Short-term exposure to cigarette smoke upregulates cathepsin S and alters expression of tight junction ZO-1.

A long-term exposure to cigarette smoke (CS) alters the integrity of airway epithelial barrier, contributes to lung dysfunction, and elicits the expression and activity of lung cathepsin S (CatS), a cysteine protease that participates in the remodeling of connective tissue and cell junctions. Here, we observed that a short-term (4 days) exposure of mice to CS increased the expression and activity of CatS, while the expression level of zonula occludens 1 (ZO-1), an epithelial tight junction protein that stabilizes barrier assembly, was reduced in lung tissue lysates. Present data support that proteolytically active CatS may contribute to the defect of ZO-1 in CS-exposed mice.

The Interplay of Glycosaminoglycans and Cysteine Cathepsins in Mucopolysaccharidosis.

Mucopolysaccharidosis (MPS) consists of a group of inherited lysosomal storage disorders that are caused by a defect of certain enzymes that participate in the metabolism of glycosaminoglycans (GAGs). The abnormal accumulation of GAGs leads to progressive dysfunctions in various tissues and organs during childhood, contributing to premature death. As the current therapies are limited and inefficient, exploring the molecular mechanisms of the pathology is thus required to address the unmet needs of MPS patients to improve their quality of life. Lysosomal cysteine cathepsins are a family of proteases that play key roles in numerous physiological processes. Dysregulation of cysteine cathepsins expression and activity can be frequently observed in many human diseases, including MPS. This review summarizes the basic knowledge on MPS disorders and their current management and focuses on GAGs and cysteine cathepsins expression in MPS, as well their interplay, which may lead to the development of MPS-associated disorders.

Binding of heparan sulfate to human cystatin C modulates inhibition of cathepsin L: Putative consequences in mucopolysaccharidosis.

Mucopolysaccharidoses (MPS) are a group of rare lysosomal storage diseases characterized by glycosaminoglycan (GAG) accumulation causing progressive multi-organs dysfunction and ultimately severe cardio-respiratory damages. Human cystatin C (hCC), a potent inhibitor of cysteine cathepsins, plays an important role in respiratory diseases. However, its regulation remained unknown in MPS. Herein, elevated hCC levels were measured in respiratory specimens from MPS-I, -II, and -III patients and were significantly correlated with severe respiratory symptoms (rs = 0.7173). Heparan sulfate (HS), a prominent GAG, dampened its inhibitory activity toward cathepsin L in a dose-dependent manner. HS and HS-oligosaccharides bound tightly hCC, in combination with a secondary structure rearrangement. Molecular modeling studies identified three HS binding regions in hCC, including the N-terminus, which is crucial in the inhibition of cathepsins. Impairment of inhibitory potential of hCC may reflect abnormal regulation of proteolytic activity of cathepsin L in lung, ultimately contributing to the severity of MPS.

Cathepsin V: Molecular characteristics and significance in health and disease.

Human cysteine cathepsins form a family of eleven proteases (B, C, F, H, K, L, O, S, V, W, X/Z) that play important roles in a considerable number of biological and pathophysiological processes. Among them, cathepsin V, also known as cathepsin L2, is a lysosomal enzyme, which is mainly expressed in cornea, thymus, heart, brain, and skin. Cathepsin V is a multifunctional endopeptidase that is involved in both the release of antigenic peptides and the maturation of MHC class II molecules and participates in the turnover of elastin fibrils as well in the cleavage of intra- and extra-cellular substrates. Moreover, there is increasing evidence that cathepsin V may contribute to the progression of diverse diseases, due to the dysregulation of its expression and/or its activity. For instance, increased expression of cathepsin V is closely correlated with malignancies (breast cancer, squamous cell carcinoma, or colorectal cancer) as well vascular disorders (atherosclerosis, aortic aneurysm, hypertension) being the most prominent examples. This review aims to shed light on current knowledge on molecular aspects of cathepsin V (genomic organization, protein structure, substrate specificity), its regulation by protein and non-protein inhibitors as well to summarize its expression (tissue and cellular distribution). Then the core biological and pathophysiological roles of cathepsin V will be depicted, raising the question of its interest as a valuable target that can open up pioneering therapeutic avenues.

Cleavage of Occludin by Cigarette Smoke-Elicited Cathepsin S Increases Permeability of Lung Epithelial Cells.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is an irreversible disease mainly caused by smoking. COPD is characterized by emphysema and chronic bronchitis associated with enhanced epithelial permeability. HYPOTHESIS: Lung biopsies from smokers revealed a decreased expression level of occludin, which is a protein involved in the cohesion of epithelial tight junctions. Moreover, the occludin level correlated negatively with smoking history (pack-years), COPD grades, and cathepsin S (CatS) activity. Thus, we examined whether CatS could participate in the modulation of the integrity of human lung epithelial barriers. METHODS AND RESULTS: Cigarette smoke extract (CSE) triggered the upregulation of CatS by THP-1 macrophages through the mTOR/TFEB signaling pathway. In a co-culture model, following the exposure of macrophages to CSE, an enhanced level of permeability of lung epithelial (16HBE and NHBE) cells towards FITC-Dextran was observed, which was associated with a decrease in occludin level. Similar results were obtained using 16HBE and NHBE cells cultured at the air-liquid interface. The treatment of THP-1 macrophages by CatS siRNAs or by a pharmacological inhibitor restored the barrier function of epithelial cells, suggesting that cigarette smoke-elicited CatS induced an alteration of epithelial integrity via the proteolytic injury of occludin. CONCLUSIONS: Alongside its noteworthy resistance to oxidative stress induced by cigarette smoke oxidants and its deleterious elastin-degrading potency, CatS may also have a detrimental effect on the barrier function of epithelial cells through the cleavage of occludin. The obtained data emphasize the emerging role of CatS in smoking-related lung diseases and strengthen the relevance of targeting CatS in the treatment of emphysema and COPD.

Upregulation of gut cathepsin L during Eimeria tenella infection.

Coccidiosis is a disease caused by Eimeria, which represents the first parasitic disease in poultry farming. Among them, E. tenella is a virulent species which specifically colonizes the caecum. The inflammatory response to infection is associated to numerous host proteases including cysteine cathepsins that can be deleterious for tissue and innate immunity integrity. Here, germ-free and conventional chickens were used as models to find out whether the microbiota could modify the intestinal expression of host cysteine cathepsins during coccidiosis. The basal caecal peptidase activity primarily relies on host proteases rather than proteases from the commensal flora. While mRNA levels of E. tenella cathepsins B and L remained unchanged in germ-free and conventional broilers, an overall increase in endopeptidase activity of cysteine cathepsins was found in E. tenella-infected caeca in both experimental models (P < 0.005). A significant decrease in avian cystatin C transcription was also observed in infected conventional, but not in infected germ-free broilers. Despite an unchanged mRNA level of avian cathepsin L (CatL), its protein expression raised following infection, in parallel with an increased transcription of antimicrobial ß-defensins (AvBD1, AvBD2, AvBD4, AvBD6, and AvBD7). Taken together, data support that host CatL is post-translationally upregulated during E. tenella infection, and thus may be involved in the alteration of the gut proteolytic balance. Furthermore, CatL may participate to inflammation occurring during coccidiosis through its known ability to proteolytically inactivates up-regulated avian ß-defensins that are key molecules of innate immunity.

Cystatin M/E (Cystatin 6): A Janus-Faced Cysteine Protease Inhibitor with Both Tumor-Suppressing and Tumor-Promoting Functions.

Alongside its contribution in maintaining skin homeostasis and its probable involvement in fetal and placental development, cystatin M/E (also known as cystatin 6) was first described as a tumor suppressor of breast cancer. This review aims to provide an update on cystatin M/E with particular attention paid to its role during tumorigenesis. Cystatin M/E, which is related to type 2 cystatins, displays the unique property of being a dual tight-binding inhibitor of both legumain (also known as asparagine endopeptidase) and cysteine cathepsins L, V and B, while its expression level is epigenetically regulated via the methylation of the CST6 promoter region. The tumor-suppressing role of cystatin M/E was further reported in melanoma, cervical, brain, prostate, gastric and renal cancers, and cystatin M/E was proposed as a biomarker of prognostic significance. Contrariwise, cystatin M/E could have an antagonistic function, acting as a tumor promoter (e.g., oral, pancreatic cancer, thyroid and hepatocellular carcinoma). Taking into account these apparently divergent functions, there is an urgent need to decipher the molecular and cellular regulatory mechanisms of the expression and activity of cystatin M/E associated with the safeguarding homeostasis of the proteolytic balance as well as its imbalance in cancer.

The abnormal accumulation of heparan sulfate in patients with mucopolysaccharidosis prevents the elastolytic activity of cathepsin V.

Mucopolysaccharidosis (MPS) are rare inherited diseases characterized by accumulation of lysosomal glycosaminoglycans, including heparan sulfate (HS). Patients exhibit progressive multi-visceral dysfunction and shortened lifespan mainly due to a severe cardiac/respiratory decline. Cathepsin V (CatV) is a potent elastolytic protease implicated in extracellular matrix (ECM) remodeling. Whether CatV is inactivated by HS in lungs from MPS patients remained unknown. Herein, CatV colocalized with HS in MPS bronchial epithelial cells. HS level correlated positively with the severity of respiratory symptoms and negatively to the overall endopeptidase activity of cysteine cathepsins. HS bound tightly to CatV and impaired its activity. Withdrawal of HS by glycosidases preserved exogenous CatV activity, while addition of Surfen, a HS antagonist, restored elastolytic CatV-like activity in MPS samples. Our data suggest that the pathophysiological accumulation of HS may be deleterious for CatV-mediated ECM remodeling and for lung tissue homeostasis, thus contributing to respiratory disorders associated to MPS diseases.

Regulation of the Proteolytic Activity of Cysteine Cathepsins by Oxidants.

Besides their primary involvement in the recycling and degradation of proteins in endo-lysosomal compartments and also in specialized biological functions, cysteine cathepsins are pivotal proteolytic contributors of various deleterious diseases. While the molecular mechanisms of regulation via their natural inhibitors have been exhaustively studied, less is currently known about how their enzymatic activity is modulated during the redox imbalance associated with oxidative stress and their exposure resistance to oxidants. More specifically, there is only patchy information on the regulation of lung cysteine cathepsins, while the respiratory system is directly exposed to countless exogenous oxidants contained in dust, tobacco, combustion fumes, and industrial or domestic particles. Papain-like enzymes (clan CA, family C1, subfamily C1A) encompass a conserved catalytic thiolate-imidazolium pair (Cys25-His159) in their active site. Although the sulfhydryl group (with a low acidic pKa) is a potent nucleophile highly susceptible to chemical modifications, some cysteine cathepsins reveal an unanticipated resistance to oxidative stress. Besides an introductory chapter and peculiar attention to lung cysteine cathepsins, the purpose of this review is to afford a concise update of the current knowledge on molecular mechanisms associated with the regulation of cysteine cathepsins by redox balance and by oxidants (e.g., Michael acceptors, reactive oxygen, and nitrogen species).

Oxidation of cathepsin S by major chemicals of cigarette smoke.

Lung cysteine cathepsin S (CatS) that is a potent elastase plays a deleterious role in alveolar remodeling during smoke-induced emphysema. Despite the presence of a reactive nucleophilic cysteine (Cys25) within its active site, most of its elastinolytic activity is preserved after exposure to cigarette smoke extract (CSE), a major source of sulfhydryl oxidants. This result led us to decipher CatS resistance to major and representative CSE oxidants: hydrogen peroxide, formaldehyde, acrolein and peroxynitrite. CatS was inactivated by hydrogen peroxide, peroxynitrite and acrolein in a time- and dose-dependent manner, while formaldehyde was a weaker oxidant. Hydrogen peroxide, but not CSE, formaldehyde, and peroxynitrite impaired the autocatalytic maturation of pro-CatS, whereas acrolein prevented the formation of mature CatS without hindering the initial step of the two-step autocatalytic process. Far-UV CD spectra analysis supported that oxidation by CSE and hydrogen peroxide did not led to a structural alteration of CatS, despite a notable increase of protein carbonylation, a major hallmark of oxidative damage. Evaluation of the oxidation status of Cys25 by specific biotinylated redox sensing probes suggested the formation of sulfenic acid followed by a slower conversion to sulfinic acid after incubation with hydrogen peroxide. Addition of reducing reagents (dithiothreitol, glutathione and N-acetyl cysteine) led to a partial recovery of CatS activity following incubation with CSE, hydrogen peroxide and peroxynitrite. Current results provide some mechanistic evidence of CatS stability and activity in the presence of CSE, supporting its harmful contribution to the pathophysiology of emphysema.

Rat cathepsin K: Enzymatic specificity and regulation of its collagenolytic activity.

Human cathepsin K (hCatK), which is highly expressed in osteoclasts, has the noteworthy ability to cleave type I and II collagens in their helical domain. Its collagenase potency depends strictly on the formation of an oligomeric complex with chondroitin 4-sulfate (C4-S). Accordingly, hCatK is a pivotal protease involved in bone resorption and is an attractive target for the treatment of osteoporosis. As rat is a common animal model for the evaluation of hCatK inhibitors, we conducted a comparative analysis of rat CatK (rCatK) and hCatK, which share a high degree of identity (88%) and similarity (93%). The pH activity profile of both enzymes displayed a similar bell-shaped curve (optimal pH: 6.4). Presence of Ser134 and Val160 in the S2 pocket of rCatK instead of Ala and Leu residues, respectively, in hCatK, led to a weaker peptidase activity, as observed for mouse CatK. Also, regardless of the presence of C4-S, rCatK cleaved in the nonhelical telopeptide regions of both type I (tail) and type II (articular joint) rat collagens. Structure-based computational analyses (electrostatic potential, molecular docking, molecular dynamics, free energy calculations) sustained that the C4-S mediated collagenolytic activity of rCatK obeys distinct molecular interactions from those of hCatK. Additionally, T-kininogen (a.k.a. thiostatin), a unique rat serum acute phase molecule, acted as a tight-binding inhibitor of hCatK (Ki = 0.11 ± 0.05 nM). Taken into account the increase of T-Kininogen level in inflamed rat sera, this may raise the question of the appropriateness to evaluate pharmacological hCatK inhibitors in this peculiar animal model.

Cigarette smoke induces overexpression of active human cathepsin S in lungs from current smokers with or without COPD.

Cigarette smoking has marked effects on lung tissue, including induction of oxidative stress, inflammatory cell recruitment, and a protease/antiprotease imbalance. These effects contribute to tissue remodeling and destruction resulting in loss of lung function in chronic obstructive pulmonary disease (COPD) patients. Cathepsin S (CatS) is a cysteine protease that is involved in the remodeling/degradation of connective tissue and basement membrane. Aberrant expression or activity of CatS has been implicated in a variety of diseases, including arthritis, cancer, cardiovascular, and lung diseases. However, little is known about the effect of cigarette smoking on both CatS expression and activity, as well as its role in smoking-related lung diseases. Here, we evaluated the expression and activity of human CatS in lung tissues from never-smokers and smokers with or without COPD. Despite the presence of an oxidizing environment, CatS expression and activity were significantly higher in current smokers (both non-COPD and COPD) compared with never-smokers, and correlated positively with smoking history. Moreover, we found that the exposure of primary human bronchial epithelial cells to cigarette smoke extract triggered the activation of P2X7 receptors, which in turns drives CatS upregulation. The present data suggest that excessive CatS expression and activity contribute, beside other proteases, to the deleterious effects of cigarette smoke on pulmonary homeostasis.

Processing and Maturation of Cathepsin C Zymogen: A Biochemical and Molecular Modeling Analysis.

Cysteine cathepsin C (CatC) is a ubiquitously expressed, lysosomal aminopeptidase involved in the activation of zymogens of immune-cell-associated serine proteinases (elastase, cathepsin G, proteinase 3, neutrophil serine proteinase 4, lymphocyte granzymes, and mast cell chymases). CatC is first synthetized as an inactive zymogen containing an intramolecular chain propeptide, the dimeric form of which is processed into the mature tetrameric form by proteolytic cleavages. A molecular modeling analysis of proCatC indicated that its propeptide displayed a similar fold to those of other lysosomal cysteine cathepsins, and could be involved in dimer formation. Our in vitro experiments revealed that human proCatC was processed and activated by CatF, CatK, and CatV in two consecutive steps of maturation, as reported for CatL and CatS previously. The unique positioning of the propeptide domains in the proCatC dimer complex allows this order of cleavages to be understood. The missense mutation Leu172Pro within the propeptide region associated with the Papillon-Lefèvre and Haim-Munk syndrome altered the proform stability as well as the maturation of the recombinant Leu172Pro proform.

Imaging of extracellular cathepsin S activity by a selective near infrared fluorescence substrate-based probe.

We designed a near-infrared fluorescent substrate-based probe (SBP), termed MG101, for monitoring extracellular cathepsin S (CatS) activity. We conceived a fused peptide hairpin loop-structure, combining a CatS recognition domain, an electrostatic zipper (with complementary charges of a polyanionic (D-Glu)5 segment and a polycationic (D-Arg)5 motif, as well as a N and C terminal Förster resonance energy transfer pair (donor: AlexaFluor680; quencher: BHQ3) to facilitate activity-dependent imaging. MG101 showed excellent stability since no fluorescence release corresponding to a self-dequenching was observed in the presence of either 2 M NaCl or after incubation at a broad range of pH (2.2-8.2). Cathepsins B, D, G, H, and K, neutrophil elastase and proteinase 3 did not cleave MG101, while CatS, and to a lesser extent CatL, hydrolysed MG101 at pH 5.5. However MG101 was fully selective for CatS at pH 7.4 (kcat/Km = 140,000 M-1 s-1) and sensitive to low concentration of CatS (<1 nM). The selectivity of MG101 was successfully endorsed ex vivo, as it was hydrolysed in cell lysates derived from wild-type but not knockout CatS murine spleen. Furthermore, application of the SBP probe with confocal microscopy confirmed the secretion of active CatS from THP-1 macrophages, which could be abrogated by pharmacological CatS inhibitors. Taken together, present data highlight MG101 as a novel near-infrared fluorescent SBP for the visualization of extracellular active CatS from macrophages and other cell types.

Curcumin inhibits the TGF-ß1-dependent differentiation of lung fibroblasts via PPARγ-driven upregulation of cathepsins B and L.

Pulmonary fibrosis is a progressive disease characterized by a widespread accumulation of myofibroblasts and extracellular matrix components. Growing evidences support that cysteine cathepsins, embracing cathepsin B (CatB) that affects TGF-ß1-driven Smad pathway, along with their extracellular inhibitor cystatin C, participate in myofibrogenesis. Here we established that curcumin, a potent antifibrotic drug used in traditional Asian medicine, impaired the expression of both α-smooth muscle actin and mature TGF-ß1 and inhibited the differentiation of human lung fibroblasts (CCD-19Lu cells). Curcumin induced a compelling upregulation of CatB and CatL. Conversely cystatin C was downregulated, which allowed the recovery of the peptidase activity of secreted cathepsins and the restoration of the proteolytic balance. Consistently, the amount of both insoluble and soluble type I collagen decreased, reaching levels similar to those observed for undifferentiated fibroblasts. The signaling pathways activated by curcumin were further examined. Curcumin triggered the expression of nuclear peroxisome proliferator-activated receptor γ (PPARγ). Contrariwise PPARγ inhibition, either by an antagonist (2-chloro-5-nitro-N-4-pyridinyl-benzamide) or by RNA silencing, restored TGF-ß1-driven differentiation of curcumin-treated CCD-19Lu cells. PPARγ response element (PPRE)-like sequences were identified in the promoter regions of both CatB and CatL. Finally, we established that the transcriptional induction of CatB and CatL depends on the binding of PPARγ to PPRE sequences as a PPARγ/Retinoid X Receptor-α heterodimer.

Selective inhibition of human cathepsin S by 2,4,6-trisubstituted 1,3,5-triazine analogs.

We report herein the synthesis and biological evaluation of a new series of 2,4,6-trisubstituted 1,3,5-triazines as reversible inhibitors of human cysteine cathepsins. The desired products bearing morpholine and N-Boc piperidine, respectively, were obtained in three to four steps from commercially available trichlorotriazine. Seventeen hitherto unknown compounds were evaluated in vitro against various cathepsins for their inhibitory properties. Among them, compound 7c (4-(morpholin-4-yl)-6-[4-(trifluoromethoxy)anilino]-1,3,5-triazine-2-carbonitrile) was identified as the most potent and selective inhibitor of cathepsin S (Ki  =  2  ±â€¯â€¯0.3 nM). Also 7c impaired the autocatalytic maturation of procathepsin S. Molecular docking studies support that 7c bound within the active site of cathepsin S, by interacting with Gly23, Cys25 and Trp26 (S1 subsite), with Asn67, Gly69 and Phe70 (S2 subsite) and with Gln19 (S1' pocket).

Substrate-derived triazolo- and azapeptides as inhibitors of cathepsins K and S.

Cathepsin (Cat) K is a critical bone-resorbing protease and is a relevant target for the treatment of osteoporosis and bone metastasis, while CatS is an attractive target for drugs in autoimmune diseases (e.g. rheumatoid arthritis), emphysema or neuropathic pain. Despite major achievements, current pharmacological inhibitors are still lacking in safety and may have damaging side effects. A promising strategy for developing safer reversible and competitive inhibitors as new lead compounds could be to insert non-cleavable bonds at the scissile P1-P1' position of selective substrates of CatS and CatK. Accordingly, we introduced a 1,4-disubstituted 1,2,3-triazole heterocycle that mimics most of the features of a trans-amide bond, or we incorporated a semicarbazide bond (azaGly residue) by replacing the α-carbon of the glycyl residue at P1 by a nitrogen atom. AzaGly-containing peptidomimetics inhibited powerfully their respective target proteases in the nM range, while triazolopeptides were weaker inhibitors (Ki in the µM range). The selectivity of the azaGly CatS inhibitor (1b) was confirmed by using spleen lysates from wild-type vs CatS-deficient mice. Alternatively, the azaGly bradykinin-derived CatK inhibitor (2b) potently inhibited CatK (Ki = 9 nM) and impaired its kininase activity in vitro. Molecular modeling studies support that the semicarbazide bond of 2b is more favorable than the 1,2,3-triazole linkage of the bradykinin-derived pseudopeptide 2a to preserve an effective affinity towards CatK, its protease target.

Discordance in cathepsin B and cystatin C expressions in bronchoalveolar fluids between murine bleomycin-induced fibrosis and human idiopathic fibrosis.

The activity of cysteine cathepsin B increased markedly in lung homogenates and in bronchoalveolar lavage fluids (BALF) of the mouse model of bleomycin-induced lung fibrosis after 14 days of challenge. In contrast the level of the cysteine cathepsin inhibitor cystatin C was unaffected in BALF of wild-type and cathepsin B-deficient mice. Therefore, murine cystatin C is not a reliable marker of fibrosis during bleomycin-induced lung fibrosis. Current data are in sharp contrast to previous analysis carried on human BALF from patients with idiopathic pulmonary fibrosis, for which the level of cathepsin B remained unchanged while cystatin C was significantly increased.

The Unusual Resistance of Avian Defensin AvBD7 to Proteolytic Enzymes Preserves Its Antibacterial Activity.

Defensins are frontline peptides of mucosal immunity in the animal kingdom, including birds. Their resistance to proteolysis and their ensuing ability to maintain antimicrobial potential remains questionable and was therefore investigated. We have shown by bottom-up mass spectrometry analysis of protein extracts that both avian beta-defensins AvBD2 and AvBD7 were ubiquitously distributed along the chicken gut. Cathepsin B was found by immunoblotting in jejunum, ileum, caecum, and caecal tonsils, while cathepsins K, L, and S were merely identified in caecal tonsils. Hydrolysis product of AvBD2 and AvBD7 incubated with a panel of proteases was analysed by RP-HPLC, mass spectrometry and antimicrobial assays. AvBD2 and AvBD7 were resistant to serine proteases and to cathepsins D and H. Conversely cysteine cathepsins B, K, L, and S degraded AvBD2 and abolished its antibacterial activity. Only cathepsin K cleaved AvBD7 and released Ile4-AvBD7, a N-terminal truncated natural peptidoform of AvBD7 that displayed antibacterial activity. Besides the 3-stranded antiparallel beta-sheet typical of beta-defensins, structural analysis of AvBD7 by two-dimensional NMR spectroscopy highlighted the restricted accessibility of the C-terminus embedded by the N-terminal region and gave a formal evidence of a salt bridge (Asp9-Arg12) that could account for proteolysis resistance. The differential susceptibility of avian defensins to proteolysis opens intriguing questions about a distinctive role in the mucosal immunity against pathogen invasion.

Straightforward synthesis of 2,4,6-trisubstituted 1,3,5-triazine compounds targeting cysteine cathepsins K and S.

The synthesis and evaluation against various cysteine cathepsins with endopeptidase activity, of two new families of hitherto unknown 1,3,5-triazines, substituted by a nitrile function and either a cyclohexylamine moiety (5-like) or a piperazine moiety (9-like) are described. The structure-activity relationship was discussed; from 16 synthesized novel compounds, 9h was the most active and selectively inhibitor of Cat K (IC50 = 28 nM) and Cat S (IC50 = 23 nM). Molecular docking of 9h to X-ray crystal structure of cathepsins K and S confirmed a common binding mode with a crucial covalent bond with Cys25. We observed for 9h that p-trifluorophenyl group is located in S2 pocket and possess hydrophobic interactions with Tyr67 and Met68. Triazine and piperazine moieties are located in S'1 pocket and interact with Gly23, Cys63, Gly64 and Gly65. Altogether, these results indicate that the new analogs can make them effective agents against some viruses for which the glycoprotein cleavage is mediated by an array of proteases.