Reactions of Medicinal Gold Compounds with Cathepsin B Explored through Electrospray Mass Spectrometry Measurements.

Medicinal gold compounds, a novel class of potential anticancer drugs, are believed to produce their pharmacological effects mainly through direct gold binding to protein targets at the level of solvent exposed cysteine (or selenocysteine) residues. We have explored therein the reactions of a panel of seven representative gold compounds with the cysteine protease cathepsin B according to an established ESI MS approach. Detailed information on the mode of protein binding of these gold compounds is gained; notably, quite distinct patterns of cathepsin B metalation have emerged from these studies. It is shown that panel gold compounds interact preferentially, often exclusively, with the free cysteine located in the active site of the enzyme.

Regulation of Peptidase Activity beyond the Active Site in Human Health and Disease.

This comprehensive review addresses the intricate and multifaceted regulation of peptidase activity in human health and disease, providing a comprehensive investigation that extends well beyond the boundaries of the active site. Our review focuses on multiple mechanisms and highlights the important role of exosites, allosteric sites, and processes involved in zymogen activation. These mechanisms play a central role in shaping the complex world of peptidase function and are promising potential targets for the development of innovative drugs and therapeutic interventions. The review also briefly discusses the influence of glycosaminoglycans and non-inhibitory binding proteins on enzyme activities. Understanding their role may be a crucial factor in the development of therapeutic strategies. By elucidating the intricate web of regulatory mechanisms that control peptidase activity, this review deepens our understanding in this field and provides a roadmap for various strategies to influence and modulate peptidase activity.

Activity-based probes trap early active intermediates during metacaspase activation.

Metacaspases are essential cysteine proteases present in plants, fungi, and protists that are regulated by calcium binding and proteolytic maturation through mechanisms not yet understood. Here, we developed and validated activity-based probes for the three main metacaspase types, and used them to study calcium-mediated activation of metacaspases from their precursors in vitro. By combining substrate-inspired tetrapeptide probes containing an acyloxymethylketone (AOMK) reactive group, with purified representatives of type-I, type-II, and type-III metacaspases, we were able to demonstrate that labeling of mature metacaspases is strictly dependent on calcium. The probe with the highest affinity for all metacaspases also labels higher molecular weight proteoforms of all three metacaspases only in the presence of calcium, displaying the active, unprocessed metacaspase intermediates. Our data suggest that metacaspase activation proceeds through previously unknown active intermediates that are formed upon calcium binding, before precursor processing.

Kinetic Characterization of Cerium and Gallium Ions as Inhibitors of Cysteine Cathepsins L, K, and S.

Heavy metal ions can disrupt biological functions via multiple molecular mechanisms, including inhibition of enzymes. We investigate the interactions of human papain-like cysteine endopeptidases cathepsins L, K, and S with gallium and cerium ions, which are associated with medical applications. We compare these results with zinc and lead, which are known to inhibit thiol enzymes. We show that Ga3+, Ce3+, and Ce4+ ions inhibit all tested peptidases with inhibition constants in the low micromolar range (between 0.5 µM and 10 µM) which is comparable to Zn2+ ions, whereas inhibition constants of Pb2+ ions are one order of magnitude higher (30 µM to 150 µM). All tested ions are linear specific inhibitors of cathepsin L, but cathepsins K and S are inhibited by Ga3+, Ce3+, and Ce4+ ions via hyperbolic inhibition mechanisms. This indicates a mode of interaction different from that of Zn2+ and Pb2+ ions, which act as linear specific inhibitors of all peptidases. All ions also inhibit the degradation of insoluble elastin, which is a common target of these peptidases in various inflammatory diseases. Our results suggest that these ions and their compounds have the potential to be used as cysteine cathepsin inhibitors in vitro and possibly in vivo.

Zinc pyrithione is a potent inhibitor of PLPro and cathepsin L enzymes with ex vivo inhibition of SARS-CoV-2 entry and replication.

Zinc pyrithione (1a), together with its analogues 1b-h and ruthenium pyrithione complex 2a, were synthesised and evaluated for the stability in biologically relevant media and anti-SARS-CoV-2 activity. Zinc pyrithione revealed potent in vitro inhibition of cathepsin L (IC50=1.88 ± 0.49 µM) and PLPro (IC50=0.50 ± 0.07 µM), enzymes involved in SARS-CoV-2 entry and replication, respectively, as well as antiviral entry and replication properties in an ex vivo system derived from primary human lung tissue. Zinc complexes 1b-h expressed comparable in vitro inhibition. On the contrary, ruthenium complex 2a and the ligand pyrithione a itself expressed poor inhibition in mentioned assays, indicating the importance of the selection of metal core and structure of metal complex for antiviral activity. Safe, effective, and preferably oral at-home therapeutics for COVID-19 are needed and as such zinc pyrithione, which is also commercially available, could be considered as a potential therapeutic agent against SARS-CoV-2.

Regioselective Synthesis of 5- and 3-Hydroxy-N-Aryl-1H-Pyrazole-4-Carboxylates and Their Evaluation as Inhibitors of Plasmodium falciparum Dihydroorotate Dehydrogenase.

In silico evaluation of various regioisomeric 5- and 3-hydroxy-substituted alkyl 1-aryl-1H-pyrazole-4-carboxylates and their acyclic precursors yielded promising results with respect to their binding in the active site of dihydroorotate dehydrogenase of Plasmodium falciparum (PfDHODH). Consequently, four ethyl 1-aryl-5-hydroxy-1H-pyrazole-4-carboxylates and their 3-hydroxy regioisomers were prepared by two-step syntheses via enaminone-type reagents or key intermediates. The synthesis of 5-hydroxy-1H-pyrazoles was carried out using the literature protocol comprising acid-catalyzed transamination of diethyl [(dimethylamino)methylene]malonate with arylhydrazines followed by base-catalyzed cyclization of the intermediate hydrazones. For the synthesis of isomeric methyl 1-aryl-3-hydroxy-1H-pyrazole-4-carboxylates, a novel two-step synthesis was developed. It comprises acylation of hydrazines with methyl malonyl chloride followed by cyclization of the hydrazines with tert-butoxy-bis(dimethylamino)methane. Testing the pyrazole derivatives for the inhibition of PfDHODH showed that 1-(naphthalene-2-yl)-5-hydroxy-1H-pyrazole-4-carboxylate and 1-(naphthalene-2-yl)-, 1-(2,4,6-trichlorophenyl)-, and 1-[4-(trifluoromethyl)phenyl]-3-hydroxy-1H-pyrazole-4-carboxylates (~30% inhibition) were slightly more potent than a known inhibitor, diethyl α-{[(1H-indazol-5-yl)amino]methylidene}malonate (19% inhibition).

Structural polymorphism of coiled-coils from the stalk domain of SARS-CoV-2 spike protein.

Spike trimer plays a key role in SARS-CoV-2 infection and vaccine development. It consists of a globular head and a flexible stalk domain that anchors the protein into the viral membrane. While the head domain has been extensively studied, the properties of the adjoining stalk are poorly understood. Here, we characterize the coiled-coil formation and thermodynamic stability of the stalk domain and its segments. We find that the N-terminal segment of the stalk does not form coiled-coils and remains disordered in solution. The C-terminal stalk segment forms a trimeric coiled-coil in solution, which becomes significantly stabilized in the context of the full-length stalk. Its crystal structure reveals a novel antiparallel tetramer coiled-coil with an unusual combination of a-d and e-a-d hydrophobic core packing. Structural analysis shows that a subset of hydrophobic residues stabilizes different coiled-coil structures: trimer, tetramer, and heterohexamer, underscoring a highly polymorphic nature of the SARS-CoV-2 stalk sequence.

Evolutionary Analysis of Dipeptidyl Peptidase I.

Human dipeptidyl peptidase I (DPPI) belongs to the family of papain-like cysteine peptidases. Its distinctive features are the unique exclusion domain which enables the eponymous activity and homotetramerization of DPPI, and its dependence on chloride ions for enzymatic activity. The oligomeric state of DPPI is unique in this family of predominantly monomeric peptidases. However, a distant DPPI ortholog from Plasmodium falciparum has been shown to be monomeric, indicating that the oligomeric state of DPPI varies between lineages. The aim of this work was to study the evolution of DPPI, with particular attention to the structural features that determine its characteristic enzymatic activity and preferences, and to reconstruct the evolution of its oligomerization. We analyzed fifty-seven selected sequences of DPPI and confirmed its presence in three lineages, namely, Amorphea (including animals and Amoebozoa), Alveolates and the metamonad Giardia. The amino acid residues that bind the chloride ion are highly conserved in all species, indicating that the dependence on chloride ions for activity is an evolutionarily conserved feature of DPPI. The number of N-glycosylation sites is significantly increased in animals, particularly vertebrates. Analysis of homology models and subunit contacts suggests that oligomerization is likely restricted to DPPIs in the Amorphea group.

Synthesis and kinetic characterization of hyperbolic inhibitors of human cathepsins K and S based on a succinimide scaffold.

Cathepsins K and S are closely related papain-like cysteine peptidases and potential therapeutic targets for metabolic and inflammatory diseases such as osteoporosis and arthritis. Here we describe the reduction of a previously characterized succinimide (2,5-dioxopyrrolidine)-containing hyperbolic inhibitor of cathepsin K (methyl (RS)-N-[1-(4-methoxyphenyl)-2,5-dioxopyrrolidin-3-yl]glycinate), to obtain a better and more selective compound (compound 4a - methyl (2,5-dioxopyrrolidin-3-yl)glycinate), which acted as a hyperbolic mixed inhibitor/activator similar to already known allosteric effectors of cathepsin K. We then investigated the potential of the succinimide scaffold as inhibitors of cathepsins K and/or S and synthesized a library of such compounds by 1,4-addition of α-amino acid esters and related compounds to N-substituted maleimides. From the generated library, we identified the first small molecule hyperbolic inhibitors of cathepsin S (methyl ((R)-2,5-dioxopyrrolidin-3-yl)-l-threoninate (compound R-4c) and 3-{[(1S,2R,3'S)-2-hydroxycyclohexyl]amino}pyrrolidine-2,5-dione (compound (1S,2R,3'S-10)). The former acted via a similar mechanism to compound 4a, while the latter was a hyperbolic specific inhibitor of cathepsin S. Given the versatility of the scaffold, the identified compounds will be used as the basis for the development of high-affinity hyperbolic inhibitors of the individual peptidases and to explore the potential of hyperbolic inhibitors for the inhibition of cysteine cathepsins in in vitro models.

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.

Interplay between tetrameric structure, enzymatic activity and allosteric regulation of human dipeptidyl-peptidase I.

Human dipeptidyl-peptidase I (DPPI) is a tetrameric enzyme from the family of papain-like cysteine peptidases. It is ubiquitously expressed and plays important roles in general protein turnover, skin homeostasis and proteolytic processing of effector peptidases in immune cells. In this work we investigate allosteric regulation of DPPI and its relation to the oligomeric structure. First, we investigate the functional significance of the tetrameric state by comparing the kinetic properties of the tetrameric form (DPPItet) with a recombinant monomeric form (DPPImono). We find that both forms have very similar kinetic properties for the hydrolysis of a commonly used synthetic substrate. In agreement with previous studies, no cooperativity is observed in the tetramer. The only significant difference between both forms is a higher catalytic rate of DPPImono. We then characterize three compounds, 3'-nitrophthalanilic acid, chlorogenic acid and caffeic acid that affect DPPI activity via kinetic mechanisms consistent with binding outside of the active site. These compounds are the first known modifiers of DPPI that do not act as specific inhibitors. Chlorogenic acid and caffeic acid act as linear mixed and linear catalytic inhibitors, respectively, and do not discriminate between both forms. In contrast, 3'-nitrophthalanilic acid is a hyperbolic inhibitor that binds DPPItet and DPPImono with different affinities and inhibits their activities via different kinetic mechanisms. Altogether, these results show that the tetrameric structure of DPPI is not necessary for enzymatic activity, however, oligomerization-related structural features can play a role in its regulation.

Tetrahydro-1H,5H-pyrazolo[1,2-a]pyrazole-1-carboxylates as inhibitors of Plasmodium falciparum dihydroorotate dehydrogenase.

The reactions between 5-substituted pyrazolidine-3-ones, aldehydes, and methyl methacrylate provided tetrahydropyrazolo[1,2-a]pyrazole-1-carboxylates as mixtures of syn- and anti-diastereomers. Testing for inhibition of dihydroorotate dehydrogenase of Plasmodium falciparum (PfDHODH) revealed high activity of some anti-isomers of the methyl esters, while the corresponding carboxylic acids and carboxamides were not active. The most active representative, methyl (1S*,3S*,5R*)-1,5-dimethyl-7-oxo-3-phenyltetrahydro-1H,5H-pyrazolo[1,2-a]pyrazole-1-carboxylate (IC50 = 2.9 ±â€¯0.3 µM), also exhibited very high selectivity of the parasite enzyme vs. the human enzyme, PfDHODH/HsDHODH > 350. According to the molecular docking score, this high activity is explainable by synergic interactions of the methyl, phenyl and the CO2Me substituent with the hydrophobic pockets in the active site of the enzyme. The carboxylic acid and carboxamides derived from this compound did not inhibit PfDHODH.

The catalytic domain of cathepsin C (dipeptidyl-peptidase I) alone is a fully functional endoprotease.

Cathepsin C is a tetrameric lysosomal protease that acts as a dipeptidyl-peptidase due to the presence of the exclusion domain that is unique among papain-like cysteine proteases. Here we describe a recombinant form of cathepsin C lacking its exclusion domain (CatCΔEx) produced in a bacterial expression system (E. coli). CatCΔEx is a monomer with endoprotease activity and affinity for hydrophobic residues such as Phe, Leu or Pro, but not Val, in the P2 position. As opposed to cathepsin C, it does not require chloride ions for its activity. Despite lower turnover rates of hydrolysis of synthetic substrates, CatCΔEx has elastolytic and gelatinolytic activity comparable to other cysteine cathepsins.

Synthesis and biological evaluation of 7-(aminoalkyl)pyrazolo[1,5-a]pyrimidine derivatives as cathepsin K inhibitors.

A series of novel 7-aminoalkyl substituted pyrazolo[1,5-a]pyrimidine derivatives were synthesized and tested for inhibition of cathepsin K. The synthetic methodology comprises cyclization of 5-aminopyrazoles with N-Boc-α-amino acid-derived ynones followed by transformation of the ester and the Boc-amino functions. It allows for easy diversification of the pyrazolo[1,5-a]pyrimidine scaffold at various positions. Molecular docking studies with pyrazolo[1,5-a]pyrimidine derivatives were also performed to elucidate the binding mode in the active site of cathepsin K. The synthesized compounds exhibited moderate inhibition activity (Ki ≥ 77 µM).

The two cathepsin B-like proteases of Arabidopsis thaliana are closely related enzymes with discrete endopeptidase and carboxydipeptidase activities.

The genome of the model plant Arabidopsis thaliana encodes three paralogues of the papain-like cysteine proteinase cathepsin B (AtCathB1, AtCathB2 and AtCathB3), whose individual functions are still largely unknown. Here we show that a mutated splice site causes severe truncations of the AtCathB1 polypeptide, rendering it catalytically incompetent. By contrast, AtCathB2 and AtCathB3 are effective proteases which display comparable hydrolytic properties and share most of their substrate specificities. Site-directed mutagenesis experiments demonstrated that a single amino acid substitution (Gly336→Glu) is sufficient to confer AtCathB2 with the capacity to tolerate arginine in its specificity-determining S2 subsite, which is otherwise a hallmark of AtCathB3-mediated cleavages. A degradomics approach utilizing proteome-derived peptide libraries revealed that both enzymes are capable of acting as endopeptidases and exopeptidases, releasing dipeptides from the C-termini of substrates. Mutation of the carboxydipeptidase determinant His207 also affected the activity of AtCathB2 towards non-exopeptidase substrates, highlighting mechanistic differences between plant and human cathepsin B. This was also noted in molecular modeling studies which indicate that the occluding loop defining the dual enzymatic character of cathepsin B does not obstruct the active-site cleft of AtCathB2 to the same extent as in its mammalian orthologues.

Computational investigation of conformational variability and allostery in cathepsin K and other related peptidases.

Allosteric targeting is progressively gaining ground as a strategy in drug design. Its success, however, depends on our knowledge of the investigated system. In the case of the papain-like cysteine peptidase cathepsin K, a major obstacle in our understanding of allostery is represented by the lack of observable conformational change at the active site. This makes it difficult to understand how binding of effectors at known allosteric sites translates into modified enzyme activity. Herein, we address this issue by a computational approach based on experimental data. We analyze the conformational space of the papain-like family and the positioning of cathepsin K within it using principal component analysis and molecular dynamics simulations. We show that human cathepsin L-like endopeptidases (cathepsins L, K, S and V) adopt similar conformations which are distinct from their non-animal counterparts and other related peptidases. Molecular dynamics simulations show that the conformation of cathepsin K is influenced by known allosteric effectors, chondroitin sulfate and the small molecules NSC13345 and NSC94914. Importantly, all effectors affect the geometry of the active site around sites S1 and S2 that represent the narrowest part of the active site cleft and the major specificity determinant in papain-like endopeptidases. The effectors act by stabilizing pre-existing conformational states according to a two-state model and thereby facilitate or hinder the binding of substrate into the active site, as shown by molecular docking simulations. Comparison with other related enzymes shows that similar conformational variability and, by implication, allostery also exist in other papain-like endopeptidases.

The papain-like cysteine proteinases NbCysP6 and NbCysP7 are highly processive enzymes with substrate specificities complementary to Nicotiana benthamiana cathepsin B.

The tobacco-related plant Nicotiana benthamiana is gaining interest as a versatile host for the production of monoclonal antibodies and other protein therapeutics. However, the susceptibility of plant-derived recombinant proteins to endogenous proteolytic enzymes limits their use as biopharmaceuticals. We have now identified two previously uncharacterized N. benthamiana proteases with high antibody-degrading activity, the papain-like cysteine proteinases NbCysP6 and NbCysP7. Both enzymes are capable of hydrolysing a wide range of synthetic substrates, although only NbCysP6 tolerates basic amino acids in its specificity-determining S2 subsite. The overlapping substrate specificities of NbCysP6 and NbCysP7 are also documented by the closely related properties of their other subsites as deduced from the action of the enzymes on proteome-derived peptide libraries. Notable differences were observed to the substrate preferences of N. benthamiana cathepsin B, another antibody-degrading papain-like cysteine proteinase. The complementary activities of NbCysP6, NbCysP7 and N. benthamiana cathepsin B indicate synergistic roles of these proteases in the turnover of recombinant and endogenous proteins in planta, thus representing a paradigm for the shaping of plant proteomes by the combined action of papain-like cysteine proteinases.

Cathepsin K cleavage of SDF-1α inhibits its chemotactic activity towards glioblastoma stem-like cells.

Glioblastoma (GBM) is the most aggressive primary brain tumor with poor patient survival that is at least partly caused by malignant and therapy-resistant glioma stem-like cells (GSLCs) that are protected in GSLC niches. Previously, we have shown that the chemo-attractant stromal-derived factor-1α (SDF-1α), its C-X-C receptor type 4 (CXCR4) and the cysteine protease cathepsin K (CatK) are localized in GSLC niches in glioblastoma. Here, we investigated whether SDF-1α is a niche factor that through its interactions with CXCR4 and/or its second receptor CXCR7 on GSLCs facilitates their homing to niches. Furthermore, we aimed to prove that SDF-1α cleavage by CatK inactivates SDF-1α and inhibits the invasion of GSLCs. We performed mass spectrometric analysis of cleavage products of SDF-1α after proteolysis by CatK. We demonstrated that CatK cleaves SDF-1α at 3 sites in the N-terminus, which is the region of SDF-1α that binds to its receptors. Confocal imaging of human GBM tissue sections confirmed co-localization of SDF-1α and CatK in GSLC niches. In accordance, 2D and 3D invasion experiments using CXCR4/CXCR7-expressing GSLCs and GBM cells showed that SDF-1α had chemotactic activity whereas CatK cleavage products of SDF-1α did not. Besides, CXCR4 inhibitor plerixafor inhibited invasion of CXCR4/CXCR7-expressing GSLCs. In conclusion, CatK can cleave and inactivate SDF-1α. This implies that CatK activity facilitates migration of GSLCs out of niches. We propose that activation of CatK may be a promising strategy to prevent homing of GSLCs in niches and thus render these cells sensitive to chemotherapy and radiation.

Synthesis of Novel 5-(N-Boc-N-Benzyl-2-aminoethyl)-7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidin-3-carboxamides and Their Inhibition of Cathepsins B and K.

Eight novel 5-(N-Boc-N-benzyl-2-aminoethyl)-7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidin-3-carboxamides were prepared in three steps from methyl 3-amino-1H-pyrazole-4-carboxylate and methyl 5-(benzyl(tert-butoxycarbonyl)amino)-3-oxopentanoate. The synthetic procedure comprises cyclocondensation of the above starting compounds, hydrolysis of the ester, and bis(pentafluorophenyl) carbonate (BPC)-mediated amidation. Title carboxamides were tested for inhibition of cathepsins K and B. The N-butylcarboxamide 5a exhibited appreciable inhibition of cathepsin K (IC50 ~ 25 µM), while the strongest inhibition of cathepsin B was achieved with N-(2-picolyl)carboxamide 5c (IC50 ~ 45 µM).

An allosteric site enables fine-tuning of cathepsin K by diverse effectors.

The cysteine peptidase cathepsin K is a potent collagenolytic enzyme and a promising target for the treatment of osteoporosis. Here, we characterize its allosteric fine-tuning via a recently identified allosteric site. We show that compound NSC94914 binds this site and acts as a specific partial inhibitor of the collagenolytic activity of cathepsin K. We link the functional differences between NSC94914 and known effectors (compound NSC11345 and glycosaminoglycans) to their different modes of interaction with the site. We characterize the allosteric site by site-directed mutagenesis and show that it is involved in specific regulation of the collagenolytic activity of cathepsin K.