RESUMO
Solid-phase synthesis of phosphinic peptides was introduced 10 years ago. A major application of this chemistry has been the development of potent synthetic inhibitors of zinc metalloproteases. Specific properties of the inhibitors produced in recent years are reviewed, supporting the notion that phosphinic pseudo-peptides are useful tools for studying the structural and functional biology of zinc proteases.
Assuntos
Metaloendopeptidases/antagonistas & inibidores , Peptídeos/metabolismo , Ácidos Fosfínicos/metabolismo , Zinco/metabolismo , Animais , Cromatografia Líquida de Alta Pressão , Humanos , Ácidos Hidroxâmicos/química , Ácidos Hidroxâmicos/metabolismo , Metaloendopeptidases/metabolismo , Peptídeos/química , Ácidos Fosfínicos/químicaRESUMO
Stromelysin-3 (ST3) is a matrix metalloproteinase (MMP-11) whose proteolytic activity plays an important role in tumorigenicity enhancement. In breast cancer, ST3 is a bad prognosis marker: its expression is associated with a poor clinical outcome. This enzyme therefore represents an attractive therapeutic target. The topology of matrix metalloproteinases (MMPs) is remarkably well conserved, making the design of highly specific inhibitors difficult. The major difference between MMPs lies in the S(1)' subsite, a well-defined hydrophobic pocket of variable depth. The present crystal structure, the first 3D-structure of the ST3 catalytic domain in interaction with a phosphinic inhibitor mimicking a (d, l) peptide, clearly demonstrates that its S(1)' pocket corresponds to a tunnel running through the enzyme. This open channel is filled by the inhibitor P(1)' group which adopts a constrained conformation to fit this pocket, together with two water molecules interacting with the ST3-specific residue Gln215. These observations provide clues for the design of more specific inhibitors and show how ST3 can accommodate a phosphinic inhibitor mimicking a (d, l) peptide. The presence of a water molecule interacting with one oxygen atom of the inhibitor phosphinyl group and the proline residue of the Met-turn suggests how the intermediate formed during proteolysis may be stabilized. Furthermore, the hydrogen bond distance observed between the methyl of the phosphinic group and the carbonyl group of Ala182 mimics the interaction between this carbonyl group and the amide group of the cleaved peptidic bond. Our crystal structure provides a good model to study the MMPs mechanism of proteolysis.
Assuntos
Metaloendopeptidases/química , Oligopeptídeos/química , Sequência de Aminoácidos , Sítios de Ligação , Cálcio/química , Cátions Bivalentes/química , Cátions Bivalentes/metabolismo , Simulação por Computador , Cristalografia por Raios X , Desenho de Fármacos , Ligação de Hidrogênio , Ligantes , Metaloproteinase 11 da Matriz , Metaloendopeptidases/antagonistas & inibidores , Metaloendopeptidases/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Oligopeptídeos/farmacologia , Inibidores de Proteases/farmacologia , Alinhamento de Sequência , Zinco/químicaRESUMO
The development of the combinatorial chemistry of phosphinic peptides has led to the discovery of both highly potent and selective inhibitors of various zinc metalloproteinases. Several properties of these compounds are reviewed, supporting the view that this class of inhibitors should represent useful tools for probing several aspects of the function of this broad family of proteases in vivo.
Assuntos
Técnicas de Química Combinatória , Metaloendopeptidases/metabolismo , Zinco/metabolismo , Aminopeptidases/química , Aminopeptidases/metabolismo , Modelos Químicos , Modelos Moleculares , Biblioteca de Peptídeos , Peptídeos/química , Peptídeos/metabolismoRESUMO
The human somatic angiotensin converting enzyme (ACE) contains two homologous domains, each bearing a zinc-dependent active site. All of the synthetic inhibitors of this enzyme used in clinical applications interact with these two active sites to a similar extent. Recently, several lines of evidence have suggested that the N-terminal active site of ACE might be involved in specific hydrolysis of some important physiological substrates, like Acetyl-Seryl-Aspartyl-Lysyl-Proline, a negative regulator of hematopoietic stem cell differentiation and proliferation. These findings have stimulated studies aimed at identifying new ACE inhibitors able to block only one of the two active sites of this enzyme. By screening phosphinic peptide libraries, we discovered a phosphinic peptide Ac-Asp-(L)Phepsi(PO2-CH2)(L)Ala-Ala-NH2, called RXP 407, which is able to differentiate the two ACE active sites, with a dissociation constant three orders of magnitude lower for the N-domain of the enzyme. The usefulness of a combinatorial chemistry approach to develop new lead structures is underscored by the unusual chemical structure of RXP 407, as compared with classical ACE inhibitors. As a highly potent and selective inhibitor of the N-terminal active site of wild ACE (Ki = 12 nM), RXP 407, which is metabolically stable in vivo, may lead to a new generation of ACE inhibitors able to block in vivo only a subset of the different functions regulated by ACE.
Assuntos
Inibidores da Enzima Conversora de Angiotensina/farmacologia , Oligopeptídeos/farmacologia , Peptidil Dipeptidase A/química , Peptidil Dipeptidase A/metabolismo , Ácidos Fosfínicos/farmacologia , Sequência de Aminoácidos , Substituição de Aminoácidos , Inibidores da Enzima Conversora de Angiotensina/síntese química , Inibidores da Enzima Conversora de Angiotensina/química , Animais , Sítios de Ligação , Células CHO , Cricetinae , Humanos , Cinética , Taxa de Depuração Metabólica , Mutagênese Sítio-Dirigida , Oligopeptídeos/síntese química , Oligopeptídeos/farmacocinética , Biblioteca de Peptídeos , Ácidos Fosfínicos/síntese química , Ácidos Fosfínicos/farmacocinética , Ratos , Relação Estrutura-Atividade , TransfecçãoRESUMO
The influence of the substrate P1' position on the specificity of two zinc matrix metalloproteases, membrane type-1 matrix metalloprotease (MT1-MMP) and stromelysin-3 (ST3), was evaluated by synthesizing a series of fluorogenic substrates of general formula dansyl-Pro-Leu-Ala-Xaa-Trp-Ala-Arg-NH2, where Xaa in the P1' position represents unusual amino acids containing either long arylalkyl or alkyl side chains. Our data demonstrate that both MT1-MMP and ST3 cleave substrates containing in their P1' position unusual amino acids with extremely long side chains more efficiently than the corresponding substrates with natural phenylalanine or leucine amino acids. In this series of substrates, the replacement of leucine by S-para-methoxybenzyl cysteine increased the kcat/Km ratio by a factor of 37 for MT1-MMP and 9 for ST3. The substrate with a S-para-methoxybenzyl cysteine residue in the P1' position displayed a kcat/Km value of 1.59 10(6) M-1 s-1 and 1.67 10(4) M-1 s-1, when assayed with MT1-MMP and ST3, respectively. This substrate is thus one of the most rapidly hydrolyzed substrates so far reported for matrixins, and is the first synthetic peptide efficiently cleaved by ST3. These unexpected results for these two matrixins suggest that extracellular proteins may be cleaved by matrixins at sites containing amino acids with unusual long side chains, like those generated in vivo by some post-translational modifications.
Assuntos
Aminoácidos/metabolismo , Metaloendopeptidases/metabolismo , Oligopeptídeos/metabolismo , Aminoácidos/química , Sítios de Ligação , Compostos Cromogênicos/metabolismo , Compostos de Dansil/metabolismo , Metaloproteinase 11 da Matriz , Metaloproteinases da Matriz Associadas à Membrana , Metaloproteínas/metabolismo , Especificidade por Substrato , ZincoRESUMO
We have determined the three-dimensional structure of a non-selfcomplementary nonanucleotide duplex which contains an abasic (apyrimidinic) site in the centre, i.e. a deoxyribose residue opposite an adenosine. The majority of the base and sugar proton resonances were assigned by NOESY, COSY and 2DQF spectra in D2O and H2O. We have measured the initial slope of buildup of NOEs in NOESY spectra at very short mixing times (25 to 50 ms), and from these were able to establish interproton distances for the central part of the duplex. We propose a different strategy for proton-proton distance determinations which takes into account the observed variations in correlation times for particular proton-proton vectors. A set of 31 measured interproton distances was incorporated into the refinement of the oligonucleotide structure by molecular mechanics calculations. Two structures were obtained which retain all aspects of a classical B DNA in which the unpaired adenine and the abasic deoxyribose lie inside the helix. We observe that the non-hydrogen bonded adenine is held well in the helix, the Tm of this base being the same as that of the A.T base pairs in the same duplex.