Activity-Based Probes for Proteases Pave the Way to Theranostic Applications.

Proteases are important enzymes in health and disease. Their activities are regulated at multiple levels. In fact, proteases are synthesized as inactive proenzymes (zymogens) that are activated by proteolytic removal of their pro-peptide sequence and can remain active or their activity can be attenuated by complex formation with specific endogenous inhibitors or by limited proteolysis or degradation. Consequently, quite often, only a fraction of the protease molecules is in the active/functional form, thus, the abundance of a protease is not always linearly proportional to the (patho)physiological function(s). Therefore, assays to determine the active forms of proteases are needed, not only in research but also in molecular diagnosis and therapy. Activity-based probes (ABPs) are chemical entities that bind covalently to the active enzyme/protease. ABPs carry a detection tag to enable localization and quantification of specific enzymatic/proteolytic activities with applications in molecular imaging and diagnosis. Moreover, ABPs act as suicide inhibitors of proteases, which can be exploited for delineation of the functional role(s) of a given protease in (patho) biological context and as potential therapeutics. In this sense, ABPs represent new theranostic agents. We outline recent developments pertaining to ABPs for proteases with potential therapeutic applications, with the aim to highlight their importance in theranostics.

Reconstructing the epidermal proteolytic cascades in health and disease.

The epidermis is the outer stratified epithelium of the skin, forming the physical barrier that is indispensable for homeostasis. Epidermal proteolysis, mainly but not exclusively executed by kallikrein-related peptidases (KLKs), is tightly regulated to ensure maintenance of physiological skin renewal and an intact skin barrier. Perturbation of epidermal proteolytic networks is implicated in a wide array of rare and common skin pathologies of diverse genetic backgrounds. Recent studies of monogenic human skin diseases and newly developed animal models have revealed new mechanisms of regulation of proteolytic pathways in epidermal physiology and in disease states. These new data have challenged some accepted views, for example the role of matriptase in epidermal desquamation, which turned out to be restricted to mouse skin. The significance of PAR2 signaling in skin inflammation should also be reconsidered in the face of recent findings. Cumulatively, recent studies necessitate a sophisticated redefinition of the proteolytic and signaling pathways that operate in human skin. We elaborate how epidermal proteolysis is finely regulated at multiple levels, and in a spatial manner that has not been taken into consideration so far, in which specific proteases are confined to distinct epidermal sublayers. Of interest, transglutaminases have emerged as regulators of epidermal proteolysis and desquamation by spatially fixing endogenous protease inhibitors, constituting regulatory factors that were not recognized before. Furthermore, new evidence suggests a link between proteolysis and lipid metabolism. By synthesis of established notions and recent discoveries, we provide an up-to-date critical evaluation and synthesis of current knowledge and the extended complexity of proteolysis regulation and signaling pathways in skin. © 2022 The Pathological Society of Great Britain and Ireland.

A chemogenomic approach is required for effective treatment of amyotrophic lateral sclerosis.

ALS is a fatal untreatable disease involving degeneration of motor neurons. Μultiple causative genes encoding proteins with versatile functions have been identified indicating that diverse biological pathways lead to ALS. Chemical entities still represent a promising choice to delay ALS progression, attenuate symptoms and/or increase life expectancy, but also gene-based and stem cell-based therapies are in the process of development, and some are tested in clinical trials. Various compounds proved effective in transgenic models overexpressing distinct ALS causative genes unfortunately though, they showed no efficacy in clinical trials. Notably, while animal models provide a uniform genetic background for preclinical testing, ALS patients are not stratified, and the distinct genetic forms of ALS are treated as one group, which could explain the observed discrepancies between treating genetically homogeneous mice and quite heterogeneous patient cohorts. We suggest that chemical entity-genotype correlation should be exploited to guide patient stratification for pharmacotherapy, that is administered drugs should be selected based on the ALS genetic background.

Cocktails of KLK5 Protease Inhibitors and Anti-TNFα Therapeutics: an Effective Treatment for Netherton Syndrome.

Netherton syndrome (NS) is a rare, severe type of ichthyosis, often lethal in neonates, for which there is no therapy. Spink5-/- mice recapitulate major NS hallmarks and die homogeneously within 5 h from birth due to severe epidermal barrier defect leading to dehydration. Spink5-/-Klk5-/- mice survive neonatal lethality, indicating that KLK5 could be a drug target for NS. Nevertheless, after a week, these mice developed epidermal inflammation and signs of barrier defect leading to lethality. Here we tested whether anti-TNFα strategy in combination with anti-KLK5 could provide a long-term effective therapy for NS. Deletion of Tnfa in Spink5-/- suppressed the inflammatory phenotype but did not rescue neonatal lethality of Spink5-/- indicating that anti-TNFα therapy alone would not be sufficient to treat NS. Interestingly, in Spink5-/-Klk5-/-Tnfa-/- mice, NS features were rescued, and mice lived normally for 16-18 months. For the first time, evidence is provided that a combination of anti-TNFα and anti-KLK5 therapeutics represents an effective therapeutic strategy for NS. Notably, anti-TNFα factors are marketed and used widely, while LMW KLK5 inhibitors are being developed.

Keratinocyte differentiation and proteolytic pathways in skin (patho) physiology.

The epidermis is a stratified epithelium that forms the barrier between the organism and its environment. It is mainly composed of keratinocytes at various stages of differentiation. The stratum corneum is the outermost layer of the epidermis and is formed of multiple layers of anucleated keratinocytes called corneocytes. We aim to highlight the roles of epidermal differentiation and proteolysis in skin diseases. Skin biopsies isolated from Spink5-/- mice, the established model of Netherton syndrome (NS), and from patients with NS, seborrheic dermatitis (SD) and psoriasis, as well as healthy controls, were analyzed by histology and immunohistochemistry. Our results showed that NS, SD, and psoriasis are all characterized by abnormal epidermal differentiation, manifested by hyperplasia, hyperkeratosis, and parakeratosis. At the molecular level, abnormal differentiation is accompanied by increased expression of involucrin and decreased expression of loricrin in NS and psoriasis. Increased epidermal proteolysis associated with increased kallikrein-related peptidases (KLKs) expression is also observed in both NS and psoriatic epidermis. Furthermore, reduced expression of desmosomal proteins is observed in NS, but increased in psoriasis. Since desmosomal proteins are proteolytic substrates and control keratinocyte differentiation, their altered expression directly links epidermal proteolysis to differentiation. In conclusion, abnormal cellular differentiation and proteolysis are interconnected and underlie the pathology of NS, SD and psoriasis.

Kallikreins emerge as new regulators of viral infections.

Kallikrein-related peptidases (KLKs) or kallikreins have been linked to diverse (patho) physiological processes, such as the epidermal desquamation and inflammation, seminal clot liquefaction, neurodegeneration, and cancer. Recent mounting evidence suggests that KLKs also represent important regulators of viral infections. It is well-established that certain enveloped viruses, including influenza and coronaviruses, require proteolytic processing of their hemagglutinin or spike proteins, respectively, to infect host cells. Similarly, the capsid protein of the non-enveloped papillomavirus L1 should be proteolytically cleaved for viral uncoating. Consequently, extracellular or membrane-bound proteases of the host cells are instrumental for viral infections and represent potential targets for drug development. Here, we summarize how extracellular proteolysis mediated by the kallikreins is implicated in the process of influenza (and potentially coronavirus and papillomavirus) entry into host cells. Besides direct proteolytic activation of viruses, KLK5 and 12 promote viral entry indirectly through proteolytic cascade events, like the activation of thrombolytic enzymes that also can process hemagglutinin, while additional functions of KLKs in infection cannot be excluded. In the light of recent evidence, KLKs represent potential host targets for the development of new antivirals. Humanized animal models to validate their key functions in viral infections will be valuable.

Generation of a quenched phosphonate activity-based probe for labelling the active KLK7 protease.

Kallikrein 7 (KLK7) is a chymotrypsin-like serine protease with established roles in skin diseases like the rare Netherton syndrome, an overdesquamating and inflammatory condition, but also common atopic dermatitis, and a potential drug target for these and possibly other diseases. Nevertheless, tools to determine the active KLK7 enzyme are not available. Here, a mixed alkyl aryl phosphonate quenched activity-based probe that detects the active KLK7 was developed and evaluated in vitro. This KLK7-qABP can potentially be used to monitor KLK7 activity in vivo.

A novel theranostic activity-based probe targeting kallikrein 7 for the diagnosis and treatment of skin diseases.

We applied a new in silico approach for using protease-substrate motifs to design a kallikrein 7 (KLK7)-specific phosphonate activity-based probe (ABP) to quantify the active KLK7 in situ. Epidermal application of the ABP-inhibitor on Spink5-/-Klk5-/- mice, a Netherton syndrome model, reversed disease hallmarks, providing preclinical proof-of-concept for using ABPs as theranostics.

Research in practice: Towards deciphering the role of epidermal proteases in recessive dystrophic epidermolysis bullosa progression.

Recessive dystrophic epidermolysis bullosa (RDEB) is an incurable severe skin disease caused by loss of collagen VII, an extracellular protein that ensures skin cohesion. It manifests in skin blistering and unresolved cycles of wounding and healing that progressively lead to dermal stiffening and early development of aggressive cutaneous squamous cell carcinomas. Inflammation and subsequent tissue fibrosis highly contribute to RDEB pathogenicity and targeting them could provide new therapeutic options. Kallikreins (KLKs) are epidermal secreted proteases, which contribute to skin desquamation and inflammation. Kallikreins are involved in the pathogenesis of several inflammatory skin disorders, but interestingly also in the initiation and progression of different cancers. Our project aims at deciphering the role of KLKs in inflammation, fibrosis, and tumor development in RDEB.

Redirecting drug repositioning to discover innovative cosmeceuticals.

Skin appearance is essential for self-esteem and quality of life; consequently, skin care products represent a huge market. In particular, cosmeceuticals constitute a hybrid category of skin care formulations, at the interphase of cosmetics and pharmaceuticals, rationally designed to target (patho) physiological mechanisms aiming to enhance skin health and appearance. Cosmeceuticals are marketed as anti-ageing, anti-wrinkle, hair regrowth, skin whitening and wound healing agents with special emphasis on scar-free healing. An overview on recent cutting-edge advances concerning the discovery and development of enhanced performance cosmeceuticals by drug repositioning approaches is presented here. In this context, we propose "target repositioning," a new term, to highlight that druggable protein targets implicated in multiple diseases (hubs in the diseasome) can be exploited to accelerate the discovery of molecularly targeted cosmeceuticals that can promote skin health as an added benefit, which is a novel concept not described before. In this direction, emphasis is placed on the role of mouse models, for often untreatable skin diseases, as well as recent breakthroughs on monogenic rare skin syndromes, in promoting compound repositioning to innovative cosmeceuticals.

Ectopic expression of KLK6 in MDA-MB-435 melanoma cells reduces tumorigenicity in vivo.

Melanoma is an aggressive form of cancer with poor prognosis therefore, identification of associated pathophysiological mechanisms is imperative towards the development of new therapeutic strategies. The KLK6 is a serine protease normally expressed in the epidermis. Recently, we found that elimination of Klk6 in mice results in enhanced resistance to chemically induced non-melanoma skin cancer. To delineate putative roles of KLK6 in melanoma, the invasive KLK6-non-expressing MDA-MB-435 melanoma cell line was stably transfected with the full-length KLK6 cDNA and expression of the corresponding RNA and protein were confirmed. Interestingly, restoration of KLK6 expression resulted in markedly suppressed growth of primary tumors when orthotopically implanted in SCID mice. Analysis of data retrieved from the human protein atlas revealed that melanomas with high KLK6 expression have a trend for longer survival. Collectively, we suggest that KLK6 inhibits growth of melanomas.

Plausible Emergence of Biochemistry in Enceladus Based on Chemobrionics.

Saturn's satellite Enceladus is proposed to have a soda-type subsurface ocean with temperature able to support life and an iron ore-based core. Here, it was demonstrated that ocean chemistry related to Enceladus can support the development of Fe-based hydrothermal vents, one of the places suggested to be the cradle of life. The Fe-based chemical gardens were characterized with Fourier-transform (FT)IR spectroscopy and XRD. The developed chemobrionic structures catalyzed the condensation polymerization of simple organic prebiotic molecules to kerogens. Further, they could passively catalyze the condensation of the prebiotic molecule formamide to larger polymers, suggesting that elementary biochemical precursors could have emerged in Enceladus.

Cathelicidin represents a new target for manipulation of skin inflammation in Netherton syndrome.

Netherton syndrome (NS) is a severe ichthyosis caused by inactivating mutations in the SPINK5 gene encoding the serine protease inhibitor LEKTI. Spink5-/- mice recapitulate NS and die perinatally from extensive dehydration as a result of a severe defect of the epidermal barrier. We showed that deletion of Klk5 in Spink5-/- rescues neonatal lethality (Furio et al., 2015). However, Spink5-/-Klk5-/- mice developed skin shedding and inflammation during the first week from birth and the majority (70%) succumbed on P7. The remaining mice lived short (i.e. mean survival was 5 months) indicating alternative inflammatory pathways. Since cathelicidin is increased in Spink5-/- epidermis, we investigated whether it could be implicated in NS pathology. Ablation of Camp in Spink5-/- suppressed epidermal inflammation and restored abnormal epidermal differentiation, nevertheless, it failed to inhibit overdesquamation and Spink5-/-Camp-/- succumbed perinatally due to skin barrier defect, similarly to Spink5-/-. Joint invalidation of Klk5 and Camp significantly extended survival of Spink5-/-Klk5-/-Camp-/- mice. We provide evidence that cathelicidin is implicated in NS-associated skin inflammation in vivo. Therefore, marketed products that are known to reduce cathelicidin expression could be repurposed for the management of NS.

Elimination of KLK5 inhibits early skin tumorigenesis by reducing epidermal proteolysis and reinforcing epidermal microstructure.

Epidermal desquamation involves a finely-tuned proteolytic cascade ensuring the regulated cleavage of desmosomes that releases old stratum corneum outermost layers. Although the roles of desmosomes in normal physiology are well-established, their putative involvement in cancer remains unexplored. The KLK5 protease is thought of having fundamental roles in epidermal proteolysis and homeostasis, and its aberrant activity has been linked to skin pathologies. We found that deletion of Klk5 results in significantly higher numbers of lengthier desmosomes and enhanced skin strength. Klk5-/- mice retained normal skin barrier function and are resistant to chemically-induced skin tumorigenesis. The resistance to tumorigenesis was not due to inhibition of inflammation, and on the contrary, absence of Klk5 increased the TPA-induced inflammatory skin response. We found that increased desmosomes and reduced proteolysis prevent oncogenic signaling by capturing ß-catenin into the cytoplasm and facilitate epidermal keratinocyte apoptosis, thus, inhibiting tumor initiation. We highlight that the skin ultrastructure affects early neoplastic transformation by modulating intracellular signaling and suggest that tissue reinforcement provides a novel mode of tumor suppression.

A proinflammatory role of KLK6 protease in Netherton syndrome.

BACKGROUND: Netherton syndrome (NS) is a rare but severe type of ichthyosis characterized by atopy, allergies, and potentially lethal skin overdesquamation associated with highly elevated proteolytic activities in LEKTI-deficient epidermis. NS symptoms are recapitulated in Spink5-/- mouse where the gene encoding Lekti has been invalidated. Spink5-/- mice die within 5h from birth due to their severe skin barrier defect leading to dehydration. Spink5-/- mice also serve as a model for atopic dermatitis. The KLK6 protease is expressed by epidermal keratinocytes and shown in vitro to cleave desmosomal components. OBJECTIVE: To investigate in vivo whether KLK6 is implicated in epidermal overdesquamation and/or inflammation associated with NS. METHODS: The role of KLK6 was evaluated by generating Spink5-/-Klk6-/- double knockout mice. The phenotype was assessed by macroscopic observation, immunohistochemistry for differentiation markers, in situ zymography for proteolysis, and quantification of proinflammatory cytokines. RESULTS: Elimination of Klk6 in Spink5-/- remarkably suppresses the expression of Tslp, a major itching-inducing factor and driver of allergic reactions. Tnfα and the Th17 promoting cytokine Il-23 were also suppressed. Spink5-/-Klk6-/- mice display normalized keratinocyte differentiation, nevertheless, epidermal proteolytic activities and the associated overdesquamation were not ameliorated, and Spink5-/-Klk6-/- still died from a severe epidermal barrier defect as the Spink5-/-. CONCLUSIONS: Ablation of Klk6 largely suppresses epidermal inflammation but cannot rescue overdesquamation leading to the lethal NS phenotype. Nonetheless, our findings demonstrate for the first time that KLK6 is implicated in skin inflammation and may represent a novel druggable target for NS and other inflammatory conditions e.g. atopic dermatitis.

Treatment of keratinocytes with 4-phenylbutyrate in epidermolysis bullosa: Lessons for therapies in keratin disorders.

BACKGROUND: Missense mutations in keratin 5 and 14 genes cause the severe skin fragility disorder epidermolysis bullosa simplex (EBS) by collapsing of the keratin cytoskeleton into cytoplasmic protein aggregates. Despite intense efforts, no molecular therapies are available, mostly due to the complex phenotype of EBS, comprising cell fragility, diminished adhesion, skin inflammation and itch. METHODS: We extensively characterized KRT5 and KRT14 mutant keratinocytes from patients with severe generalized EBS following exposure to the chemical chaperone 4-phenylbutyrate (4-PBA). FINDINGS: 4-PBA diminished keratin aggregates within EBS cells and ameliorated their inflammatory phenotype. Chemoproteomics of 4-PBA-treated and untreated EBS cells revealed reduced IL1ß expression- but also showed activation of Wnt/ß-catenin and NF-kB pathways. The abundance of extracellular matrix and cytoskeletal proteins was significantly altered, coinciding with diminished keratinocyte adhesion and migration in a 4-PBA dose-dependent manner. INTERPRETATION: Together, our study reveals a complex interplay of benefits and disadvantages that challenge the use of 4-PBA in skin fragility disorders.

Biochemical pathways mediated by KLK6 protease in breast cancer.

Kallikrein-related peptidase 6 (KLK6) is a serine protease normally expressed in mammary tissue and aberrantly regulated in breast cancer. At physiological levels, KLK6 functions as a suppressor of breast cancer, while its aberrant overexpression (> 50-fold higher than normal) is characteristic of a subset of breast cancers and has been linked to accelerated growth of primary breast tumors in severe combined immunodeficiency mice (Pampalakis et al. Cancer Res 2009, 69, 3779). Here, we investigated the molecular mechanisms underlying the concentration-dependent functions of KLK6 by comparing MDA-MB-231 stable transfectants expressing increasing levels of KLK6 in in vitro and in vivo tumorigenicity assays (soft agar, xenograft growth, tail vein metastasis). Quantitative proteomics was applied to identify proteins that are altered upon re-expression of KLK6 in MDA-MB-231 at normal or constitutive levels. Overexpression of KLK6 is associated with increased metastatic ability of breast cancer cells into lungs, increased expression of certain S100 proteins (S100A4, S100A11) and keratins (KRT), and downregulation of the apoptosis-related proteases CASP7 and CASP8, and RABs. On the other hand, KLK6 re-expression at physiological levels leads to inhibition of lung metastases associated with suppression of S100 proteins (S100A4, S100A10, S100A13, S100A16) and induced CASP7 and CASP8 expression. As this is the first report that KLK6 expression is associated with S100 proteins, caspases, RABs, and KRTs, we validated this finding in clinical datasets. By integrating proteomics and microarray data from breast cancer patients, we generated two composite scores, KLK6 + S100B-S100A7 and KLK6 + S100B-S100A14-S100A16, to predict long-term survival of breast cancer patients. We present previously unknown pathways implicating KLK6 in breast cancer. The findings promise to aid our understanding of the functional roles of KLK6 in breast cancer and may yield new biomarkers for the cancer types in which KLK6 is known to be aberrantly upregulated.