Growth and development of Colorado potato beetle larvae, Leptinotarsa decemlineata, on potato plants expressing the oryzacystatin II proteinase inhibitor.

Plant proteinase inhibitors (PIs) are attractive tools for crop improvement and their heterologous expression can enhance insect resistance in transgenic plants. PI oryzacystatin II (OCII), isolated from rice, showed potential in controlling pests that utilize cysteine proteinases for protein digestion. To evaluate the applicability of the OCII gene in enhancing plant defence, OCII-transformed potatoes were bioassayed for resistance to Colorado potato beetle (Leptinotarsa decemlineata Say). Feeding on transformed leaves of potato cultivars Desiree and Jelica significantly affected larval growth and development, but did not change mortality rates. During the L2 and L3 developmental stages larvae consumed the OCII-transformed foliage faster as compared to the nontransformed control. Also these larvae reached the prepupal stage (end of L4 stage) 2 days earlier than those fed on control leaves. However, the total amounts of consumed OCII-transformed leaves were up to 23% lower than of control, and the maximal weights of prepupal larvae were reduced by up to 18% as compared to larvae fed on nontransformed leaves. The reduction in insect fitness reported in this study in combination with other control measures, could lead to improved CPB resistance management in potato.

(-)-epigallocatechin-3-gallate inhibits fibrillogenesis of chicken cystatin.

Previous studies have reported that (-)-epigallocatechin-3-gallate (EGCG), the most abundant flavonoid in green tea, can bind to unfolded native polypeptides and prevent conversion to amyloid fibrils. To elucidate whether this antifibril activity is specific to disease-related target proteins or is more generic, we investigated the ability of EGCG to inhibit amyloid fibril formation of amyloidogenic mutant chicken cystatin I66Q, a generic amyloid-forming model protein that undergoes fibril formation through a domain swapping mechanism. We demonstrated that EGCG was a potent inhibitor of amyloidogenic cystatin I66Q amyloid fibril formation in vitro. Computational analysis suggested that EGCG prevented amyloidogenic cystatin fibril formation by stabilizing the molecule in its native-like state as opposed to redirecting aggregation toward disordered and amorphous aggregates. Therefore, although EGCG appears to be a generic inhibitor of amyloid-fibril formation, the mechanism by which it achieves such inhibition may be specific to the target fibril-forming polypeptide.

Fungal gene-encoded peptidase inhibitors.

Peptidases can be inhibited by natural or synthetic small-molecule compounds, or by gene-encoded, proteinaceous inhibitors. Small-molecule peptidase inhibitors have been in the spotlight of researchers and pharmaceutical companies for many years. The studies concerning gene-encoded inhibitors are less frequent. The last decade has seen a boom of fungal genomics followed by extensive bioinformatic analyses focused particularly on those species that can cause infections in humans, animals or crops. Many sequences of putative inhibitors have been identified on the basis of homology with gene-encoded peptidase inhibitors of Saccharomyces cerevisiae, mammals or other organisms. However, characterization of the respective proteins is often missing. Gene-encoding peptidase inhibitors are rather diverse in size, mode of action, type of the target peptidase and localization. While some of the inhibitors are secreted to extracellular space and participate in host-pathogen interactions, others act intracellularly and their precise role in fungal physiology is not fully understood. However, most of the gene-encoded peptidase inhibitors are rather selective and efficient, and may be an inspiration for future directions of antimycotic research.

Aggregation and inactivation of pancreatic cystatin by riboflavin-derived singlet oxygen and flavin triplet state: polyphenols as preventive agents.

Caprine pancreatic thiol proteinase inhibitor (PTPI) a cystatin superfamily variant has high affinity for cysteine proteinases providing tight regulation of their proteolytic potential. Oxidative stress buildup in various pancreatic pathologies worsens the disease course often by disturbing the delicate balance between proteinases and their inhibitors. We aimed to study the effect of reactive oxygen species (ROS) on PTPI and to determine the potency of caffeic acid, curcumin and quercetin as agents against the inflicted damage. Fluorescence spectroscopy, polyacrylamide gel electrophoresis, and papain inhibitory assay revealed that photoilluminated riboflavin severely challenged the functional and structural integrity of the inhibitor. Three hundred and fifty micromolar affeic acid or quercetin prevented the damage. Curcumin, however, failed to reverse the changes completely. Conclusively, PTPI rendered dysfunctional by ROS may explain the increased necrotic damage to the host tissue. Also, dietary antioxidants can reverse the riboflavin-induced protein damage providing an economic and safe anti-ROS therapy.

The green tea polyphenol (-)-epigallocatechin gallate precipitates salivary proteins including alpha-amylase: biochemical implications for oral health.

Green tea is a popular drink throughout the world, and it contains various components, including the green tea polyphenol (-)-epigallocatechin gallate (EGCG). Tea interacts with saliva upon entering the mouth, so the interaction between saliva and EGCG interested us, especially with respect to EGCG-protein binding. SDS-PAGE revealed that several salivary proteins were precipitated after adding EGCG to saliva. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) peptide mass fingerprinting indicated that the major proteins precipitated by EGCG were alpha-amylase, S100, and cystatins. Surface plasmon resonance revealed that EGCG bound to alpha-amylase at dissociation constant (K(d)) = 2.74 × 10(-6) M, suggesting that EGCG interacts with salivary proteins with a relatively strong affinity. In addition, EGCG inhibited the activity of alpha-amylase by non-competitive inhibition, indicating that EGCG is effective at inhibiting the formation of fermentable carbohydrates involved in caries formation. Interestingly, alpha-amylase reduced the antimicrobial activity of EGCG against the periodontal bacterium Aggregatibacter actinomycetemcomitans. Therefore, we considered that EGCG-salivary protein interactions might have both protective and detrimental effects with respect to oral health.

Inhibition of invasion and metastasis of MHCC97H cells by expression of snake venom cystatin through reduction of proteinases activity and epithelial-mesenchymal transition.

Snake venom cystatin (sv-cystatin) is a member of the cystatin family of cysteine protease inhibitors. To further evaluate the possibility of sv-cystatin in cancer therapy, this study examined the effects of sv-cystatin on the invasion and metastasis of liver cancer cells (MHCC97H) in vitro and in vivo as well as the underlying mechanism. sv-cystatin cDNA was transfected into MHCC97H cells and the anti-invasion and antimetastasis effects of sv-cystatin were determined using migration and matrigel invasion assays and a lung-metastasis mice model. The results suggest that sv-cyst clone (sv-cystatin expression in MHCC97H cells) delayed the invasion and metastasis in vitro and in vivo compared to the parental, mock and si-sv-cyst clone cells (inhibited sv-cystatin expression by siRNA). The decreased activities of cathepsin B, MMP-2 and MMP-9 and EMT change index including higher E-cadherin, lower N-cadherin and decreased Twist activity were observed in the sv-cyst clone, which contributes to the change in invasion and metastasis ability of MHCC97H cells. This study provides evidence that expression of the sv-cystatin gene in MHCC97H cells inhibits tumor cell invasion and metastasis through the reduction of the proteinases activity and Epithelial-Mesenchymal Transition (EMT), which might contribute to the anticancer research of the sv-cystatin protein.

Preventive effect of curcumin and quercetin against nitric oxide mediated modification of goat lung cystatin.

Cysteine proteinase inhibitors are of prime physiologic importance inside the cells, controlling the activities of lysosomal cysteine proteases. The present work aimed to realize the effects of nitric oxide on the structure and function of goat lung cystatin (GLC) and to evaluate antinitrostative efficacy of curcumin and quercetin. Nitric oxide induced structural modifications were followed by fluorescence spectroscopy and PAGE and functional inactivation by monitoring the inhibition of caseinolytic activity of papain. Ten millimolar sodium nitroprusside (SNP) caused time dependent inactivation of GLC-I with complete functional loss precipitating at 180 min. Curcumin (50 microM) and quercetin (250 microM) opposed such loss in papain inhibitory activity of GLC-I. Loss in tertiary structure of GLC-I (fluorescence quenching and 15 nm red shift) was observed on SNP treatment. Inhibition of functional and structural SNP mediated damage of GLC-I by curcumin (50 microM) and quercetin (250 microM) reaffirms their NO scavenging potency.

DNA accelerates the inhibition of human cathepsin V by serpins.

A balance between proteolytic activity and protease inhibition is crucial to the appropriate function of many biological processes. There is mounting evidence for the presence of both papain-like cysteine proteases and serpins with a corresponding inhibitory activity in the nucleus. Well characterized examples of cofactors fine tuning serpin activity in the extracellular milieu are known, but such modulation has not been studied for protease-serpin interactions within the cell. Accordingly, we present an investigation into the effect of a DNA-rich environment on the interaction between model serpins (MENT and SCCA-1), cysteine proteases (human cathepsin V and human cathepsin L), and cystatin A. DNA was indeed found to accelerate the rate at which MENT inhibited cathepsin V, a human orthologue of mammalian cathepsin L, up to 50-fold, but unexpectedly this effect was primarily effected via the protease and secondarily by the recruitment of the DNA as a "template" onto which cathepsin V and MENT are bound. Notably, the protease-mediated effect was found to correspond both with an altered substrate turnover and a conformational change within the protease. Consistent with this, cystatin inhibition, which relies on occlusion of the active site rather than the substrate-like behavior of serpins, was unaltered by DNA. This represents the first example of modulation of serpin inhibition of cysteine proteases by a co-factor and reveals a mechanism for differential regulation of cathepsin proteolytic activity in a DNA-rich environment.

[Usefulness of equations based on serum cystatin C concentration in the study of renal function]. / Utilidad de las ecuaciones basadas en la concentracion serica de cistatina C en el estudio de la funcion renal.

Serum creatinine is an insensitive marker to identify early changes in glomerular filtration rate (GFR), for this reason alternative methods to estimate renal function result of great clinical importance. Forty-one patients were studied using creatinine clearance modified with cimetidina (Clcrc) as surrogate of GFR, cystatin C-based equations (i.e. Larsson and Hoek formulas), Cockroft-Gault and MDRD abbreviated equations. In the whole group, as well as in those patients with serum creatinine < or =1.2 mg/dl--but reduced renal function: Clcrc 62.01 +/- 17.33 ml/ min/1.73 m(2)-, Larsson and Hoek equations showed higher correlations and lower bias than creatinine-based formulas. Abbreviated MDRD equation showed good performance just in those patients with evident alteration of renal function (serum creatinine > 1.2 mg/dl). We concluded that in patients with different stages of renal function, cystatin C-based equations detect reduction of renal function earlier than the serum creatinine-based formulas.

[Carminerin contributes to chondrocyte calcification during pathological endochondral ossification].

Endochondral ossification is an essential process not only for physiological skeletal development and growth, but also for pathological disorders such as osteophyte formation in osteoarthritic joints and ectopic ossification with ageing. We recently identified a novel cartilage-specific molecule carminerin. Although the carminerin-deficient mice developed and grew normally, pathological endochondral ossification were suppressed. We conclude that carminerin contributes to chondrocyte calcification during pathological endochondral ossification without affecting physiological skeletal conditions, suggesting that this molecule could be a therapeutic target for these disorders.

Down-regulation of human extracellular cysteine protease inhibitors by the secreted staphylococcal cysteine proteases, staphopain A and B.

Of seven human cystatins investigated, none inhibited the cysteine proteases staphopain A and B secreted by the human pathogen Staphylococcus aureus. Rather, the extracellular cystatins C, D and E/M were hydrolyzed by both staphopains. Based on MALDI-TOF time-course experiments, staphopain A cleavage of cystatin C and D should be physiologically relevant and occur upon S. aureus infection. Staphopain A hydrolyzed the Gly11 bond of cystatin C and the Ala10 bond of cystatin D with similar Km values of approximately 33 and 32 microM, respectively. Such N-terminal truncation of cystatin C caused >300-fold lower inhibition of papain, cathepsin B, L and K, whereas the cathepsin H activity was compromised by a factor of ca. 10. Similarly, truncation of cystatin D caused alleviated inhibition of all endogenous target enzymes investigated. The normal activity of the cystatins is thus down-regulated, indicating that the bacterial enzymes can cause disturbance of the host protease-inhibitor balance. To illustrate the in vivo consequences, a mixed cystatin C assay showed release of cathepsin B activity in the presence of staphopain A. Results presented for the specificity of staphopains when interacting with cystatins as natural protein substrates could aid in the development of therapeutic agents directed toward these proteolytic virulence factors.

Frequent epigenetic inactivation of cystatin M in breast carcinoma.

Cystatin M is a potent endogenous inhibitor of lysosomal cysteine proteases. In breast carcinoma, cystatin M expression is frequently downregulated. It has been shown that cystatin M expression suppressed growth and migration of breast cancer cells. We examined the methylation status of the CpG island promoter of cystatin M in four breast cancer cell lines (MDAMB231, ZR75-1, MCF7 and T47D), in 40 primary breast carcinoma and in corresponding normal tissue probes by combined bisulphite restriction analysis. To investigate the effects of cystatin M expression on the growth of breast carcinoma, cystatin M was transfected in T47D. The cystatin M promoter was highly methylated in all four-breast cancer cell lines. Primary breast tumours were significantly more frequently methylated compared to normal tissue samples (60 vs 25%; P=0.006 Fisher's exact test). Treatment of breast cancer cells with 5-aza-2'-deoxycytidine (5-Aza-CdR), reactivated the transcription of cystatin M. Transfection of breast carcinoma cells with cystatin M caused a 30% decrease in colony formation compared to control transfection (P=0.002). Our results show that cystatin M is frequently epigenetically inactivated during breast carcinogenesis and cystatin M expression suppresses the growth of breast carcinoma. These data suggest that cystatin M may encode a novel epigenetically inactivated candidate tumour suppressor gene.

Utilidad de las ecuaciones basadas en la concentración sérica de cistatina C en el estudio de la función renal / Usefulness of equations based on serum cystatin C concentration in the study of renal function

La creatinina sérica es un marcador poco sensible para identificar reducciones leves del índice de filtración glomerular (IFG); por ello resulta de gran importancia clínica disponer de métodos alternativos para estimar la función renal. Con este objetivo estudiamos la función renal de 41 pacientes -grupo completo y divididos según la creatinina sérica (menor o igual 1.2 mg/dl o mayores)- usando el clearance de creatinina modificado con cimetidina (Clcrc) como aproximación al IFG, las ecuaciones de Larsson y Hoek que incluyen el uso de cistatina C sérica y las tradicionales fórmulas de Cockroft-Gault y MDRD abreviada. En el grupo completo de pacientes y especialmente en aquellos con creatinina sérica menor o igual 1.2 mg/dl - con reducción de la función renal: Clcrc: 62.01 mas o menos 17.33 ml/min/1.73 m2-, las ecuaciones de Larsson y Hoek mostraron mejores correlaciones y menores diferencias promedio respecto a las fórmulas basadas en la creatinina sérica. La ecuación MDRD abreviada mostró buen rendimiento sólo en el grupo con evidente alteración de la función renal (creatinina sérica > 1.2 mg/dl). Concluimos que en pacientes con diferentes estadios de función renal, las fórmulas que emplean la cistatina C sérica detectan la reducción del IFG más precozmente respecto a aquellas basadas en la creatinina sérica.

Serum creatinine is an insensitive marker to identify early changes in glomerular filtration rate (GFR), for this reason alternative methods to estimate renal function result of great clinical importance. Forty-one patients were studied using creatinine clearance modified with cimetidina (Clcrc) as surrogate of GFR, cystatin C-based equations (i.e. Larsson and Hoek formulas), Cockroft-Gault and MDRD abbreviated equations. In the whole group, as well as in those patients with serum creatinine less than or equal to 1.2 mg/dl -but reduced renal function: Clcrc 62.01 more or less 17.33 ml/min/1.73 m2-, Larsson and Hoek equations showed higher correlations and lower bias than creatinine-based formulas. Abbreviated MDRD equation showed good performance just in those patients with evident alteration of renal function (serum creatinine > 1.2 mg/dl). We concluded that in patients with different stages of renal function, cystatin C-based equations detect reduction of renal function earlier than the serum creatinine-based formulas.

Utilidad de las ecuaciones basadas en la concentración sérica de cistatina C en el estudio de la función renal / Usefulness of equations based on serum cystatin C concentration in the study of renal function

La creatinina sérica es un marcador poco sensible para identificar reducciones leves del índice de filtración glomerular (IFG); por ello resulta de gran importancia clínica disponer de métodos alternativos para estimar la función renal. Con este objetivo estudiamos la función renal de 41 pacientes -grupo completo y divididos según la creatinina sérica (menor o igual 1.2 mg/dl o mayores)- usando el clearance de creatinina modificado con cimetidina (Clcrc) como aproximación al IFG, las ecuaciones de Larsson y Hoek que incluyen el uso de cistatina C sérica y las tradicionales fórmulas de Cockroft-Gault y MDRD abreviada. En el grupo completo de pacientes y especialmente en aquellos con creatinina sérica menor o igual 1.2 mg/dl - con reducción de la función renal: Clcrc: 62.01 mas o menos 17.33 ml/min/1.73 m2-, las ecuaciones de Larsson y Hoek mostraron mejores correlaciones y menores diferencias promedio respecto a las fórmulas basadas en la creatinina sérica. La ecuación MDRD abreviada mostró buen rendimiento sólo en el grupo con evidente alteración de la función renal (creatinina sérica > 1.2 mg/dl). Concluimos que en pacientes con diferentes estadios de función renal, las fórmulas que emplean la cistatina C sérica detectan la reducción del IFG más precozmente respecto a aquellas basadas en la creatinina sérica.(AU)

Serum creatinine is an insensitive marker to identify early changes in glomerular filtration rate (GFR), for this reason alternative methods to estimate renal function result of great clinical importance. Forty-one patients were studied using creatinine clearance modified with cimetidina (Clcrc) as surrogate of GFR, cystatin C-based equations (i.e. Larsson and Hoek formulas), Cockroft-Gault and MDRD abbreviated equations. In the whole group, as well as in those patients with serum creatinine less than or equal to 1.2 mg/dl -but reduced renal function: Clcrc 62.01 more or less 17.33 ml/min/1.73 m2-, Larsson and Hoek equations showed higher correlations and lower bias than creatinine-based formulas. Abbreviated MDRD equation showed good performance just in those patients with evident alteration of renal function (serum creatinine > 1.2 mg/dl). We concluded that in patients with different stages of renal function, cystatin C-based equations detect reduction of renal function earlier than the serum creatinine-based formulas.(AU)

Purification and characterization of high molecular mass and low molecular mass cystatin from goat brain.

Cystatin are thiol proteinase inhibitors ubiquitously present in mammalian body and serve various important physiological functions. In the present study two cystatins were isolated from goat brain using alkaline treatment, ammonium sulphate fractionation, gel filtration and ion exchange chromatography. The high molecular mass cystatin of 70.8 kDa was named as HM-GBC (high molecular mass goat brain cystatin) and the low molecular mass cystatin of 12.72 kDa was named as LM-GBC (low molecular mass goat brain cystatin). The molecular mass determined by SDS-PAGE was found to be 70.8 and 12.88 kDa for HM-GBC and LM-GBC, respectively, however with gel filtration the masses were found to be 70.8 and 12.58 kDa. Both the cystatins were found to be stable in broad range of pH and temperature. HM-GBC was found to have 2% carbohydrate content while LM-GBC lacks any carbohydrate content. Both cystatins were found to be devoid of any sulphydryl content. Stoke's radii of 36 and 16 A, and diffusion coefficient of 6.189 x 10(-15) and 1.392 x 10(-14) cm(2)/s were calculated for HM-GBC and LM-GBC. K (i) values with papain were found to be 1.875 x 10(-8) and 3.125 x 10(-8) M for HM-GBC and LM-GBC, respectively. K (+1), K (-1) and half-life calculated along with K (i) values obtained showed that HM-GBC inhibited papain more specifically as compared to LM-GBC. The IC(50) values obtained for HM-GBC and LM-GBC also showed that HM-GBC binds more effectively to papain than LM-GBC. Ultraviolet and fluorescence spectra indicated that upon formation of papain-HM-GBC/LM-GBC complex there is significant conformational change after interaction in one or both the proteins of the complex.

Cystatin B as an intracellular modulator of bone resorption.

Degradation of organic bone matrix requires proteinase activity. Cathepsin K is a major osteoclast proteinase needed for bone resorption, although osteoclasts also express a variety of other cysteine- and matrix metalloproteinases that are involved in bone remodellation. Cystatin B, an intracellular cysteine proteinase inhibitor, exhibits a lysosomal distribution preferentially in osteoclasts but it's role in osteoclast physiology has remained unknown. The current paper describes a novel regulatory function for cystatin B in bone-resorbing osteoclasts in vitro. Rat osteoclasts were cultured on bovine bone and spleen-derived cystatin B was added to the cultures. Nuclear morphology was evaluated and the number of actively resorbing osteoclasts and resorption pits was counted. Intracellular cathepsin K and tartrate-resistant acid phosphatase (TRACP) activities were monitored using fluorescent enzyme substrates and immunohistology was used to evaluate distribution of cystatin B in rat metaphyseal bone. Microscopical evaluation showed that cystatin B inactivated osteoclasts, thus resulting in impaired bone resorption. Cathepsin K and TRACP positive vesicles disappeared dose-dependently from the cystatin B-treated osteoclasts, indicating a decreased intracellular trafficking of bone degradation products. At the same time, cystatin B protected osteoclasts from experimentally induced apoptosis. These data show for the first time that, in addition to regulating cysteine proteinase activity and promoting cell survival in the nervous system, cystatin B inhibits bone resorption by down-regulating intracellular cathepsin K activity despite increased osteoclast survival.

Neuronal cell death induced by cystatin C in vivo and in cultured human CNS neurons is inhibited with cathepsin B.

Cystatin C, a cysteine protease inhibitor, is implicated in pathogenesis of late-onset Alzheimer's disease and other neurological disorders. Our recent study showed that cystatin C injection into rat hippocampus induced neuronal cell death in granule cell layer of dentate gyrus in vivo. We further confirmed that cystatin C neurotoxicity was inhibited by simultaneous coapplication of cathepsin B, a cysteine protease. In vitro cytotoxicity was also studied in cultures of human CNS neurons, mixed cultures with astrocytes and A1 human hybrid neurons. Cystatin C induced neuronal cell death in a dose-dependent manner, which accompanied increased number of TUNEL (+) cells, up-regulation of active caspase-3 and DNA ladder. The results of the present study indicate that cystatin C participates in the process of apoptotic neuronal cell death in experimental conditions by means of inhibitory activity of cysteine proteases, and that cystatin C might be involved in the pathogenesis in human neurological disorders including Alzheimer's disease.

Biochemical and biophysical changes induced by fungicide sodium diethyl dithiocarbamate (SDD), in phytocystatin purified from Phaseolus mungo (Urd): a commonly used Indian legume.

Phytocystatins are the plant thiol protease inhibitors involved in several reaction mechanisms of the plant system like regulation of proteolytic activity and storage of proteins. Biochemical and biophysical changes induced by fungicide SDD in phytocystatin purified from Phaseolus mungo have been investigated in terms of mass spectroscopy, Fourier transform infrared spectroscopy, and fluorescence spectroscopy, at pH 7.0, with varying fungicide concentrations (1-9 mM) and a time of incubation ranging from 2 to 8 h at 37 degrees C, with a fixed cystatin concentration (1.5 mM). Reactive oxygen species responsible for inhibitor damage were also investigated, and thiourea was found to scavenge the free radicals generated by SDD. FTIR analysis indicates a significant conformational transition from alpha-helix to beta-sheet structure; quenching of fluorescence is evident by fluorescence spectroscopy. The activity assay showed a decrease in inhibitory activity, as well as a fragmentation of the inhibitor was observed in electrophoresis. Results obtained implicate that exposure of phytocystatins to SDD involves physicochemical changes in cystatins leading to damage and a decrease in the activity of the inhibitor.

Azapeptides structurally based upon inhibitory sites of cystatins as potent and selective inhibitors of cysteine proteases.

A series of azapeptides as potential inhibitors of cysteine proteases were synthesized. Their structures, based on the binding center of cystatins, contain an azaglycine residue (Agly) in place of the evolutionarily conserved glycine residue in the N-terminal part of the enzyme binding region of cystatins. Incorporation of Agly should lead to deactivation of the acyl-enzyme complex formed against nucleophilic attack by water molecules in the final step of peptide bond hydrolysis. The majority of synthesized azapeptides shows high inhibitory potency toward the investigated cysteine proteases, papain, cathepsin B, and cathepsin K. One of them, Z-Arg-Leu-Val-Agly-Ile-Val-OMe (compound 17), which contains in its sequence the amino acid residues from the N-terminal binding segment as well as the hydrophobic residues from the first binding loop of human cystatin C, proved to be a highly potent and selective inhibitor of cathepsin B. It inhibits cathepsin B with a K(i) value of 0.088 nM. To investigate the influence of the structure of compound 17 for its inhibitory properties, we determined its conformation by means of NMR studies and theoretical calculations. The Z-Arg-Leu-Val-Agly fragment, covalently linked to Cys29 of cathepsin B, was also developed and modeled, in the catalytic pocket of the enzyme, through a molecular dynamics approach, to analyze ligand-protein interactions in detail. Analysis of the simulation trajectories generated using the AMBER force field provided us with atomic-level understanding of the conformational variability of this inhibitor, which is discussed in the context of other experimental and theoretical data.