TY - JOUR
T1 - A potent, proteolysis-resistant inhibitor of kallikrein-related peptidase 6 (KLK6) for cancer therapy, developed by combinatorial engineering
AU - Sananes, Amiram
AU - Cohen, Itay
AU - Shahar, Anat
AU - Hockla, Alexandra
AU - De Vita, Elena
AU - Miller, Aubry K.
AU - Radisky, Evette S.
AU - Papo, Niv
N1 - Funding Information:
2 Supported by National Institutes of Health Grant R01CA154387.
Funding Information:
This work was supported in part by the European Research Council “Ideas Program” ERC-2013-StG Contract Grant 336041, the Prostate Cancer Foundation (to N. P.), and the DKFZ-MOST Contract Grant GR2495 (to N. P. and A. K. M.). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. 1 Supported by United States–Israel Binational Science Foundation. 2 Supported by National Institutes of Health Grant R01CA154387. We thank Dr. Alon Zilka and Dr. Uzi Hadad for their technical assistance. FACS experiments were performed at the Ilse Katz Institute for Nanoscale Science and Technology, BGU. The structural studies were performed on beamlines ID23-1 and ID30-B at the European Synchrotron Radiation Facility (ESRF), Grenoble, France. We are grateful to Christoph Mueller-Dieckmann for providing assistance in using those beamlines. We thank Eitan Rabinovich for providing KLK4.
Funding Information:
1 Supported by United States–Israel Binational Science Foundation.
Funding Information:
This work was supported in part by the European Research Council “Ideas Program” ERC-2013-StG Contract Grant 336041, the Prostate Cancer Foun-dation (to N. P.), and the DKFZ-MOST Contract Grant GR2495 (to N. P. and A. K. M.). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Publisher Copyright:
© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.
PY - 2018/8/17
Y1 - 2018/8/17
N2 - Human tissue kallikrein (KLK) proteases are hormone-like signaling molecules with important functions in cancer patho-physiology. 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.
AB - Human tissue kallikrein (KLK) proteases are hormone-like signaling molecules with important functions in cancer patho-physiology. 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.
UR - http://www.scopus.com/inward/record.url?scp=85051712634&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85051712634&partnerID=8YFLogxK
U2 - 10.1074/jbc.RA117.000871
DO - 10.1074/jbc.RA117.000871
M3 - Article
C2 - 29934309
AN - SCOPUS:85051712634
VL - 293
SP - 12663
EP - 12680
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 33
ER -