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

Abstract

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.

Inhibitors of kallikrein‐related peptidases: An overview

“…By exchanging individual amino acids, it was possible to alter target specificity and design potent inhibitors of several KLK‐related peptidases. Whereas the natural SFTI‐1 possesses only weak to moderate inhibitory activities against KLKs, several groups reported that substituting preferred substrate sequences into the P4, P2, P1, and P2’ residues of SFTI‐1 (Table ) yielded several potent inhibitors of KLK5 ( K i = 2.1 nM), KLK4 ( K i = 39 pM), KLK7 ( K i = 0.8 nM), and KLK14 (0.4 nM) . Swedberg et al.…”

“…) is known to inhibit KLK1, KLK2, KLK6, KLK8, KLK11, and KLK12 . p ‐Amino‐benzamidine 8b (PABA) inhibits KLK4 and KLK5 . Several co‐crystallizations of benzamidine or PABA with KLKs were described, giving crucial information about the key interactions involved in the reversible inhibition of the enzymes.…”

“…37 p-Amino-benzamidine 8b (PABA) inhibits KLK4 34,37,113 and KLK5. 111 Several co-crystallizations of benzamidine or PABA with KLKs were described, giving crucial information about the key interactions involved in the reversible inhibition of the enzymes. Benzamidine derivatives bind in the S1 pocket ( Fig.…”

Going Round in Circles with N→S Acyl Transfer

“…57 An identical process was adopted to produce variants of 14 residue SFTI-1 which were ultimately tested as inhibitors of Kalikrein 5 (KLK5), a protease involved in skin barrier homeostasis. 45,58 Overall, rather than constituting a severe limitation, the differing responses of Xaa-Cys motifs toward thioloysis and the reduced reactivity at internal Cys residues has in fact proved very beneficial on several occasions. A rather general strategy has been adopted for producing SFTI and cyclotide analogues from linear precursors and it is likely that this approach could be extended to other classes of cyclic peptides and combined with unnatural amino acids using an expanded genetic code, 59 in order to introduce novel functionality.…”

Молекулярные Аспекты Иммунитета Растений И Их Коэволюции С Насекомыми

“…Особенно интенсивно работают с ингибитором из подсолнечника в области медицины, где на его основе разрабатывают препараты для терапии опухолей, заболеваний крови, кожи, анальгетики и т. д. [9,29,30,61,102].…”