Recombinant scFv-Fc Anti-kallikrein 7 Antibody-Loaded Thermosensitive Hydrogels Against Skin Desquamation Disorders.

Human tissue kallikrein-related peptidase 7 (KLK7) is a serine protease implicated in the physiology of skin desquamation, and its uncontrolled activity can lead to chronic diseases such as psoriasis, atopic dermatitis, and Netherton syndrome. For this reason, kallikrein 7 has been identified as a potential therapeutic target. This work aimed to evaluate Pluronic (PL) hydrogels as topical carriers of four specific scFv-Fc antibodies to inhibit KLK7. The hydrogels comprised PL F127 (30% w/v) alone and a binary F127/P123 (28-2% w/v) system. Each formulation was loaded with 1 µg/mL of each antibody and characterized by physicochemical and pharmaceutical techniques, considering antibody-micelle interactions and hydrogel behavior as smart delivery systems. Results showed that the antibodies were successfully loaded into the PL-based systems, and the sol-gel transition temperature was shifted to high values after the P123 addition. The antibodies released from the gels preserved their rheological properties (G' > G'', 35- to 41-fold) and inhibitory activity against KLK7, even after 24 h. This work presented potential agents targeting KLK7 that may provide strategies for treating skin abnormalities.

Human Tissue Kallikreins-Related Peptidases Are Targets for the Treatment of Skin Desquamation Diseases.

Human tissue Kallikrein-related peptidases (hKLKs) are serine proteases distributed in several tissues that are involved in several biological processes. In skin, many are responsible for skin desquamation in the Stratum Corneum (SC) of the epidermis, specially hKLK5, hKLK7, hKLK6, hKLK8, and hKLK14. In SC, hKLKs cleave proteins of corneodesmosomes, an important structure responsible to maintain corneocytes attached. As part of skin desquamation, hKLKs are also involved in skin diseases with abnormal desquamation and inflammation, such as Atopic Dermatitis (AD), psoriasis, and the rare disease Netherton Syndrome (NS). Many studies point to hKLK overexpression or overactive in skin diseases, and they are also part of the natural skin inflammation process, through the PAR2 cleavage pathway. Therefore, the control of hKLK activity may offer successful treatments for skin diseases, improving the quality of life in patients. Diseases like AD, Psoriasis, and NS have an impact on social life, causing pain, itchy and mental disorders. In this review, we address the molecular mechanisms of skin desquamation, emphasizing the roles of human tissue Kallikrein-related peptidases, and the promising therapies targeting the inhibition of hKLKs.

Generation of recombinant antibodies against human tissue kallikrein 7 to treat skin diseases.

Human tissue kallikreins (KLKs) constitute a family of 15 serine proteases that are distributed in various tissues and implicated in several pathological disorders. KLK7 is an unusual serine protease that presents both trypsin-like and chymotrypsin-like specificity and appears to be upregulated in pathologies that are related to skin desquamation processes, such as atopic dermatitis, psoriasis and Netherton syndrome. In recent years, various groups have worked to develop specific inhibitors for this enzyme, as KLK7 represents a potential target for new therapeutic procedures for diseases related to skin desquamation processes. In this work, we selected nine different single-chain variable fragment antibodies (scFv) from a human naïve phage display library and characterized their inhibitory activities against KLK7. The scFv with the lowest IC50 against KLK7 was affinity maturated, which resulted in the generation of four new scFv-specific antibodies for the target protease. These new antibodies were expressed in the scFv-Fc format in HEK293-6E cells, and the characterization of their inhibitory activities against KLK7 showed that three of them presented IC50 values lower than that of the original antibody. The cytotoxicity analysis of these recombinant antibodies demonstrated that they can be safely used in a cellular model. In conclusion, our research showed that in our case, a phage-display methodology in combination with enzymology assays can be a very suitable tool for the development of inhibitors for KLKs, suggesting a new strategy to identify therapeutic protease inhibitors for diseases related to uncontrolled kallikrein activity.

Functional and structural characterization of an ecotin-like serine protease inhibitor from Trypanosoma cruzi.

Ecotin, a serine peptidase inhibitor (ISP), discovered in Escherichia coli, inhibit a wide range of trypsin-like serine peptidases, protecting microorganisms from the host's immune response. In eukaryotes, ISPs encoding genes were found only in Trypanosomatidae protozoa, including the genus Trypanosoma, which harbors Trypanosoma cruzi, the ethiological agent of Chagas' disease. T. cruzi encodes the ISP2 Trypanosomatidae orthologous, which in Leishmania species present inhibitory activity on mammalian proteases from S1A family suggesting its role in vertebrate-host-parasite interactions. In this study, the structural and biochemical characterization of the recombinant T. cruzi ISP2 (rTcISP2), produced in E. coli was purified in soluble form and analyzed by circular dichroism, fluorescence spectroscopy, native electrophoresis, dynamic light scattering, low X-ray scattering and homology modeling. The obtained data revealed that rTcISP2 was biologically active and forms homodimers in solution. Furthermore, inhibitory activity of rTcISP2 against human neutrophil elastase (HNE) is the highest among ISP2 orthologous from bacteria and trypanosomatids. The role of NE to control T. cruzi parasites through modulation of cellular and humoral innate immune responses in vertebrate hosts, make TcISP2 a key molecular component for parasite infection efficiency, providing a useful basis for investigation of host-parasite interactions and the potential of TcISP2 for biotechnological applications.

3-Acyltetramic acids as a novel class of inhibitors for human kallikreins 5 and 7.

Human kallikreins 5 and 7 (KLK5 and KLK7) exhibit trypsin- and chymotrypsin-like activities and are involved in pathologies related to skin desquamation process. A series of new 3-acyltetramic acids were developed as a novel class of inhibitors of KLK5, KLK7 and trypsin enzymes. The nature and length of the acyl chain is crucial to the KLK5, KLK7 and trypsin inhibition activities, and the most potent compounds (but not the most selective) 2b, 2c and 2g showed low micromolar IC50 values. While very few of the compounds were selective for KLK5, the selective inhibition of trypsin against chymotrypsin was achieved. Our molecular modelling studies revealed that the double bond in 2g provided the best fit in the binding site of KLK5, while the hydrogen bonding interactions modulated the best fit of 2c in the binding site of KLK7 due to the hydrophobicity of the cavity.

The tick-derived rBmTI-A protease inhibitor attenuates the histological and functional changes induced by cigarette smoke exposure.

INTRODUCTION: Smoking is the main risk factor for chronic obstructive pulmonary disease development and cigarette smoke (CS) exposure is considered an important approach to reproduce in rodents this human disease. We have previously shown that in an elastase-induced model of emphysema, the administration of a protease inhibitor (rBmTI-A) prevented and attenuated tissue destruction in mice. Thus, in this study we aimed to verify the effects of rBmTI-A administration on the physiopathological mechanisms of CS-induced emphysema. METHODS: Mice (C57BL/6) were exposed to CS or room air for 12 weeks. In this period, 3 nasal instillations of rBmTI-A inhibitor or its vehicle were performed. After euthanasia, respiratory mechanics were evaluated and lungs removed for analysis of mean linear intercept, volume proportion of collagen and elastic fibers, density of polymorphonuclear cells, macrophages, and density of positive cells for MMP-12, MMP-9, TIMP-1 and gp91phox. RESULTS: The rBmTI-A administration improved tissue elastance, decreased alveolar enlargement and collagen fibers accumulation to control levels and attenuated elastic fibers accumulation in animals exposed to CS. There was an increase of MMP-12, MMP-9 and macrophages in CS groups and the rBmTIA only decreased the number of MMP-12 positive cells. Also, we demonstrated an increase in gp91phox in CS treated group and in TIMP-1 levels in both rBmTI-A treated groups. CONCLUSION: In summary, the rBmTI-A administration attenuated emphysema development by an increase of gp91phox and TIMP-1, accompanied by a decrease in MMP-12 levels.

Functional and Evolutionary Characterization of a UDP-Xylose Synthase Gene from the Plant Pathogen Xylella fastidiosa, Involved in the Synthesis of Bacterial Lipopolysaccharide.

Xylella fastidiosa is a plant-infecting bacillus, responsible for many important crop diseases, such as Pierce's disease of vineyards, citrus variegated chlorosis, and coffee leaf scorch (CLS), among others. Recent genomic comparisons involving two CLS-related strains, belonging to X. fastidiosa subsp. pauca, revealed that one of them carries a frameshift mutation that inactivates a gene encoding an oxidoreductase of the short-chain dehydrogenase/reductase (SDR) superfamily, which may play important roles in determining structural variations in bacterial glycans and glycoconjugates. However, the exact nature of this SDR has been a matter of controversy, as different annotations of X. fastidiosa genomes have implicated it in distinct reactions. To confirm the nature of this mutated SDR, a comparative analysis was initially performed, suggesting that it belongs to a subgroup of SDR decarboxylases, representing a UDP-xylose synthase (Uxs). Functional assays, using a recombinant derivative of this enzyme, confirmed its nature as XfUxs, and carbohydrate composition analyses, performed with lipopolysaccharide (LPS) molecules obtained from different strains, indicate that inactivation of the X. fastidiosa uxs gene affects the LPS structure among CLS-related X. fastidiosa strains. Finally, a comparative sequence analysis suggests that this mutation is likely to result in a morphological and evolutionary hallmark that differentiates two subgroups of CLS-related strains, which may influence interactions between these bacteria and their plant and/or insect hosts.

Discovery of a new isomannide-based peptidomimetic synthetized by Ugi multicomponent reaction as human tissue kallikrein 1 inhibitor.

Human kallikrein 1 (KLK1) is the most extensively studied member of this family and plays a major role in inflammation processes. From Ugi multicomponent reactions, isomannide-based peptidomimetic 10 and 13 where synthesized and showed low micromolar values of IC50 for KLK1 The most active compound (10) presented competitive mechanism, with three structural modifications important to interact with active site residues which corroborates its KLK1 inhibition. Finally, the most active compound also showed good ADMET profile, which indicates compound 10 as a potential hit in the search for new KLK1 inhibitors with low side effects.

Vioserpin, a serine protease inhibitor from Gloeobacter violaceus possibly regulated by heparin.

Serine peptidase inhibitor (serpin) is the name given to the superfamily of proteins with wide range of biological functions, and that the main feature is the inhibition of serine proteases. Here we describe the inhibitory characterization of a serpin from Gloeobacter violaceus that we named vioserpin. The serpin presented a high specificity to inhibit trypsin-like enzymes with a rapid inhibition rate constant (2.1 × 10(6) M(-1) s(-1)). We also demonstrated that the inhibitory activity of the vioserpin is influenced by the concentration of heparin, and this finding may throw a new light on understanding the molecular evolution of serpins.

Synthesis, biological evaluation and molecular modeling of pseudo-peptides based statine as inhibitors for human tissue kallikrein 5.

Human kallikrein 5 (KLK5) is a potential target for the treatment of skin inflammation and cancer. A new series of statine based peptidomimetic compounds were designed and synthesized through simple and efficient reactions. Some KLK5 inhibitors (2a-c compounds) were identified with nanomolar affinity showing Ki values of 0.12-0.13 µM. Our molecular modeling studies suggest that the inhibitors binding at the KLK5 through H-bond interactions with key residues (mainly His108, Gln242, Gly243, Ser245, and Ser260), disrupting the correlated motions mainly among the Ile67-Tyr127, Glu128-Val187, and Gly237-Ser293 subdomains, which seems to be crucial for KLK5 activity. Therefore, we believe that these findings will significantly facilitate our understanding of the conformational dynamics in the course of KLK5 inhibition and, consequently, the development of more potent molecules as alternative for cancer treatment.

The natural flavone fukugetin as a mixed-type inhibitor for human tissue kallikreins.

The human tissue kallikreins (KLK1-KLK15) comprise a family of 15 serine peptidases detected in almost every tissue of the human body and that actively participate in many physiological and pathological events. Some kallikreins are involved in diseases for which no effective therapy is available, as for example, epithelial disorders, bacterial infections and in certain cancers metastatic processes. In recent years our group have made efforts to find inhibitors for all kallikreins, based on natural products and synthetic molecules, and all the inhibitors developed by our group presented a competitive mechanism of inhibition. Here we describe fukugetin, a natural product that presents a mixed-type mechanism of inhibition against KLK1 and KLK2. This type of inhibitor is gaining importance today, especially for the development of exosite-type inhibitors, which present potential to selectively inhibit the enzyme activity only against specific substrate.

Isomannide-based peptidomimetics as inhibitors for human tissue kallikreins 5 and 7.

Human kallikrein 5 (KLK5) and 7 (KLK7) are potential targets for the treatment of skin inflammation and cancer. Previously, we identified isomannide derivatives as potent and competitive KLK7 inhibitors. The introduction of N-protected amino acids into the isomannide-based scaffold was studied. Some KLK5 inhibitors with submicromolar affinity (K i values of 0.3-0.7 µM) were identified, and they were 6- to 13-fold more potent than our previous hits. Enzyme kinetics studies and the determination of the mechanism of inhibition confirmed that the new isomannide-based derivatives are competitive inhibitors of both KLK5 and KLK7. Molecular docking and MD simulations of selected inhibitors into the KLK5 binding site provide insight into the molecular mechanism by which these compounds interact with the enzyme. The promising results obtained in this study open new prospects on the design and synthesis of highly specific KLK5 and KLK7 inhibitors.

Human tissue kallikreins 3 and 5 can act as plasminogen activator releasing active plasmin.

Human tissue kallikreins (KLKs) are a group of serine proteases found in many tissues and biological fluids and are differentially expressed in several specific pathologies. Here, we present evidences of the ability of these enzymes to activate plasminogen. Kallikreins 3 and 5 were able to induce plasmin activity after hydrolyzing plasminogen, and we also verified that plasminogen activation was potentiated in the presence of glycosaminoglycans compared with plasminogen activation by tPA. This finding can shed new light on the plasminogen/plasmin system and its involvement in tumor metastasis, in which kallikreins appear to be upregulated.

Isomannide derivatives as new class of inhibitors for human kallikrein 7.

Human kallikrein 7 (KLK7) is a potential target for the treatment of skin inflammation and cancer. Despite its potential, few KLK7-specific small-molecule inhibitors have been reported in the literature. As an extension of our program to design serine protease inhibitors, here we describe the in vitro assays and the investigation of the binding mechanism by molecular dynamics simulation of a novel class of pseudo-peptide inhibitors derived from isomannide. Of the inhibitors tested, two inhibited KLK7 with K(i) values in the low micromolar range (9g=1.8µM; 9j=3.0µM). Eadie-Hofstee and Dixon plots were used to evaluate the competitive mechanism of inhibition for the molecules. Calculated binding free energies using molecular MM/PB(GB)SA approach are in good agreement with experimental results, suggesting that the inhibitors share the same binding mode, which is stabilized by hydrophobic interactions and by a conserved network of hydrogen bonds. The promising results obtained in this study make these compounds valid leads for further optimization studies aiming to improve the potency of this new class of kallikrein inhibitors.

Biological evaluation and docking studies of natural isocoumarins as inhibitors for human kallikrein 5 and 7.

Human kallikrein 5 and 7 (KLK5 and KLK7) are trypsin-like and chymotrypsin-like serine proteases, respectively, and promising targets for the treatment of skin desquamation, inflammation and cancer. In an effort to develop new inhibitors for these enzymes, we carried out enzymatic inhibition assays and docking studies with three isocoumarin compounds. Some promising inhibitors were uncovered, with vioxanthin and 8,8'-paepalantine being the most potent competitive inhibitors of KLK5 (K(i)=22.9 µM) and KLK7 (K(i)=12.2 µM), respectively. Our docking studies showed a good correlation with the experimental results, and revealed a distinct binding mode for the inhibitors at the binding sites of KLK5 and KLK7. In addition, the docking results suggested that the formation of hydrogen bonds at the oxyanion hole is essential for a good inhibitor.

Leviserpin: a serine peptidase inhibitor (Serpin) from the Sugarcane Weevil Sphenophorus levis.

Serine peptidase inhibitors (serpins) form a superfamily of proteins covering abroad spectrum of different biological functions. Here we describe the inhibitory characterization of leviserpin, the first serpin from the sugar cane weevil Sphenophorus levis. Leviserpin was able to inhibit bovine trypsin by the formation of the covalent complex serpin-peptidase, demonstrated by SDS-PAGE and mass spectroscopy analysis. We also have determined the cleavage site at the reactive center loop, by the analysis of the polypeptides released from de C-terminus of leviserpin. Moreover we investigated the mRNA expression of leviserpin in different stages of S. levis development. Thus the specificity of leviserpin, in addition with its mRNA coding being transcribed through all lifecycle of the insect, can suggest a possible role in defense mechanism by regulating the action of prophenoloxidase (proPO) activating enzyme.

Plasminogen hydrolysis by cathepsin S and identification of derived peptides as selective substrate for cathepsin V and cathepsin L inhibitor.

Plasminogen is a glycoprotein implicated in angiogenesis and fibrin clot degradation associated with the release of angiostatin and plasmin activation, respectively. We have recently reported that cathepsin V, but not cathepsins L, B, and K, can release angiostatin-like fragments from plasminogen. Here, we extended the investigation to cathepsin S which has been implicated in angiogenesis and tumor cell proliferation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of plasminogen hydrolysis by cathepsin S revealed generation of two fragments (60 and 38 kDa). Amino-terminal sequencing indicated that cleavage occurs at the Leu469-Leu470 peptide bond. In contrast to cathepsin V, which possesses antiangiogenic activity, cathepsin S plasminogen cleavage products were not capable of inhibiting angiogenesis on endothelial cells. Moreover, we explored the different selectivities presented by cathepsins V and S towards plasminogen and synthesized fluorescence resonance energy transfer peptides encompassing the hydrolyzed peptide bonds by both enzymes. The peptide Abz-VLFEKKQ-EDDnp (Abz=ortho-aminobenzoic acid; EDDnp= N-[2,4-dinitrophenyl]ethylenediamine), hydrolyzed by cath-epsin V at the Phe-Glu bond, is a selective substrate for the enzyme when compared with cathepsins B, L, and S, whereas Abz-VLFEKKVYLQ-EDDnp is an efficient cathepsin L inhibitor. The demonstrated importance of the S(3)'-P(3)' interaction indicates the significance of the extended subsites for enzyme specificity and affinity.

Cathepsin V, but not cathepsins L, B and K, may release angiostatin-like fragments from plasminogen.

Abstract Cathepsin V is a lysosomal cysteine peptidase highly expressed in corneal epithelium; however, its function in the eye is still unknown. Here, we describe the capability of cathepsin V to hydrolyze plasminogen, which is also expressed in human cornea at levels high enough to produce physiologically relevant amounts of angiostatin-related molecules. The co-localization of these two proteins suggests an important role for the enzyme in the maintenance of corneal avascularity, essential for optimal visual performance. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of plasminogen digestion by cathepsin V revealed the generation of three major products of 60, 50 and 40 kDa, which were electrotransferred to polyvinylidene difluoride membranes and excised for characterization. NH(2)-terminal amino acid sequencing of these fragments revealed the sequences EKKVYL, TEQLAP and LLPNVE, respectively. These data are compatible with cleavage sites at plasminogen F94-E95, S358-T359 and V468-L469 peptide bonds generating fragments of the five-kringle domains. In contrast, we did not detect any plasminogen degradation by cathepsins B, K and L. Using a Matrigel assay, we confirmed the angiogenesis inhibition activity on endothelial cells caused by plasminogen processing by cathepsin V. Our results suggest a novel physiological role for cathepsin V related to the control of neovascularization in cornea.

Recombinant expression and characterization of a Xylella fastidiosa cysteine protease differentially expressed in a nonpathogenic strain.

Xylella fastidiosa is a xylem-limited, Gram-negative bacterium responsible for citrus variegated chlorosis (CVC) in sweet oranges. In the present study, we present the recombinant expression, purification and characterization of an X. fastidiosa cysteine protease (dubbed Xylellain). The recombinant Xylellain ((HIS)Xylellain) was able to hydrolyze carbobenzoxy-Phe-Arg-7-amido-4-methylcoumarin (Z-FR-MCA) and carbobenzoxy-Arg-Arg-7-amido-4-methylcoumarin (Z-RR-MCA) with similar catalytic efficiencies, suggesting that this enzyme presents substrate specificity requirements similar to cathepsin B. The immunization of mice with (HIS)Xylellain provided us with antibodies, which recognized a protein of c. 31 kDa in the X. fastidiosa pathogenic strains 9a5c, and X. fastidiosa isolated from coffee plants. However, these antibodies recognized no protein in the nonpathogenic X. fastidiosa J1a12, suggesting the absence or low expression of this protein in the strain. These findings enabled us to identify Xylellain as a putative target for combating CVC and other diseases caused by X. fastidiosa strains.

Recombinant human cathepsin X is a carboxymonopeptidase only: a comparison with cathepsins B and L.

The S1 and S2 subsite specificity of recombinant human cathepsins X was studied using fluorescence resonance energy transfer (FRET) peptides with the general sequences Abz-Phe-Xaa-Lys(Dnp)-OH and Abz-Xaa-Arg-Lys(Dnp)-OH, respectively (Abz=ortho-aminobenzoic acid and Dnp=2,4-dinitrophenyl; Xaa=various amino acids). Cathepsin X cleaved all substrates exclusively as a carboxymonopeptidase and exhibited broad specificity. For comparison, these peptides were also assayed with cathepsins B and L. Cathepsin L hydrolyzed the majority of them with similar or higher catalytic efficiency than cathepsin X, acting as an endopeptidase mimicking a carboxymonopeptidase (pseudo-carboxymonopeptidase). In contrast, cathepsin B exhibited poor catalytic efficiency with these substrates, acting as a carboxydipeptidase or an endopeptidase. The S1' subsite of cathepsin X was mapped with the peptide series Abz-Phe-Arg-Xaa-OH and the enzyme preferentially hydrolyzed substrates with hydrophobic residues in the P1' position.