| Περίληψη: | Kallikrein-related peptidase 6 (KLK6 for human; Klk6 for mouse) is a trypsin-like serine protease, member of the family of kallikrein-related (KLKs) serine proteases. Certain KLKs have been linked to various diseases, including autoimmune and inflammatory skin diseases, neurodegeneration, and multiple types of cancer. In particular KLK6 was found dysregulated in several cancer types, as well as in Parkinson disease and Alzheimer’s disease. Until now, little progress has been achieved in establishing specific inhibitors for the KLK6 protease. On the other hand, Activity-Based Probes (ABPs) are chemical molecules that bind specifically to the active form of an enzyme but not to its inactive forms, e.g. the corresponding pro-enzyme, etc. Thus, ABPs are unique tools for detection and quantification of active enzymes with various applications as research and molecular diagnostic tools. ABPs can also act as inhibitors their specific protease, thus, they provide opportunities for the development of theranostic agents. Typically, ABPs are comprised of [a] a reactive group that binds to the active site of the enzyme, [b] a recognition sequence or linker, which interacts selectively with the side pockets of the recognized protease, and [c] a detection tag, typically a fluorophore or biotin. The main aim of this study was to evaluate two KLK6-ABP-inhibitors that are phosphonate derivatives of arginine for their inhibitory activity and selectivity against active KLK6 but not against other KLK proteases that are co-expressed in human skin (and other tissues) like KLK5 and KLK13, for example. For this, rKLK6 R80Q, rKlk6 and rKLK5 were produced in the yeast Pichia pastoris and purified to homogeneity by a two-step chromatography protocol, i.e. by hydrophobic interaction chromatography followed by cation exchange chromatography. The activity of the purified recombinant proteins was confirmed by gel zymography and also against the fluorogenic protease substrate Z-Arg-Phe-AMC. Following, the inhibitory activity of two synthetic compounds, i.e. benzyl(1-(diphenoxyphosphoryl)-4guanidino butyl)carbamate (Z-Arg(P)-(OPh)2) and diphenyl(1-amino4guanidinobutyl) phosphonate (H2N-Arg(P)-(OPh)2) against the purified recombinant proteases was assayed and kinetic constants were calculated. Indeed, we found that both these compounds can inhibit KLK6 activity efficiently, nevertheless, they could also inhibit trypsin, suggesting lack of selectivity for KLK6. The benzyl(1-(diphenoxy phosphoryl)-4-guanidinobutyl)carbamate was a more potent inhibitor than the diphenyl(1-amino-4-guanidinobutyl)phosphonate, and the calculated kinact/KI constants were 1987.43 M-1 s-1 and 65 M-1 s-1, respectively.
As part of the current study, a sandwich ELISA specific for KLK6 was developed using three commercial α-KLK6 antibodies and one α-KLK6 IgYs polyclonal produced in-house (Sotiropoulou et al., 2012), that were compared in parallel to optimize the assay. The established ELISA exhibited a dynamic range of 3-35 ng/ml and a lower detection limit of 1.3 ng/ml. This ELISA assay will find wide applications for the quantification of KLK6 in biological fluids but also in clinical samples given that aberrant KLK6 expression/activity has been associated with various human diseases. While total, i.e. active + inactive KLK6 in a given sample can be quantified by the developed ELISA, the % corresponding to the active KLK6 enzyme in the same sample can be quantified by a KLK6-ABP based ELISA. In conclusion, synthetic phosphonate analogs of arginine proved potent but not selective inhibitors of KLK6. Modification of these compounds is ongoing to achieve selectivity, which may eventually lead to an optimized KLK6-ABP-inhibitor for theranostic purposes.
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