Tissue Kallikrein Inhibitors Based on the Sunflower Trypsin Inhibitor Scaffold - A Potential Therapeutic Intervention for Skin Diseases

• Published in: PloS one 2016-11, Vol.11 (11), p.e0166268-e0166268
• Main Authors: Chen, Wenjie, Kinsler, Veronica A, Macmillan, Derek, Di, Wei-Li
• Format: Article
• Language: English
• Subjects: Amino acids; Analogs; Biology and Life Sciences; Calcium; Calcium (intracellular); Calorimetry; Cell activation; Cell culture; Cell Line; Childrens health; Enzymes; Gene therapy; Health aspects; Helianthus; Helianthus - metabolism; Humans; Inflammation; Inhibition; Inhibitors; Interleukin; Interleukin 8; Interleukin-8 - metabolism; Interleukins; Kallikrein; Medicine and Health Sciences; Mutation; Netherton syndrome; Netherton Syndrome - drug therapy; Netherton Syndrome - metabolism; Pathogenesis; Peptides; Peptides, Cyclic - antagonists & inhibitors; Peptides, Cyclic - metabolism; Physical Sciences; Protease; Protease inhibitors; Proteinase Inhibitory Proteins, Secretory - pharmacology; Proteins; Receptor, PAR-2 - metabolism; Research and Analysis Methods; Resins; Shedding; Signaling; Skin; Skin - drug effects; Skin - metabolism; Skin diseases; Skin Diseases - drug therapy; Skin Diseases - metabolism; Soybeans; Tissue kallikrein; Tissue Kallikreins - antagonists & inhibitors; Titration; Titration calorimetry; Trypsin; Trypsin inhibitors; Trypsin Inhibitors - pharmacology
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0166268

Tissue kallikreins (KLKs), in particular KLK5, 7 and 14 are the major serine proteases in the skin responsible for skin shedding and activation of inflammatory cell signaling. In the normal skin, their activities are controlled by an endogenous protein protease inhibitor encoded by the SPINK5 gene. Loss-of-function mutations in SPINK5 leads to enhanced skin kallikrein activities and cause the skin disease Netherton Syndrome (NS). We have been developing inhibitors based on the Sunflower Trypsin Inhibitor 1 (SFTI-1) scaffold, a 14 amino acids head-to-tail bicyclic peptide with a disulfide bond. To optimize a previously reported SFTI-1 analogue (I10H), we made five analogues with additional substitutions, two of which showed improved inhibition. We then combined those substitutions and discovered a variant (Analogue 6) that displayed dual inhibition of KLK5 (tryptic) and KLK7 (chymotryptic). Analogue 6 attained a tenfold increase in KLK5 inhibition potency with an Isothermal Titration Calorimetry (ITC) Kd of 20nM. Furthermore, it selectively inhibits KLK5 and KLK14 over seven other serine proteases. Its biological function was ascertained by full suppression of KLK5-induced Protease-Activated Receptor 2 (PAR-2) dependent intracellular calcium mobilization and postponement of Interleukin-8 (IL-8) secretion in cell model. Moreover, Analogue 6 permeates through the cornified layer of in vitro organotypic skin equivalent culture and inhibits protease activities therein, providing a potential drug lead for the treatment of NS.