RESUMO
We report a systematic analysis of the P1' and P2' substrate specificity of TNF-alpha converting enzyme (TACE) using a peptide library and a novel analytical method, and we use the substrate specificity information to design novel reverse hydroxamate inhibitors. Initial truncation studies, using the amino acid sequence around the cleavage site in precursor-TNF-alpha, showed that good turnover was obtained with the peptide DNP-LAQAVRSS-NH2. Based on this result, 1000 different peptide substrates of the form Biotin-LAQA-P1'-P2'-SSK(DNP)-NH2 were prepared, with 50 different natural and unnatural amino acids at P1' in combination with 20 different amino acids at P2'. The peptides were pooled, treated with purified microsomal TACE, and the reaction mixtures were passed over a streptavidin affinity column to remove unreacted substrate and the N-terminal biotinylated product. C-terminal cleavage products not binding to streptavidin were subjected to liquid chromatography/mass spectrometry analysis where individual products were identified and semiquantitated. 25 of the substrates were resynthesized as discrete peptides and assayed with recombinant TACE. The experiments show that recombinant TACE prefers lipophilic amino acids at the P1' position, such as phenylglycine, homophenylalanine, leucine and valine. At the P2' position, TACE can accommodate basic amino acids, such as arginine and lysine, as well as certain non-basic amino acids such as citrulline, methionine sulfoxide and threonine. These substrate preferences were used in the design of novel reverse hydroxamate TACE inhibitors with phenethyl and 5-methyl-thiophene-methyl side-chains at P1', and threonine and nitro-arginine at P2'.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Metaloendopeptidases/antagonistas & inibidores , Metaloendopeptidases/metabolismo , Inibidores de Proteases/química , Inibidores de Proteases/farmacologia , Mapeamento de Interação de Proteínas/métodos , Proteínas ADAM , Proteína ADAM17 , Sítios de Ligação , Biotina/química , Cromatografia Líquida/métodos , Desenho de Fármacos , Processamento de Imagem Assistida por Computador , Espectrometria de Massas/métodos , Metaloendopeptidases/genética , Modelos Moleculares , Biblioteca de Peptídeos , Peptídeos/química , Peptídeos/farmacologia , Conformação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Relação Estrutura-Atividade , Especificidade por Substrato , Fator de Necrose Tumoral alfa/metabolismoRESUMO
TNF alpha converting enzyme (TACE) processes precursor TNF alpha between Ala76 and Val77, yielding a correctly processed bioactive 17 kDa protein. Genetic evidence indicates that TACE may also be involved in the shedding of other ectodomains. Here we show that native and recombinant forms of TACE efficiently processed a synthetic substrate corresponding to the TNF alpha cleavage site only. For all other substrates, conversion occurred only at high enzyme concentrations and prolonged reaction times. Often, cleavage under those conditions was accompanied by nonspecific reactions. We also compared TNF alpha cleavage by TACE to cleavage by those members of the matrix metalloproteinase (MMP) family previously implied in TNF alpha release. The specificity constants for TNF alpha cleavage by the MMPs were approximately 100-1000-fold slower relative to TACE. MMP 7 also processed precursor TNF alpha at the correct cleavage site but did so with a 30-fold lower specificity constant relative to TACE. In contrast, MMP 1 processed precursor TNF alpha between Ala74 and Gln75, in addition to between Ala76 and Val77, while MMP 9 cleaved this natural substrate solely between Ala74 and Gln75. Additionally, the MMP substrate Dnp-PChaGC(Me)HK(NMA)-NH(2) was not cleaved at all by TACE, while collagenase (MMP 1), gelatinase (MMP 9), stromelysin 1 (MMP 3), and matrilysin (MMP 7) all processed this substrate efficiently. All of these results indicate that TACE is unique in terms of its specificity requirements for substrate cleavage.
