Selective Substrates and Inhibitors for Kallikrein-Related Peptidase 7 (KLK7) Shed Light on KLK Proteolytic Activity in the Stratum Corneum.

Proteases have pivotal roles in the skin's outermost layer, the epidermis. In the stratum corneum, serine proteases from the kallikrein-related peptidase (KLK) family have been implicated in several key homeostatic processes, including desquamation. However, the precise contribution of specific KLKs to each process remains unclear. To address this, we used a chemical biology approach and designed selective substrates and inhibitors for KLK7, the most abundant KLK protease in the stratum corneum. The resulting KLK7 inhibitor is the most potent inhibitor of this protease reported to date (Ki = 140 pM), and displays at least 1,000-fold selectivity over several proteases that are related by function (KLK5 and KLK14) or specificity (chymotrypsin). We then used substrates and inhibitors for KLK5, KLK7, and KLK14 to explore the activity of each protease in the stratum corneum using casein zymography and an ex vivo desquamation assay. These experiments provide the most detailed assessment of each KLK's contribution to corneocyte shedding in the plantar stratum corneum, revealing that inhibition of KLK7 alone is sufficient to block shedding, whereas KLK5 is also a major contributor. Collectively, these findings unveil chemical tools for studying KLK activity and demonstrate their potential for characterizing KLK biological functions in epidermal homeostasis.

Mechanism-based selection of a potent kallikrein-related peptidase 7 inhibitor from a versatile library based on the sunflower trypsin inhibitor SFTI-1.

Potent and specific enzyme inhibition is a key goal in the development of therapeutic inhibitors targeting proteolytic activity. The backbone-cyclized peptide, Sunflower Trypsin Inhibitor (SFTI-1) affords a scaffold that can be engineered to achieve both these aims. SFTI-1's mechanism of inhibition is unusual in that it shows fast-on/slow-off kinetics driven by cleavage and religation of a scissile bond. This phenomenon was used to select a nanomolar inhibitor of kallikrein-related peptidase 7 (KLK7) from a versatile library of SFTI variants with diversity tailored to exploit distinctive surfaces present in the active site of serine proteases. Inhibitor selection was achieved through the use of size exclusion chromatography to separate protease/inhibitor complexes from unbound inhibitors followed by inhibitor identification according to molecular mass ascertained by mass spectrometry. This approach identified a single dominant inhibitor species with molecular weight of 1562.4 Da, which is consistent with the SFTI variant SFTI-WCTF. Once synthesized individually this inhibitor showed an IC50 of 173.9 ± 7.6 nM against chromogenic substrates and could block protein proteolysis. Molecular modeling analysis suggested that selection of SFTI-WCTF was driven by specific aromatic interactions and stabilized by an enhanced internal hydrogen bonding network. This approach provides a robust and rapid route to inhibitor selection and design.

Mechanism-based selection of a potent kallikrein-related peptidase 7 inhibitor from a versatile library based on the sunflower trypsin inhibitor SFTI-1.

Potent and specific enzyme inhibition is a key goal in development of therapeutic inhibitors targeting proteolytic activity. The backbone-cyclised peptide, Sunflower Trypsin Inhibitor (SFTI-1) affords a scaffold that can be engineered to achieve both these aims. SFTI-1's mechanism of inhibition is unusual in that it shows fast-on/slow-off kinetics driven by cleavage and religation of a scissile bond. This phenomenon was used to select a nanomolar inhibitor of kallikrein related peptidase 7 (KLK7) from a versatile library of SFTI variants with diversity tailored to exploit distinctive surfaces present in the active site of serine proteases. Inhibitor selection was achieved through use of size exclusion chromatography to separate protease/inhibitor complexes from unbound inhibitors followed by inhibitor identification according to molecular mass ascertained by mass spectrometry. This approach identified a single dominant inhibitor species with molecular weight of 1562.4 Da, which is consistent with the SFTI variant SFTI-WCTF. Once synthesised individually this inhibitor showed an IC50 of 173.9±7.6 nM against chromogenic substrates and could block protein proteolysis. Molecular modelling analysis suggested that selection of SFTI-WCTF was driven by specific aromatic interactions and stabilised by an enhanced internal hydrogen bonding network. This approach provides a robust and rapid route to inhibitor selection and design. © 2013 Wiley Periodicals, Inc. Biopolymers, 2013.