Abstract
Pancreatic cancer has the lowest survival rate of all common cancers due to late diagnosis and limited treatment options. Serine hydrolases are known to mediate cancer progression and metastasis through initiation of signaling cascades and cleavage of extracellular matrix proteins, and the kallikrein-related peptidase (KLK) family of secreted serine proteases have emerging roles in pancreatic ductal adenocarcinoma (PDAC). However, the lack of reliable activity-based probes (ABPs) to profile KLK activity has hindered progress in validation of these enzymes as potential targets or biomarkers. Here, we developed potent and selective ABPs for KLK6 by using a positional scanning combinatorial substrate library and characterized their binding mode and interactions by X-ray crystallography. The optimized KLK6 probe IMP-2352 (kobs/I = 11,000 M-1 s-1) enabled selective detection of KLK6 activity in a variety of PDAC cell lines, and we observed that KLK6 inhibition reduced the invasiveness of PDAC cells that secrete active KLK6. KLK6 inhibitors were combined with N-terminomics to identify potential secreted protein substrates of KLK6 in PDAC cells, providing insights into KLK6-mediated invasion pathways. These novel KLK6 ABPs offer a toolset to validate KLK6 and associated signaling partners as targets or biomarkers across a range of diseases.
| Original language | English |
|---|---|
| Pages (from-to) | 22493-22504 |
| Number of pages | 12 |
| Journal | Journal of the American Chemical Society |
| Volume | 144 |
| Issue number | 49 |
| Early online date | 22 Nov 2022 |
| DOIs | |
| Publication status | Published - 14 Dec 2022 |
Bibliographical note
The authors thank Cancer Research UK (grants C24523/A25192, C29637/A20183, and C29637/A21451), the EPSRC Centre for Doctoral Training in Physical Sciences Innovation in Chemical Biology for Bioindustry and Healthcare (grant EP/LO15498/1) for financial support, the European Union Horizon 2020 Program (MSCA-IF fellowship 890900 to E.D.V.), Worldwide Cancer Research (grant 19-0059), and the EPSRC for a Doctoral Prize fellowship to S.L. The authors thank Dr. Lisa Haigh (Department of Chemistry Mass Spectrometry Facility, Imperial College London) for assistance in acquiring high-resolution mass spectrometry (HRMS) and LC–MS/MS Orbitrap data. BioRender.com was used in the generation of Figures 1, 5B and 6. Molecular graphics performed with UCSF Chimera, developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco, with support from NIH P41-GM103311.Funding Information:
The authors thank Cancer Research UK (grants C24523/A25192, C29637/A20183, and C29637/A21451), the EPSRC Centre for Doctoral Training in Physical Sciences Innovation in Chemical Biology for Bioindustry and Healthcare (grant EP/LO15498/1) for financial support, the European Union Horizon 2020 Program (MSCA-IF fellowship 890900 to E.D.V.), Worldwide Cancer Research (grant 19-0059), and the EPSRC for a Doctoral Prize fellowship to S.L. The authors thank Dr. Lisa Haigh (Department of Chemistry Mass Spectrometry Facility, Imperial College London) for assistance in acquiring high-resolution mass spectrometry (HRMS) and LC–MS/MS Orbitrap data. BioRender.com was used in the generation of , B and . Molecular graphics performed with UCSF Chimera, developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco, with support from NIH P41-GM103311.
Data Availability Statement
The crystal structures have been submitted to the Protein Data Bank under accession code 7QFT and 7QFV. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifier PXD035111.
