Discovery and structure-activity relationship of imidazolinylindole derivatives as kallikrein 7 inhibitors.

A series of imidazolinylindole derivatives were discovered as novel kallikrein 7 (KLK7, stratum corneum chymotryptic enzyme) inhibitors. Structure-activity relationship (SAR) studies led to the identification of potent human KLK7 inhibitors. By further modification of the benzenesulfonyl moiety to overcome species differences in inhibitory activity, potent inhibitors against both human and mouse KLK7 were identified. Furthermore, the complex structure of 25 with mouse KLK7 could explain the SAR and the cause of the species differences in inhibitory activity.

Basic Residues of ß-Sheet A Contribute to Heparin Binding and Activation of Vaspin (Serpin A12).

Many members of the serine protease inhibitor (serpin) family are activated by glycosaminoglycans (GAGs). Visceral adipose tissue-derived serpin (vaspin), serpin A12 of the serpin family, and its target protease kallikrein 7 (KLK7) are heparin-binding proteins, and inhibition of KLK7 by vaspin is accelerated by heparin. However, the nature of GAG binding to vaspin is not known. Here, we measured vaspin binding of various glycosaminoglycans and low molecular weight heparins by microscale thermophoresis and analyzed acceleration of protease inhibition by these molecules. In addition, basic residues contributing to heparin binding and heparin activation were identified by a selective labeling approach. Together, these data show that vaspin binds heparin with high affinity (KD = 21 ± 2 nm) and that binding takes place at a basic patch on top of ß-sheet A and is different from other heparin-binding serpins. Mutation of basic residues decreased heparin binding and activation of vaspin. Similarly, reactive center loop insertion into sheet A decreased heparin binding because it disturbs the basic cluster. Finally, using vaspin-overexpressing keratinocyte cells, we show that a significant part of secreted vaspin is bound in the extracellular matrix on the cell surface. Together, basic residues of central ß-sheet A contribute to heparin binding and activation of vaspin. Thus, binding to GAGs in the extracellular matrix can direct and regulate vaspin interaction with target proteases or other proteins and may play an important role in the various beneficial functions of vaspin in different tissues.

Structure-based drug design of 1,3,6-trisubstituted 1,4-diazepan-7-ones as selective human kallikrein 7 inhibitors.

A novel series of 1,3,6-trisubstituted 1,4-diazepan-7-ones were investigated as human kallikrein 7 (KLK7, stratum corneum chymotryptic enzyme) inhibitors. Based on the X-ray co-crystal structure of compound 1 bound to human KLK7, the derivatives of this scaffold were designed, synthesized, and evaluated. Through structure-activity relationship studies focused on the side chain located in the prime site region of the enzyme, representative compounds 15, 33a, and 35a were identified as highly potent and selective inhibitors of human KLK7.

Proteomic analysis of filaggrin deficiency identifies molecular signatures characteristic of atopic eczema.

BACKGROUND: Atopic eczema (AE) is characterized by skin barrier and immune dysfunction. Null mutations in filaggrin (FLG), a key epidermal barrier protein, strongly predispose to AE; however, the precise role of FLG deficiency in AE pathogenesis remains incompletely understood. OBJECTIVES: We sought to identify global proteomic changes downstream of FLG deficiency in human epidermal living skin-equivalent (LSE) models and validate findings in skin of patients with AE. METHODS: Differentially expressed proteins from paired control (nontargeting control short hairpin RNA [shNT]) and FLG knockdown (FLG knockdown short hairpin RNA [shFLG]) LSEs were identified by means of proteomic analysis (liquid chromatography-mass spectrometry) and Ingenuity Pathway Analysis. Expression of key targets was validated in independent LSE samples (quantitative RT-PCR and Western blotting) and in normal and AE skin biopsy specimens (immunofluorescence). RESULTS: Proteomic analysis identified 17 (P ≤ .05) differentially expressed proteins after FLG knockdown, including kallikrein-7 (KLK7; 2.2-fold), cyclophilin A (PPIA; 0.9-fold), and cofilin-1 (CFL1, 1.3-fold). Differential protein expression was confirmed in shNT/shFLG LSEs; however, only KLK7 was transcriptionally dysregulated. Molecular pathways overrepresented after FLG knockdown included inflammation, protease activity, cell structure, and stress. Furthermore, KLK7 (1.8-fold) and PPIA (0.65-fold) proteins were differentially expressed in lesional biopsy specimens from patients with AE relative to normal skin. CONCLUSIONS: For the first time, we show that loss of FLG in the absence of inflammation is sufficient to alter the expression level of proteins relevant to the pathogenesis of AE. These include proteins regulating inflammatory, proteolytic, and cytoskeletal functions. We identify PPIA as a novel protein with levels that are decreased in clinically active AE skin and show that the characteristic upregulation of KLK7 expression in patients with AE occurs downstream of FLG loss. Importantly, we highlight disconnect between the epidermal proteome and transcriptome, emphasizing the utility of global proteomic studies.

[The expression of KLK7 in pancreatic cancer and the effects on the biological behavior of pancreatic cancer cells].

Objective: To investigate the expression of KLK7 in pancreatic cancer and its clinical significance. Methods: Immunohistochemistry was used to detect the expression of KLK7 protein in pancreatic cancer tissue microarray with 92 samples. Statistical analysis of the relationship between KLK7 and clinicopathological characteristics was finished. Pancreatic cancer cell lines were infected with lentiviuses in order to get cells with KLK7 stable overexpression.KLK7-siRNA was transfected into pancreatic cancer cells to knock down KLK7.Cell proliferation and chemosensitivity were detected by CCK-8 assay; Cell invasion and migration abilities were detected by Transwell assay. At the same time, subcutaneous xenograft tumor models were established in nude mice to observe the effect of KLK7 on tumor growth in nude mice. Data were statistically analyzed by rank sum test, χ(2) test and Logistic regression analysis. Results: The expression level of KLK7 in pancreatic cancer tissues was higher than that in paired adjacent tissues (P<0.05). KLK7 expression was correlated with vascular invasion(χ(2)=7.535, P<0.05). Further univariate and multivariate analysis showed that KLK7 expression was an independent risk factor for vascular invasion of pancreatic cancer(χ(2)=7.535, P<0.05). The overexpression of KLK7 in pancreatic cancer cell lines BxPC-3 and CFPAC can increase their proliferation abilities, reduce the chemosensitivity and promote their migration and invasion behaviour; The results of in vivo experiments showed that the volume of subcutaneously transplanted tumors in the overexpressing KLK7 group was significantly larger than that in the control group (t=4.479, P<0.05). The group of overexpressing KLK7 showed greater tumor weight than the control group(t=2.831, P<0.05). Conclusions: The expression level of KLK7 in pancreatic ductal adenocarcinoma was higher than that in paired adjacent tissues and it is an independent risk factor for vascular invasion of pancreatic cancer.KLK7 can promote the proliferation of pancreatic cancer cells, reduce the chemosensitivity and increase the invasion and migration of pancreatic cancer cells.

Glycosylation of human vaspin (SERPINA12) and its impact on serpin activity, heparin binding and thermal stability.

Vaspin is a glycoprotein with three predicted glycosylation sites at asparagine residues located in proximity to the reactive center loop and close to domains that play important roles in conformational changes underlying serpin function. In this study, we have investigated the glycosylation of human vaspin and its effects on biochemical properties relevant to vaspin function. We show that vaspin is modified at all three sites and biochemical data demonstrate that glycosylation does not hinder inhibition of the target protease kallikrein 7. Although binding affinity to heparin is slightly decreased, the protease inhibition reaction is still significantly accelerated in the presence of heparin. Glycosylation did not affect thermal stability.

Inhibition of Human Kallikrein 5 Protease by Triterpenoids from Natural Sources.

Stratum corneum tryptic enzyme kallikrein 5 (KLK5) is a serine protease that is involved in the cell renewal and maintenance of the skin barrier function. The excessive activation of KLK5 causes an exacerbation of dermatoses, such as rosacea and atopic dermatitis. Some triterpenoids are reported to suppress the serine proteases. We aimed to investigate whether bioactive triterpenoids modulate the KLK5 protease. Nineteen triterpenoids occurring in medicinal crude drugs were evaluated using an enzymatic assay to measure the anti-KLK5 activity. The KLK5-dependent cathelicidin peptide LL-37 production in human keratinocytes was examined using immunoprecipitation and Western blotting. Screening assays for evaluating the anti-KLK5 activity revealed that ursolic acid, oleanolic acid, saikosaponin b1, tumulosic acid and pachymic acid suppressed the KLK5 protease activity, although critical molecular moieties contributing to anti-KLK5 activity were unclarified. Ursolic acid and tumulosic acid suppressed the proteolytic processing of LL-37 in keratinocytes at ≤10 µM; no cytotoxicity was observed. Both triterpenoids were detected in the plasma of rats administered orally with triterpenoid-rich crude drug Jumihaidokuto. Our study reveals that triterpenoids, such as ursolic acid and tumulosic acid, modulate the KLK5 protease activity and cathelicidin peptide production. Triterpenoids may affect the skin barrier function via the regulation of proteases.

A unique serpin P1' glutamate and a conserved ß-sheet C arginine are key residues for activity, protease recognition and stability of serpinA12 (vaspin).

SerpinA12 (vaspin) is thought to be mainly expressed in adipose tissue and has multiple beneficial effects on metabolic, inflammatory and atherogenic processes related to obesity. KLK7 (kallikrein 7) is the only known protease target of vaspin to date and is inhibited with a moderate inhibition rate. In the crystal structure, the cleavage site (P1-P1') of the vaspin reactive centre loop is fairly rigid compared with the flexible residues before P2, possibly supported by an ionic interaction of P1' glutamate (Glu(379)) with an arginine residue (Arg(302)) of the ß-sheet C. A P1' glutamate seems highly unusual and unfavourable for the protease KLK7. We characterized vaspin mutants to investigate the roles of these two residues in protease inhibition and recognition by vaspin. Reactive centre loop mutations changing the P1' residue or altering the reactive centre loop conformation significantly increased inhibition parameters, whereas removal of the positive charge within ß-sheet C impeded the serpin-protease interaction. Arg(302) is a crucial contact to enable vaspin recognition by KLK7 and it supports moderate inhibition of the serpin despite the presence of the detrimental P1' Glu(379), which clearly represents a major limiting factor for vaspin-inhibitory activity. We also show that the vaspin-inhibition rate for KLK7 can be modestly increased by heparin and demonstrate that vaspin is a heparin-binding serpin. Noteworthily, we observed vaspin as a remarkably thermostable serpin and found that Glu(379) and Arg(302) influence heat-induced polymerization. These structural and functional results reveal the mechanistic basis of how reactive centre loop sequence and exosite interaction in vaspin enable KLK7 recognition and regulate protease inhibition as well as stability of this adipose tissue-derived serpin.

Skin Barrier Function Is Not Impaired and Kallikrein 7 Gene Polymorphism Is Frequently Observed in Korean X-linked Ichthyosis Patients Diagnosed by Fluorescence in Situ Hybridization and Array Comparative Genomic Hybridization.

X-linked ichthyosis (XLI) is a recessively inherited ichthyosis. Skin barrier function of XLI patients reported in Western countries presented minimally abnormal or normal. Here, we evaluated the skin barrier properties and a skin barrier-related gene mutation in 16 Korean XLI patients who were diagnosed by fluorescence in situ hybridization and array comparative genomic hybridization analysis. Skin barrier properties were measured, cytokine expression levels in the stratum corneum (SC) were evaluated with the tape stripped specimen from skin surface, and a genetic test was done on blood. XLI patients showed significantly lower SC hydration, but normal basal trans-epidermal water loss and skin surface pH as compared to a healthy control group. Histopathology of ichthyosis epidermis showed no acanthosis, and levels of the pro-inflammatory cytokines in the corneal layer did not differ between control and lesional/non-lesional skin of XLI patients. Among the mutations in filaggrin (FLG), kallikrein 7 (KLK7), and SPINK5 genes, the prevalence of KLK7 gene mutations was significantly higher in XLI patients (50%) than in controls (0%), whereas FLG and SPINK5 prevalence was comparable. Korean XLI patients exhibited unimpaired skin barrier function and frequent association with the KLK7 gene polymorphism, which may differentiate them from Western XLI patients.

Vaspin (serpinA12) in obesity, insulin resistance, and inflammation.

While genome-wide association studies as well as candidate gene studies have revealed a great deal of insight into the contribution of genetics to obesity development and susceptibility, advances in adipose tissue research have substantially changed the understanding of adipose tissue function. Its perception has changed from passive lipid storage tissue to active endocrine organ regulating and modulating whole-body energy homeostasis and metabolism and inflammatory and immune responses by secreting a multitude of bioactive molecules, termed adipokines. The expression of human vaspin (serpinA12) is positively correlated to body mass index and insulin sensitivity and increases glucose tolerance in vivo, suggesting a compensatory role in response to diminished insulin signaling in obesity. Recently, considerable insight has been gained into vaspin structure, function, and specific target tissue-dependent effects, and several lines of evidence suggest vaspin as a promising candidate for drug development for the treatment of obesity-related insulin resistance and inflammation. These will be summarized in this review with a focus on molecular mechanisms and pathways.

Plasma-Soluble Biomarkers for Fibrodysplasia Ossificans Progressiva (FOP) Reflect Acute and Chronic Inflammatory States.

Fibrodysplasia ossificans progressiva (FOP) is a progressive, debilitating genetic disease in which skeletal muscle and connective tissue is episodically replaced by heterotopic bone. Discovery of surrogate biomarkers of disease (genotype)-related and flare-up-associated activity of FOP in a readily accessible matrix, such as plasma, would facilitate an understanding of the complex pathophysiology of FOP, aid patient care, and provide a valuable tool for the development and monitoring of potential therapeutics. In a case-control study, using a carefully collected and curated set of plasma samples from 40 FOP patients with the classic ACVR1R206H mutation and 40 age- and sex-matched controls, we report the identification of disease-related and flare-up-associated biomarkers of FOP using a multiplex analysis of 113 plasma-soluble analytes. Adiponectin (implicated in hypoxia, inflammation, and heterotopic ossification) as well as tenascin-C (an endogenous activator of innate immune signaling through the TLR4 pathway and a substrate for kallikrein-7) were highly correlated with FOP genotype, while kallikrein-7 was highly correlated with acute flare-up status. Plasma-soluble biomarkers for FOP support a flare-up-related acute inflammatory phase of disease activity superimposed on a genotypic background of chronic inflammation. © 2021 American Society for Bone and Mineral Research (ASBMR).

The Role of Kallikrein 7 in Tumorigenesis.

Kallikrein 7 (KLK7) is a secreted serine protease with chymotrypsic protease activity. Abnormally high expression of KLK7 is closely related to the occurrence and development of various types of cancer. Therefore, KLK7 has been identified as a potential target for cancer drug development design in recent years. KLK7 mediates various biological and pathological processes in tumorigenesis, including cell proliferation, migration, invasion, angiogenesis, and cell metabolism, by hydrolyzing a series of substrates such as membrane proteins, extracellular matrix proteins, and cytokines. This review mainly introduces the downstream cell signaling pathways involved in the activation of KLK7 and its substrate-related proteins. This review will not only help us to better understand the mechanisms of KLK7 in regulating biological and pathological processes of cancer cells but also lay a solid foundation for the design of inhibitors targeting KLK7.

NFAT5 Controls the Integrity of Epidermis.

The skin protects the human body against dehydration and harmful challenges. Keratinocytes (KCs) are the most abundant epidermal cells, and it is anticipated that KC-mediated transport of Na+ ions creates a physiological barrier of high osmolality against the external environment. Here, we studied the role of NFAT5, a transcription factor whose activity is controlled by osmotic stress in KCs. Cultured KCs from adult mice were found to secrete more than 300 proteins, and upon NFAT5 ablation, the secretion of several matrix proteinases, including metalloproteinase-3 (Mmp3) and kallikrein-related peptidase 7 (Klk7), was markedly enhanced. An increase in Mmp3 and Klk7 RNA levels was also detected in transcriptomes of Nfat5-/- KCs, along with increases of numerous members of the 'Epidermal Differentiation Complex' (EDC), such as small proline-rich (Sprr) and S100 proteins. NFAT5 and Mmp3 as well as NFAT5 and Klk7 are co-expressed in the basal KCs of fetal and adult epidermis but not in basal KCs of newborn (NB) mice. The poor NFAT5 expression in NB KCs is correlated with a strong increase in Mmp3 and Klk7 expression in KCs of NB mice. These data suggests that, along with the fragile epidermis of adult Nfat5-/- mice, NFAT5 keeps in check the expression of matrix proteases in epidermis. The NFAT5-mediated control of matrix proteases in epidermis contributes to the manifold changes in skin development in embryos before and during birth, and to the integrity of epidermis in adults.

Immunohistochemical expression of kallikrein 7 in oral squamous cell carcinoma.

BACKGROUND AND OBJECTIVES: The kallikrein (KLK) family of genes consists of 15 members, many of which are highly expressed in number of cancers compared to their normal parent tissues. KLK7 was initially characterized as an enzyme implicated in the degradation of intercellular cohesive structures in the stratum corneum of stratified squamous epithelia, preceding desquamation in the skin. It catalyzes the degradation of desmosomes in the outermost layer of skin and permits cell shedding to take place at the skin surface. Overexpression of KLK7 in tumor cells has been reported to significantly enhance the invasive potential in intracranial malignancies and ovarian cancer cells. Thus, KLK7 could contribute to the degradation of extracellular matrices in oral squamous cell carcinoma (OSCC) tissues, promoting invasion of neoplastic cells locally and facilitating metastasis to regional lymph nodes. The objectives of the present study were to compare the expression of KLK 7 in normal subjects and patients with OSCC, to correlate the expression of KLK 7 with respect to the clinical staging of OSCC and to evaluate the expression of KLK 7with respect to different histopathological grades of OSCC. MATERIALS AND METHODS: Thirty cases of OSCC were staged clinically and graded histopathologically. The immunohistochemical method was used to detect the expression of KLK 7 in OSCC. The scores obtained were documented and compared statistically. RESULTS: KLK 7 immunoreactivity was noticed in all cases of OSCC. A statistically significant difference was observed in immunoreactivity of KLK 7 between the normal and OSCC (P = 0.0001*) and in different histopathological grades (P = 0.0001*) and in different clinical stages (P = 0.0127*) of OSCC using Kruskal-Wallis analysis of variance test. CONCLUSION: The KLK 7 immunoexpression histopathologically increased from low grade to high grade and clinically from Stage 1 to Stage 4 in OSCC. Hence, increased expression of KLK 7 may be related to poor prognosis in patients with OSCC.

Experiment research of cisplatin implants inhibiting transplantation tumor growth and regulating the expression of KLK7 and E-cad of tumor-bearing mice with gastric cancer.

OBJECTIVE: To study the influence of cisplatin implants on transplantation tumor growth and the expression of tissue kallikrein-7 (KLK7) and E-cadherin (E-cad) in tumor-bearing mice with gastric cancer. METHODS: BALB/c nude mice were collected as experimental animal and were randomly divided into model control group (Group A), tail intravenous injection of cisplatin group (Group B), intratumor injection of cisplatin group (Group C) and cisplatin implants treatment group (Group D). After the drugs intervening, the weight and volume of transplantation tumors were measured on Day 20, Day 30 and Day 40 and serum and KLK7 and E-cad contents in transplanted tumor tissue were examined. RESULTS: On Day 20, Day 30 and Day 40 after treatment, the weight and volume of transplantation tumors of tumor-bearing mice in four groups were different (Group A > Group B > Group C > Group D). The contents of KLK-7 and E-cad in tumor tissue and serum of tumor-bearing mice in four groups were different (Group A > Group B > Group C > Group D in KLK-7) and (Group A < Group B < Group C < Group D in E-cad). The weight and volume, and KLK7 and E-cad contents of transplantation tumors in four groups were significant difference (P < 0.05). CONCLUSION: Cisplatin implants can inhibit the growth of transplanted tumor tissue and down-regulated KLK7 expression and up-regulated E-cad expression of tumor-bearing mice with gastric cancer.

Pyrido-imidazodiazepinones as a new class of reversible inhibitors of human kallikrein 7.

The human tissue kallikrein-7 (KLK7) is a chymotryptic serine protease member of tissue kallikrein family. KLK7 is involved in skin homeostasis and inflammation. Excess of KLK7 activity is also associated with tumor metastasis processes, especially in ovarian carcinomas, prostatic and pancreatic cancers. Development of Kallikrein 7 inhibitors is thus of great interest in oncology but also for treating skin diseases. Most of the developed synthetic inhibitors present several drawbacks such as poor selectivity and unsuitable physico-chemical properties for in vivo use. Recently, we described a practical sequence for the synthesis of imidazopyridine-fused [1,3]-diazepines. Here, we report the identification of pyrido-imidazodiazepinone core as a new potential scaffold to develop selective and competitive inhibitors of kallikrein-related peptidase 7. Structure-activity relationships (SAR), inhibition mechanisms and selectivity as well as cytotoxicity against selected cancer cell lines were investigated.