Chondroitin Sulfate Promotes Activation of Cathepsin K

Cathepsin K (CatK), a major lysosomal collagenase produced by osteoclasts, plays an important role in bone resorption. Evidence exists that the collagenase activity of CatK is promoted by chondroitin sulfate (CS), a sulfated glycosaminoglycan. This study examines the role of CS in facilitating CatK activation. We have demonstrated that chondroitin 4-sulfate (C4-S) promotes autoprocessing of the pro-domain of CatK at pH ≤ 5, leading to a fully matured enzyme with collagenase and peptidase activities. We present evidence to demonstrate this autoactivation process is a trans-activation event that is efficiently inhibited by both the covalent cysteine protease inhibitor E-64 and the reversible selective CatK inhibitor L-006,235. During bone resorption, CatK and C4-S are co-localized at the ruffled border between osteoclast bone interface, supporting the proposal that CatK activation is accomplished through the combined action of the acidic environment together with the presence of a high concentration of C4-S. Formation of a multimeric complex between C4-S and pro-CatK has been speculated to accelerate CatK autoactivation and promote efficient collagen degradation. Together, these results demonstrate that CS plays an important role in contributing to the enhanced efficiency of CatK collagenase activity in vivo.     

Design,synthesis and biological evaluation of inhibitors of cathepsin K on dedifferentiated chondrocytes

Selective proteinase inhibitors have demonstrated utility in the investigation of cartilage degeneration mechanisms and may have clinical use in the management of osteoarthritis. The cysteine protease cathepsin K (CatK) is an attractive target for arthritis therapy. Here we report the synthesis of two cathepsin K inhibitors (CKIs): racemic azanitrile derivatives CKI-E and CKI-F, which have better inhibition properties on CatK than the commercial inhibitor odanacatib (ODN). Their IC₅₀ values and inhibition constants (K_i) have been determined in vitro. Inhibitors demonstrate differential selectivity for CatK over cathepsin B, L and S in vitro, with K_i amounting to 1.14 and 7.21?nM respectively. We analyzed the effect of these racemic inhibitors on viability in different cell types. The human osteoblast-like cell line MG63, MOVAS cells (a murine vascular smooth muscle cell line) or murine primary chondrocytes, were treated either with CKI-E or with CKI-F, which were not toxic at doses of up to 5?µM. Primary chondrocytes subjected to several passages were used as a model of phenotypic loss of articular chondrocytes, occurring in osteoarthritic cartilage. The efficiency of CKIs regarding CatK inhibition and their specificity over other proteases were validated in primary chondrocytes subjected to several passages. Racemic CKI-E and CKI-F at 0.1 and 1?µM significantly inhibited CatK activity in dedifferentiated chondrocytes, even better than the commercial CatK inhibitor ODN. The enzymatic activity of other proteases such as matrix metalloproteinases or aggrecanases were not affected. Taken together, these findings support the possibility to design CatK inhibitors for preventing cartilage degradation in different pathologies.     

Discovery of potent selective bioavailable phosphodiesterase 2 (PDE2) inhibitors active in an osteoarthritis pain model. Part II: Optimization studies and demonstration of in vivo efficacy

Selective phosphodiesterase 2 (PDE2) inhibitors are shown to have efficacy in a rat model of osteoarthritis (OA) pain. We identified potent, selective PDE2 inhibitors by optimizing residual PDE2 activity in a series of phosphodiesterase 4 (PDE4) inhibitors, while minimizing PDE4 inhibitory activity. These newly designed PDE2 inhibitors bind to the PDE2 enzyme in a cGMP-like binding mode orthogonal to the cAMP-like binding mode found in PDE4. Extensive structure activity relationship studies ultimately led to identification of pyrazolodiazepinone, 22, which was >1000-fold selective for PDE2 over recombinant, full length PDEs 1B, 3A, 3B, 4A, 4B, 4C, 7A, 7B, 8A, 8B, 9, 10 and 11. Compound 22 also retained excellent PDE2 selectivity (241-fold to 419-fold) over the remaining recombinant, full length PDEs, 1A, 4D, 5, and 6. Compound 22 exhibited good pharmacokinetic properties and excellent oral bioavailability (F = 78%, rat). In an in vivo rat model of OA pain, compound 22 had significant analgesic activity 1 and 3 h after a single, 10 mg/kg, subcutaneous dose.     

A new xylanase from thermoacidophilic Alicyclobacillus sp. A4 with broad-range pH activity and pH stability

We have identified a highly pH-adaptable and stable xylanase (XynA4) from the thermoacidophilic Alicyclobacillus sp. A4, a strain that was isolated from a hot spring in Yunnan Province, China. The gene (xynA4) that encodes this xylanase was cloned, sequenced, and expressed in Escherichia coli. It encodes a 338-residue polypeptide with a calculated molecular mass of 42.5 kDa. The deduced amino acid sequence is most similar to (53% identity) an endo-1,4-β-xylanase from Geobacillus stearothermophilus that belongs to family 10 of the glycoside hydrolases. Purified recombinant XynA4 exhibited maximum activity at 55°C and pH 7.0, had broad pH adaptability (>40% activity at pH 3.8–9.4) and stability (retaining >80% activity after incubation at pH 2.6–12.0 for 1 h at 37°C), and was highly thermostable (retaining >90% activity after incubation at 60°C for 1 h at pH 7.0). These properties make XynA4 promising for application in the paper industry. This is the first report that describes cloning and expression of a xylanase gene from the genus Alicyclobacillus.     

The crystal and molecular structures of a cathepsin K:chondroitin sulfate complex

Cathepsin K is the major collagenolytic enzyme produced by bone-resorbing osteoclasts. We showed earlier that the unique triple-helical collagen-degrading activity of cathepsin K depends on the formation of complexes with bone-or cartilage-resident glycosaminoglycans, such as chondroitin 4-sulfate (C4-S). Here, we describe the crystal structure of a 1:n complex of cathepsin K:C4-S inhibited by E64 at a resolution of 1.8 Å. The overall structure reveals an unusual “beads-on-a-string”-like organization. Multiple cathepsin K molecules bind specifically to a single cosine curve-shaped strand of C4-S with each cathepsin K molecule interacting with three disaccharide residues of C4-S. One of the more important sets of interactions comes from a single turn of helix close to the N terminus of the proteinase containing a basic amino acid triplet (Arg8-Lys9-Lys10) that forms multiple hydrogen bonds either to the caboxylate or to the 4-sulfate groups of C4-S. Altogether, the binding sites with C4-S are located in the R-domain of cathepsin K and are distant from its active site. This explains why the general proteolytic activity of cathepsin K is not affected by the binding of chondroitin sulfate. Biochemical analyses of cathepsin K and C4-S mixtures support the presence of a 1:n complex in solution; a dissociation constant, K_d, of about 10 nM was determined for the interaction between cathepsin K and C4-S.     

Endogenous reduction of miR‐185 accelerates cardiac function recovery in mice following myocardial infarction via targeting of cathepsin K

Angiogenesis is critical for re‐establishing the blood supply to the surviving myocardium after myocardial infarction (MI) in patients with acute coronary syndrome (ACS). MicroRNAs are recognised as important epigenetic regulators of endothelial function. The aim of this study was to determine the roles of microRNAs in angiogenesis. Eighteen circulating microRNAs including miR‐185‐5p were differently expressed in plasma from patients with ACS by high‐throughput RNA sequencing. The expressional levels of miR‐185‐5p were dramatically reduced in hearts isolated from mice following MI and cultured human umbilical vein endothelial cells (HUVECs) under hypoxia, as determined by fluorescence in situ hybridisation and quantitative RT‐PCR. Evidence from computational prediction and luciferase reporter gene activity indicated that cathepsin K (CatK) mRNA is a target of miR‐185‐5p. In HUVECs, miR‐185‐5p mimics inhibited cell proliferations, migrations and tube formations under hypoxia, while miR‐185‐5p inhibitors performed the opposites. Further, the inhibitory effects of miR‐185‐5p up‐regulation on cellular functions of HUVECs were abolished by CatK gene overexpression, and adenovirus‐mediated CatK gene silencing ablated these enhancive effects in HUVECs under hypoxia. In vivo studies indicated that gain‐function of miR‐185‐5p by agomir infusion down‐regulated CatK gene expression, impaired angiogenesis and delayed the recovery of cardiac functions in mice following MI. These actions of miR‐185‐5p agonists were mirrored by in vivo knockdown of CatK in mice with MI. Endogenous reductions of miR‐185‐5p in endothelial cells induced by hypoxia increase CatK gene expression to promote angiogenesis and to accelerate the recovery of cardiac function in mice following MI.     

Optimization of culture conditions and properties of lipase from Penicillium camembertii Thom PG-3

The culture medium including nitrogen source, carbon source and metal ions, for lipase from Penicillium camembertii Thom PG-3 was optimized and the optimal medium consisted of soybean meal (fat free) 4%, Jojoba oil 0.5%, (NH₄)₂HPO₄, 0.1% Tween 60, initial pH 6.4 and the inoculation was at 28 °C for 96 h. The lipase activity produced was enhanced 3.9-fold and reached 500 U/ml. The lipase was purified 19.8-fold by pH precipitation, ethanol precipitation and ammonium sulphate precipitation as well as DEAE-cellulose chromatography. The purified lipase showed one polypeptide band in SDS-polyacrylamide gel electrophoreses (SDS-PAGE) with molecular weight 28.18 kDa. The optimal pH and temperature for activity of lipase were 6.4 and 48 °C, respectively, which are higher than those lipases from other penicillium sources. The P. camembertii Thom lipase is 1,3-positional specificity for hydrolysis of triglyceride and hydrolyses plant oil preferentially to animal oil. The lipase can be used in short chain ester synthesis with an esterification degree of 95%.     

Highly potent inhibitors of cathepsin K with a differently positioned cyanohydrazide warhead: structural analysis of binding mode to mature and zymogen-like enzymes

Cathepsin K (CatK) is a target for the treatment of osteoporosis, arthritis, and bone metastasis. Peptidomimetics with a cyanohydrazide warhead represent a new class of highly potent CatK inhibitors; however, their binding mechanism is unknown. We investigated two model cyanohydrazide inhibitors with differently positioned warheads: an azadipeptide nitrile Gü1303 and a 3-cyano-3-aza-β-amino acid Gü2602. Crystal structures of their covalent complexes were determined with mature CatK as well as a zymogen-like activation intermediate of CatK. Binding mode analysis, together with quantum chemical calculations, revealed that the extraordinary picomolar potency of Gü2602 is entropically favoured by its conformational flexibility at the nonprimed-primed subsites boundary. Furthermore, we demonstrated by live cell imaging that cyanohydrazides effectively target mature CatK in osteosarcoma cells. Cyanohydrazides also suppressed the maturation of CatK by inhibiting the autoactivation of the CatK zymogen. Our results provide structural insights for the rational design of cyanohydrazide inhibitors of CatK as potential drugs.     

3D-QSAR and docking studies of aldehyde inhibitors of human cathepsin K

In order to better understand the structural and chemical features of human cathepsin K (CatK), which is an important cysteine protease in the pathogenesis of osteoporosis, the 3D-QSAR (CoMFA) studies were conducted on recently explored aldehyde compounds with known CatK inhibitory activities. The genetic algorithm of GOLD2.2 has been employed to position 59 aldehyde compounds into the active sites of CatK to determine the probable binding conformation. Good correlations between the predicted binding free energies and the experimental inhibitory activities suggested that the identified binding conformations of these potential inhibitors are reliable. The docking results also provided a reliable conformational alignment scheme for 3D-QSAR model. Based on the docking conformations, highly predictive comparative molecular field analysis (CoMFA) was performed with q2 value of 0.723. The predictive ability was validated by some compounds that were not included in the training set. Furthermore, the CoMFA model was mapped back to the binding sites of CatK, to get a better understanding of vital interactions between the aldehyde compounds and the protease. The CoMFA field distributions are in good agreement with the structural characteristics of the binding groove of the CatK, which suggested that the n-Bu in R4 position is the favor group substitute at P1 and moderate groups in R2 group are required on P2 substitute. In addition, 3D-QSAR results also demonstrated that aldehyde is an important pharmacophore because of electrostatic effect. These results, together with the good correlations between the inhibitory activities and the binding free energies predicted by GOLD2.2, demonstrated the power of combining docking/QSAR approach to explore the probable binding conformations of compounds at the active sites of the protein target, and further provided useful information in understanding the structural and chemical features of CatK in designing and finding new potential inhibitors.     

Purification and Characterization of Trypsin-like Enzymes from <Emphasis Type="Italic">Neomysis japonica</Emphasis> Using BApNA as Substrate

Using N-α-benzoyl-l-arginine p-nitroanilide (BApNA) as substrate, trypsin-like enzymes (TLEs) were purified from mysis (Neomysis japonica) following two chromatographic steps, Sephacryl S100 HR gel filtration and Benzamidine-Sepharose 4B affinity. They presented a high stability in the raw material, retaining over 45% of the initial activity after 30 days of storage at pH 8.0, 45 °C. The purified TLEs had relative molecular mass between 32 kDa and 33 kDa. With higher stability and greater activity, they had similar stability and activity profiles (pH 6.0–11.0, 15–65 °C) as bovine trypsin but had a different optimum temperature (35 °C for trypsin and 45 °C for TLEs). Similar to bovine trypsin, the purified TLEs could be activated by Ca²⁺ and Mg²⁺. And the purified TLEs also showed similar inhibitory profiles as bovine trypsin with the exception of chicken egg ovomucoid (CEOM), an effective inhibitor of bovine trypsin but less so for purified TLEs. Having TLEs with physiological efficiency 3.6 times that of bovine trypsin, the use of mysis as a source for commercial production of TLEs is discussed.     

Properties of a partially purified acid phosphatase from pathogenic Nocardia brasiliensis

Like many other bacteria, Nocardia sp. possess acid phosphatase activity. In N. brasiliensis, a human and animal pathogen, this activity was resolved into two enzyme forms by native gel electrophoresis. One (isozyme I) was partially purified and characterized. It exhibited an estimated molecular weight on SDS-PAGE of 50 kDa, a pH optimum of 5.2, and a Km value of 1.25 mM for p-nitrophenylphosphate. The N. brasiliensis enzyme was stable at 4 °C for at least 24 h, but readily inactivated at 60 °C. Ammonium molybdate, sodium fluoride and L-(+)-tartrate were found to be potent inhibitors of the enzyme. Although its function is presently unknown, by analogy to other bacterial systems it could be envisioned to play an important role in the physiology and pathogenicity of the microorganism.     

Synthesis and evaluation of NLRP3-inhibitory sulfonylurea [11C]MCC950 in healthy animals

The diaryl sulfonylurea MCC950/CRID3 is a potent NLRP3 inhibitor (IC₅₀ = 8 nM) and, in animal models, MCC950 protects against numerous NLRP3-related neurodegenerative disorders. To evaluate the brain uptake and investigate target engagement of MCC950, we synthesised [¹¹C-urea]MCC950 via carrier added [¹¹C]CO₂ fixation chemistry (activity yield = 237 MBq; radiochemical purity >99%; molar activity = 7 GBq/µmol; radiochemical yield (decay-corrected from [¹¹C]CO₂) = 1.1%; synthesis time from end-of-bombardment = 31 min; radiochemically stable for >1 h). Despite preclinical efficacy in neurodegeneration studies, preclinical positron emission tomography (PET) imaging studies in mouse, rat and rhesus monkey revealed poor brain uptake of low molar activity [¹¹C]MCC950 and rapid washout. In silico prediction tools suggest efflux transporter liabilities for MCC950 at microdoses, and this information should be taken into account when developing next generation NLRP3 inhibitors and/or PET radiotracers.     

Purification and characterization of a periplasmic laccase produced by Sinorhizobium meliloti

Sinorhizobium meliloti CE52G strain produces a periplasmic laccase that has been purified by a two-step procedure involving heat treatment and immobilized metal affinity chromatography (IMAC). The fraction with laccase activity retained its original activity after 24 h of incubation at pH between 4.0 and 8.0 and after 3 h of incubation at 70 °C, pH 7.2 and supplemented with 1.3 M (NH₄)₂SO₄. It proved to be a homodimeric protein with an apparent molecular mass of 45 kDa each subunit and an isoelectric point of 6.2. CE52G laccase was inhibited by halides (NaF and NaCl), ions (Fe³⁺, Mn²⁺, and Cu²⁺), sulfhydryl organic compounds (β-mercaptoethanol and reduced glutathione), and electron flow inhibitors (NaCN and NaF). Laccase activity was strongly enhanced by (NH₄)₂SO₄, Na₂SO₄, and K₂SO₄. The effects of all these agents, as well as the probability of a partially unfolding polypeptide chain to enhance the interaction between the substrate and the active site, are discussed. CE52G laccase is a pH- and thermo-stable protein with promising biotechnological applications.     

Ascorbic acid enhances the expression of type 1 and type 4 collagen and SVCT2 in cultured human skin fibroblasts

Ascorbic acid (AA) is essential for collagen biosynthesis as a cofactor for prolyl and lysyl hydroxylase and as a stimulus for collagen gene expression. Many studies have evaluated the relationship between AA and collagen expression in short- and long-term effects on cells after a single administration of AA into the culture medium. However, no such study has monitored in detail the stability of AA in medium or the alterations of intracellular AA levels during a protracted interval. Therefore, we examined here intracellular AA levels and stability throughout its exposure to human skin fibroblasts in vitro. Moreover, we determined the effects on type 1 and type 4 collagen and sodium-dependent vitamin C transporter (SVCT) gene expression when medium containing 100 μM AA was replaced every 24 h for 5 days to avoid depletion of AA. Throughout this long-term culture, intracellular AA levels remained constant; the expression of type 1 and type 4 collagens and SVCT2 mRNA was enhanced, and type 1 procollagen synthesis increased. Thus, these results indicate that human skin fibroblasts exposed to AA over time had rising levels of type 1/type 4 collagens and SVCT2 mRNA expression and type 1 procollagen synthesis.     

Three-phase partitioning of trypsin inhibitor from legume seeds

Three-phase partitioning (TPP) was used to isolate trypsin inhibitors from navy bean (NB), red kidney bean (RK) and adzuki bean (AZ) from the Royal Project Foundation in Thailand. The method was to mix the crude extract with solid ammonium sulfate (30% saturation, w/v) and tert-butanol (t-butanol) in order to obtain the three phases. The trypsin inhibitor was purified to 5-, 14- and 7-fold with 315%, 441% and 228% recovery for NB, RK and AZ, respectively. The SDS-PAGE showed the major inhibitor band with the molecular weights (MWs) of 132, 118 and 13 kDa for NB, RK and AZ, respectively. The fractions from NB and AZ showed higher pH stability compared to that of the RK, and they had the optimum pH ranges of 7–9. The highest relative inhibitory activity of the fractions of NB and RK were found at 50 °C, and all fractions were quite stable at 90 °C for 60 min of incubation. Increasing the concentration of salt (up to 3%, w/v) did not significantly decrease the inhibitory activity of all fractions (p > 0.05). The trypsin inhibitors from the three legumes were unable to inhibit the autolysis of Pacific whiting and arrowtooth flounder muscles.     

A novel xylanase,XynA4-2, from thermoacidophilic <Emphasis Type="Italic">Alicyclobacillus</Emphasis> sp. A4 with potential applications in the brewing industry

A xylanase gene, xynA4-2, was obtained from the genome sequence of thermoacidophilic Alicyclobacillus sp. A4 and expressed in Escherichia coli BL21 (DE3). xynA4-2 encodes a mature protein of 411 residues with a calculated molecular weight of 46.8 kDa. Based on the amino acid sequence similarities (highest identity of 61%), the enzyme was confined into glycoside hydrolase family 10. The purified recombinant XynA4-2 exhibited maximum activity at pH 6.2 and 55°C. The enzyme was stable over a broad pH range, retaining more than 90% of the original activity at pH 5.8–12.0, 37°C for 1 h. The substrate specificity of XynA4-2 was relatively narrow, exhibiting 100, 93, and 35% of the relative activity towards birchwood xylan, oat spelt xylan, and wheat arabinoxylan, respectively. Supplementation of XynA4-2 to mash caused the reduction of mash filtration rate (5.6%) and viscosity (4.0%). When combined with the commercial glucanase from Sunson, higher reduction was detected in the filtration rate (12.0%) and viscosity (17.2%). These favorable properties make XynA4-2 a good candidate in the brewing industry.     

Identification of (4-(9H-fluoren-9-yl) piperazin-1-yl) methanone derivatives as falcipain 2 inhibitors active against Plasmodium falciparum cultures

Background

Falcipain 2 (FP-2) is the hemoglobin-degrading cysteine protease of Plasmodium falciparum most extensively targeted to develop novel antimalarials. However, no commercial antimalarial drugs based on FP-2 inhibition are available yet due to the low selectivity of most FP-2 inhibitors against the human cysteine proteases.

Purification and characterization of trypsin inhibitor from seeds of faba bean (Vicia faba L.)

A trypsin inhibitor from seeds of faba bean (Vicia faba L.) was purified to near homogeneity as judged by native-PAGE with about 11 % recovery using ammonium sulphate fractionation, ion-exchange chromatography on DEAE-cellulose and gel filtration through Sephadex G-100. The inhibitor had a molecular weight of 18 kD as determined by SDS-PAGE and Sephadex G-100. The inhibitor inhibited trypsin and chymotrypsin to the extent of 48 and 12 %, respectively. The inhibtion was of non-competitive type with dissociation constant for the enzyme inhibitor complex in the region of 0.07 mg·ml⁻¹. The inhibtor was stable between pH 4 and 5. It completely lost its activity when heated at 125 °C for 1 h or at 100 °C for 2 h. The inhibitor also lost its activity on exposure to 2-mercaptoethanol. Based on these properties, it could be concluded that Vicia faba trypsin inhibitor belongs to Bowman-Birk type of inhibitors, as it has molecular weight lower than generally observed for Kunitz type inhibitors.     

Green asymmetric synthesis of epoxypeptidomimetics and evaluation as human cathepsin K inhibitors

Cathepsin K (CatK) is a cysteine protease known for its potent collagenolytic activity, being recognized as an important target to the development of therapies for the treatment of bone disorders. Epoxypeptidomimetics have been reported as potent inhibitors of cathepsins, thus in this work we present a green synthesis of new peptidomimetics by using a one-pot asymmetric epoxidation/Ugi multicomponent reaction. The compounds were evaluated against CatK showing selectivity when compared with cathepsin L, with an inhibition profile in the low micromolar IC₅₀ range. Investigation of the mechanism of action carried out for compounds LSPN428 and LSPN694 suggested a mixed inhibition mode and docking studies allowed a better understanding about interactions of inhibitors with the enzyme.     

The collagen substrate specificity of rat uterus collagenase

The collagen substrate specificity of rat uterus collagenase was studied as a function of both collagen type and species of substrate origin. For each collagen examined, values for the basic kinetic parameters, Km and Vmax (kcat), were determined on collagen in solution at 25 degrees C. In all cases, Lineweaver-Burk plots were linear and rat uterus collagenase behaved as a normal Michaelis-Menten enzyme. Collagen types I, II, and III of all species tested were degraded by rat uterus collagenase. Collagen types IV and V were resistant to enzymatic attack. Both enzyme-substrate affinity and catalytic rates were very similar for all susceptible collagens (types I-III). Values for Km ranged from 0.9 to 2.5 X 10(-6) M. Values for kcat varied from 10.7 to 28.1 h-1. The homologous rat type I collagen was no better a substrate than the other animal species type I collagens. The ability of rat uterus collagenase to degrade collagen types I, II, and III with essentially the same catalytic efficiency is unlike the action of human skin fibroblast collagenase or any other interstitial collagenase reported to date. The action of rat uterus collagenase on type I collagen was compared to that of human skin fibroblast collagenase, with regard to their capacity to cleave collagen as solution monomers versus insoluble fibrils. Both enzymes had essentially equal values for kcat on monomeric collagen, yet the specific activity of the rat uterus collagenase was 3- to 6-fold greater on collagen fibrils than the skin fibroblast enzyme. Thus, in spite of their similar activity on collagen monomers in solution, the rat uterus collagenase can degrade collagen aggregated into fibrils considerably more readily than can human skin fibroblast collagenase.