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

Abstract

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 (K_i = 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.

Figure 1

Structures of the selected cathepsin X inhibitors.

Figure 2

Selected inhibitors bind reversibly to cathepsin X. (a) Cathepsin X activity was measured immediately (dark grey) after 1-hour pre-incubation with selected compounds, or after 200000-fold dilution in activation buffer using a Centricon (10 kDa cut-off; Millipore) for washing out the compounds (light grey). After removing the compounds almost all cathepsin X activity was regained according to the control (DMSO) except in the case of irreversible inhibitor E-64. Values are means of two independent experiments, performed in duplicate ± S.E.M. (b) After rapid 100-fold dilution of (pre-incubated) cathepsin X (100-fold concentration) and compound (10-fold concentration) cathepsin X activity was measured. 90% of the recovered cathepsin X activity points toward reversible inhibitors with rapid dissociation. Inset: graph representation of activity measurements - control (green), E-64 (blue) and tested compounds (other colors).

Figure 3

Kinetic evaluation of cathepsin X inhibitors. (a) Progress curves of the product formation by cathepsin X in the absence and presence of compound 22 at different concentrations of the compound (0 – 200 µM). (b) Reaction scheme describing the inhibition mechanism for the second group of compounds. (c) The system of differential equations for the reaction mechanism for the second group of inhibitors. k_cat, K_m, k_activ, k_on and k_off are the kinetic constants, A is acylated substrate.

Figure 4

The new inhibitors affect cell processes associated with cathepsin X activity. (a) The level of PC-3 migration was assessed by increases in the curve slopes (1/h) that represent cell migration ability. Slopes were calculated from cell index values in the chosen time interval. Results are normalized to cells treated with DMSO (the control) with 100% migration ability. Cells treated with AMS36 served as a positive control. Right panel: representative migration curves of control and inhibitor 22 treated cells. The time interval used for calculating slopes is marked with blue lines. Assays were carried out three times, each in triplicate. Results are means ± S.E.M. *P ≤ 0.01 (T test). (b) The level of neurite outgrowth was determined after 48 hours of treatment with inhibitors. Results are expressed relative to the cell count and the control group (DMSO) was considered as 100% of neurite outgrowth. Values are means of two independent experiments, each performed in quadruplicate ± S.E.M. *P < 0.014 (T test). (c) Representative images of increased actin polymerization in the presence of cathepsin X inhibitors are shown using fluorescence microscopy and staining with TRITC-conjugated phalloidin. White arrows indicate rapid formation of neurites after 2-hour presence of cathepsin X inhibitors. Bars, 10 μm.