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Extracellular cysteine proteases, in particular cathepsin B, have been implicated in a variety of pathological processes. Selectively targeting labels of this enzyme are important tools to gain more detailed understanding of its specific roles. Starting from our recently developed irreversible epoxysuccinyl‐based inhibitor (R‐Gly‐Gly‐Leu‐(2S,3S)‐tEps‐Leu‐Pro‐OH, R=OMe), we have synthesized two affinity labels, R=NH‐(CH2)6‐NH‐rhodamine B and R=NH‐(CH2)6‐NH‐biotin. Using MCF‐7 cells, the labeled inhibitors were shown to be virtually non‐cell‐permeant. Moreover, affinity blot analysis with the biotinylated inhibitor allowed a highly sensitive and selective non‐radioactive detection of active cathepsin B.

The three-dimensional structure of the two hepatotoxic microcystins LR and LY has been determined by two-dimensional nuclear magnetic resonance (2D NMR) spectroscopy and distance geometry calculations. For the microcystin LY a single family of highly convergent structures was obtained. This family is characterized by a relatively compact boat-like ring structure with the large side chain of the Adda residue protruding from the concave side, in close proximity to the Tyr side chain. Conversely, for the microcystin LR the calculations result in three conformational families characterized by an even more compact ring structure. The Adda and Arg side chains protrude from the ring distal from one another caused by the repulsion between the guanido function of Arg and the hydrophobic Adda. The lower toxicity of the LY microcystin could result from the restricted access of the Adda side chain, an essential residue for activity, which results from the close proximity of the aromatic Tyr residue. A significant enthalpic cost would be expected for disturbance of this hydrophobic collapse and correspondingly lower binding affinity to receptor molecules would be predicted. From the structures of the two related microcystins, and homology with other known toxins, we propose a working hypothesis of the Adda side chain interacting with a hydrophobic pore of the phosphatases while the rest of the microcystin acts as a scaffold to help stabilize the interdigitation of the Adda with additional intermolecular interactions.

A human minigastrin‐II analog was prepared by conventional methods in solution using N‐benzyloxycarbonyltyrosine O‐sulfate as starting material in the synthetic route. Upon removal of the acid‐labile protecting groups and purification by preparative reversed‐phase chromatography the sulfated gastrin peptide was obtained in satisfactory overall yields as homogeneous material. It was found to be about twice as active as the non‐sulfated form in stimulating gastric acid secretion and to exhibit a 10‐fold higher affinity to gastrin receptors of purified parietal cells.

A human minigastrin-II analog was prepared by conventional methods in solution using N-benzyloxycarbonyltyrosine O-sulfate as starting material in the synthetic route. Upon removal of the acid-labile protecting groups and purification by preparative reversed-phase chromatography the sulfated gastrin peptide was obtained in satisfactory overall yields as homogeneous material. It was found to be about twice as active as the non-sulfated form in stimulating gastric acid secretion and to exhibit a 10-fold higher affinity to gastrin receptors of purified parietal cells.

A heterotrimeric collagen peptide was designed and synthesized which contains the collagenase cleavage site ( P4- P′ 910) of type I collagen linked to a C-terminal cystine-knot, and N-terminally extended with (Gly-Pro-Hyp) 5triplets for stabilization of the triple-helical conformation. By employing a newly developed regioselective cysteine pairing strategy based exclusively on thiol disulfide exchange reactions, we succeeded in assembling in high yields and in a reproducible manner the triple-stranded cystine peptide. While the single chains showed no tendency to self-association into triple helices, the heterotrimer (α1α2α1′) was found to exhibit a typical collagen-like CD spectrum at room temperature and a melting temperature ( Tm) of 33°C. This triple-helical collagen-like peptide is cleaved by the full-length human neutrophil collagenase (MMP-8) at a single locus fully confirming the correct raster of the heterotrimer. Its digestion proceeds at rates markedly higher than that of a single α1′ chain. In contrast, opposite digestion rates were measured with the catalytic Phe 79-MMP-8 domain of HNC. Moreover, the full-length enzyme exhibits Kmvalues of 5 μM and 1 mM for the heterotrimer and the single α1′ chain, respectively, which compare well with those reported for collagen type I (∼ 1 μM), gelatine (∼ 10 μM) and for octapeptides of the cleavage sequence (≥1 mM). The high affinity of the MMP-8 for the triple-helical heterotrimer and the fast digestion of this collagenous peptide confirm the decisive role of the hemopexin domain in recognition and possibly, partial unfolding of collagen.

Mono(6‐succinylamido‐6‐deoxy)‐β‐cyclodextrin was synthesized by classical carbohydrate chemistry and used as a template mono‐functionalized with the linear, fully flexible 4C‐spacer carboxylate for covalent linkage of the calpain inhibitor leucyl‐leucyl‐norleucinal. Spectroscopic analyses of the conjugate do not support a self‐inclusion of part of the hydrophobic peptide tail, but confirm its intra‐ or intermolecular interaction with the template moiety that leads to full water solubility. The inhibitory potency of the β‐cyclodextrin/peptide aldehyde construct was compared with that of the parent Ac‐Leu‐Leu‐Nle‐H against cathepsin B and calpain. Despite the large size of the template the inhibition of cathepsin B was only slightly reduced in full agreement with the X‐ray structure of this enzyme which shows full accessibility of the S‐subsites. For this enzyme the 4C‐spacer is apparently sufficient to guarantee optimal interaction of the peptide tail with the binding cleft. Conversely, for μ‐calpain a significantly decreased inhibitory potency was obtained with the conjugate suggesting steric interference of the template in the binding process. These results show that the beneficial properties of the cyclodextrin template can be retained in conjugates with bioactive peptides if attention is paid to optimize in each case the size and nature of the spacer for optimal recognition of the grafted biomolecule.

The three‐dimensional structure of the two hepatotoxic microcystins LR and LY has been determined by two‐dimensional nuclear magnetic resonance (2D NMR) spectroscopy and distance geometry calculations. For the microcystin LY a single family of highly convergent structures was obtained. This family is characterized by a relatively compact boat‐like ring structure with the large side chain of the Adda residue protruding from the concave side, in close proximity to the Tyr side chain. Conversely, for the microcystin LR the calculations result in three conformational families characterized by an even more compact ring structure. The Adda and Arg side chains protrude from the ring distal from one another caused by the repulsion between the guanido function of Arg and the hydrophobic Adda. The lower toxicity of the LY microcystin could result from the restricted access of the Adda side chain, an essential residue for activity, which results from the close proximity of the aromatic Tyr residue. A significant enthalpic cost would be expected for disturbance of this hydrophobic collapse and correspondingly lower binding affinity to receptor molecules would be predicted. From the structures of the two related microcystins, and homology with other known toxins, we propose a working hypothesis of the Adda side chain interacting with a hydrophobic pore of the phosphatases while the rest of the microcystin acts as a scaffold to help stabilize the interdigitation of the Adda with additional intermolecular interactions.

A heterotrimeric collagen peptide was designed and synthesized which contains the collagenase cleavage site () of type I collagen linked to a C‐terminal cystine‐knot, and N‐terminally extended with (Gly‐Pro‐Hyp)5triplets for stabilization of the triple‐helical conformation. By employing a newly developed regioselective cysteine pairing strategy based exclusively on thiol disulfide exchange reactions, we succeeded in assembling in high yields and in a reproducible manner the triple‐stranded cystine peptide. While the single chains showed no tendency to self‐association into triple helices, the heterotrimer (α1α2α1′) was found to exhibit a typical collagen‐like CD spectrum at room temperature and a melting temperature (Tm) of 33°C. This triple‐helical collagen‐like peptide is cleaved by the full‐length human neutrophil collagenase (MMP‐8) at a single locus fully confirming the correct raster of the heterotrimer. Its digestion proceeds at rates markedly higher than that of a single α1′ chain. In contrast, opposite digestion rates were measured with the catalytic Phe79‐MMP‐8 domain of HNC. Moreover, the full‐length enzyme exhibits Kmvalues of 5 μM and 1 mM for the heterotrimer and the single α1′ chain, respectively, which compare well with those reported for collagen type I (∼ 1 μM), gelatine (∼ 10 μM) and for octapeptides of the cleavage sequence (≥1 mM). The high affinity of the MMP‐8 for the triple‐helical heterotrimer and the fast digestion of this collagenous peptide confirm the decisive role of the hemopexin domain in recognition and possibly, partial unfolding of collagen.

The cDNAs of several snake venom zinc endopeptidases code for a putative propeptide, which includes the conserved cysteine‐containing sequence PKMCGVT. It has been suggested that binding of the cysteine thiol function to the active‐site zinc, resulting in inactivation of the catalytic domain, occurs in a mode similar to the ‘cysteine switch’ mechanism proposed for matrix metalloproteinases. In order to confirm this hypothesis, inhibition kinetics have been performed on the metalloproteinase adamalysin II of the venom of the snake Crotalus adamanteususing several cysteine peptides. Among these the synthetic hexapeptide PKMCGV‐NH2, corresponding to the conserved sequence portion of the known propeptides, was found to be by far the strongest inhibitor of this proteinase with a Kiof 3.4 μM. The inhibitory potencies of an equivalent peptide with the l‐Cys replaced by a d‐Cys or by an l‐Ser as well as of reduced glutathione, cysteine and two unrelated cysteine peptides were by one to two orders of magnitudes lower. These findings strongly support a cysteine switch‐like mechanism even for activation of the snake venom metalloproteinases.

GRF (10 −8–10 −5M) is shown to inhibit competitively the binding of [ 125I]VIP to human and rat intestinal epithelial membranes. The affinity of GRF for VIP receptor is 700–800-times lower than that of VIP in both species. The order of affinity of different peptides is VIP > PHI > secretin > GRF in rat, and VIP > GRF > PHI > secretin in man. The important species specificity of VIP receptors in recognizing PHI and secretin does not occur in the case of GRF. GRF stimulates adenylate cyclase through its interaction with VIP receptors in rat and human membranes. However, while GRF behaves as a VIP agonist in human tissue, it is a partial agonist/antagonist of VIP in the rat.

GRF (10−8–10−5M) is shown to inhibit competitively the binding of [125I]VIP to human and rat intestinal epithelial membranes. The affinity of GRF for VIP receptor is 700–800‐times lower than that of VIP in both species. The order of affinity of different peptides is VIP > PHI > secretin > GRF in rat, and VIP > GRF > PHI > secretin in man. The important species specificity of VIP receptors in recognizing PHI and secretin does not occur in the case of GRF. GRF stimulates adenylate cyclase through its interaction with VIP receptors in rat and human membranes. However, while GRF behaves as a VIP agonist in human tissue, it is a partial agonist/antagonist of VIP in the rat.

Based on recent information about the anti‐substrate binding mode of the propeptide portion of procathepsin B and the well established substrate‐like binding of epoxysuccinyl‐dipeptide carboxylates to the S′ subsites of cathepsin B a new endo‐trans‐epoxysuccinyl peptide was synthesized that contains the dipeptide moiety Leu‐Pro‐OH for the P1′–P2′ substrate positions and the tripeptide moiety Leu‐Gly‐Gly‐OMe (sequence portion 46–48 of the propeptide) for the P2–P4 positions in anti‐substrate orientation. With an unequivocal (2S,3S) configuration this new trans‐epoxysuccinyl peptide derivative was found to inhibit cathepsin B with an apparent second‐order rate constant of 1 520 000 M−1s−1which represents so far the most potent inhibitor among E‐64‐derived compounds. Conversely, the (2R,3R) diastereomer exhibited a significantly lower inhibition potency. This observation fully agrees with our previous findings that inhibitor/enzyme interactions at the S subsites are favored by the (2S,3S) and reverse interactions at the S′ subsites by the (2R,3R) configuration of the trans‐epoxysuccinyl moiety.

Based on recent information about the anti-substrate binding mode of the propeptide portion of procathepsin B and the well established substrate-like binding of epoxysuccinyl-dipeptide carboxylates to the S′ subsites of cathepsin B a new endo- trans-epoxysuccinyl peptide was synthesized that contains the dipeptide moiety Leu-Pro-OH for the P1′–P2′ substrate positions and the tripeptide moiety Leu-Gly-Gly-OMe (sequence portion 46–48 of the propeptide) for the P2–P4 positions in anti-substrate orientation. With an unequivocal (2 S,3 S) configuration this new trans-epoxysuccinyl peptide derivative was found to inhibit cathepsin B with an apparent second-order rate constant of 1 520 000 M −1s −1which represents so far the most potent inhibitor among E-64-derived compounds. Conversely, the (2 R,3 R) diastereomer exhibited a significantly lower inhibition potency. This observation fully agrees with our previous findings that inhibitor/enzyme interactions at the S subsites are favored by the (2 S,3 S) and reverse interactions at the S′ subsites by the (2 R,3 R) configuration of the trans-epoxysuccinyl moiety.