Abstract B055: New potential therapeutic applications of WX-UK1 as a specific and potent inhibitor of human trypsin-2 and human trypsin-3
Authors: Oldenberg Emil , Schar Christine R. , Lange Eva L. , Plasse Terry F. , Abramson Danielle T. , Levitt Mark L. , Fathi Reza , Jensen Jan K. 
Published: August 2018

WX-UK1, a multi-serine protease inhibitor, was believed to function primarily as a synthetic small-molecule inhibitor of urokinase (uPA). We now reveal that WX-UK1 is a potent and rather specific inhibitor of human trypsin-2 and human trypsin-3, and propose that these new molecular targets offer potential for new indications in oncology and other diseases. Initially, we performed a bioinformatic analysis of the ~200 human trypsin-like serine proteases, most of which play crucial roles in homeostasis and disease. Among these we selected a subset for biochemical analysis based on an inspection of modeled 3D structures of WX-UK1:protease complexes and sequence alignment of binding site residues. Samples of the selected proteases were prepared and characterized biochemically with respect to inhibitory constant (Ki) of inhibition by WX-UK1-1 and dissociation constant (Kd). The Ki values were determined from enzymatic activity assays (37°C), and Kd values (affinity) by surface plasmon resonance (BIACORE) (25°C). Serine protease(human)Ki (nM)Mean ± SD (n)Kd (nM)Mean ± SD (n)Urokinase (uPA)874 ± 95 (3)720 ± 300 (3)Trypsin(-1)190 ± 10 (6)74 ± 18 (3)Trypsin-275 ± 3 (6)N.D.Trypsin-319 ± 4 (6)6 ± 4 (11)

While previously described as an inhibitor of bovine trypsin, we now show WX-UK1 to be a specific competitive inhibitor of both human trypsin-2 and human trypsin-3 with Ki of 75 and 19 nM, respectively. With a Kd of 6 nM, WX-UK-1 binds human trypsin-3 with a &gt;100-fold binding specificity relative to most other trypsin-like serine proteases tested so far, including uPA, and 10-fold over the classical human trypsin. WX-UK1 (and its oral prodrug, upamostat) was originally developed as an inhibitor of uPA and other members of the S1 family of trypsin-like serine proteases, essential to aspects of tissue remodeling associated with tumor invasion and metastasis. However, with the identification of human trypsin-3 and human trypsin-2 as high-affinity molecular targets, new disease indications on top of oncology indications may be feasible. As a low-nanomolar inhibitor of human trypsin-3, WX-UK1 may have potential utility in the treatment of inflammatory digestive diseases, including irritable bowel syndrome, inflammatory bowel disease, and pancreatitis, for the latter of which there are currently no approved therapies. Furthermore, WX-UK1, as a human trypsin-2 inhibitor, could potentially be used in the treatment of inflammatory lung diseases, including acute respiratory distress syndrome, acute lung injury, α-1 antitrypsin deficiency, and chronic obstructive pulmonary disease (COPD)–a major unmet medical need.

Citation Format: Emil Oldenberg, Christine R. Schar, Eva L. Lange, Terry F. Plasse, Danielle T. Abramson, Mark L. Levitt, Reza Fathi, Jan K. Jensen. New potential therapeutic applications of WX-UK1 as a specific and potent inhibitor of human trypsin-2 and human trypsin-3 [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B055.