Development of recombinant inhibitors specific to human kallikrein 2 using phage‐display selected substrates

Abstract

The reactive site loop of serpins undoubtedly defines in part their ability to inhibit a particular enzyme. Exchanges in the reactive loop of serpins might reassign the targets and modify the serpin-protease interaction kinetics. Based on this concept, we have developed a procedure to change the specificity of known serpins. First, reactive loops are very good substrates for the target enzymes. Therefore, we have used the phage-display technology to select from a pentapeptide phage library the best substrates for the human prostate kallikrein hK2 [Cloutier, S.M., Chagas, J.R., Mach, J.P., Gygi, C.M., Leisinger, H.J. & Deperthes, D. (2002) Eur. J. Biochem. 269, 2747Biochem. 269, -2754. Selected substrates were then transplanted into the reactive site loop of a1-antichymotrypsin to generate new variants of this serpin, able to inhibit the serine protease. Thus, we have developed some highly specific a1-antichymotrypsin variants toward human kallikrein 2 which also show high reactivity. These inhibitors might be useful to help elucidate the importance of hK2 in prostate cancer progression.Keywords: phage-display; protease; human kallikrein; inhibitor; a1-antichymotrypsin.Prostate cancer is currently the most commonly diagnosed cancer in American men. This pathology is the second leading cause of cancer death after lung cancer and the majority of the patients with locally advanced prostate cancer have an increased risk for disease progression. In this progression, proteases are believed to play a pivotal role in the malignant behaviour of cancer cells, including rapid tumor growth, invasion and metastasis. Human glandular kallikrein (hK2) protein is a trypsin-like serine protease expressed predominantly in the prostate epithelium. First isolated from human seminal plasma [1], hK2 has emerged recently as a diagnostic marker for prostate cancer. When tested in combination with assays for various forms of prostate specific antigen (PSA), hK2 seemed to be better suited to distinguish malignant from benign prostate disease than the well established marker PSA (prostate specific antigen or hK3) [2][3][4]. In addition to its role as a marker, the proteolytic activities suggest that hK2 could contribute to cancer progression. Several potential functions for this enzyme have been proposed, including the activation of urokinase-type plasminogen activator Taking into account its prostate tissue-specific expression and the involvement of all its potential substrates in cancer development, hK2 can be considered as a potential therapeutic target.The serpins (serine protease inhibitors) are a large family of proteins implicated in the regulation of complex physiological processes. These proteins of about 45 kDa can be subdivided into two groups, one being inhibitory and the other noninhibitory. Serpins contain an exposed flexible reactive-site loop (RSL), which is implicated in the interaction with the putative target protease. Following the binding to the enzyme and cleavage of the P1-P'1 scissile bond of the RSL, a covalent com...

Unleashing the therapeutic potential of human kallikrein-related serine proteases

“…In a similar manner, serpins have been used as starting scaffolds for the development of more specific KLK inhibitors. On the basis of their preferred substrate specificities, α1-antitrypsin and α1-antichymotrypsin were modified through adjustment of their reactive loops to generate potent new inhibitors of KLK14 and KLK2, respectively 25,34,199,200 . The therapeutic usefulness of KLK-based antibodies has also been reported.…”

Evolution of cyclic peptide protease inhibitors

“…We next utilized a bacterial selection to identify cyclic peptides that inhibit a protease of interest. Several such schemes have been described in the literature for protease inhibitors (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35) in which the viability of E. coli is linked to protease activity (32,33). For example, Block and Grafstrom (32) introduced an HIV protease recognition sequence into the tetracycline resistance gene (Tn10), a metal-tetracycline/ H þ antiporter that contains two functional, homologous integral membrane domains (TetA and TetB) linked by a central cytosolic hinge (36).…”

Activation and enzymatic characterization of recombinant human kallikrein 8

“…Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG was purchased from Jackson ImmunoResearch Inc. (West Grove, PA, USA). Recombinant pro-hK8 (Kishi et al, 2003b) and the a1-antichymotrypsin variant ACT(PCI) (Cloutier et al, 2004) were prepared as described elsewhere. All other reagents used were of analytical grade.…”

“…Recombinant ACT(PCI), an a1-antichymotrypsin variant containing a reactive site loop modification derived from protein C inhibitor, has been described as an inhibitor of hK2 (Cloutier et al, 2004). Besides its inhibitory activity on hK2, ACT(PCI) also inhibits other human tissue kallikreins, including hK8, by forming stable complexes (unpublished data).…”