Monash University
20170316-Ilyichova-Thesis.pdf (91.57 MB)

Structural Studies of Kallikrein-Related Peptidase 4 in Complex with Inhibitors

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posted on 2019-03-08, 00:08 authored by Olga Ilyichova
Human kallikrein-related peptidases (KLKs) are a family of 15 secreted serine proteases with either trypsin or chymotrypsin activity, that are found in diverse tissues and biological fluids. Abnormalities in expression levels of KLKs have been implicated in pathogenesis of many diseases including different types of cancer.

   Kallikrein related peptidase 4 (KLK4) is a member of the KLKs family. Under normal physiological conditions, KLK4 is known to process amelogenin during tooth development. It is also highly expressed in basal and secretory cells of the prostate gland. Over-expression of KLK4 is associated with aggressive forms of hormone-dependent prostate, ovarian and breast cancers. Recent studies show that KLK4 may be involved in a number of cancer-related proteolytic activities, namely activation of stromal protease activated receptors 1 and 2 (PAR-1 and PAR-2), activation of urokinase plasminogen activator receptor (uPAR) and single chain urokinase plasminogen activator (uPA), inhibition of promyelocytic leukemia zinc finger protein and others. Furthermore, it was demonstrated that KLK4 is localized in both prostate cancer cells and osteoblasts. This evidence, along with the physiological role of KLK4 in processing extracellular matrix proteins in teeth, suggest that KLK4 may facilitate cancer progression and bone metastasis.
   Given the spectrum of pathophysiological activities that KLK4 might be involved in when dysregulated, it can be easily appreciated that the use of selective KLK4 inhibitors presents a promising therapeutic approach. Sunflower trypsin inhibitor (SFTI-1), a member of the Bowman-Birk protease inhibitor family, is a 14 amino acid bicyclic peptide that is stabilized by both backbone cyclisation and a disulfide bridge. Another important characteristic of SFTI-1 is that proteolytic cleavage between P1 and P1’ residues does not induce conformational changes, allowing the scissile bond to be reformed with an equilibrium of 1:9 in favour of the intact bond. This phenomenon was characterized in a large number of inhibitors and was named the Laskowski mechanism (also known as the standard or canonical mechanism). The rigid double-cyclic structure of SFTI-1, with an extensive internal hydrogen bond network, has been utilized in the development of various selective inhibitors of tryptic and chymotryptic serine proteases, e.g. matriptase, KLK5, KLK14 and others. Although it has been shown to effectively inhibit KLK4 with an IC50 of 221 nM, SFTI-1 is not a specific inhibitor of KLK4. Therefore, the attractive template of SFTI-1 was used as a platform for the development of new potent and highly KLK4 selective inhibitors.
   The first generation re-engineered SFTI-1 variant – FCQR (amino acids 2-5 RCTK mutated to FCQR), achieved an IC50 = 7.97 ± 1.08 nM. FCQR showed improved selectivity against off-target serine proteases and other closely related KLKs, blocked protease activated receptor signalling in vitro and showed remarkable stability in cell culture (t1/2 = 4 days). The second generation inhibitor, FCQR(Asn14) (Asp14 mutated to Asn14) was designed to strengthen the internal hydrogen bonding network of FCQR and gave a further 100-fold (IC50 = 0.0635 ± 0.0024 nM) improvement in potency without sacrificing selectivity.
   In this study a new high-yield refolding protocol was developed that produced sufficient quantities of KLK4 for structural studies. KLK4 was crystallized in the presence of SFTI-1 and three engineered SFTI-1 variants. The X-ray crystal structures of these complexes were determined to atomic resolution (1.0 – 1.3 Å). These data provide a detailed structural description of the KLK4-inhibitor interactions. Furthermore, the structure of the complex between KLK4 and acyclic[1,14]-FCQR(Asn14) variant reveals a cleaved form of the peptide. To the best of my knowledge, this is the first report of a crystal structure of an active serine protease bound to a cleaved Laskowski mechanism inhibitor bound in the active site upon cleavage. Therefore, these structures also provide insight into the hydrolysis resistance mechanism of Bowman-Birk Inhibitors.


Principal supervisor

Ashley Buckle

Year of Award


Department, School or Centre

Biochemistry and Molecular Biology

Campus location



Doctor of Philosophy

Degree Type



Faculty of Medicine Nursing and Health Sciences