Inhibitors Of Human Cathepsin Research Articles

Inhibition of Human Cathepsins B and L by Caffeic Acid and Its Derivatives.

Caffeic acid (CA) and its derivatives caffeic acid phenethyl ester (CAPE) and chlorogenic acid (CGA) are phenolic compounds of plant origin with a wide range of biological activities. Here, we identify and characterize their inhibitory properties against human cathepsins B and L, potent, ubiquitously expressed cysteine peptidases involved in protein turnover and homeostasis, as well as pathological conditions, such as cancer. We show that CAPE and CGA inhibit both peptidases, while CA shows a preference for cathepsin B, resulting in the strongest inhibition among these combinations. All compounds are linear (complete) inhibitors acting via mixed or catalytic mechanisms. Cathepsin B is more strongly inhibited at pH 7.4 than at 5.5, and CA inhibits its endopeptidase activity preferentially over its peptidyl-dipeptidase activity. Altogether, the results identify the CA scaffold as a promising candidate for the development of cathepsin B inhibitors, specifically targeting its endopeptidase activity associated with pathological proteolysis of extracellular substrates.

N-Sulfonyl dipeptide nitriles as inhibitors of human cathepsin S: In silico design, synthesis and biochemical characterization

A library of cathepsin S inhibitors of the dipeptide nitrile chemotype, bearing a bioisosteric sulfonamide moiety, was synthesized. Kinetic investigations were performed at four human cysteine proteases, i.e. cathepsins S, B, K and L. Compound 12 with a terminal 3-biphenyl sulfonamide substituent was the most potent (Ki = 4.02 nM; selectivity ratio cathepsin S/K = 5.8; S/L = 67) and 24 with a 4′-fluoro-4-biphenyl sulfonamide substituent the most selective cathepsin S inhibitor (Ki = 35.5 nM; selectivity ratio cathepsin S/K = 57; S/L = 31). In silico design and biochemical evaluation emphasized the impact of the sulfonamide linkage on selectivity and a possible switch of P2 and P3 substituents with respect to the occupation of the corresponding binding sites of cathepsin S.

What comparisons of natural and chimeric contacts reveal about inhibition of human cathepsins K, L and S by their prosegments

Human cathepsins K, L, and S, which are involved in the development of several serious diseases, are strongly inhibited by their related prosegments, to which they are covalently bound or simply forming complexes. In this work, three-dimensional structures of the three natural complexes of these enzymes with their related proregions were constructed, as well as six chimeric complexes of the same three prosegments with their non-cognate enzymes. We made a comparative study of the contacts in all nine structures throughout their active sites. The analysis was performed looking for a structural parameter that could agree with the values of the inhibition constants reported experimentally for each of the nine complexes. We found that this correlating parameter was the difference of the electrostatic energy (involving hydrogen bonds and ion pairs) at the binding interface of a 13-amino acid fragment of the prosegments. We used the results of this work, on the one hand, to identify the key residues involved in the electrostatic intermolecular recognition in each studied complex and, on the other, to explain some results achieved by different research groups on the inhibition of the same enzymes analyzed here. It was found that the natural cathepsin L complex showed a higher number of electrostatic interactions, some of them interconnected, when compared to the other two natural complexes. In addition, the chimeric contacts revealed binding sites that could be used to achieve a more potent inhibition of these cathepsins, avoiding cross-interactions.

Manduca sexta serpin-12 controls the prophenoloxidase activation system in larval hemolymph

Insect prophenoloxidase activation is coordinated by a serine protease network, which is regulated by serine protease inhibitors of the serpin superfamily. The enzyme system also leads to proteolytic processing of a Spätzle precursor. Binding of Spätzle to a Toll receptor turns on a signaling pathway to induce the synthesis of defense proteins. Previous studies of the tobacco hornworm Manduca sexta have revealed key members of the protease cascade, which generates phenoloxidase for melanogenesis and Spätzle to induce immunity-related genes. Here we provide evidence that M. sexta serpin-12 regulates hemolymph protease-14 (HP14), an initiating protease of the cascade. This inhibitor, unlike the other serpins characterized in M. sexta, has an amino-terminal extension rich in hydrophilic residues and an unusual P1 residue (Leu429) right before the scissile bond cleaved by a target protease. Serpins with similarities to serpin-12, including Drosophila Necrotic, were identified in a wide range of insects including flies, moths, wasps, beetles, and two hemimetabolous species. The serpin-12 mRNA is present at low, constitutive levels in larval fat body and hemocytes and becomes more abundant after an immune challenge. We produced the serpin-12 core domain (serpin-12ΔN) in insect cells and in Escherichia coli and demonstrated its inhibition of human cathepsin G, bovine α-chymotrypsin, and porcine pancreatic elastase. MALDI-TOF analysis of the reaction mixtures confirmed the predicted P1 residue of Leu429. Supplementation of larval plasma samples with the serpin-12ΔN decreased prophenoloxidase activation elicited by microbial cells and reduced the proteolytic activation of the protease precursors of HP6, HP8, PAPs, and other serine protease-related proteins. After incubation of plasma stimulated with peptidoglycan, a 72 kDa protein appeared, which was recognized by polyclonal antibodies against both serpin-12 and HP14, suggesting that a covalent serpin-protease complex formed when serpin-12 inhibited HP14. Together, these data suggest that M. sexta serpin-12 inhibits HP14 to regulate melanization and antimicrobial peptide induction.

Optimization of dipeptidic inhibitors of cathepsin L for improved Toxoplasma gondii selectivity and CNS permeability

The neurotropic protozoan Toxoplasma gondii is the second leading cause of death due to foodborne illness in the US, and has been designated as one of five neglected parasitic infections by the Center for Disease Control and Prevention. Currently, no treatment options exist for the chronic dormant-phase Toxoplasma infection in the central nervous system (CNS). T. gondii cathepsin L (TgCPL) has recently been implicated as a novel viable target for the treatment of chronic toxoplasmosis. In this study, we report the first body of SAR work aimed at developing potent inhibitors of TgCPL with selectivity vs the human cathepsin L. Starting from a known inhibitor of human cathepsin L, and guided by structure-based design, we were able to modulate the selectivity for Toxoplasma vs human CPL by nearly 50-fold while modifying physiochemical properties to be more favorable for metabolic stability and CNS penetrance. The overall potency of our inhibitors towards TgCPL was improved from 2 μM to as low as 110 nM and we successfully demonstrated that an optimized analog 18b is capable of crossing the BBB (0.5 brain/plasma). This work is an important first step toward development of a CNS-penetrant probe to validate TgCPL as a feasible target for the treatment of chronic toxoplasmosis.

Synthesis and biological evaluation of novel peptidomimetics as rhodesain inhibitors

Novel rhodesain inhibitors were developed by combining an enantiomerically pure 3-bromoisoxazoline warhead with a 1,4-benzodiazepine scaffold as specific recognition moiety. All compounds were proven to inhibit rhodesain with Ki values in the low-micromolar range. Their activity towards rhodesain was found to be coupled to an in vitro antitrypanosomal activity, with IC50 values ranging from the mid-micromolar to a low-micromolar value for the most active rhodesain inhibitor (R,S,S)-3. All compounds showed a good selectivity against the target enzyme since all of them were proven to be poor inhibitors of human cathepsin L.

Cathepsin K inhibitors increase distal femoral bone mineral density in rapidly growing rabbits

BackgroundSelective and reversible inhibitors of human Cathepsin K (CatK), including odanacatib (ODN), have been developed as potential therapeutics for the treatment of osteoporosis. Inhibitors of human CatK show significantly less potency for the rodent enzymes compared with that for the human or rabbit enzymes; thus the Schenk model in growing rabbit was developed as a screening assay for the in vivo activity of CatK inhibitors in blocking bone resorption.MethodsIn this study, the efficacy of the selective inhibitors L-833905, L-006235, L-873724, and L-1037536 (ODN) of human CatK in the rapidly growing rabbit ‘Schenk’ model (age seven weeks) was compared to vehicle, using the bisphosphonate, alendronate (ALN), as a positive control, to assess inhibition of bone resorption. An enzyme inhibition assay (EIA) and an in vitro bone resorption assay using rabbit osteoclasts on bovine cortical bone slices were performed to evaluate the potency of these CatK inhibitors. Bone mineral density of the distal femur (DFBMD) was measured after ten days of treatment using ex vivo DXA densitometry.ResultsResults of the EIA using rabbit CatK and the rabbit bone resorption assay showed that three of the four compounds (L-006235, L-873724, and ODN) had similar potencies in the reduction of collagen degradation. L-833905 appeared to be a weaker inhibitor of CatK. Taking into account the respective in vitro potencies and pharmacokinetic profiles via oral administration, the efficacy of these four CatK inhibitors was demonstrated in a dose-related manner in the growing rabbit. Significant increases in DFBMD in animals dosed with the CatK inhibitors compared to vehicle were seen.ConclusionsEfficacy of the CatK inhibitors in the Schenk rabbit correlated well with that in the in vitro rabbit bone resorption assay and in the ovariectomized rabbit model as previously published. Hence, these studies validated the rabbit Schenk assay as a rapid and reliable in vivo model for prioritizing human CatK inhibitors as potential therapeutic agents.

Chemical introduction of the green fluorescence: imaging of cysteine cathepsins by an irreversibly locked GFP fluorophore

An activity-based probe, containing an irreversibly locked GFP-like fluorophore, was synthesized and evaluated as an inhibitor of human cathepsins and, as exemplified with cathepsin K, it proved to be suitable for ex vivo imaging and quantification of cysteine cathepsins by SDS-PAGE.

New Cathepsin D Inhibitor Library Utilizing Hydroxyethyl Isosteres with Cyclic Tertiary Amines

The design and synthesis of hydroxyethylamine isosteres as inhibitors of cathepsin D based on SAR data have been accomplished. A library of 96 of these hydroxyethylamine isosteres are described and many have proven to be very potent inhibitors of human cathepsin D activity as measured using a fluorometric assay technique, via peptide substrate Ac-Glu-Glu(Edans)-Lys-Pro-Ile-Cys-Phe-Phe-Arg-Leu-Gly-Lys(Methyl Red)-Glu-NH(2). Compounds showing strongest inhibition of cathepsin D activity were those that contain a hydroxyethyl-N'-2- or N'-(4-chlorophenyl)piperazine moiety (IC(50) values range from 0.55 to 8.5 nM), with N'-(2-pyrimidyl)piperizine (IC(50) values range from 0.5 to 21.6 nM), with N-N'- L-piperazinocolinamide (IC(50) values range from 0.001 - 0.25 nM), or N-N'-L-piperazinocolin-N-methylamide (IC(50) values range from 0.015 - 7.3 nM).

Balicatib, a cathepsin K inhibitor, stimulates periosteal bone formation in monkeys

Balicatib, an inhibitor of the osteoclastic enzyme cathepsin K, was tested in ovariectomized monkeys, a model for osteoporosis. As expected, ovariectomy-induced bone mass changes were partially prevented by balicatib treatment. Bone turnover was significantly decreased at most sites, but unlike most bone resorption inhibitors, periosteal bone formation rates were increased. Selective inhibitors of the osteoclastic enzyme cathepsin K have potential in osteoporosis treatment. This study evaluated the efficacy of balicatib (AAE581), a novel inhibitor of human cathepsin K, on bone mass and dynamic histomorphometric endpoints in ovariectomized monkeys. Eighty adult female Macaca fascicularis underwent bilateral ovariectomies and were dosed twice daily by oral gavage with balicatib at 0, 3, 10, and 50 mg/kg for 18 months (groups O, L, M, H, respectively). Approximately 1 month after treatment initiation, the 50 mg/kg dose was decreased to 30 mg/kg. Twenty animals underwent sham-ovariectomies (group S). Bone mass was measured at 3-6 month intervals. At 18 months, vertebra and femur were collected for histomorphometry. In both spine and femur, group O animals lost BMD and all other groups gained BMD between 0 and 18 months. In balicatib-treated animals, BMD change in the spine was intermediate between group S and O, with groups L and M significantly different from group O. In femur, all three doses of balicatib significantly increased BMD gain relative to group O and group mean values were also higher than group S. Most histomorphometric indices of bone turnover in vertebra and femoral neck were significantly lower than group O with balicatib treatment, except that periosteal bone formation rates (Ps.BFR) were significantly higher. Ps.BFR in mid-femur was also significantly increased by treatment. Balicatib partially prevented ovariectomy-induced changes in bone mass, inhibited bone turnover at most sites, and had an unexpected stimulatory effect on periosteal bone formation.

Balicatib, a cathepsin K inhibitor, stimulates periosteal bone formation in monkeys

Balicatib, an inhibitor of the osteoclastic enzyme cathepsin K, was tested in ovariectomized monkeys, a model for osteoporosis. As expected, ovariectomy-induced bone mass changes were partially prevented by balicatib treatment. Bone turnover was significantly decreased at most sites, but unlike most bone resorption inhibitors, periosteal bone formation rates were increased. Selective inhibitors of the osteoclastic enzyme cathepsin K have potential in osteoporosis treatment. This study evaluated the efficacy of balicatib (AAE581), a novel inhibitor of human cathepsin K, on bone mass and dynamic histomorphometric endpoints in ovariectomized monkeys. Eighty adult female Macaca fascicularis underwent bilateral ovariectomies and were dosed twice daily by oral gavage with balicatib at 0, 3, 10, and 50 mg/kg for 18 months (groups O, L, M, H, respectively). Approximately 1 month after treatment initiation, the 50 mg/kg dose was decreased to 30 mg/kg. Twenty animals underwent sham-ovariectomies (group S). Bone mass was measured at 3-6 month intervals. At 18 months, vertebra and femur were collected for histomorphometry. In both spine and femur, group O animals lost bone mineral density (BMD), and all other groups gained BMD between 0 and 18 months. In balicatib-treated animals, BMD change in the spine was intermediate between group S and O, with groups L and M significantly different from group O. In femur, all three doses of balicatib significantly increased BMD gain relative to group O, and group mean values were also higher than group S. Most histomorphometric indices of bone turnover in vertebra and femoral neck were significantly lower than group O with balicatib treatment, except that periosteal bone formation rates (Ps.BFR) were significantly higher. Ps.BFR in mid-femur was also significantly increased by treatment. Balicatib partially prevented ovariectomy-induced changes in bone mass, inhibited bone turnover at most sites, and had an unexpected stimulatory effect on periosteal bone formation.

Molecular determinants of improved cathepsin B inhibition by new cystatins obtained by DNA shuffling

BackgroundCystatins are inhibitors of cysteine proteases. The majority are only weak inhibitors of human cathepsin B, which has been associated with cancer, Alzheimer's disease and arthritis.ResultsStarting from the sequences of oryzacystatin-1 and canecystatin-1, a shuffling library was designed and a hybrid clone obtained, which presented higher inhibitory activity towards cathepsin B. This clone presented two unanticipated point mutations as well as an N-terminal deletion. Reversing each point mutation independently or both simultaneously abolishes the inhibitory activity towards cathepsin B. Homology modeling together with experimental studies of the reverse mutants revealed the likely molecular determinants of the improved inhibitory activity to be related to decreased protein stability.ConclusionA combination of experimental approaches including gene shuffling, enzyme assays and reverse mutation allied to molecular modeling has shed light upon the unexpected inhibitory properties of certain cystatin mutants against Cathepsin B. We conclude that mutations disrupting the hydrophobic core of phytocystatins increase the flexibility of the N-terminus, leading to an increase in inhibitory activity. Such mutations need not affect the inhibitory site directly but may be observed distant from it and manifest their effects via an uncoupling of its three components as a result of increased protein flexibility.

Natural inhibitors targeting osteoclast-mediated bone resorption

Human cathepsin K, matrix metalloproteinase 9, and α Vβ 3 integrin are the key regulators in osteoclast-mediated bone resorption. In this paper, we found natural inhibitors 1– 10 for them by enzyme inhibition assays. Inhibitors 1– 7, 8– 9, and 10 are novel inhibitors of human cathepsin K, matrix metalloproteinase 9, and α Vβ 3, respectively.

Trifluoroethylamines as amide isosteres in inhibitors of cathepsin K

The P2-P3 amide of dipeptide cathepsin K inhibitors can be replaced by the metabolically stable trifluoroethylamine group. The non-basic nature of the nitrogen allows the important hydrogen bond to Gly66 to be made. The resulting compounds are 10- to 20-fold more potent than the corresponding amide derivatives. Compound 8 is a 5 pM inhibitor of human cathepsin K with >10,000-fold selectivity over other cathepsins.

Tellurium-based cysteine protease inhibitors: evaluation of novel organotellurium(IV) compounds as inhibitors of human cathepsin B

New organotellurium(IV) compounds with specific cysteine protease inhibitory activity were synthesized. Serine and aspartic protease activity were not affected by any of these compounds. All Te(IV) compounds tested exhibited high specific second-order constant for cathepsin B inactivation. Tellurium(IV) compound 6 was the best inhibitor of the series, showing a second-order constant of 36,000 M −1 s −1. This value is about 100-fold higher than the second-order rate for cysteine protease inactivation shown by the historic Te(IV) compound AS 101 ( 1). The inhibition was irreversible and time and concentration dependent; no saturation kinetics were observed, suggesting a direct bimolecular reaction. The results described in this paper show that the new organotellurium(IV) compounds are powerful inhibitors of cathepsin B, constituting promising potential anti-metastatic agents.

Synthesis of peptide fluoromethyl ketones and the inhibition of human cathepsin B

Peptide fluoromethyl ketones have been synthesized for the first time. The inhibitor 3-( N-benzyloxycarbonylphenylalanylamido)- dl-1-fluoro-2-butanone (Z-Phe-AlaCH 2F) was found to be a 30-fold more potent inactivator of human cathepsin B than 3-( N-benzyloxycarbonylphenylalanylamido)- l-1-diazo-2-butanone (Z-Phe-AlaCHN 2), but less reactive than 3-( N-benzyloxycarbonylphenylalanylamido)- l-1-chloro-2-butanone (Z-Phe-AlaCH 2Cl). The fluoromethyl ketone's increased potency over the diazomethyl ketone is mainly due to its tighter binding to cathepsin B, with little difference between their respective k 3 values. Both Z-Phe-AlaCH 2 and Z-Phe-AlaCH 2F were quite stable to high concentrations of dithiothreitol, while Z-Phe-AlaCH 2Cl was rapidly destroyed by the thiol.