[Alteration of pulmonary epithelial permeability by cathepsin S in chronic obstructive pulmonary disease]. / Altération de la perméabilité épithéliale pulmonaire par la cathepsine S au cours de la bronchopneumopathie chronique obstructive.

Smoking is accountable for most of the chronic obstructive pulmonary disease (COPD) cases. COPD, which is characterized by the development of chronic bronchitis, could be associated with emphysema. In active smokers, there is an overexpression of cathepsin S, a cysteine protease, which participates in the development of emphysema via its elastinolytic activity. Likewise, we demonstrated that cathepsin S could degrade one or more protein constituents of cell junctions. This deleterious proteolytic activity leads to an alteration of the integrity of the lung epithelial barrier, which in turn could aggravate chronic inflammation and promote the exacerbation phases associated with infections.

In silico and in vitro mapping of specificity patterns of glycosaminoglycans towards cysteine cathepsins B, L, K, S and V.

Human cysteine cathepsins are lysosomal proteases, which are involved in different biological processes. Their enzymatic activity can be regulated by glycosaminoglycans (GAGs): long linear periodic negatively charged polysaccharides, which dimeric building blocks consist of uronic acid and hexosamine monosaccharide units. In this study, molecular docking simulations of chondroitin 4-sulfate, chondroitin 6-sulfate, heparin, heparan sulfate, dermatan sulfate and hyaluronic acid of various chain lengths were performed with cathepsins B, L, K, S and V and followed by molecular dynamics-based refinement and binding free energy analysis. We concluded that electrostatics might be a driving force for cathepsin-GAG interactions; indeed as in most of characterised systems, the increase of GAG chain length consequently leads to a more pronounced effect on the strength of cathepsin-GAG interactions. Results also suggest that binding of GAGs at different regions on cathepsins surface affect differently their enzymatic activity and could is dependent on cathepsin and GAG type. Present data contribute to systematic description of cathepsin-GAG interactions, which is helpful in understanding the subtle molecular mechanisms of protease regulation behind their biological functions.

Modulation of the expression and activity of cathepsin S in reconstructed human skin by neohesperidin dihydrochalcone.

Dysregulation of cathepsin S (Cat S), a cysteine protease involved in extracellular-matrix and basement membrane (BM) degradation, is a concomitant feature of several inflammatory skin diseases. Therefore, Cat S has been suggested as a potential therapeutic target. Flavonoids, which were identified as regulatory molecules of various proteolytic enzymes, exert beneficial effects on skin epidermis. Herein, thirteen flavonoid compounds were screened in vitro and in silico and neohesperidin dihydrochalcone (NHDC) was identified as a potent, competitive, and selective inhibitor (Ki=8±1 µM) of Cat S. Furthermore, Cat S-dependent hydrolysis of nidogen-1, a keystone protein of BM architecture, as well elastin, collagens I and IV was impaired by NHDC, while both expression and activity of Cat S were significantly reduced in NHDC-treated human keratinocytes. Moreover, a reconstructed human skin model showed a significant decrease of both mRNA and protein levels of Cat S after NHDC treatment. Conversely, the expression of nidogen-1 was significantly increased. NHDC raised IL-10 expression, an anti-inflammatory cytokine, and mediated STAT3 signaling pathway, which in turn dampened Cat S expression. Our findings support that NHDC may represent a valuable scaffold for structural improvement and development of Cat S inhibitors to preserve the matrix integrity and favor skin homeostasis during inflammatory events.

Evaluation of a peptide ELISA for the detection of rituximab in serum.

Pharmacokinetic studies of therapeutic monoclonal antibodies necessitate the measurement of their biologically active fraction. The aim of this work was to develop an enzyme-linked immunosorbent assay (ELISA) for rituximab, a chimeric anti-CD20 monoclonal antibody, based on its binding to a 20-mer peptide (P20) derived from the extracellular loop of human CD20 (residues 165-184). Derivatives of P20 were prepared by conjugation to bovine serum albumin (BSA-P20ACM) or biotin (Biot-P20ACM). Interactions of P20 and its derived peptides with rituximab were analyzed by surface plasmon resonance (SPR) and by ELISA. A monoclonal anti-idiotype antibody (MB2A4) was used as the reference in each case. SPR analysis showed that P20 (conjugated or unconjugated) had a lower affinity for rituximab than MB2A4. ELISA methods based on P20 or MB2A4 were both highly accurate and reproducible for rituximab measurement in spiked samples, but the MB2A4-based assay had a lower limit of quantification. Interestingly, discrepant results were obtained with the two ELISA methods when analyzing pharmacokinetic samples, with the rituximab concentrations obtained with the MB2A4-based method being systematically higher than those determined by the P20-based method. Possible interference of circulating CD20 with the P20-based method was supported by competition experiments. Rituximab aggregation in the bloodstream may also account for the bias observed in samples from healthy mice. The P20-based ELISA is far less sensitive than the MB2A-based ELISA, thus limiting its utility for pharmacokinetic studies. However, the discrepancy observed between two different approaches for rituximab measurement indicates that data from different studies should be interpreted with care.

Revisiting the S2 specificity of papain by structural analogs of Phe.

Papain characteristically has a strong preference for encoded L-aromatic amino acids (Phe > Tyr) at P2 position. We re-examined papain S2 specificity using structural analogs of Phe, in fluorogenic substrates of the series: dansyl-Xaa-Arg-Ala-Pro-Trp (Xaa = P2 residue). Kinetic analyses showed that the S2 pocket accommodates a broad spectrum of Phe derivatives. Papain is poorly stereoselective towards Dns-(D/L)-Phe-Arg-Ala-Pro-Trp and binding is not critically affected by replacement of the benzyl ring by the non-aromatic lateral chain of cyclohexylalanine. The Km was significantly improved by mono- and di-chlorination of Phe, or by its substitution by an electronegative group-like NO2, but the specificity constant was unchanged. Shortening or lengthening the side chain by adding or removing a methylene group impairs the P2/S2 interactions significantly, as do constrained structural analogs of Phe. Incorporation of benzyl-substituted phenylalanyl amino acid could help to design peptide-derived inhibitors with greater affinity and bioavailability.

Inhibition of cathepsin B by its propeptide: use of overlapping peptides to identify a critical segment.

Ten overlapping 15-mer peptides (peptidyl amides) spanning the proregion of rat cathepsin B (residues 1p-60p) were constructed to identify minimal segments having inhibitory activity towards the mature enzyme, that could be used to develop a new generation of peptide-derived inhibitors specifically targeting the active site of the corresponding proteinase. Three synthetic peptides, containing the pentapeptide Leu-Cys-Gly-Thr-Val (residues 41p-45p) in their sequence, inhibited cathepsin B with Ki values in the micromolar range. Alkylation of the thiol group of Cys-42p of peptide PB8 (36p-50p) resulted in its rapid proteolytic degradation, suggesting that this residue is essential for inhibition. The inhibition constant was slightly improved (Ki = 2 microM) using a longer peptide (26p-50p) which was completely resistant to cleavage even after a prolonged incubation. Alkylation of its cysteinyl residue also resulted in rapid cleavage of the peptide chain. Peptides derived from the rat cathepsin B prosequence also inhibited human cathepsin B with similar Ki values. Unlike rat cathepsin B, which cleaves peptide PB8 at the G47p-G48p bond after prolonged incubation, the human enzyme cleaved both PB8 and PB11 at the Lys-40p-Leu-41p bond, in agreement with the different kinetic properties of these two proteinases. New probes with improved specificity for cysteine proteinases may therefore be designed based on the sequences of their propeptides.

Reversible inhibition of cathepsin L-like proteases by 4-mer pseudopeptides.

A library of 121 pseudopeptides was designed to develop reversible inhibitors of trypanosomal enzymes (cruzain from Trypanosoma cruzi and congopain from Trypanosoma congolense). The peptides share the framework: Cha-X1-X2-Pro (Cha=cyclohexyl-alanine, X1 and X2 were phenylalanyl analogs), based on a previous report [Lecaille, F., Authié, E., Moreau, T., Serveau, C., Gauthier, F. and Lalmanach, G. (2001) Eur. J. Biochem. 268, 2733-2741]. Five peptides containing a nitro-substituted aromatic residue (Tyr/Phe) and one a 4-chloro-phenylalanine at the X1 position, and 3-(2-naphthyl)-alanine, homocyclohexylalanine or 3-nitro-tyrosine (3-NO(2)-Tyr) at the X2 position, were selected. They inhibited congopain more effectively than cruzain, except Cha-4-NO(2)-Phe-3-NO(2)-Tyr-Pro which bound the two parasitic enzymes similarly. Among this series, Cha-3-NO(2)-Tyr-HoCha-Pro and Cha-4-NO(2)-Phe-3-NO(2)-Tyr-Pro are the most selective for congopain relative to host cathepsins. No hydrolysis occurred upon prolonged incubation time with purified enzymes. In addition introduction of non-proteogenic residues in the peptidyl backbone greatly enhanced resistance to proteolysis by mammalian sera.

An immunochemical approach to investigating the mechanism of inhibition of cysteine proteinases by members of the cystatin superfamily.

Antibodies were raised against a synthetic dodecameric peptide KGAGQVVAGPWK (K12K), encompassing sequences thought to be important for the function of the cysteine proteinase inhibitors of the cystatin superfamily. These antibodies specifically recognized molecules of family 3, i.e., kininogens, in the serum of seven mammalian species tested in this study. The only notable exception was that of rat thiostatin (T kininogen) which is structurally related to the kininogen family. The antibodies also discriminated between family 2 (cystatins) and family 3 (kininogens) of the cystatin superfamily, since neither chicken cystatin nor human and rat cystatins C and S, which all belong to family 2 were recognized. The cystatin-like inhibitory domains resulting from fragmentation of human low molecular weight kininogen by bovine trypsin, were still recognized by antibodies, indicating that discrimination does not require two neighbouring inhibitory sites on the kininogen heavy chain. The antibodies blocked the capacity of kininogens to inhibit papain, suggesting that they recognize a conformational epitope at or near the kininogen inhibitory sites. The inhibitory properties of family 2 cystatins remained unchanged, confirming that members of this family do not interact with anti K12K antibodies. These antibodies are thus a new tool able to discriminate functionally and structurally between the members of the cystatin superfamily.

A comparison of the enzymatic properties of the major cysteine proteinases from Trypanosoma congolense and Trypanosoma cruzi.

Congopain and cruzipain, the major cysteine proteinases from Trypanosoma congolense and Trypanosoma cruzi, were compared for their activities towards a series of new, sensitive fluorogenic substrates of the papain family of cysteine proteinases and for their sensitivity to inhibition by cystatins and related biotinylated peptidyl diazomethanes. Low Ki values, in the 10 pM range, were found for the interaction of both proteinases with natural cystatin inhibitors. The kinetic constants for the hydrolysis of cystatin-derived substrates, and the inhibition by related diazomethanes were essentially identical. Unlike cathepsins B and L, the related mammal papain family proteinases, congopain and cruzipain accomodate a prolyl residue in P2'. Substrates having the sequence VGGP from P2 to P2' were hydrolysed by both congopain and cruzipain with a k(cat)/Km greater than 4.10(3) mM(-1) s(-1). Irreversible diazomethane inhibitors, deduced from the unprime sequence of cystatin-derived substrates, inhibited the two parasite proteinases. N-terminal labelling of diazomethanes with a biotin group did not alter the rate of inhibition significantly, which provides a useful tool for examining the distribution of these enzymes in the parasite and in the host. Despite their similar activities on cystatin-derived substrates, congopain and cruzipain had significantly different pH-activity profiles when assayed with a cystatin-derived substrate. They were correlated with structural differences, especially at the presumed S2 subsites.

Altered expression of cruzipain and a cathepsin B-like target in a Trypanosoma cruzi cell line displaying resistance to synthetic inhibitors of cysteine-proteinases.

The therapeutic potential of synthetic inhibitors to the major cysteine-proteinase from Trypanosoma cruzi (cruzain or cruzipain) was recently demonstrated in animal models of Chagas' disease. A possible limitation of this strategy would be the emergence of parasite populations developing resistance to cysteine-proteinase inhibitors. Here, we describe the properties of a phenotypically stable T. cruzi cell line (R-Dm28) that displays increased resistance to Z-(SBz)Cys-Phe-CHN2, an irreversible cysteine-proteinase inhibitor which preferentially inactivates cathepsin L-like enzymes. Isolated from axenic cultures of the parental cells (IC50 1.5 microM), R-Dm28 epimastigotes exhibited 13-fold (IC50) 20 microM) higher resistance to this inhibitor and did not display cross-resistance to unrelated trypanocidal drugs, such as benznidazol and nifurtimox. Western blotting (with mAb), affinity labeling (with biotin-LVG-CHN2) and FACS analysis of R-Dm28 log-phase epimastigotes revealed that the cruzipain target was expressed at lower levels, as compared with Dm28c. Interestingly, this deficit was paralleled by increased expression of an unrelated Mr 30 000 cysteine-proteinase whose activity was somewhat refractory to inhibition by Z-(SBz)Cys-Phe-CHN,. N-terminal sequencing of the affinity-purified biotin-LVG-proteinase complex allowed its identification as a cathepsin B-like enzyme. Increased antigenic deposits of this proteinase were found in the grossly enlarged and electron dense reservosomes from R-Dm28 epimastigotes. Our data suggest that R-Dm28 resistance to toxic effects induced by the synthetic inhibitor may result from decreased availability of the most sensitive cysteine-proteinase target, cruzipain. The deficit in metabolic functions otherwise mediated by this cathepsin L-like proteinase is likely compensated by increased expression/accumulation of a cathepsin B-like target.

Cysteine protease isoforms from Trypanosoma cruzi, cruzipain 2 and cruzain, present different substrate preference and susceptibility to inhibitors.

Cysteine-proteinases from parasitic protozoa have been recently characterized as factors of virulence and pathogenicity in several human and veterinary diseases. In Chagas' disease, the chronic infection caused by Trypanosoma cruzi, structure-functional studies on cysteine proteases were thus far limited to the parasite's major isoform, a cathepsin L-like lysosomal protease designated as cruzipain, cruzain or GP57/51. Encoded by a large gene family, cruzipain is efficiently targeted by synthetic inhibitors, which prevent parasite intracellular growth and differentiation. We have previously demonstrated that the multicopy cruzipain gene family includes polymorphic sequences, which could encode functionally different isoforms. We report here a comparative kinetic study between cruzain, the archetype of the cruzipain family, and an isoform, termed cruzipain 2, which is expressed preferentially by the mammalian stages of T. cruzi. Heterologous expression of the catalytic domain of cruzipain 2 in Saccharomyces cerevisae yielded an enzyme that differs markedly from cruzain with respect to pH stability, substrate specificity and sensitivity to inhibition by natural and synthetic inhibitors of cysteine proteases. We suggest that the structural-functional diversification imparted by genetic polymorphism of cruzipain genes may have contributed to T. cruzi adaptation to vertebrate hosts.

Immunisation of cattle with cysteine proteinases of Trypanosoma congolense: targetting the disease rather than the parasite.

In order to test the hypothesis that trypanosome cysteine proteinases (CPs) contribute to pathology of trypanosomosis, cattle were immunised with CP1 and/or CP2, the major CPs of Trypanosoma congolense, and subsequently challenged with T. congolense. Immunisation had no effect on the establishment of infection and the development of acute anaemia. However, immunised cattle, unlike control cattle, maintained or gained weight during infection. Their haematocrit and leukocyte counts showed a tendency to recovery after 2-3 months of infection. Cattle immunised with CP2 mounted early and prominent IgG responses to CPs and to the variable surface glycoprotein following challenge. Thus trypanosome CPs may play a role in anaemia and immunosuppression; conversely, anti-CP antibody may modulate the trypanosome-induced pathology.

Functional expression of the catalytic domains of two cysteine proteinases from Trypanosoma congolense.

The catalytic domains of two closely related cysteine proteinases (CP1 and CP2) from Trypanosoma congolense, referred to as C1 and C2, were expressed as proforms in Escherichia coli (C1) and in the baculovirus system (C1 and C2). While the bacterial expression system did not allow recovery of active C1, the baculovirus system led to secretion of inactive zymogens which could be processed at acidic pH into mature enzymes. Active C1 and C2 were purified from serum-free culture supernatants by anion-exchange chromatography and characterised. Their kinetic parameters and pH activity profiles confirmed the relatedness between C2 and native CP2 (congopain). These properties also underline major functional differences between C1 and C2, that appear to relate to discrete but essential sequence differences. It is likely that these two enzymes perform distinct roles in vivo, in the parasite and/or in the host-parasite relationships.

The occluding loop of cathepsin B prevents its effective inhibition by human kininogens.

Kininogens, the major plasma cystatin-like inhibitors of cysteine cathepsins, are degraded at sites of inflammation, and cathepsin B has been identified as a prominent mediator of this process. Cathepsin B, in contrast to cathepsins L and S, is poorly inhibited by kininogens. This led us to delineate the molecular interactions between this protease and kininogens (high molecular weight kininogen and low molecular weight kininogen) and to elucidate the dual role of the occluding loop in this weak inhibition. Cathepsin B cleaves high molecular weight kininogen within the N-terminal region of the D2 and D3 cystatin-like domains and close to the consensus QVVAG inhibitory pentapeptide of the D3 domain. The His110Ala mutant, unlike His111Ala cathepsin B, fails to hydrolyze kininogens, but rather forms a tight-binding complex as observed by gel-filtration analysis. K(i) values (picomolar range) as well as association rate constants for the His110Ala cathepsin B variant compare to those reported for cathepsin L for both kininogens. Homology modeling of isolated inhibitory (D2 and D3) domains and molecular dynamics simulations of the D2 domain complexed with wild-type cathepsin B and its mutants indicate that additional weak interactions, due to the lack of the salt bridge (Asp22-His110) and the subsequent open position of the occluding loop, increase the inhibitory potential of kininogens on His110Ala cathepsin B.

A virus essential for insect host-parasite interactions encodes cystatins.

Cotesia congregata is a parasitoid wasp that injects its eggs in the host caterpillar Manduca sexta. In this host-parasite interaction, successful parasitism is ensured by a third partner: a bracovirus. The relationship between parasitic wasps and bracoviruses constitutes one of the few known mutualisms between viruses and eukaryotes. The C. congregata bracovirus (CcBV) is injected at the same time as the wasp eggs in the host hemolymph. Expression of viral genes alters the caterpillar's immune defense responses and developmental program, resulting in the creation of a favorable environment for the survival and emergence of adult parasitoid wasps. Here, we describe the characterization of a CcBV multigene family which is highly expressed during parasitism and which encodes three proteins with homology to members of the cystatin superfamily. Cystatins are tightly binding, reversible inhibitors of cysteine proteases. Other cysteine protease inhibitors have been described for lepidopteran viruses; however, this is the first description of the presence of cystatins in a viral genome. The expression and purification of a recombinant form of one of the CcBV cystatins, cystatin 1, revealed that this viral cystatin is functional having potent inhibitory activity towards the cysteine proteases papain, human cathepsins L and B and Sarcophaga cathepsin B in assays in vitro. CcBV cystatins are, therefore, likely to play a role in host caterpillar physiological deregulation by inhibiting host target proteases in the course of the host-parasite interaction.

Cathepsin L, but not cathepsin B, is a potential kininogenase.

Although papain-like enzymes are strongly inhibited by their natural tight-binding inhibitors of the cystatin superfamily, cathepsins B and L may still retain some residual proteolytic activity toward Z-Phe-Arg-AMC in the presence of an excess of kininogen. This activity is abolished by adding E-64 or chicken cystatin. Cathepsins B and L show a single band of gelatinolytic activity when subjected to gelatin-SDS-PAGE. Adding high Mr kininogen, low Mr kininogen, T-kininogen, or chicken cystatin to cathepsin L results in additional intense bands of enzyme activity corresponding to the protease-inhibitor complexes. Cathepsin B does not produce these additional bands. This gelatinolytic activity was inhibited by E-64, but not by EDTA, PMSF or Pefabloc. Cathepsin L also specifically generated kinins from high and low molecular weight kininogens in vitro, but cathepsin B did not. T-kininogen did not release any immunoreactive kinins when complexed with cathepsin L, as previously observed using tissue kallikreins. The ability of cathepsin L to generate vasoactive peptides raises the question of the physiological significance of this mechanism during inflammation.

Discrimination between rat thiostatin (T-kininogen) and one of its cystatin-like inhibitory fragments by a monoclonal antibody, and localization of the epitope.

A monoclonal antibody (mAb D3) raised against rat thiostatin (T-kininogen) strongly interacted with a fragment, identified as cystatin-like domain 3, which inhibits cysteine proteinases but did not recognize intact, native thiostatin. The antigen-antibody reaction requires cleavage of the single peptide chain of thiostatin in its inter-domain 2-3 region. This mAb can also differentiate between the two molecular varieties of thiostatin, reacting only with immobilized domain 3 from T1 thiostatin, which differs from the T2 variety by only 10 out of 125 residues. mAb D3 did not react with an N-terminally truncated domain 3 of T1 thiostatin prepared by submaxillary gland kallikrein k10 proteolysis. This suggests that the epitope, or an essential part of it, is located on a stretch of 12 residues at the N-terminal of the T1 thiostatin domain 3. This sequence in T1 thiostatin differs from that in T2 thiostatin by four amino acids, two of which are arginyl residues in T1. Chemical modification of these residues located at positions 246 and 250 decreased the reactivity of T1 domain 3 towards the antibody, suggesting that at least one of them is a critical residue of the epitope. Arginine 246 is part of a small disulfide loop between cysteines 245 and 248 which is also necessary for antibody recognition. This antibody does not change the inhibitory properties of purified domain 3 towards papain or rat liver cathepsin L, indicating that the N-terminal part of domain 3 is not involved in inhibition. mAb D3 was used to demonstrate the presence of inhibitory thiostatin fragments in ascites fluid but not in plasma from normal or turpentine-injected rats.

Cystatin mimicry by synthetic peptides.

Synthetic peptides which tentatively mimic the cystatin inhibitory surface were used to study the mechanism of inhibition of cysteine proteinases by their natural inhibitors. The inhibitory properties of these peptides depend mainly on the presence of the QxVxG consensus sequence. N and C-terminal peptide derivatives bearing large hydrophobic groups showed dramatically improved inhibition. Molecular dynamic studies after energy minimization showed that the non covalent interaction between these hydrophobic groups induced the formation of a loop structure which probably favours inhibition. Antibodies were raised against one of these peptides, which recognized kininogens in the serum of all mammal species tested, but not cystatins from family two.

Investigation of the substrate specificity of cruzipain, the major cysteine proteinase of Trypanosoma cruzi, through the use of cystatin-derived substrates and inhibitors.

A panel of intramolecularly quenched fluorogenic substrates containing the conserved QVVA and LVG inhibitory sequences of cystatin inhibitors was used to describe the specificity of the major cysteine proteinase of Trypanosoma cruzi (cruzipain or cruzain). This approach was based on the observations that: (1) cruzipain is strongly inhibited by chicken cystatin and rat T-kininogen, two representative members of cystatin families 2 and 3; (2) the QVVA- and LVG-containing substrates are specifically hydrolysed by papain-like proteinases; and (3) the cystatin-like motifs are similar to the proteolytically sensitive sequences in cruzipain that separate the pro-region and/or the C-terminal extension from the catalytic domain. Specificity constants (kcat/Km) were determined and compared with those of mammalian cathepsins B and L from rat liver lysosomes. Cruzipain and the mammalian proteinases cleaved cystatin-derived substrates at the same site, but their specificities differed significantly. Increased specificity for cruzipain was obtained by replacing amino acids at critical positions on both sides of the cleavage sites, especially at position P2'. The specificity constants (k(cat)/Km) obtained for the two substrates with a prolyl residue at P2' (O-aminobenzoyl-QVVAGP-ethylenediamine 2-4-dinitrophenyl and O-aminobenzoyl-VVGGP-ethylenediamine 2-4-dinitrophenyl) were about 50 times higher for cruzipain than for rat cathepsin L and about 100 times higher than for cathepsin B. Diazomethylketone derivatives, based on the non-prime sequence of cystatin-derived substrates, inhibited cruzipain irreversibly, but their inactivation rate constants were considerably lower than those for mammalian cathepsins B and L, confirming the importance of P' residues for cruzipain specificity.

Biotin-labelled peptidyl diazomethane inhibitors derived from the substrate-like sequence of cystatin: targeting of the active site of cruzipain, the major cysteine proteinase of Trypanosoma cruzi.

Biotin-labelled peptidyl diazomethane inhibitors of cysteine proteinases, based on the N-terminal substrate-like segment of human cystatin C, a natural inhibitor of cysteine proteinases, were synthesized. These synthetic derivatives were tested as irreversible inhibitors of cruzipain, the major cysteine proteinase of Trypanosoma cruzi, to compare the kinetics of the inhibition of the parasite proteinase with that of the mammalian cathepsins B and L. The accessibility of the active sites of these proteinases to these probes was also investigated. The inhibition of cruzipain by Biot-LVG-CHN2 (where Biot represents biotinyl and L,V and G are single-letter amino acid residue abbreviations) and Biot-Ahx-LVG-CHN2 (where Ahx represents 6-aminohexanoic acid) was similar to that of unlabelled inhibitor. Biotin labelling of the inhibitor slowed the inhibition of both cathepsin B and cathepsin L. Adding a spacer arm (Ahx) between the biotin and the peptide moiety of the derivative increased the inhibition of cathepsin B but not that of cathepsin L. The discrimination provided by this spacer is probably due to differences in the topologies of the binding sites of proteinases, a feature that can be exploited to improve targeting of individual cysteine proteinases. Analysis of the blotted proteinases revealed marked differences in the accessibility of extravidin-peroxidase conjugate to the proteinase-bound biotinylated inhibitor. Cruzipain molecules exposed to Biot-LVG-CHN2 or Biot-Ahx-LVG-CHN2 were readily identified, but the reaction was much stronger when the enzyme was treated with the spacer-containing inhibitor. In contrast with the parasite enzyme, rat cathepsin B and cathepsin L treated with either Biot-LVG-CHN2 or Biot-Ahx-LVG-CHN2 produced no detectable bands. Papain, the archetype of this family of proteinases, was poorly labelled with Biot-LVG-CHN2, but strong staining was obtained with Biot-Ahx-LVG-CHN2. These findings suggest that optimized biotinylated diazomethanes might considerably improve their selectivity for the T. cruzi target enzyme.