Interaction of phosphorylated FcepsilonRIgamma immunoglobulin receptor tyrosine activation motif-based peptides with dual and single SH2 domains of p72syk. Assessment of binding parameters and real time binding kinetics.

To examine the characteristics of the interaction of the FcepsilonRIgamma ITAM with the SH2 domains of p72(syk), the binding of an 125I-labeled dual phosphorylated FcepsilonRIgamma ITAM-based peptide to the p72(syk) SH2 domains was monitored utilizing a novel scintillation proximity based assay. The Kd for this interaction, determined from the saturation binding isotherm, was 1.4 nM. This high affinity binding was reflected in the rapid rate of association for the peptide binding to the SH2 domains. Competition studies utilizing a soluble C-terminal SH2 domain knockout and N-terminal SH2 domain knockouts revealed that both domains contribute cooperatively to the high affinity binding. Unlabeled dual phosphorylated peptide competed with the 125I-labeled peptide for binding to the dual p72(syk) SH2 domains with an IC50 value of 4.8 nM. Monophosphorylated 24-mer FcepsilonRIgamma ITAM peptides, and phosphotyrosine also competed for binding, but with substantially higher IC50 values. This, and other data discussed, suggest that high affinity binding requires both tyrosine residues to be phosphorylated and that the preferred binding orientation of the ITAM is such that the N-terminal phosphotyrosine occupies the C-terminal SH2 domain and the C-terminal phosphotyrosine occupies the N-terminal SH2 domain.

Potent peptide inhibitors of stromelysin based on the prodomain region of matrix metalloproteinases.

Stromelysin is secreted as an inactive zymogen that is activated in the extracellular space by cleavage of the His81-Phe82 bond with the release of the 81-amino acid propeptide domain. This segment contains a 12-amino acid sequence (MRKPRC75GVPDVG) that is highly conserved in all matrix metalloproteinases. Previous studies have shown that the hexapeptide, Ac-RCGVPD-NH2, and the pentapeptide, Ac-RCGVP-NH2, based on this region retain significant inhibitory activity. This new structure-activity relationship study of both peptides has shown that only Cys75 and Val77 are essential for inhibitory activity. Peptides based on this series inhibited stromelysin and collagenase with equal potency. Additional peptides spanning this region were synthesized in order to focus on these two sites. Significantly, isocysteine was substituted for Cys75 without significant loss of inhibitory activity. Tyr-(2,6-dichlorobenzyl) was substituted for Val77. The introduction of these 2 new residues into Ac-CGVP-NH2 produced a very potent inhibitor, Ac-isoCGY-(2,6 dichlorobenzyl)-P-NH2 with an IC50 of 3 microM. The following factors, acting in combination, determine the inhibitory activity of peptides in this series: distance between the sulfur atom and the peptide backbone, coordination geometry of the thiol side chain with the active-site zinc, and conformational flexibility of the side-chain.

Inhibition of human stromelysin by peptides based on the N-terminal domain of tissue inhibitor of metalloproteinases-1.

The tissue inhibitors of metalloproteinases (TIMPs) represent a family of naturally occurring protein inhibitors of stromelysin and other members of the family of matrix metalloproteinases. A series of peptides based on the N-terminal sequence of natural TIMP-1 was synthesized and assessed for inhibitory activity against purified human stromelysin. Inhibitor peptides were identified in the loop (bounded by the disulfide bonds [C3-C99] and [C13-C124]), e.g., [C3(Acm)-C13], (IC50, 42 microM). It was established that inhibition was due to the free sulfhydryl group of either C13 or C124. However, peptides within [C70(Acm)-C98(Acm)] inhibited stromelysin independently of zinc co-ordination by cysteine. The binding epitope in TIMP-1 may be discontinuous and comprised of sequences from at least 2 loops.

Peptides based on the conserved predomain sequence of matrix metalloproteinases inhibit human stromelysin and collagenase.

Prostromelysin, a member of the family of matrix metalloproteinases, is secreted as a zymogen which is activated after cleavage of the His81-Phe82 bond. The 82 amino acid propeptide that is removed during activation contains 12 amino acids, MRKPRC75GVPDVG, that are highly conserved in all MMPs. We evaluated a series of peptides that span this region for their ability to inhibit stromelysin. The hexapeptide, Ac-RCGVPD, and the pentapeptide, Ac-RCGVP had IC50 values of approx. 10 microM. The tetrapeptide, Ac-RCGV, was somewhat less potent with an IC50 of 60 microM. Smaller peptides, e.g. Ac-RCG, were significantly less potent as inhibitors. Substitutions of Cys75 with Ser resulted in a complete loss of inhibitory activity. The peptides in this series also inhibited human fibroblast collagenase.