Abstract
Human tissue kallikrein (KLK) proteases are hormone-like signaling molecules with important functions in cancer pathophysiology. KLK-related peptidase 6 (KLK6), specifically, is highly up-regulated in several types of cancer, where its increased activity promotes cancer invasion and metastasis. This characteristic suggests KLK6 as an attractive target for therapeutic interventions. However, inhibitors that specifically target KLK6 have not yet been reported, possibly because KLK6 shares a high sequence homology and structural similarity with other serine proteases and resists inhibition by many polypeptide inhibitors. Here, we present an innovative combinatorial approach to engineering KLK6 inhibitors via flow cytometry-based screening of a yeast-displayed mutant library of the human amyloid precursor protein Kunitz protease inhibitor domain (APPI), an inhibitor of other serine proteases, such as anionic and cationic trypsins. On the basis of this screening, we generated APPIM17L,I18F,S19F,F34V (APPI-4M), an APPI variant with a KLK6 inhibition constant (Ki ) of 160 pm and a turnover time of 10 days. To the best of our knowledge, APPI-4M is the most potent KLK6 inhibitor reported to date, displaying 146-fold improved affinity and 13-fold improved proteolytic stability compared with WT APPI (APPIWT). We further demonstrate that APPI-4M acts as a functional inhibitor in a cell-based model of KLK6-dependent breast cancer invasion. Finally, the crystal structures of the APPIWT/KLK6 and APPI-4M/KLK6 complexes revealed the structural and mechanistic bases for the improved KLK6 binding and proteolytic resistance of APPI-4M. We anticipate that APPI-4M will have substantial translational potential as both imaging agent and therapeutic.
Highlights
Human tissue kallikrein (KLK) proteases are hormone-like signaling molecules with important functions in cancer pathophysiology
To the best of our knowledge, amyloid precursor protein Kunitz protease inhibitor domain (APPI)-4M is the most potent KLK-related peptidase 6 (KLK6) inhibitor reported to date, displaying 146fold improved affinity and 13-fold improved proteolytic stability compared with WT APPI (APPIWT)
KLK6 appears to be relevant for breast cancer, as high KLK6 expression levels were recently shown to promote the invasiveness of breast cancer cells; KLK6 serum levels were found to be significantly higher in patients with invasive breast cancer, and patients with high KLK6 expression showed poorer survival rates [22,23,24]
Summary
A potent, proteolysis-resistant inhibitor of kallikrein-related peptidase 6 (KLK6) for cancer therapy, developed by combinatorial engineering. We employed the powerful yeast surface display (YSD) technology [53, 54] to screen combinatorial libraries of APPI variants containing mutations in both the active site and the scaffold Using these libraries, we engineered an APPI that, as compared with its WT counterpart, demonstrates a 14-fold increase in proteolytic stability and a 146-fold higher binding affinity to KLK6, without increasing its binding affinity toward other KLK proteases. This novel KLK6 inhibitor offers promising avenues for cancer therapy and imaging applications, as well as an extremely valuable laboratory reagent for deciphering the specific mechanisms by which KLK6 drives cancer progression
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