Human Cysteine Cathepsins Research Articles

Three Decades of Targeting Falcipains to Develop Antiplasmodial Agents: What have we Learned and What can be Done Next?

Malaria is a devastating infectious disease that affects large swathes of human populations across the planet's tropical regions. It is caused by parasites of the genus Plasmodium, with Plasmodium falciparum being responsible for the most lethal form of the disease. During the intraerythrocytic stage in the human hosts, malaria parasites multiply and degrade hemoglobin (Hb) using a battery of proteases, which include two cysteine proteases, falcipains 2 and 3 (FP-2 and FP-3). Due to their role as major hemoglobinases, FP-2 and FP-3 have been targeted in studies aiming to discover new antimalarials and numerous inhibitors with activity against these enzymes, and parasites in culture have been identified. Nonetheless, cross-inhibition of human cysteine cathepsins remains a serious hurdle to overcome for these compounds to be used clinically. In this article, we have reviewed key functional and structural properties of FP-2/3 and described different compound series reported as inhibitors of these proteases during decades of active research in the field. Special attention is also paid to the wide range of computer-aided drug design (CADD) techniques successfully applied to discover new active compounds. Finally, we provide guidelines that, in our understanding, will help advance the rational discovery of new FP-2/3 inhibitors.

The Role of Cysteine Protease Cathepsins B, H, C, and X/Z in Neurodegenerative Diseases and Cancer.

Papain-like cysteine proteases are composed of 11 human cysteine cathepsins, originally located in the lysosomes. They exhibit broad specificity and act as endopeptidases and/or exopeptidases. Among them, only cathepsins B, H, C, and X/Z exhibit exopeptidase activity. Recently, cysteine cathepsins have been found to be present outside the lysosomes and often participate in various pathological processes. Hence, they have been considered key signalling molecules. Their potentially hazardous proteolytic activities are tightly regulated. This review aims to discuss recent advances in understanding the structural aspects of these four cathepsins, mechanisms of their zymogen activation, regulation of their activities, and functional aspects of these enzymes in neurodegeneration and cancer. Neurodegenerative effects have been evaluated, particularly in Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, multiple sclerosis, and neuropsychiatric disorders. Cysteine cathepsins also participate in tumour progression and metastasis through the overexpression and secretion of proteases, which trigger extracellular matrix degradation. To our knowledge, this is the first review to provide an in-depth analysis regarding the roles of cysteine cathepsins B, H, C, and X in neurodegenerative diseases and cancer. Further advances in understanding the functions of cysteine cathepsins in these conditions will result in the development of novel, targeted therapeutic strategies.

Structure-Based Optimization of Protease-Inhibitor Interactions to Enhance Specificity of Human Stefin-A against Falcipain-2 from the Plasmodium falciparum 3D7 Strain.

The emergence of resistance in Plasmodium falciparum to frontline artemisinin-based combination therapies has raised global concerns and emphasized the identification of new drug targets for malaria. Cysteine protease falcipain-2 (FP2), involved in host hemoglobin degradation and instrumental in parasite survival, has long been proposed as a promising malarial drug target. However, designing active-site-targeted small-molecule inhibitors of FP2 becomes challenging due to their off-target specificity toward highly homologous human cysteine cathepsins. The use of proteinaceous inhibitors, which have nonconserved exosite interactions and larger interface area, can effectively circumvent this problem. In this study, we report for the first time that human stefin-A (STFA) efficiently inhibits FP2 with Ki values in the nanomolar range. The FP2-STFA complex crystal structure, determined in this study, and sequence analyses identify a unique nonconserved exosite interaction, compared to human cathepsins. Designing a mutation Lys68 > Arg in STFA amplifies its selectivity garnering a 3.3-fold lower Ki value against FP2, and the crystal structure of the FP2-STFAK68R complex shows stronger electrostatic interaction between side-chains of Arg68 (STFAK68R) and Asp109 (FP2). Comparative structural analyses and molecular dynamics (MD) simulation studies of the complexes further confirm higher buried surface areas, better interaction energies for FP2-STFAK68R, and consistency of the newly developed electrostatic interaction (STFA-R68-FP2-D109) in the MD trajectory. The STFA-K68R mutant also shows higher Ki values against human cathepsin-L and stefin, a step toward eliminating off-target specificity. Hence, this work underlines the design of host-based proteinaceous inhibitors against FP2, with further optimization to render them more potent and selective.

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

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

The Effect of Dipeptidyl Nitrile Derivatives on Pancreatic Ductal Adenocarcinoma Cells In Vitro

Aims: This study aims to evaluate the bioactivity of dipeptidyl nitrile inhibitors of human cysteine cathepsins that could work as anticancer agents in a drug discovery and development project. Background: Human lysosomal cysteine proteases promote cancer progression, migration, and metastasis, targeted by inhibitors. Objective: Here, 19 cysteine protease inhibitors known as dipeptidyl nitriles were tested using MIA PaCa-2 pancreatic cancer cells and Balb/3T3 clone A31 non-tumoral mouse fibroblasts. Method: In vitro assays evaluated cell migration, colony formation, inhibition of the enzymatic activity in cell lysates, and combination therapy with gemcitabine. Result: There were mixed results; the inhibitors reduced the number of colonies but did not affect the total area. Cells migrated despite enzyme inhibition by Neq0709 and Neq0712. As expected, the compounds were non-cytotoxic; they improved the potency of gemcitabine in the combined therapy assay, especially for Neq0707. Conclusion: In summary, our findings revealed the complexity of dealing with the translation from biochemical to cell-based assays in the hit-to-lead step.

Water Bridges Play a Key Role in Affinity and Selectivity for Malarial Protease Falcipain-2.

Falcipain-2 (FP-2) is hemoglobinase considered an attractive drug target of Plasmodium falciparum. Recently, it has been shown that peptidomimetic nitriles containing a 3-pyridyl (3Pyr) moiety at P2 display high affinity and selectivity for FP-2 with respect to human cysteine cathepsins (hCats), outperforming other P2-Pyr isomers and analogs. Further characterization demonstrated that certain P3 variants of these compounds possess micromolar inhibition of parasite growth in vitro and no cytotoxicity against human cell lines. However, the structural determinants underlying the selectivity of the 3Pyr-containing nitriles for FP-2 remain unknown. In this work, we conduct a thorough computational study combining MD simulations and free energy calculations to decipher the bases of the selectivity of the aforementioned nitriles. Our results reveal that water bridges involving the nitrogen and one carboxyl oxygen of I85 and D234 of FP-2, respectively, and the nitrogen of the neutral 3Pyr moiety, which are either less prevalent or nonexistent in the other complexes, explain the experimental activity profiles. The presence of crystallographic waters close to the bridging water positions in the studied proteases strongly supports the occurrence of such interactions. Overall, our findings suggest that selective FP-2 inhibitors can be designed by promoting water bridge formation at the bottom of the S2 subsite and/or by introducing complementary groups that displace the bridging water.

Novel broad-spectrum activity-based probes to profile malarial cysteine proteases

Clan CA cysteine proteases, also known as papain-like proteases, play important roles throughout the malaria parasite life cycle and are therefore potential drug targets to treat this disease and prevent its transmission. In order to study the biological function of these proteases and to chemically validate some of them as viable drug targets, highly specific inhibitors need to be developed. This is especially challenging given the large number of clan CA proteases present in Plasmodium species (ten in Plasmodium falciparum), and the difficulty of designing selective inhibitors that do not cross-react with other members of the same family. Additionally, any efforts to develop antimalarial drugs targeting these proteases will also have to take into account potential off-target effects against the 11 human cysteine cathepsins. Activity-based protein profiling has been a very useful tool to determine the specificity of inhibitors against all members of an enzyme family. However, current clan CA proteases broad-spectrum activity-based probes either target endopeptidases or dipeptidyl aminopeptidases, but not both subfamilies efficiently. In this study, we present a new series of dipeptydic vinyl sulfone probes containing a free N-terminal tryptophan and a fluorophore at the P1 position that are able to label both subfamilies efficiently, both in Plasmodium falciparum and in mammalian cells, thus making them better broad-spectrum activity-based probes. We also show that some of these probes are cell permeable and can therefore be used to determine the specificity of inhibitors in living cells. Interestingly, we show that the choice of fluorophore greatly influences the specificity of the probes as well as their cell permeability.

Fluorescent probes towards selective cathepsin B detection and visualization in cancer cells and patient samples.

Human cysteine cathepsins constitute an 11-membered family of proteases responsible for degradation of proteins in cellular endosomal-lysosomal compartments as such, they play important roles in antigen processing, cellular stress signaling, autophagy, and senescence. Moreover, for many years these enzymes were also linked to tumor growth, invasion, angiogenesis and metastasis when upregulated. Individual biological roles of each cathepsin are difficult to establish, because of their redundancy and similar substrate specificities. Selective chemical tools that enable imaging of individual cathepsin activities in living cells, tumors, and the tumor microenvironment may provide a better insight into their functions. In this work, we used HyCoSuL technology to profile the substrate specificity of human cathepsin B. The use of unnatural amino acids in the substrate library enabled us to uncover the broad cathepsin B preferences that we utilized to design highly-selective substrates and fluorescent activity-based probes (ABPs). We further demonstrated that Cy5-labeled MP-CB-2 probe can selectively label cathepsin B in eighteen cancer cell lines tested, making this ABP highly suitable for other biological setups. Moreover, using Cy5-labelled MP-CB-2 we were able to demonstrate by fluorescence microscopy that in cancer cells cathepsins B and L share overlapping, but not identical subcellular localization.

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 ± 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).

Synthesis and kinetic evaluation of ethyl acrylate and vinyl sulfone derived inhibitors for human cysteine cathepsins

A series of inhibitors targeting human cathepsins have been designed and synthesized following a combinatorial approach. The compounds bear an α,β-unsaturated phenyl vinyl sulfone or ethyl acrylate warhead and a peptidomimetic portion aligned to the non-primed binding region. Biochemical evaluation toward four human cathepsins was carried out and the kinetic characterization confirmed an irreversible mode of inhibition. Compound 6c combining the most advantageous building blocks for cathepsin S inhibition was identified as a potent cathepsin S inactivator exhibiting a second-order rate constant of 30600 M−1 s−1.

Computational predictions of cysteine cathepsin-mediated fibrinogen proteolysis.

Fibrin clot formation is a proteolytic cascade of events with thrombin and plasmin identified as the main proteases cleaving fibrinogen precursor, and the fibrin polymer, respectively. Other proteases may be involved directly in fibrin(ogen) cleavage, clot formation, and resolution, or in the degradation of fibrin-based scaffolds emerging as useful tools for tissue engineered constructs. Here, cysteine cathepsins are investigated for their putative ability to hydrolyze fibrinogen, since they are potent proteases, first identified in lysosomal protein degradation and known to participate in extracellular proteolysis. To further explore this, we used two independent computational technqiues, molecular docking and bioinformatics sequence analysis (PACMANS), to predict potential binding interactions and sites of hydrolysis between cathepsins K, L, and S and fibrinogen. By comparing the results from these two objective, computational methods, it was determined that cathepsins K, L, and S do bind and cleave fibrinogen α, β, and γ chains at similar and unique sites. These differences were visualized experimentally by the unique cleaved fibrinogen banding patterns after incubation with each of the cathepsins, separately. In conclusion, human cysteine cathepsins K, L, and S are a new class of proteases that should be considered during fibrin(ogen) degradation studies both for disease processes where coagulation is a concern, and also in the implementation and design of bioengineered systems.

Antimicrobial proteins and peptides in human lung diseases: A friend and foe partnership with host proteases.

Lung antimicrobial proteins and peptides (AMPs) are major sentinels of innate immunity by preventing microbial colonization and infection. Nevertheless bactericidal activity of AMPs against Gram-positive and Gram-negative bacteria is compromised in patients with chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF) and asthma. Evidence is accumulating that expression of harmful human serine proteases, matrix metalloproteases and cysteine cathepsins is markedely increased in these chronic lung diseases. The local imbalance between proteases and protease inhibitors compromises lung tissue integrity and function, by not only degrading extracellular matrix components, but also non-matrix proteins. Despite the fact that AMPs are somewhat resistant to proteolytic degradation, some human proteases cleave them efficiently and impair their antimicrobial potency. By contrast, certain AMPs may be effective as antiproteases. Host proteases participate in concert with bacterial proteases in the degradation of key innate immunity peptides/proteins and thus may play immunomodulatory activities during chronic lung diseases. In this context, the present review highlights the current knowledge and recent discoveries on the ability of host enzymes to interact with AMPs, providing a better understanding of the role of human proteases in innate host defense.

Active Site Mapping of Human Cathepsin F with Dipeptide Nitrile Inhibitors.

Cleavage of the invariant chain is the key event in the trafficking pathway of major histocompatibility complex class II. Cathepsin S is the major processing enzyme of the invariant chain, but cathepsin F acts in macrophages as its functional synergist which is as potent as cathepsin S in invariant chain cleavage. Dedicated low-molecular-weight inhibitors for cathepsin F have not yet been developed. An active site mapping with 52 dipeptide nitriles, reacting as covalent-reversible inhibitors, was performed to draw structure-activity relationships for the non-primed binding region of human cathepsin F. In a stepwise process, new compounds with optimized fragment combinations were designed and synthesized. These dipeptide nitriles were evaluated on human cysteine cathepsins F, B, L, K and S. Compounds 10 (N-(4-phenylbenzoyl)-leucylglycine nitrile) and 12 (N-(4-phenylbenzoyl)leucylmethionine nitrile) were found to be potent inhibitors of human cathepsin F, with Ki values <10 nM. With all dipeptide nitriles from our study, a 3D activity landscape was generated to visualize structure-activity relationships for this series of cathepsin F inhibitors.

Cysteine cathepsins: their role in tumor progression and recent trends in the development of imaging probes.

Papain-like cysteine proteases bear an enormous potential as drug discovery targets for both infectious and systemic human diseases. The considerable progress in this field over the last two decades has also raised interest in the visualization of these enzymes in their native context, especially with regard to tumor imaging. After a short introduction to structure and general functions of human cysteine cathepsins, we highlight their importance for drug discovery and development and provide a critical update on the current state of knowledge toward their involvement in tumor progression, with a special emphasis on their role in therapy response. In accordance with a radiopharmaceutical point of view, the main focus of this review article will be the discussion of recently developed fluorescence and radiotracer-based imaging agents together with related molecular probes.

Cysteine cathepsins and cystatins: from ancillary tasks to prominent status in lung diseases.

Human cysteine cathepsins (family C1, clan CA) have long been regarded as ubiquitous household enzymes, primarily involved in the recycling and degradation of proteins in lysosomes. This opinion has changed considerably during recent decades, however, with the demonstration of their involvement in various physiological processes. A growing body of evidence supports the theory that cathepsins play specific functions in lung homeostasis and pathophysiological events such as asthma, lung fibrosis (including idiopathic pulmonary fibrosis), chronic obstructive pulmonary disease (embracing emphysema and chronic bronchitis), silicosis, bronchopulmonary dysplasia or tumor invasion. The objective of this review is to provide an update on the current knowledge of the role of these enzymes in the lung. Particular attention has been paid to the understanding of the role of these proteases and their natural inhibitors, cystatins (family I25, clan IH), in TGF-β1-driven fibrotic processes with an emphasis on lung fibrosis.

3-Cyano-3-aza-β-amino Acid Derivatives as Inhibitors of Human Cysteine Cathepsins

Nitrile-type inhibitors are known to interact with cysteine proteases in a covalent-reversible manner. The chemotype of 3-cyano-3-aza-β-amino acid derivatives was designed in which the N-cyano group is centrally arranged in the molecule to allow for interactions with the nonprimed and primed binding regions of the target enzymes. These compounds were evaluated as inhibitors of the human cysteine cathepsins K, S, B, and L. They exhibited slow-binding behavior and were found to be exceptionally potent, in particular toward cathepsin K, with second-order rate constants up to 52 900 × 10(3) M(-1) s(-1).

Regulation of TGF-β1-driven Differentiation of Human Lung Fibroblasts: EMERGING ROLES OF CATHEPSIN B AND CYSTATIN C

Lung matrix homeostasis partly depends on the fine regulation of proteolytic activities. We examined the expression of human cysteine cathepsins (Cats) and their relative contribution to TGF-β1-induced fibroblast differentiation into myofibroblasts. Assays were conducted using both primary fibroblasts obtained from patients with idiopathic pulmonary fibrosis and human lung CCD-19Lu fibroblasts. Pharmacological inhibition and genetic silencing of Cat B diminished α-smooth muscle actin expression, delayed fibroblast differentiation, and led to an accumulation of intracellular 50-kDa TGF-β1. Moreover, the addition of Cat B generated a 25-kDa mature form of TGF-β1 in Cat B siRNA-pretreated lysates. Inhibition of Cat B decreased Smad 2/3 phosphorylation but had no effect on p38 MAPK and JNK phosphorylation, indicating that Cat B mostly disturbs TGF-β1-driven canonical Smad signaling pathway. Although mRNA expression of cystatin C was stable, its secretion, which was inhibited by brefeldin A, increased during TGF-β1-induced differentiation of idiopathic pulmonary fibrosis and CCD-19Lu fibroblasts. In addition, cystatin C participated in the control of extracellular Cats, because its gene silencing restored their proteolytic activities. These data support the notion that Cat B participates in lung myofibrogenesis as suggested for stellate cells during liver fibrosis. Moreover, we propose that TGF-β1 promotes fibrosis by driving the effective cystatin C-dependent inhibition of extracellular matrix-degrading Cats.

Kinetics and molecular dynamic modeling of a potent and selective marine natural product inhibitor of human cathepsin L (975.3)

Protease inhibitors are an important drug target class due to their wide and varied roles in both physiological and pathological states. In particular, the human cysteine cathepsin proteases have been implicated in the progression and metastasis of cancer, participation in the development of neurodegenerative diseases, and are important processing enzymes for the production of multiple neuropeptides. Past marine natural products screening efforts have successfully yielded potent and selective inhibitors of proteases, hence, marine cyanobacterial extracts were assessed for inhibitory activity to human cathepsin L. Gallinamide A, a depsipeptide secondary metabolite isolated from a collection of filamentous marine cyanobacteria, was identified as a hit structure with potent activity. With 30 minutes of preincubation, gallinamide A displayed an IC50 of 5.0 nM, and kinetic analysis demonstrated an inhibition constant of ki = 9009 ± 262 M‐1 s‐1. A preincubation‐dilution experiment revealed an irreversible mode of inhibition, and comparative IC50 values display a 28‐ to 331‐ fold greater selectivity toward cathepsin L than closely related human cysteine cathepsins V or B. Gallinamide A shows no inhibitory activity against the exopeptidase cathepsin H, further exemplifying its selectivity. Molecular docking and molecular dynamics simulations were used to determine the pose of gallinamide in the active site of cathepsin L. These data resulted in the identification of a pose characterized by high stability, a consistent hydrogen bond network, and the reactive Michael acceptor enone functional group of gallinamide A positioned near the active site cysteine of the protease, leading to a proposed mechanism of covalent inhibition. These data reveal and characterize the novel activity of gallinamide A as a potent inhibitor of human cathepsin L.Grant Funding Source: Supported by NIH T32GM007752, NIH FIC U01 TW006634, and NIH R01MH077305

A Coumarin‐Labeled Vinyl Sulfone as Tripeptidomimetic Activity‐Based Probe for Cysteine Cathepsins

A coumarin-tetrahydroquinoline hydride 8 was synthesized as a chemical tool for fluorescent labeling. The rigidified tricyclic coumarin structure was chosen for its suitable fluorescence properties. The connection of 8 with a vinyl sulfone building block was accomplished by convergent synthesis thereby leading to the coumarin-based, tripeptidomimetic activity-based probe 10, containing a Gly-Phe-Gly motif. Probe 10 was evaluated as inactivator of the therapeutically relevant human cysteine cathepsins S, L, K, and B: it showed particularly strong inactivation of cathepsin S. The detection of recombinant and native cathepsin S was demonstrated by applying 10 to in-gel fluorescence imaging.

Specific cleavage of the lung surfactant protein A by human cathepsin S may impair its antibacterial properties

Human cysteine cathepsins (Cats) are implicated in lung injuries and tissue remodeling and have recently emerged as important players in pulmonary inflammations. The proteolytic activities of Cat B, L, K, S and H are dramatically increased in the sputum of patients with cystic fibrosis (CF), suggesting a possible involvement in the CF pathophysiology. We found that pulmonary surfactant protein A (SP-A) that participates to innate host defense is extensively degraded in CF expectorations. Breakdown of SP-A was markedly decreased in CF sputum by E-64 and Mu-Leu-Hph-VSPh, a Cat S inhibitor. Cat S cleaved efficiently and specifically SP-A within critical residues of the solvent-exposed loop of its carbohydrate recognition (C-type lectin) domain that allows binding to pathogens. Cat S decreased aggregation properties of SP-A (self-aggregation, aggregation of phospholipid vesicles and rough LPS). Moreover cleavage of SP-A by Cat S reduced binding to yeast mannan and impaired agglutination of Escherichia coli and Pseudomonas aeruginosa, a foremost detrimental pathogen colonizing the lungs of CF patients. Besides human neutrophil serine proteases and bacterial proteases, we propose that Cat S may participate in the pathophysiology of CF by weakening the antibacterial activity of SP-A. More broadly, present results provide further indication that Cat S, along with Cats B and L, could display immuno-modulatory functions by inactivating key proteins involved in the innate immunity defense.