Identification and characterization of the novel reversible and selective cathepsin X inhibitors

Cathepsin X is a cysteine peptidase involved in the progression of cancer and neurodegenerative diseases. Targeting this enzyme with selective inhibitors opens a new possibility for intervention in several therapeutic areas. In this study triazole-based reversible and selective inhibitors of cathepsin X have been identified. Their selectivity and binding is enhanced when the 2,3-dihydrobenzo[b][1,4]dioxine moiety is present as the R1 substituent. Of a series of selected triazole-benzodioxine derivatives, compound 22 is the most potent inhibitor of cathepsin X carboxypeptidase activity (Ki = 2.45 ± 0.05 μM) with at least 100-fold greater selectivity in comparison to cathepsin B or other related cysteine peptidases. Compound 22 is not cytotoxic to prostate cancer cells PC-3 or pheochromocytoma PC-12 cells at concentrations up to 10 μM. It significantly inhibits the migration of tumor cells and increases the outgrowth of neurites, both processes being under the control of cathepsin X carboxypeptidase activity. Compound 22 and other characterized triazole-based inhibitors thus possess a great potential for further development resulting in several in vivo applications.


MS/MS analysis
PC-3 cells were treated for 1 hour with compound 22 (50 µM). Cells were washed twice with cold PBS, trypsinized, washed once more and centrifuged. A pellet of cells was resuspended in 200 µL of PBS (or in 200 µL of PBS containing 5µM inhibitor for standard addition) and transferred to 1.5 mL Eppendorf vials prefilled with 0.1 mm and 0.5 mm glass beads (NextAdvance, USA). All cell samples were then homogenized with a BulletBlender (NextAdvance, USA) for 5 min at speed setting »6«. The homogenates were mixed with triple volume of ice-cold acetonitrile containing 1% formic acid and stored at -20 °C for 18 hours. The same protein precipitation procedure was applied to 300 µL samples of media sampled before and after the cellular uptake experiment. After centrifugation at 15000×g for 10 min at 4 °C the supernatants were transferred to vials for analysis by an Agilent 1290 Infinity LC coupled to an Agilent 6460 triple quadrupole mass spectrometer  Supplementary Table S4.

Labeling of cathepsin X with DCG-04
PC-3 cell lysate was diluted in cathepsin X assay buffer (1 mg/ml protein concentration). Inhibitor was added (10 µM) and after 10 minutes at room temperature biotinylated probe DCG-04 was added (10 µM). Reaction was stopped at several time points by boiling the sample in SDS-loading buffer for 5 minutes 1 . Proteins were separated on 12% Tris-glycine gel and transferred to a Hybond-N nitrocellulose membrane using iBlot 2 dry blotting system (Thermo Scientific). After blocking with 5% skimmed milk powder in TBS with 0.05% Tween 20, membrane was incubated with streptavidin-HRP (1:5000; Jackson ImmunoResearch) for 1 hour and proteins were detected with GeneSnap software (Syngene) using SuperSignal West Dura Extended Duration Substrate chemiluminescence kit (Thermo Scientific).

Supplementary Figures
Supplementary Figure S1. Relative inhibition of cathepsin X.

Supplementary Figure S3. Alternative setting of migration assay using cell invasion and migration (CIM) plates which excludes measurement of cell adhesion shows that compound 22 inhibits migration of PC-3 cells.
Since cathepsin X participates also in cell adhesion another setting of migration assay was performed using CIM plates. No inhibitor was added to the complete media of the lower compartments whereas the inhibitor was added to the serum-free media of the upper compartments Cell lysate was pre-treated with AMS36 (a) or compound 22 (b) and then labeled with biotinylated activity-based probe DCG-04. DMSO was used as a control. The reaction was stopped at different time points and labeling determined with SDS-PAGE and streptavidin-HRP western blot. a) Cathepsin X was strongly inhibited by irreversible inhibitor AMS36 through-out the experiment.
AMS36 also inhibited cathepsin B. b) Cathepsin X labeling was delayed for approximately 20 minutes with compound 22 as is typical for reversible inhibitors. A weak inhibition of cathepsin B was also observed, but to a lesser extent as with AMS36. A representative image of two performed experiments is shown. Due to the chemiluminescent detection, size marker (SeeBlue® Pre-stained Protein Standard, Life Technologies) is added as a separate strip.

X inhibitors.
Theoretical partition coefficient was calculated using ChemDraw software (PerkinElmer) and expressed as logP which represents theoretical prediction of the hydrophilic/lipophilic properties of the compounds. From the obtained values for membrane non-permeable inhibitor E64, its membrane permeable analogue E64d and our compounds, we can predict good membrane permeability for the selected inhibitors.