Substrate-Guided Design of a Potent and Selective Kallikrein-Related Peptidase Inhibitor for Kallikrein 4

Abstract

Human kallikrein-related peptidase 4 (KLK4/prostase), a trypsin-like serine protease, is a potential target for prostate cancer treatment because of its proteolytic ability to activate many tumorigenic and metastatic pathways including the protease activated receptors (PARs). Currently there are no KLK4-specific small-molecule inhibitors available for therapeutic development. Here we re-engineer the naturally occurring sunflower trypsin inhibitor to selectively block the proteolytic activity of KLK4 and prevent stimulation of PAR activity in a cell-based system. The re-engineered inhibitor was designed using a combination of molecular modeling and sparse matrix substrate screening.

High-affinity Cyclic Peptide Matriptase Inhibitors

“…Naturally occurring peptides with cyclic backbones have significant promise in drug design (14,17,43), and in this study we have highlighted the potential of the frameworks of cyclic trypsin inhibitors from seeds. In particular, we have discovered that MCoTI-II is a potent inhibitor of matriptase and also generated substantial structure-activity data regarding MCoTI-II and SFTI-1 that has provided insights on how to modulate affinity toward matriptase over the prototypic trypsin.…”

“…them ideal scaffolds for pharmaceutical design (14,17). In the current study we have discovered that a member of the trypsin inhibitory subfamily of cyclotides, Momordica cochinchinensis trypsin inhibitor-II (MCoTI-II) (26), a 34-residue cyclic peptide, is also a potent inhibitor of matriptase.…”

“…Despite this limitation, the size, versatility, and stability of the SFTI-I backbone makes it an appealing scaffold for the design of drugs (16). Swedberg et al (17), for example, enhanced both the affinity and specificity of the SFTI-1 backbone against kallikrein 4 (KLK4) through modifications of the inhibitor. They used positional scanning of a synthetic combinatorial library to study protease specificity and generated a mutant of SFTI-1 with an inhibition constant of 3.59 nM against KLK4 compared with no inhibition by native SFTI-1 (17).…”

“…Swedberg et al (17), for example, enhanced both the affinity and specificity of the SFTI-1 backbone against kallikrein 4 (KLK4) through modifications of the inhibitor. They used positional scanning of a synthetic combinatorial library to study protease specificity and generated a mutant of SFTI-1 with an inhibition constant of 3.59 nM against KLK4 compared with no inhibition by native SFTI-1 (17). Although this mutant was not tested in vivo it retained high inhibitory potential against KLK4 after days of incubation in serum, highlighting the advantages of the SFTI-1 scaffold in the design of drugs.…”

Novel Detection Methods Used in Conjunction with Affinity Chromatography for the Identification and Purification of Hydrolytic Enzymes or Enzyme Inhibitors from Insects and Plants

“…It was revealed that the inhibitor was extremely robust, resistant to proteolysis (no N-or C-ends) which, coupled to its high potency of inhibition for such proteinases as matripatse, thrombin, kallikrein 4 etc. made it an excellent candidate for use as a template for further development of drugs for therapy against cancer, thromboembolism and other proteinase-related human pathologies (Li, et al, 2007;Luckett et al, 1999;Swedberg et al, 2009;Białas & Kafarski, 2009) or agents for plant protection against pests and pathogens.…”