Structure-based design and in vivo anti-arthritic activity evaluation of a potent dipeptidyl cyclopropyl nitrile inhibitor of cathepsin C.

Cathepsin C (CatC) is a dipeptidyl-exopeptidase which activates neutrophil serine protease precursors (elastase, proteinase 3, cathepsin G and NSP4) by removing their N-terminal propeptide in bone marrow cells at the promyelocytic stage of neutrophil differentiation. The resulting active proteases are implicated in chronic inflammatory and autoimmune diseases. Hence, inhibition of CatC represents a therapeutic strategy to suppress excessive protease activities in various neutrophil mediated diseases. We designed and synthesized a series of dipeptidyl cyclopropyl nitrile compounds as putative CatC inhibitors. One compound, IcatCXPZ-01 ((S)-2-amino-N-((1R,2R)-1-cyano-2-(4'-(4-methylpiperazin-1-ylsulfonyl)biphenyl-4-yl)cyclopropyl)butanamide)) was identified as a potent inhibitor of both human and rodent CatC. In mice, pharmacokinetic studies revealed that IcatCXPZ-01 accumulated in the bone marrow reaching levels suitable for CatC inhibition. Subcutaneous administration of IcatCXPZ-01 in a monoclonal anti-collagen antibody induced mouse model of rheumatoid arthritis resulted in statistically significant anti-arthritic activity with persistent decrease in arthritis scores and paw thickness.

Consequences of cathepsin C inactivation for membrane exposure of proteinase 3, the target antigen in autoimmune vasculitis.

Membrane-bound proteinase 3 (PR3m) is the main target antigen of anti-neutrophil cytoplasmic autoantibodies (ANCA) in granulomatosis with polyangiitis, a systemic small-vessel vasculitis. Binding of ANCA to PR3m triggers neutrophil activation with the secretion of enzymatically active PR3 and related neutrophil serine proteases, thereby contributing to vascular damage. PR3 and related proteases are activated from pro-forms by the lysosomal cysteine protease cathepsin C (CatC) during neutrophil maturation. We hypothesized that pharmacological inhibition of CatC provides an effective measure to reduce PR3m and therefore has implications as a novel therapeutic approach in granulomatosis with polyangiitis. We first studied neutrophilic PR3 from 24 patients with Papillon-Lefèvre syndrome (PLS), a genetic form of CatC deficiency. PLS neutrophil lysates showed a largely reduced but still detectable (0.5-4%) PR3 activity when compared with healthy control cells. Despite extremely low levels of cellular PR3, the amount of constitutive PR3m expressed on the surface of quiescent neutrophils and the typical bimodal membrane distribution pattern were similar to what was observed in healthy neutrophils. However, following cell activation, there was no significant increase in the total amount of PR3m on PLS neutrophils, whereas the total amount of PR3m on healthy neutrophils was significantly increased. We then explored the effect of pharmacological CatC inhibition on PR3 stability in normal neutrophils using a potent cell-permeable CatC inhibitor and a CD34+ hematopoietic stem cell model. Human CD34+ hematopoietic stem cells were treated with the inhibitor during neutrophil differentiation over 10 days. We observed strong reductions in PR3m, cellular PR3 protein, and proteolytic PR3 activity, whereas neutrophil differentiation was not compromised.

Therapeutic targeting of cathepsin C: from pathophysiology to treatment.

Cathepsin C (CatC) is a highly conserved tetrameric lysosomal cysteine dipeptidyl aminopeptidase. The best characterized physiological function of CatC is the activation of pro-inflammatory granule-associated serine proteases. These proteases are synthesized as inactive zymogens containing an N-terminal pro-dipeptide, which maintains the zymogen in its inactive conformation and prevents premature activation, which is potentially toxic to the cell. The activation of serine protease zymogens occurs through cleavage of the N-terminal dipeptide by CatC during cell maturation in the bone marrow. In vivo data suggest that pharmacological inhibition of pro-inflammatory serine proteases would suppress or attenuate deleterious effects mediated by these proteases in inflammatory/auto-immune disorders. The pathological deficiency in CatC is associated with Papillon-Lefèvre syndrome (PLS). The patients however do not present marked immunodeficiency despite the absence of active serine proteases in immune defense cells. Hence, the transitory pharmacological blockade of CatC activity in the precursor cells of the bone marrow may represent an attractive therapeutic strategy to regulate activity of serine proteases in inflammatory and immunologic conditions. A variety of CatC inhibitors have been developed both by pharmaceutical companies and academic investigators, some of which are currently being employed and evaluated in preclinical/clinical trials.

Prolonged pharmacological inhibition of cathepsin C results in elimination of neutrophil serine proteases.

Cathepsin C (CatC) is a tetrameric cysteine dipeptidyl aminopeptidase that plays a key role in activation of pro-inflammatory serine protease zymogens by removal of a N-terminal pro-dipeptide sequence. Loss of function mutations in the CatC gene is associated with lack of immune cell serine protease activities and cause Papillon-Lefèvre syndrome (PLS). Also, only very low levels of elastase-like protease zymogens are detected by proteome analysis of neutrophils from PLS patients. Thus, CatC inhibitors represent new alternatives for the treatment of neutrophil protease-driven inflammatory or autoimmune diseases. We aimed to experimentally inactivate and lower neutrophil elastase-like proteases by pharmacological blocking of CatC-dependent maturation in cell-based assays and in vivo. Isolated, immature bone marrow cells from healthy donors pulse-chased in the presence of a new cell permeable cyclopropyl nitrile CatC inhibitor almost totally lack elastase. We confirmed the elimination of neutrophil elastase-like proteases by prolonged inhibition of CatC in a non-human primate. We also showed that neutrophils lacking elastase-like protease activities were still recruited to inflammatory sites. These preclinical results demonstrate that the disappearance of neutrophil elastase-like proteases as observed in PLS patients can be achieved by pharmacological inhibition of bone marrow CatC. Such a transitory inhibition of CatC might thus help to rebalance the protease load during chronic inflammatory diseases, which opens new perspectives for therapeutic applications in humans.

Reprint of: Current strategies for the use of affinity tags and tag removal for the purification of recombinant proteins.

Affinity tags are highly efficient tools for protein purification. They allow the purification of virtually any protein without any prior knowledge of its biochemical properties. The use of affinity tags has therefore become widespread in several areas of research e.g., high throughput expression studies aimed at finding a biological function to large numbers of yet uncharacterized proteins. In some cases, the presence of the affinity tag in the recombinant protein is unwanted or may represent a disadvantage for the projected application of the protein, like for clinical use. Therefore, an increasing number of approaches are available at present that are designed for the removal of the affinity tag from the recombinant protein. Most of these methods employ recombinant endoproteases that recognize a specific sequence. These process enzymes can subsequently be removed from the process by affinity purification, since they also include a tag. Here, a survey of the most common affinity tags and the current methods for tag removal is presented, with special emphasis on the removal of N-terminal histidine tags using TAGZyme, a system based on exopeptidase cleavage. In the quest to reduce the significant costs associated with protein purification at large scale, relevant aspects involved in the development of downstream processes for pharmaceutical protein production that incorporate a tag removal step are also discussed. A comparison of the yield of standard vs. affinity purification together with an example of tag removal using TAGZyme is also included.

Reprint of: Current strategies for the use of affinity tags and tag removal for the purification of recombinant proteins.

Affinity tags are highly efficient tools for protein purification. They allow the purification of virtually any protein without any prior knowledge of its biochemical properties. The use of affinity tags has therefore become widespread in several areas of research e.g., high throughput expression studies aimed at finding a biological function to large numbers of yet uncharacterized proteins. In some cases, the presence of the affinity tag in the recombinant protein is unwanted or may represent a disadvantage for the projected application of the protein, like for clinical use. Therefore, an increasing number of approaches are available at present that are designed for the removal of the affinity tag from the recombinant protein. Most of these methods employ recombinant endoproteases that recognize a specific sequence. These process enzymes can subsequently be removed from the process by affinity purification, since they also include a tag. Here, a survey of the most common affinity tags and the current methods for tag removal is presented, with special emphasis on the removal of N-terminal histidine tags using TAGZyme, a system based on exopeptidase cleavage. In the quest to reduce the significant costs associated with protein purification at large scale, relevant aspects involved in the development of downstream processes for pharmaceutical protein production that incorporate a tag removal step are also discussed. A comparison of the yield of standard vs. affinity purification together with an example of tag removal using TAGZyme is also included.

Biochemical characterization of Plasmodium falciparum dipeptidyl aminopeptidase 1.

Dipeptidyl aminopeptidase 1 (DPAP1) is an essential food vacuole enzyme with a putative role in hemoglobin catabolism by the erythrocytic malaria parasite. Here, the biochemical properties of DPAP1 have been investigated and compared to those of the human ortholog cathepsin C. To facilitate the characterization of DPAP1, we have developed a method for the production of purified recombinant DPAP1 with properties closely resembling those of the native enzyme. Like cathepsin C, DPAP1 is a chloride-activated enzyme that is most efficient in catalyzing amide bond hydrolysis at acidic pH values. The monomeric quaternary structure of DPAP1 differs from the homotetrameric structure of cathepsin C, which suggests that tetramerization is required for a cathepsin C-specific function. The S1 and S2 subsite preferences of DPAP1 and cathepsin C were profiled with a positional scanning synthetic combinatorial library. The S1 preferences bore close similarity to those of other C1-family cysteine peptidases. The S2 subsites of both DPAP1 and cathepsin C accepted aliphatic hydrophobic residues, proline, and some polar residues, yielding a distinct specificity profile. DPAP1 efficiently catalyzed the hydrolysis of several fluorogenic dipeptide substrates; surprisingly, however, a potential substrate with a P2-phenylalanine residue was instead a competitive inhibitor. Together, our biochemical data suggest that DPAP1 accelerates the production of amino acids from hemoglobin by bridging the gap between the endopeptidase and aminopeptidase activities of the food vacuole. Two reversible cathepsin C inhibitors potently inhibited both recombinant and native DPAP1, thereby validating the use of recombinant DPAP1 for future inhibitor discovery and characterization.

The use of TAGZyme for the efficient removal of N-terminal His-tags.

The use of affinity tags and especially histidine tags (His-tags) has become widespread in molecular biology for the efficient purification of recombinant proteins. In some cases, the presence of the affinity tag in the recombinant protein is unwanted or may represent a disadvantage for the projected use of the protein, like in clinical, functional or structural studies. For N-terminal tags, the TAGZyme system represents an ideal approach for fast and accurate tag removal. TAGZyme is based on engineered aminopeptidases. Using human tumor necrosis factor alpha as a model protein, we describe here the steps involved in the removal of a His-tag using TAGZyme. The tag used (UZ-HT15) has been optimized for expression in Escherichia coli and for TAGZyme efficiency. The UZ-HT15 tag and the method can be applied to virtually any protein. A description of the cloning strategy for the design of the genetic construction, two alternative approaches and a simple test to assess the performance of the tag removal process are also included.

The crystal structure of human dipeptidyl peptidase I (cathepsin C) in complex with the inhibitor Gly-Phe-CHN2.

hDDPI (human dipeptidyl peptidase I) is a lysosomal cysteine protease involved in zymogen activation of granule-associated proteases, including granzymes A and B from cytotoxic T-lymphocytes and natural killer cells, cathepsin G and neutrophil elastase, and mast cell tryptase and chymase. In the present paper, we provide the first crystal structure of an hDPPI-inhibitor complex. The inhibitor Gly-Phe-CHN2 (Gly-Phe-diazomethane) was co-crystallized with hDPPI and the structure was determined at 2.0 A (1 A=0.1 nm) resolution. The structure of the native enzyme was also determined to 2.05 A resolution to resolve apparent discrepancies between the complex structure and the previously published structure of the native enzyme. The new structure of the native enzyme is, within the experimental error, identical with the structure of the enzyme-inhibitor complex presented here. The inhibitor interacts with three subunits of hDPPI, and is covalently bound to Cys234 at the active site. The interaction between the totally conserved Asp1 of hDPPI and the ammonium group of the inhibitor forms an essential interaction that mimics enzyme-substrate interactions. The structure of the inhibitor complex provides an explanation of the substrate specificity of hDPPI, and gives a background for the design of new inhibitors.

Papaya glutamine cyclotransferase shows a singular five-fold beta-propeller architecture that suggests a novel reaction mechanism.

Cyclisation of N-terminal glutamine and/or glutamate to yield pyroglutamate is an essential posttranslational event affecting a plethora of bioactive peptides and proteins. It is directly linked with pathologies ranging from neurodegenerative diseases to inflammation and several types of cancers. The reaction is catalysed by ubiquitous glutaminyl cyclotransferases (QCs), which present two distinct prototypes. Mammalian QCs are zinc-dependent enzymes with an alpha/beta-hydrolase fold. Here we present the 1.6-A-resolution structure of the other prototype, the plant analogue from Carica papaya (PQC). The hatbox-shaped molecule consists of an unusual five-fold beta-propeller traversed by a central channel, a topology that has hitherto been described only for some sugar-binding proteins and an extracellular nucleotidase. The high resistance of the enzyme to denaturation and proteolytic degradation is explained by its architecture, which is uniquely stabilised by a series of tethering elements that confer rigidity. Strikingly, the N-terminus of PQC specifically interacts with residues around the entrance to the central channel of a symmetry-related molecule, suggesting that this location is the putative active site. Cyclisation would follow a novel general-acid/base working mechanism, pivoting around a strictly conserved glutamate. This study provides a lead structure not only for plant QC orthologues, but also for bacteria, including potential human pathogens causing diphtheria, plague and malaria.

Current strategies for the use of affinity tags and tag removal for the purification of recombinant proteins.

Affinity tags are highly efficient tools for protein purification. They allow the purification of virtually any protein without any prior knowledge of its biochemical properties. The use of affinity tags has therefore become widespread in several areas of research e.g., high throughput expression studies aimed at finding a biological function to large numbers of yet uncharacterized proteins. In some cases, the presence of the affinity tag in the recombinant protein is unwanted or may represent a disadvantage for the projected application of the protein, like for clinical use. Therefore, an increasing number of approaches are available at present that are designed for the removal of the affinity tag from the recombinant protein. Most of these methods employ recombinant endoproteases that recognize a specific sequence. These process enzymes can subsequently be removed from the process by affinity purification, since they also include a tag. Here, a survey of the most common affinity tags and the current methods for tag removal is presented, with special emphasis on the removal of N-terminal histidine tags using TAGZyme, a system based on exopeptidase cleavage. In the quest to reduce the significant costs associated with protein purification at large scale, relevant aspects involved in the development of downstream processes for pharmaceutical protein production that incorporate a tag removal step are also discussed. A comparison of the yield of standard vs. affinity purification together with an example of tag removal using TAGZyme is also included.

Cloning strategy, production and purification of proteins with exopeptidase-cleavable His-tags.

Here, we present a cloning strategy for the production of recombinant proteins tagged with a polyhistidine sequence that can be cleaved by the exopeptidase, DAPase. The method can be used with most commonly available vectors and results in the expression of a His-tag protein that can be purified in its native form regardless of its natural sequence. This approach takes advantage of the TAGZyme system for the removal of amino-terminal affinity tags. Tag removal is accomplished either with DAPase (a recombinant dipeptidyl peptidase) alone or in combination with two accessory enzymes, Qcyclase and pGAPase. The system has been used for the production of intracellular proteins in Escherichia coli and can be applied to other expression hosts for the production of secreted proteins or proteins that require post-translational modification. The production of human interleukin 1beta in E. coli is used as an example to illustrate this method. The complete protocol from initial PCR to the production of a detagged protein with its authentic N terminus can be performed within 5 days.

Metal binding sites in proteins: identification and characterization by paramagnetic NMR relaxation.

A method is presented that allows the identification and quantitative characterization of metal binding sites in proteins using paramagnetic nuclear magnetic resonance spectroscopy. The method relies on the nonselective longitudinal relaxation rates of the amide protons and their dependence on the paramagnetic metal ion concentration and the pH, and on the three-dimensional structure of the protein. The method is demonstrated using Escherichia coli thioredoxin as a model protein and Ni(2+) as the paramagnetic metal ion. Through a least-squares analysis of the relaxation rates, it is found that Ni(2+) binds to a series of specific sites on the surface of thioredoxin. The strongest binding site is found near the N-terminus of the protein, where the metal ion is coordinated to the free NH(2) group of the N-terminal serine residue and the side chain carboxylate group of the aspartic acid residue in position 2. In addition, Ni(2+) binds specifically but more weakly to the surface-exposed side chain carboxylate groups of residues D10, D20, D47, and E85.

Novel semicarbazide-derived inhibitors of human dipeptidyl peptidase I (hDPPI).

Human dipeptidyl peptidase I (hDPPI, cathepsin C, EC 3.4.14.1) is a novel putative drug target for the treatment of inflammatory diseases. Using 1 as a starting point (IC50>10 microM), we have improved potency by more than 500-fold and successfully identified novel inhibitors of DPPI via screening of a one-bead-two-compounds library of semicarbazide derivatives. Selected compounds were shown to inhibit intracellular DPPI in RBL-2H3 cells. These compounds were further characterized for adverse effects on HepG2 cells (cytotoxicity and viability) and their metabolic stability in rat liver microsomes was estimated. One of the most potent inhibitors, 8 (IC50=31+/-3 nM; Ki=45+/-2 nM, competitive inhibition), is selective for DPPI over other cysteine and serine proteases, has a half-life of 24 min in rat liver microsomes, shows approximately 50% inhibition of intracellular DPPI at 20 microM and is noncytotoxic.

Binding ability of a HHP-tagged protein towards Ni2+ studied by paramagnetic NMR relaxation: the possibility of obtaining long-range structure information.

The binding ability of a protein with a metal binding tag towards Ni(2+) was investigated by longitudinal paramagnetic NMR relaxation, and the possibility of obtaining long-range structure information from the paramagnetic relaxation was explored. A protein with a well-defined solution structure (Escherichia coli thioredoxin) was used as the model system, and the peptide His-His-Pro (HHP) fused to the N-terminus of the protein was used as the metal binding tag. It was found that the tag forms a stable dimer complex with the paramagnetic Ni(2+) ion, where each metal ion binds two HHP-tagged protein molecules. However, it was also found that additional sites in the protein compete with the HHP-tag for the binding of the metal ion. These binding sites were identified as the side chain carboxylate groups of the aspartic and glutamic acid residues. Yet, the carboxylate groups bind the Ni(2+) ions considerably weaker than the HHP-tag, and only protons spatially close to the carboxylate sites are affected by the Ni(2+) ions bound to these groups. As for the protons that are unaffected by the carboxylate-bound Ni(2+) ions, it was found that the long-range distances derived from the paramagnetic relaxation enhancements are in good agreement with the solution structure of thioredoxin. Specifically, the obtained long-range paramagnetic distance constraints revealed that the dimer complex is asymmetric with different orientations of the two protein molecules relative to the Ni(2+) ion.