadsorção Enzimática , Humanos , Técnicas In Vitro , Cinética , Modelos Químicos , Coelhos , Ratos , Proteínas Recombinantes/antagonistas & inibidores , Relação Estrutura-Atividade , Substância P/metabolismo , SulfonamidasRESUMO
Human proteoglycan was aggregated to an immobilized hyaluronan solid phase on a 96-well ELISA plate. This complex was then degraded by recombinant human stromelysin. The remaining proteoglycan fragments were detected using a monoclonal antibody probe directed against the chondroitin sulfate (CS) region of the core protein. Stromelysin degraded the aggregate in a time and dose dependent manner as reflected by the loss of the CS epitope. Assay sensitivity was 0.125 U/well with total loss of the CS epitope occurring at 4 U/well. o-phenanthroline (IC50 = 52 microM) and U24522 (IC50 = 9 microM) inhibited degradation, while phosphoramidon did not. Serine and cysteine protease inhibitors had no effect. A comparative analysis of this assay with a reference method, substance P assay, gave similar inhibitor profiles. The use of aggregated human proteoglycan (native conformation) as a substrate, may better reflect how stromelysin inhibitors behave in the presence of complex substrates such as cartilage matrix.
Assuntos
Metaloendopeptidases/metabolismo , Proteoglicanas/metabolismo , Animais , Anticorpos Monoclonais , Cartilagem Articular/metabolismo , Sulfatos de Condroitina , Ensaio de Imunoadsorção Enzimática , Fêmur , Glicopeptídeos/farmacologia , Humanos , Ácido Hialurônico/química , Ácido Hialurônico/imunologia , Metaloproteinase 3 da Matriz , Metaloendopeptidases/antagonistas & inibidores , Camundongos , Fenantrolinas/farmacologia , Proteoglicanas/química , Proteoglicanas/imunologia , Proteínas Recombinantes/metabolismo , Substância P/metabolismoRESUMO
The specific activity of cyclic adenosine 3':5'-monophosphate phosphodiesterase was measured in lymphocytes isolated from the blood of normal subjects, from patients with chronic lymphocytic leukemia, and from tonsil tissue. The mean specific activity of cyclic adenosine 3':5'-monophosphate phosphodiesterase in the lymphocytes from patients with untreated chronic lymphocytic leukemia was lower than that in lymphocytes from the blood of normal subjects or from tonsils. Cyclic adenosine 3':5'-monophosphate phosphodiesterase levels did not correlate with differences in B- and T-cell lymphocyte subpopulations or with peripheral blood lymphocyte counts.
