Structure of cruzipain/cruzain inhibitors isolated from Bauhinia bauhinioides seeds

The saline extract of Bauhinia bauhinioides dry seeds was shown to inhibit cruzipain, a cysteine proteinase from Trypanosoma cruzi. The inhibitory activity was assigned to a protein with 164 amino acid residues and molecular mass of 18 034 Da that was purified by chromatography on DEAE-Sephadex, trypsin-Sepharose (removal of trypsin inhibitors), Mono Q and a reversed-phase C4 column. The primary structure is homologous to other plant Kunitz-type inhibitors, but it lacks cysteine residues and therefore the disulfide bridges. No methionine residue was identified by amino acid sequencing. The inhibition of cruzipain fits into a slow-tight binding mechanism with a low dissociation constant (Ki 1.2 nM). The studied Bauhinia protein also inhibits cruzain (Ki 0.3 nM), a C-terminally truncated recombinant species of cruzipain. Cathepsin L, a cysteine proteinase with high homology to cruzipain, is also inhibited (Ki 0.22 nM), but not cathepsin B, papain, bromelain or ficin.     

Novel aromatic inhibitors of influenza virus neuraminidase make selective interactions with conserved residues and water molecules in the active site.

The active site of type A or B influenza virus neuraminidase is composed of 11 conserved residues that directly interact with the substrate, sialic acid. An aromatic benzene ring has been used to replace the pyranose of sialic acid in our design of novel neuraminidase inhibitors. A bis(hydroxymethyl)pyrrolidinone ring was constructed in place of the N-acetyl group on the sialic acid. The hydroxymethyl groups replace two active site water molecules, which resulted in the high affinity of the nanomolar inhibitors. However, these inhibitors have greater potency for type A influenza virus than for type B influenza virus. To resolve the differences, we determined the X-ray crystal structure of three benzoic acid substituted inhibitors bound to the active site of B/Lee/40 neuraminidase. The investigation of a hydrophobic aliphatic group and a hydrophilic guanidino group on the aromatic inhibitors shows changes in the interaction with the active site residue Glu275. The results provide an explanation for the difference in efficacy of these inhibitors against types A and B viruses, even though the 11 active site residues of the neuraminidase are conserved.     

Trypanosoma cruzi: cruzipain and membrane-bound cysteine proteinase isoform(s) interacts with human alpha(2)-macroglobulin and pregnancy zone protein

Plasmatic levels of pregnancy zone protein (PZP) increase in children with acute Chagas disease. PZP, as well as alpha2-macroglobulin (alpha2-M), are able to interact with Trypanosoma cruzi proteinases. The interaction of alpha2-M and PZP with cruzipain, the major cysteine proteinase of T. cruzi, was investigated. Several molecular changes on both alpha-M inhibitors under reaction with cruzipain were found. PAGE analysis showed: (i) formation of complexes of intermediate mobility and tetramerization of native alpha2-M and PZP, respectively; (ii) limited proteolysis of bait region in alpha2-M and PZP, and (iii) covalent binding of cruzipain to PZP and alpha2-M. Conformational and structural changes experimented by alpha-Ms correlate with modifications of the enzyme electrophoretic mobility and activity. Cruzipain-alpha-M complexes were also detected by gelatin SDS-PAGE and immunoblotting using polyclonal anti-cruzipain antibodies. Concomitantly, alpha2-M and PZP impaired the activity of cruzipain towards Bz-Pro-Phe-Arg-pNA substrate. In addition, alpha-Ms were able to form covalent complexes with membrane isoforms of cysteine proteinases cross-reacting with cruzipain. The present study suggests that both human alpha-macroglobulin inhibitors could prevent or minimize harmful action of cruzipain on host's molecules and hypothetically regulate parasite functions controlled by cruzipain.     

The propeptide of cruzipain--a potent selective inhibitor of the trypanosomal enzymes cruzipain and brucipain, and of the human enzyme cathepsin F

Papain-like cysteine proteases of pathogenic protozoa play important roles in parasite growth, differentiation and host cell invasion. The main cysteine proteases of Trypanosoma cruzi (cruzipain) and of Trypanosoma brucei (brucipain) are validated targets for the development of new chemotherapies. These proteases are synthesized as precursors and activated upon removal of the N-terminal prodomain. Here we report potent and selective inhibition of cruzipain and brucipain by the recombinant full-length prodomain of cruzipain. The propeptide did not inhibit human cathepsins S, K or B or papain at the tested concentrations, and moderately inhibited human cathepsin V. Human cathepsin F was very efficiently inhibited (K(i) of 32 pm), an interesting finding indicating that cruzipain propeptide is able to discriminate cathepsin F from other cathepsin L-like enzymes. Comparative structural modeling and analysis identified the interaction between the beta1p-alpha3p loop of the propeptide and the propeptide-binding loop of mature enzymes as a plausible cause of the observed inhibitory selectivity.     

One-bead, one-compound peptide library sequencing via high-pressure ammonia cleavage coupled to nanomanipulation/nanoelectrospray ionization mass spectrometry

Biological screening of one-bead, one-compound (OBOC) combinatorial peptide libraries is routinely carried out with the peptide remaining bound to the resin bead during screening. After a hit is identified, the bead is isolated, the peptide is cleaved from the bead, and its sequence is determined. We have developed a new technique for cleavage of peptides from resin beads whereby exposure of a 4-hydroxymethyl benzoic acid (HMBA)-linked peptide to high-pressure ammonia gas led to efficient cleavage in as little as 5 min. Here we also report a new method of extracting peptide from individual library beads for its introduction into a mass spectrometer that uses nanomanipulation combined with nanoelectrospray ionization mass spectrometry (NSI MS). Single beads analyzed by nanomanipulation/NSI MS were found to give identical MS results to those of bulk samples. Detection of 18 unique cleaved peptides 1 to 8 amino acids in length, and sequencing of 14 different peptide sequences 4 to 8 amino acids in length, was demonstrated on a combination of bulk samples and ones from individual beads of an OBOC library. The method was highly reproducible, with 100% of attempts to extract peptide resulting in high-quality MS data. This new collection of techniques allows rapid, reliable, environmentally responsible sequencing of hit beads from combinatorial peptide libraries.     

Identification of potent and reversible cruzipain inhibitors for the treatment of Chagas disease

Identification of potent and reversible cruzipain inhibitors for the treatment of Chagas disease is described. The identified inhibitors bearing an amino nitrile warhead in P1 exhibit low nanomolar in vitro potency against cruzipain. Further SAR in P2 portion led to the identification of compounds, such as 26, that have a unique selectivity profile against other cysteine proteases and offering new opportunities for safer treatment of Chagas disease.     

Comparison of the specificity, stability and individual rate constants with respective activation parameters for the peptidase activity of cruzipain and its recombinant form, cruzain, from Trypanosoma cruzi.

The Trypanosoma cruzi cysteine protease cruzipain contains a 130-amino-acid C-terminal extension, in addition to the catalytic domain. Natural cruzipain is a complex of isoforms, because of the simultaneous expression of several genes, and the presence of either high mannose-type, hybrid monoantennary-type or complex biantenary-type oligosacharide chains at Asn255 of the C-terminal extension. Cruzipain and its recombinant form without this extension (cruzain) were studied comparatively in this work. S2 to S2' subsite specificities of these enzymes were examined using four series of substrates derived from the internally quenched fluorescent peptide Abz-KLRFSKQ-EDDnp (Abz, ortho-aminobenzoic acid; EDDnp, N-(2,4-dinitrophenyl)-ethylenediamine). Large differences in the kinetic parameters were not observed between the enzymes; however, Km values were consistently lower for the hydrolysis of most of the substrates by cruzain. No difference in the pH-activity profile between the two enzymes was found, but in 1 m NaCl cruzipain presented a kcat value significantly higher than that of cruzain. The activation energy of denaturation for the enzymes did not differ significantly; however, a negative entropy value was observed for cruzipain denaturation whereas the value for cruzain was positive. We determined the individual rate constants (k1, substrate diffusion; k-1, substrate dissociation; k2, acylation; k3, deacylation) and the respective activation energies and entropies for hydrolysis of Abz-KLRFSKQ-EDDnp determining the temperature dependence of the Michaelis-Menten parameters kcat/Km and kcat as previously described [Ayala, Y.M. & Di Cera, E. (2000) Protein Sci. 9, 1589-1593]. Differences between the two enzymes were clearly detected in the activation energies E1 and E-1, which are significantly higher for cruzipain. The corresponding DeltaS1 and DeltaS-1 were positive and significantly higher for cruzipain than for cruzain. These results indicate the presence of a larger energy barrier for cruzipain relating to substrate diffusion and dissociation, which could be related to the C-terminal extension and/or glycosylation state of cruzipain.     

The defensive functions of plant inhibitors are not restricted to insect enzyme inhibition

Three plant proteinase inhibitors BbKI (kallikrein inhibitor) and BbCI (cruzipain inhibitor) from Bauhinia bauhinioides, and a BrTI (trypsin inhibitor) from B. rufa, were examined for other effects in Callosobruchus maculatus development; of these only BrTI affected bruchid emergence. BrTI and BbKI share 81% identities in their primary sequences and the major differences between them are the regions comprising the RGD and RGE motifs in BrTI. These sequences were shown to be essential for BrTI insecticidal activity, since a modified BbKI [that is a recombinant form (BbKIm) with some amino acid residues replaced by those found in BrTI sequence] also strongly inhibited insect development. By using synthetic peptides related to the BrTI sequence, YLEAPVARGDGGLA-NH₂ (RGE) and IVYYPDRGETGL-NH₂ (RGE), it was found that the peptide with an RGE sequence was able to block normal development of C. maculatus larvae (ED₅₀ 0.16% and LD₅₀ 0.09%), this being even more effective than the native protein.     

Identification of multiple HLA-A*0201-restricted cruzipain and FL-160 CD8+ epitopes recognized by T cells from chronically Trypanosoma cruzi-infected patients

Chronic Chagas disease occurs in 16 million individuals chronically infected by the protozoan Trypanosoma cruzi in Latin America, and may lead to a dilated cardiomyopathy in 10-30% of patients. A vigorous cellular immune response holds parasitism in check. However, up to now, few T. cruzi proteins have been shown to be recognized by CD8+ T cells from Chagas disease patients. In this study, we designed 94 peptides derived from T. cruzi proteins cruzipain and FL-160, predicted to bind to HLA-A2 molcules. After in vitro binding assays to HLA-A*0201, 26 peptides were selected, and their recognition by PBMC from Chagas disease patients was tested with the IFN-gamma ELISPOT assay. All 26 peptides were recognized by PBMC from at least one patient. Furthermore, a tetrameric HLA-A*0201 complex built with the cruzipain 60-68 peptide that was frequently recognized in the periphery also bound to CD8+ T cells from a heart-infiltrating T cell line obtained from a single patient with Chagas disease cardiomyopathy. Thus, our results suggest that the recognition of CD8+ T cell epitopes in cruzipain and FL-160 may have a pathogenic or protective role in chronic Chagas disease.     

Cathepsin S and cruzipain are inhibited by equistatin from Actinia equina.

Cathepsin S has been isolated for the first time from human tissue. It has a molecular mass of 24 kDa and an isoelectric point in the range of 8.2 to 8.6. The enzyme is inhibited by equistatin, which belongs to the thyropins, a new family of protein inhibitors, with an inhibition constant of Ki = 0.40 +/- 0.07 nM. Cruzipain, a cathepsin L-like enzyme sharing a 130 amino acid long C-terminal extension, is also strongly inhibited by equistatin (Ki = 0.028 +/- 0.006 nM). Together with previously reported data, these results further indicate that a functional heterogeneity exists among thyropin inhibitors, as demonstrated by their interaction with cathepsin S and cruzipain.     

Digestion of human immunoglobulin G by the major cysteine proteinase (cruzipain) from Trypanosoma cruzi

The major cysteine proteinase (cruzipain) from Trypanosoma cruzi was able to digest human IgG, as shown by polyacrylamide gel electrophoresis in the presence of SDS, and by gel filtration on a Superose 12 column, in a FPLC system. The Fab fragment of IgG was only slightly degraded, but Fc was extensively hydrolyzed to small peptides. The results suggest that cruzipain might be involved in the defense mechanisms of the parasite against the immune response of the host.     

Expression in insect cells of active mature cruzipain from Trypanosoma cruzi,containing its C-terminal domain

Cruzipain, the major cysteine proteinase from Trypanosoma cruzi, is a member of the papain family that contains a C-terminal domain in the mature enzyme, in addition to a catalytic moiety homologous to papain and some mammalian cathepsins. The native enzyme is expressed as a complex mixture of isoforms and has not been crystallized. Previous attempts to express recombinant mature cruzipain containing the C-terminal domain have failed. For this reason, the three-dimensional structure of the complete mature enzyme is not known, although the structure of a recombinant truncated molecule lacking the C-terminal domain (cruzaindeltac) has been determined. We report here the expression of active, N-glycosylated, complete mature cruzipain in an insect cell/baculovirus system. The purified recombinant enzyme, obtained with a yield of about 0.2 mg/100 ml of culture supernatant, has an apparent molecular mass similar, and an identical N-terminal sequence, compared with the native enzyme. The expressed protein is able to process itself by self-proteolysis, leaving the isolated C-terminal domain, and has kinetic properties similar to those of native cruzipain, although some differences in substrate specificity were found. These results open up the possibility of obtaining recombinant intact mature cruzipain of a quality and in quantity suitable for X-ray crystallography.     

Screening Peptide Bead Libraries for Enzyme Inhibitors

Recent developments in the technology of solid-phase peptide synthesis have made it possible to create libraries of peptides mounted on solid supports, which may then be screened for biological activity. By inserting such a randomized library region into a synthetic peptide mounted on resin beads, a full range of potentially useful mutants may be screened simultaneously. This article describes the use of this approach to generate variants of CMTI-I, a conformationally constrained proteinaceous inhibitor of trypsin and chymotrypsin. The result is a library containing mutants for the P′₁-P′₄ reactive-loop sequence of CMTI-I. This is equilibrated with a fluorescentiy labeled target enzyme (in this case human leukocyte elastase), which binds to those beads containing the most potent inhibitors. These are identified and isolated. The method utilizes the conformational rigidity of CMTI-I to improve inhibitory activity. By this means the most potent inhibitors of HLE may be distinguished and then synthesized on a preparative scale for kinetic analysis. This approach may be applied directly to other serine proteinases.     

Expression in insect cells of active mature cruzipain from Trypanosoma cruzi, containing its C-terminal domain

Cruzipain, the major cysteine proteinase from Trypanosoma cruzi, is a member of the papain family that contains a C-terminal domain in the mature enzyme, in addition to a catalytic moiety homologous to papain and some mammalian cathepsins. The native enzyme is expressed as a complex mixture of isoforms and has not been crystallized. Previous attempts to express recombinant mature cruzipain containing the C-terminal domain have failed. For this reason, the three-dimensional structure of the complete mature enzyme is not known, although the structure of a recombinant truncated molecule lacking the C-terminal domain (cruzainΔc) has been determined. We report here the expression of active, N-glycosylated, complete mature cruzipain in an insect cell/baculovirus system. The purified recombinant enzyme, obtained with a yield of about 0.2 mg/100 ml of culture supernatant, has an apparent molecular mass similar, and an identical N-terminal sequence, compared with the native enzyme. The expressed protein is able to process itself by self-proteolysis, leaving the isolated C-terminal domain, and has kinetic properties similar to those of native cruzipain, although some differences in substrate specificity were found. These results open up the possibility of obtaining recombinant intact mature cruzipain of a quality and in quantity suitable for X-ray crystallography.     

Phage display screening for peptidic chitinase inhibitors

A phage display library with disulfide-cyclized peptides was screened for peptides binding to chitinases from Serratia marcescens. One of those peptides was found to efficiently inhibit chitinase A and two others were inhibitors of chitinase B. Complete substitutional analysis of all three peptides using cellulose-bound peptide spot synthesis revealed key interaction positions and allowed optimization of the chitinase B inhibitory peptides towards higher affinity, with inhibitory constants in the lower nanomolar range. Inhibition by all peptides proved to be competitive and highly specific for the chitinase used to select them, as shown with a series of chitinases from different organisms.     

Small-molecule inhibitors of cathepsin L incorporating functionalized ring-fused molecular frameworks

Cathepsin L is a cysteine protease that is upregulated in a variety of malignant tumors and plays a significant role in cancer cell invasion and migration. It is an attractive target for the development of small-molecule inhibitors, which may prove beneficial as treatment agents to limit or arrest cancer metastasis. We have previously identified a structurally diverse series of thiosemicarbazone-based inhibitors that incorporate the benzophenone and thiochromanone molecular scaffolds. Herein we report an important extension of this work designed to explore fused aryl–alkyl ring molecular systems that feature nitrogen atom incorporation (dihydroquinoline-based) and carbon atom exclusivity (tetrahydronaphthalene-based). In addition, analogues that contain oxygen (chromanone-based), sulfur (thiochroman-based), sulfoxide, and sulfone functionalization have been prepared in order to further investigate the structure–activity relationship aspects associated with these compounds and their ability to inhibit cathepsins L and B. From this small-library of 30 compounds, five were found to be strongly inhibitory (IC₅₀ <500 nM) against cathepsin L with the most active compound (7-bromodihydroquinoline thiosemicarbazone 48) demonstrating an IC₅₀ = 164 nM. All of the compounds evaluated were inactive (IC₅₀ >10,000 nM) as inhibitors of cathepsin B, thus establishing a high degree (>20-fold) of selectivity (cathepsin L vs. cathepsin B) for the most active cathepsin L inhibitors in this series.     

Trypanosoma cruzi: partial characterization of minor cruzipain isoforms non-adsorbed to Concanavalin A-Sepharose

The present paper reports the partial characterization of a subset of atypical cruzipain molecules which do not bind to Concanavalin A-Sepharose column. They are present in different strains of epimastigote forms of Trypanosoma cruzi and represent a 2-4% of total cruzipain. They were purified by affinity chromatography on Cystatin-Sepharose, recognized by the polyclonal anti-cruzipain serum, and their activity in gelatin-containing gels was completely abolished by E-64, TLCK, leupeptin, and aprotinin but not by PMSF, pepstatin A, EDTA or 1,10-phenantroline. These cysteine proteinases, as well as cruzipain showed to be endoproteinases able to hydrolize azocasein, hemoglobin, and bovine serum albumin at acidic pHs. However, evidences are presented indicating that this subset of cruzipain isoforms were also able to use the same blocked chromogenic peptidyl substrates than cruzipain at similar optimal alkaline pH values although with a different order of preference. Moreover, they showed a different oligosaccharide pattern after enzymatic treatment by high pH anion exchange chromatography, suggesting that this structural difference may account for the atypical behaviour in the lectin column.     

Total solid-phase synthesis of bombesin analogs with different functional groups at the C-terminus.

Five bombesin analogs with different functional groups at the C-terminus were synthesized using a solid-phase strategy. The protocols were optimized using 4-(hydroxymethyl)benzoic acid (HMBA) resin to synthesize a common precursor followed by nucleophilic cleavage of the base sensitive peptide ester linkage. The C-terminal modifications included ethylamide, butylamide, methyl ester, propyl ester and hydrazide. Cleavage from the resin was possible with the fully protected or deprotected precursor peptide; however, higher purity of the final products was achieved when cleavage protocols were conducted after side-chain deprotection. The synthesized peptides were analyzed and characterized using reverse phase HPLC and ESI-MS. The peptides were obtained in 13-32% overall recovery, calculated from the coupling efficiency of the first amino acid residue, and in 91-97% purity.     

Design,synthesis, and biological evaluation of potent thiosemicarbazone based cathepsin L inhibitors

A small library of 36 functionalized benzophenone thiosemicarbazone analogs has been prepared by chemical synthesis and evaluated for their ability to inhibit the cysteine proteases cathepsin L and cathepsin B. Inhibitors of cathepsins L and B have the potential to limit or arrest cancer metastasis. The six most active inhibitors of cathepsin L (IC₅₀ < 85 nM) in this series incorporate a meta-bromo substituent in one aryl ring along with a variety of functional groups in the second aryl ring. These six analogs are selective for their inhibition of cathepsin L versus cathepsin B (IC₅₀ > 10,000 nM). The most active analog in the series, 3-bromophenyl-2′-fluorophenyl thiosemicarbazone 1, also efficiently inhibits cell invasion of the DU-145 human prostate cancer cell line.     

Polymorphisms of the genes encoding cruzipain, the major cysteine proteinase of Trypanosoma cruzi, in the region encoding the C-terminal domain

Forty-eight cDNA clones obtained from different developmental stages of Trypanosoma cruzi and all encoding the C-terminal domain of the major cysteine proteinase (cruzipain) have been sequenced. A number of polymorphisms were detected, seven of them resulting in amino acid replacements. The predicted pI values of the corresponding gene products varied between 7.05 and 8.12. These changes in amino acid sequence, together with previously reported variations in carbohydrate composition at the only N-glycosylation site in the C-terminal domain, may account for most of the heterogeneities found in the mature enzyme.