Carboxypeptidase Inhibitor Research Articles

NMR Structural Characterization and Computational Predictions of the Major Intermediate in Oxidative Folding of Leech Carboxypeptidase Inhibitor

The III-A intermediate constitutes the major rate-determining step in the oxidative folding of leech carboxypeptidase inhibitor (LCI). In this work, III-A has been directly purified from the folding reaction and structurally characterized by NMR spectroscopy. This species, containing three native disulfides, displays a highly native-like structure; however, it lacks some secondary structure elements, making it more flexible than native LCI. III-A represents a structurally determined example of a disulfide-insecure intermediate; direct oxidation of this species to the fully native protein seems to be restricted by the burial of its two free cysteine residues inside a native-like structure. We also show that theoretical approaches based on topological constraints predict with good accuracy the presence of this folding intermediate. Overall, the derived results suggest that, as it occurs with non-disulfide bonded proteins, native-like interactions between segments of secondary structure rather than the crosslinking of disulfide bonds direct the folding of LCI.

Functional Screening of Serine Protease Inhibitors in the Medical Leech Hirudo medicinalis Monitored by Intensity Fading MALDI-TOF MS

The blood-feeding invertebrates are a rich biological source of drugs and lead compounds to treat cardiovascular diseases because they have evolved highly efficient mechanisms to feed on their hosts by blocking blood coagulation. In this work, we focused our attention on the leech Hirudo medicinalis. We performed, by "intensity fading" MALDI-TOF mass spectrometry, a comprehensive detection and functional analysis of pre-existent peptides and small proteins with the capability of binding to trypsin-like proteases related to blood coagulation. Combining "intensity fading MS" and off-line LC prefractionation allowed us to detect more than 75 molecules present in the leech extract that interact specifically with a trypsin-like protease over a sample profile of nearly 2,000 different peptides/proteins in the 2-20-kDa range. Moreover we resolved 232 individual components from the complex mixture, 13 of which have high sequence homology with previously described serine protease inhibitors. Our findings indicate that such extracts are much more complex than expected. Additionally, intensity fading MS, when complemented with LC separation strategies, seems to be a useful tool to investigate complex biological samples, establishing a new bridge between profiling, functional peptidomics, and subsequent drug discovery.

2-MPPA, a selective inhibitor of PSMA, delays prostate cancer growth and attenuates taxol-induced neuropathy in mice

8054 Background: Docetaxel is a standard of care treatment for patients with progression castration resistant prostate cancer, from which peripheral neuropathy may be dose limiting. A selective inhibitor of PSMA, termed 2-(3-mercaptopropyl) pentanedioic acid (2-MPPA), both prevents and reverses taxol-induced neuropathy in murine models, as indicated by deficits in sensory nerve conduction velocity (SNCV). 2-MPPA, a potent (IC50 of 90 nM) and selective inhibitor of glutamate carboxypeptidase (GCPII), also known as PSMA or NAALADase was also able to delay tumor growth in a prostate cancer model expressing high levels of PSMA. Of importance, there was no change in efficacy when taxol was administered with 2-MPPA in this model. Methods: Neuropathy: Female DBA2 mice received 5 mg/kg taxol (5 i.p. doses in two weeks) or 25 mg/kg taxol (4 i.p. doses in two weeks) with daily 30 mg/kg 2-MPPA or vehicle. Neuropathy was indicated by a SNCV deficit compared to baseline (pre-chemotherapy) values. Cancer: nu/nu mice with or with out testosterone supplements were inoculated with CWR22 or CWR22RS tumor. When tumors reached 50mm3, mice were dosed with 2-MPPA (10 to 750 mg/kg) bid PO for two weeks and tumor volume was monitored. Results: 2-MPPA significantly prevented the deficit in SNCV induced by both 5 and 25 mg/kg taxol by 96.3 ± 4.4% and 98.3± 11.6% respectively (p<0.001). Moreover, 7-day p.o. treatment with 30 mg/kg 2-MPPA given after a persistent SNCV deficit was established reversed the decline in SNCV by 98.3±8.0% (p<0.05). Peripheral nerve histopathology evaluations are ongoing. In a separate study, 10 mg/kg 2-MPPA inhibited tumor growth in the high expressor PSMA model CWR22RS by 70% (p<0.03). Lastly, 2-MPPA was dosed concomitantly with a therapeutic dose of taxol in the hormone-dependent model of prostate cancer, CWR22, and no effect on taxol efficacy was observed. Conclusions: Inhibition of GCPII/PSMA may have a therapeutic potential both as an anti-cancer agent, as well as to reduce chemotherapy-induced neuropathy No significant financial relationships to disclose.

A new functional assay of thrombin activatable fibrinolysis inhibitor.

New thrombin activatable fibrinolysis inhibitor (TAFI) assays are necessary for studying the role of this fibrinolysis inhibitor in cardiovascular disease. The identification of a functional single nucleotide polymorphism (SNP) (1040C/T) leading to a TAFI-variant with increased stability but lower antigen levels has made the determination of functional activity even more essential. Therefore, we developed a new assay for the functional activity of TAFI in citrated plasma samples. This assay is based on the retardation of plasma clot lysis by TAFIa. TAFI activation was induced simultaneously with fibrin formation and lysis was mediated by rt-PA. The variability of other plasma components was minimized by a 20-fold dilution of the samples in TAFI-depleted plasma. Lysis times (-/+ potato carboxypeptidase inhibitor) and the TAFI-related retardation of clot lysis, the functional parameter of the assay, were determined in a group of 92 healthy volunteers, as well as TAFI antigen levels (electroimmunoassay) and two TAFI SNPs (-438A/G and 1040C/T). TAFI-related retardation was 19.8 +/- 5.6 min (mean +/- SD) and was correlated with the antigen level. The specific antifibrinolytic activity of TAFI was associated with the -438A/G and 1040C/T genotypes. Individuals with the 325Ile-variant had on average a 34% higher TAFI-specific antifibrinolytic activity than individuals with the 325Thr-isoform. The TAFI-related retardation in the two groups of individuals did not differ, as a lower level compensated for the higher specific antifibrinolytic activity of the 325Ile-isoform. This assay provides valuable information about the performance of different TAFI isoforms and constitutes a new method for studying the role of TAFI in cardiovascular disease.

The Three-Dimensional Structures of Tick Carboxypeptidase Inhibitor in Complex with A/B Carboxypeptidases Reveal a Novel Double-headed Binding Mode

The tick carboxypeptidase inhibitor (TCI) is a proteinaceous inhibitor of metallo-carboxypeptidases present in the blood-sucking tick Rhipicephalus bursa. The three-dimensional crystal structures of recombinant TCI bound to bovine carboxypeptidase A and to human carboxypeptidase B have been determined and refined at 1.7 A and at 2.0 A resolution, respectively. TCI consists of two domains that are structurally similar despite the low degree of sequence homology. The domains, each consisting of a short alpha-helix followed by a small twisted antiparallel beta-sheet, show a high level of structural homology to proteins of the beta-defensin-fold family. TCI anchors to the surface of mammalian carboxypeptidases in a double-headed manner not previously seen for carboxypeptidase inhibitors: the last three carboxy-terminal amino acid residues interact with the active site of the enzyme in a way that mimics substrate binding, and the N-terminal domain binds to an exosite distinct from the active-site groove. The structures of these complexes should prove valuable in the applications of TCI as a thrombolytic drug and as a basis for the design of novel bivalent carboxypeptidase inhibitors.

Mechanism of action of potato carboxypeptidase inhibitor (PCI) as an EGF blocker

The epidermal growth factor receptor (EGFR) signal transduction pathway plays a prominent role in the development of carcinomas, and is an interesting target for antitumoral therapy. We have previously described how potato carboxypeptidase inhibitor (PCI), a 39-amino acid protease inhibitor with a T-Knot motif, binds to EGFR receptor and inhibits the activation of receptor protein tyrosine kinase. In this paper it is shown that PCI interferes with EGFR activation through inhibition of receptor dimerization and receptor transphosphorylation induced by epidermal growth factor (EGF) and by transforming growth factor alpha (TGF-α). Moreover, PCI blocks the formation and activation of ErbB1/ErbB-2 heterodimers that have a prominent role in carcinoma development. As a result of these effects, PCI interferes in the EGFR signal transduction pathway by reversing the effects of EGF on the growth of two tumoral cell lines, A431 and MDA-MB-453, and promotes EGFR down-regulation. These results show that PCI acts as an EGF/TGF-α antagonist, which suggests its therapeutic potential in the treatment of carcinomas.

Cyclen‐Containing Inhibitors of Carboxypeptidase A Synthesized in Search of Target‐Selective Artificial Proteases.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Determination of potato carboxypeptidase inhibitor in African Green Monkey plasma using 96-well SPE and LC–MS/MS

Potato carboxypeptidase inhibitor (CPI), a peptide with multiple isoforms (MW > 4000 Da) was determined from African Green Monkey plasma using a PE Sciex API-3000 LC–MS/MS in the positive ionization mode with the turbo ionspray interface (450 °C). Samples were prepared using an Oasis MCX 96-well solid phase extraction plate and chromatographed on an Allure C 18 HPLC Column (50 mm × 1.0 mm, 5 μm) using gradient elution. Upon analysis of the extracts using LC–MS/MS, the concentration of CPI was calculated using a single MS/MS transition ( m/z 830.5 → 221.0) that was reflective of the mass concentration (μg/mL) of main the CPI isoforms present in plasma from monkeys after they were given an intravenous dose of CPI. The assay was linear for CPI over concentrations of 0.05–10 μg/mL when extracting 200-μL aliquots of African Green Monkey plasma. The assay was applied to the determination of CPI in African Green Monkey plasma samples in two separate analytical runs (correlation of standard curves, r 1 = 0.9991 and r 2 = 0.9953). Quality control (QC) samples were run at 0.05, 0.1, 0.2, 0.5, 1.0, 2.0 and 5.0 μg/mL for each assay. Average ranges ( n = 12) for accuracy and precision for all concentrations of QCs during the two runs were 92.0–102.0% of expected potency and 10.4–21.8% (coefficient of variations), respectively.

Stabilization of the C-Terminal Part of Pig and Horse Colipase by Carboxypeptidase and Trypsin Inhibitors

Pig and horse colipases have been purified by a common procedure using trypsin and carboxypeptidase inhibitors as stabilizers. Two forms of pig colipase were identified: a predominant A1 form with about 103-105 residues, and a minor slightly degraded A2 form in which the last two C-terminal residues, Asp and Ser, were lacking. This type of degradation is considerably slowed down by carboxypeptidase inhibitors. A total of four forms of the horse cofactor were characterized: two (A1 and B1) were probably isocolipases which differed by only a few substitutions. Both contained the same number of residues (about 96), an N-terminal valine and an Arg-Ser-Glu-(Glx)1,2-ArgC-terminal sequence. A2 and B2 were slightly degraded forms probably resulting from tryptic cleavage of the Arg-Ser bond in the above sequence. The presence of methionine in the horse cofactor allowed fragmentation by cyanogen bromide. The C-terminal fragment was composed of 16 or 17 residues and contained no histidine. The single histidine of horse B1 was found in the intermediary fragment between Met-18 and Met-(n-17). These data show that the C-terminal parts of both pig and horse colipases are still more exposed to proteolytic degradations than the N-terminal parts. Preliminary attempts to crystallize B1 were carried out.

Structure of human carboxypeptidase A4 with its endogenous protein inhibitor, latexin.

The only endogenous protein inhibitor known for metallocarboxypeptidases (MCPs) is latexin, a 25-kDa protein discovered in the rat brain. Latexin, alias endogenous carboxypeptidase inhibitor, inhibits human CPA4 (hCPA4), whose expression is induced in prostate cancer cells after treatment with histone deacetylase inhibitors. hCPA4 is a member of the A/B subfamily of MCPs and displays the characteristic alpha/beta-hydrolase fold. Human latexin consists of two topologically equivalent subdomains, reminiscent of cystatins, consisting of an alpha-helix enveloped by a curved beta-sheet. These subdomains are packed against each other through the helices and linked by a connecting segment encompassing a third alpha-helix. The enzyme is bound at the interface of these subdomains. The complex occludes a large contact surface but makes rather few contacts, despite a nanomolar inhibition constant. This low specificity explains the flexibility of latexin in inhibiting all vertebrate A/B MCPs tested, even across species barriers. In contrast, modeling studies reveal why the N/E subfamily of MCPs and invertebrate A/B MCPs are not inhibited. Major differences in the loop segments shaping the border of the funnel-like access to the protease active site impede complex formation with latexin. Several sequences ascribable to diverse tissues and organs have been identified in vertebrate genomes as being highly similar to latexin. They are proposed to constitute the latexin family of potential inhibitors. Because they are ubiquitous, latexins could represent for vertebrate A/B MCPs the counterparts of tissue inhibitors of metalloproteases for matrix metalloproteinases.

An Inflammatory Role for the Mammalian Carboxypeptidase Inhibitor Latexin: Relationship to Cystatins and the Tumor Suppressor TIG1

Latexin, the only known mammalian carboxypeptidase inhibitor, has no detectable sequence similarity with plant and parasite inhibitors, but it is related to a human putative tumor suppressor protein, TIG1. Latexin is expressed in the developing brain, and we find that it plays a role in inflammation, as it is expressed at high levels and is inducible in macrophages in concert with other protease inhibitors and potential protease targets. The crystal structure of mouse latexin, solved at 1.83 A resolution, shows no structural relationship with other carboxypeptidase inhibitors. Furthermore, despite a lack of detectable sequence duplication, the structure incorporates two topologically analogous domains related by pseudo two-fold symmetry. Surprisingly, these domains share a cystatin fold architecture found in proteins that inhibit cysteine proteases, suggesting an evolutionary and possibly functional relationship. The structure of the tumor suppressor protein TIG1 was modeled, revealing its putative membrane binding surface.

A Carboxypeptidase Inhibitor from the Tick Rhipicephalus bursa: ISOLATION, cDNA CLONING, RECOMBINANT EXPRESSION, AND CHARACTERIZATION

A novel proteinaceous metallo-carboxypeptidase inhibitor, named tick carboxypeptidase inhibitor (TCI), was isolated from the ixodid tick Rhipicephalus bursa and N-terminally sequenced. The complete cDNA encoding this protein was cloned from tick mRNA by reverse transcription-PCR and rapid amplification of cDNA ends techniques. The full-length TCI cDNA contains an open reading frame coding for a precursor protein of 97 amino acid residues that consists of a predicted signal peptide of 22 residues and of mature TCI, a 75-residue cysteine-rich protein (12 Cys). The deduced amino acid sequence shows no homology to other known proteins; the C terminus, however, resembles those of other protein metallo-carboxypeptidase inhibitors, suggesting a common mechanism of inhibition. Recombinant TCI expressed in Escherichia coli is fully functional and inhibits carboxypeptidases of the A/B subfamily with equilibrium dissociation constants in the nanomolar range. Structural analyses by circular dichroism and nuclear magnetic resonance indicate that TCI is a protein strongly constrained by disulfide bonds, unusually stable over a wide pH range and highly resistant to denaturing conditions. As a tight binding inhibitor of plasma carboxypeptidase B, also known as thrombin-activatable fibrinolysis inhibitor, recombinant TCI stimulates fibrinolysis in vitro and thus may have potential for applications to prevent or treat thrombotic disorders.

A Method for Screening Enzyme Inhibitors Using Size Exclusion Chromatography and ESI-LC-MS/MS

A pilot study was performed for the development of a method to screen compound libraries using an electrospray mass spectrometer interfaced with liquid chromatography (LC). The mixture of compounds was obtained by combining low-molecular weight inhibitors of carboxypeptidase A (CPA), a representative zinc-containing proteolytic enzyme. After the incubation of the mixture with CPA, the enzyme-bound compounds were separated by size exclusion chromatography (SEC) from unbound compounds. The separation of compounds was affected by LC. Three compounds were identified, which represent the tight binding inhibitors of the library. These compounds were quantitated using an automatic switching valve to avoid the interference of buffer salts with the detection of analytes. The quantitated amounts of the compounds were found to be in good accordance with the K(i) values.

Proteolysis of Non-phosphorylated and Phosphorylated Tau by Thrombin

The microtubule-associated protein tau aggregates intracellularly by unknown mechanisms in Alzheimer's disease and other tauopathies. A contributing factor may be a failure to break down free cytosolic tau, thus creating a surplus for aggregation, although the proteases that degrade tau in brain remain unknown. To address this issue, we prepared cytosolic fractions from five normal human brains and from perfused rat brains and incubated them with or without protease inhibitors. D-Phenylalanyl-L-prolylarginyl chloromethyl ketone, a thrombin-specific inhibitor, prevented tau breakdown in these fractions, suggesting that thrombin is a brain protease that processes tau. We next exposed human recombinant tau to purified human thrombin and analyzed the fragments by N-terminal sequencing. We found that thrombin proteolyzed tau at multiple arginine and lysine sites. These include Arg(155)-Gly(156), Arg(209)-Ser(210), Arg(230)-Thr(231), Lys(257)-Ser(258), and Lys(340)-Ser(341) (numbering according to the longest human tau isoform). Temporally, the initial cleavage occurred at the Arg(155)-Gly(156) bond. Proteolysis of the resultant C-terminal tau fragment then proceeded bidirectionally. When tau was phosphorylated by glycogen synthase kinase-3beta, most of these proteolytic processes were inhibited, except for the first cleavage at the Arg(155)-Gly(156) bond. Furthermore, paired helical filament tau prepared from Alzheimer's disease brain was more resistant to thrombin proteolysis than following dephosphorylation by alkaline phosphatase. The results suggest a possible role for thrombin in proteolysis of tau under physiological and/or pathological conditions in human brains. They are consistent with the hypothesis that phosphorylation of tau inhibits proteolysis by thrombin or other endogenous proteases, leading to aggregation of tau into insoluble fibrils.

Inhibition of carboxypeptidase U (TAFIa) activity improves rt-PA induced thrombolysis in a dog model of coronary artery thrombosis

The objective of this study was to test the hypothesis if thrombolysis induced by recombinant tissue-type plasminogen activator, (rt-PA) could be facilitated by inhibiting carboxypeptidase U (CPU, active Thrombin Activatable Fibrinolysis Inhibitor, TAFIa) activity. The efficacy of rt-PA alone, or in combination with the carboxypeptidase inhibitor MERGETPA, was compared in a dog model of coronary artery thrombosis. Twenty dogs were randomised in two groups, one received rt-PA, 1 mg kg −1, as intravenous infusion over 20 min starting 30 min after thrombus formation, and the other group received rt-PA, 1 mg kg −1, as group one with the addition of MERGEPTA 5 mg kg −1 starting 25 min prior to coronary artery occlusion and followed by infusion of 5 mg kg −1 h −1 until the end of experiment. Efficacy was assessed by determination of time to lysis, duration of patency and blood flow during patency. Both groups had similar baseline characteristics with respect to haemodynamic parameters, i.e., heart rate, blood pressure and coronary artery blood flow. Coadministration of rt-PA and MERGETPA resulted in significant decrease in time to lysis (15±1.5 min vs. 20±1.7 min, p=0.03), increased patency time (87±16 min vs. 46±12 min, p=0.047) and increased coronary blood flow during patency (1131 mL h −1 vs. 405 mL h −1, p=0.015), compared to rt-PA alone. These results indicate that an inhibitor of CPU activity may have a beneficial effect in patients undergoing thrombolytic therapy by attaining shorter time to reperfusion and improved coronary patency.

Multiple insect resistance in transgenic tomato plants over-expressing two families of plant proteinase inhibitors

Protease inhibitors have been proposed as potential defense molecules for increased insect resistance in crop plants. Compensatory over-production of insensitive proteases in the insect, however, has limited suitability of these proteins in plant protection, with very high levels of inhibitor required for increased plant resistance. In this study we have examined whether combined used of two inhibitors is effective to prevent this compensatory response. We show that leaf-specific over-expression of the potato PI-II and carboxypeptidase inhibitors (PCI) results in increased resistance to Heliothis obsoleta and Liriomyza trifolii larvae in homozygote tomato lines expressing high levels (>1% the total soluble proteins) of the transgenes. Leaf damage in hemizygous lines for these transformants was, however, more severe than in the controls, thus evidencing a compensation response of the larvae to the lower PI concentrations in these plants. Development of comparable adaptive responses in both insects suggests that insect adaptation does not entail specific recognition of the transgene, but rather represents a general adaptive mechanism triggered in response to the nutritional stress imposed by sub-lethal concentrations of the inhibitors. Combined expression of defense genes with different mechanisms of action rather than combinations of inhibitors may then offer a better strategy in pest management as it should be more effective in overcoming this general adaptive response in the insect.

Inhibition of glutamate carboxypeptidase II (NAALADase) protects against dynorphin A-induced ischemic spinal cord injury in rats

Glutamate carboxypeptidase (GCP) II (EC 3.4.17.21), which is also known as N-acetylated-α-linked acidic dipeptidase (NAALADase), hydrolyses the endogenous acidic dipeptide N-acetylaspartylglutamate (NAAG), yielding N-acetyl-aspartate and glutamate. Inhibition of this enzyme by 2-(phosphonomethyl) pentanedioic acid (2-PMPA) has been shown to protect against ischemic injury to the brain and hypoxic and metabolic injury to neuronal cells in culture, presumably by increasing and decreasing the extracellular concentrations of NAAG and glutamate, respectively. Since both NAAG and GCP II are found in especially high concentrations in the spinal cord, injuries to the spinal cord involving pathophysiological elevations in extracellular glutamate might be particularly responsive to GCP II inhibition. Lumbar subarachnoid injections of dynorphin A in rats cause ischemic spinal cord injury, elevated extracellular glutamate and a persistent hindlimb paralysis that is mediated through excitatory amino acid receptors. We therefore used this injury model to evaluate the protective effects of 2-PMPA. When coadministered with dynorphin A, 2-PMPA significantly attenuated the dynorphin A-induced elevations in cerebrospinal fluid glutamate levels and by 24 h postinjection caused significant dose-dependent improvements in motor scores that were associated with marked histopathological improvements. These results indicate that 2-PMPA provides effective protection against excitotoxic spinal cord injury.

The profibrinolytic effect of plasma thrombomodulin in factor XI deficiency and its implications in hemostasis

Bleeding tendency in factor (F)XI deficiency may result from premature clot lysis due to insufficient thrombin activatable fibrinolysis inhibitor (TAFI) activation. Thrombomodulin (TM), upon binding to thrombin, is capable of modulating TAFI activation. In this study, we investigated the effects of plasma TM on fibrinolysis in FXI-deficient patients. A clot lysis assay showed the defective down-regulation of fibrinolysis in FXI-deficient patients as compared with normal controls. To evaluate the effects of plasma TM on fibrinolysis, a monoclonal anti-TM IgG was preincubated with plasma for 30 min. The presence of anti-TM IgG significantly prolonged the clot lysis times both in the FXI-deficient and normal plasma, indicating that plasma TM stimulated fibrinolysis. Furthermore, the presence of anti-TM IgG not only reduced protein C activation, but also increased thrombin generation and TAFI activation. The profibrinolytic effect of plasma TM was inhibited in the assay by including either a monoclonal anti-TAFI IgG or a specific TAFI inhibitor--carboxypeptidase inhibitor (CPI). Our results indicate that the impaired thrombin generation in FXI-deficient patients leads to the defective down-regulation of fibrinolysis, and that plasma TM stimulates fibrinolysis through APC pathway which inhibits TAFI activation. The profibrinolytic effect of plasma TM may contribute to the bleeding tendency observed in some FXI-deficient patients.

Proteolytic activities in body and faecal extracts of the storage mite, Acarus farris

Trypsin, chymotrypsin, cathepsins B and D, aminopeptidase and carboxypeptidases A and B were detected in body extracts of the storage mite Acarus farris (Oudemans) (Astigmata: Acaridae). Faeces-enriched medium exhibited higher (10-50-fold) specific protease activity rates than those measured with mite body extracts for trypsin, chymotrypsin and carboxypeptidases A and B, suggesting that they are involved in mite digestion. However, the activity of cathepsin B was only three-fold higher in faecal than in body extracts, indicating that its presence in the lumen of the digestive tract is low compared to that of serine proteases. The activity of aminopeptidases was higher in mite bodies, indicating that they might be membrane bound. Cathepsin D activity was only detected in body extracts, indicating that this enzyme is not a digestive protease in this species. Zymograms resolved three major bands of gelatinolytic activity, but at least one protease form was only present in body extracts. Protease inhibitors of different specificity were tested in vivo to establish their potential as control agents. The development of A. farris was significantly retarded when the immature stages were fed on artificial diet containing inhibitors of serine and cysteine proteases and aminopeptidases, whereas no such effect was found with inhibitors of aspartyl proteases and carboxypeptidases. Interestingly, the most significant effects on A. farris occurred when a combination of inhibitors targeting different enzyme classes was supplied mixed in the diet, suggesting a synergistic toxicity. Several plant lectins were also tested, but only wheat germ agglutinin and concanavalin-A affected development.

Cyclen-Containing Inhibitors of Carboxypeptidase A Synthesized in Search of Target-Selective Artificial Proteases

Previously, we reported the first substrate-selective artificial protease by using myoglobin as the substrate. Target-selective peptide-cleaving catalysts can be used as drugs if the target is a protein or an oligopeptide related to a disease. For example, a peptide-cleaving catalyst specifically recognizing HIV protease and rapidly cleaving it into two pieces can be used as a new drug for AIDS. Since only a catalytic amount of the drug is needed, the drug dosage and the side effects can be reduced by using the peptide-cleaving catalysts. The myoglobin-cleaving catalysts reported previously were designed by attaching a catalytic group to a binding site that recognizes the surface of myoglobin. The Cu(II) or Co(III) complex of cyclen (Cyc) was used as the catalytic group in view of their catalytic activity in peptide hydrolysis. Thus, connection of a catalytic group to a binding site can be considered as a general method for creation of targetselective peptide-cleaving catalysts. The binding site may recognize either a certain portion on the surface of the target protein as in the case of the myoglobin-cleaving catalyst mentioned above or the active site of the target protein. In view of a vast amount of ligand molecules reported to have high affinity toward many disease-related proteins, the known ligand molecules may be utilized as the binding site of the peptide-cleaving catalysts. In this regard, we undertook synthesis of peptide-cleaving catalysts selective for carboxypeptidase A (CPA) by using CPA inhibitors as the binding site of the catalysts. The idea of designing a CPA-selective peptide-cleaving catalyst based on a known CPA inhibitor is illustrated in Scheme 1.