| Περίληψη: | The generation of a novel activity-based probe (ABP) and inhibitor specific for kallikrein 7 (KLK7) and validation of its therapeutic effects in vivo is described here. KLK7 is a protease with well-established functions in skin inflammation and overdesquamation and in cancer. ABPs, on the other hand, are small molecules that recognise the active enzyme specifically. Thus, in contrast to antibody-based assays, ABPs are uniquely valuable tools for the detection and quantification of the active fraction of a given enzyme, as well as for in vivo imaging enzymatic activities.
To generate the KLK7-ABP, an in silico approach was applied for the identification of a new KLK7-specific substrate. The substrate was modified to yield a specific and selective KLK7 phosphonate inhibitor (Boc-Phe-PheP-(OPh)2) that was biotinylated to yield an ABP (biotin-X-X-Phe-PheP-(OPh)2). In addition, a fluorescently quenched ABP (KLK7-qABP) was synthesized and validated in vitro by established analytical methods, as well as a new histochemical assay, named activography (Pampalakis et al., Chem Commun (Camb) 53: 3246-3248, 2017). It is shown that the developed KLK7-ABP recognizes the active form of human KLK7 in vitro, but also the endogenous human KLK7 and mouse Klk7 proteins in corresponding cell extracts and skin biopsies.
The therapeutic potential of the KLK7-ABP and KLK7-inhibitor was demonstrated in vivo using Spink5-/-Klk5-/- mice. Spink5-/- mice represent an established preclinical model of the rare disease Netherton syndrome, but also for common atopic dermatitis. Netherton syndrome is a severe (potentially lethal) rare ichthyosis characterized by pathological skin ovedesquamation and constitutive inflammation. It is known that KLK7 is aberrantly high in the skin of Spink5-/- mice upon ablation of the Klk5 protease, i.e., in the studied Spink5-/-Klk5-/- mice, and is functionally implicated in pathology (Furio et al., 2015; Kasparek et al., 2017). Epidermal administration of both the KLK7 inhibitor and the ABP attenuated skin inflammation and overdesquamation significantly. These novel results provide preclinical proof-of-concept for potential therapeutic and diagnostic (theranostic) applications of the developed KLK7-ABP in skin pathologies and, potentially, in ovarian and pancreatic cancer, in which KLK7 has been implicated.
Further, a KLK6-ABP (biotin-dPEG®4-His-Ile-Val-ArgP-(OPh)2) was generated with the same strategy to determine the active KLK6 protease in biological and clinical specimens, since it is well-established that KLK6 is implicated in Alzheimer's and Parkinson's disease and in different types of cancer.
Until now, the roles of theranostic ABPs targeting multiple cathepsin cysteine proteases have been assessed for cancer and cardiovascular diseases (Ben-Nun et al., 2015; Weiss-Sadan et al., 2019). In terms of this thesis, novel ABP-inhibitors with dual functions accommodated on a single chemical scaffold were developed and validated against the KLK7 (Bisyris et al., 2021a and 2021b) and KLK6 serine proteases. Evidence is provided for their potential analytical, diagnostic, and therapeutic applications, which expands the use of ABPs as theranostic agents by demonstrating their application in targeting the kallikreins, the largest family of serine proteases in the human genome.
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