Identification and pre-clinical testing of a reversible cathepsin protease inhibitor reveals anti-tumor efficacy in a pancreatic cancer model

Abstract

Proteolytic activity is required for several key processes in cancer development and progression, including tumor growth, invasion and metastasis. Accordingly, high levels of protease expression and activity have been found to correlate with malignant progression and poor patient prognosis in a wide variety of human cancers. Members of the papain family of cysteine cathepsins are among the protease classes that have been functionally implicated in cancer. Therefore, the discovery of effective cathepsin inhibitors has considerable potential for anti-cancer therapy. In this study we describe the identification of a novel, reversible cathepsin inhibitor, VBY-825, which has high potency against cathepsins B, L, S and V. VBY-825 was tested in a pre-clinical model of pancreatic islet cancer and found to significantly decrease tumor burden and tumor number. Thus, the identification of VBY-825 as a new and effective anti-tumor drug encourages the therapeutic application of cathepsin inhibitors in cancer.

Figure 1. Identification and characterization of the selective cathepsin inhibitor VBY-825

(A) Chemical structure of VBY-825. (B) Analysis of VBY-825 inhibition on cathepsin B and L activity in intact HUVECs using a radioiodinated diazomethylketone-Tyr-Ala (¹²⁵I-DMK) activity-based probe. Cells were incubated with varying concentrations of VBY-825 followed by a short incubation with ¹²⁵I-DMK, and IC₅₀ values of inhibition were determined. The IC₅₀ values for inhibition of the two heavy chain isoforms of cellular cathepsin L were 0.5 nM and 3.3 nM. The IC₅₀ value for inhibition of cathepsin B was 4.3 nM. (C) Iip10 accumulation demonstrating cathepsin S inhibition in spleen, and VBY-825 plasma concentrations in mice following 3 days of subcutaneous QD administration of VBY-825 at 10 mg/kg. Times indicated are timepoints following the third dose, and plasma levels indicate the average plasma concentration of VBY-825 (nM) in plasma from three mice at each time point. Veh=D5W vehicle.

Figure 2. VBY-825 treatment reduces tumor incidence and tumor growth in a RT2 intervention trial

(A) Tumor number was decreased by 33% in VBY-825 treated RT2 mice compared to the D5W vehicle control-treated cohort, * P<0.05. Mice were treated with D5W or VBY-825 (10 mg/kg/day) once daily by subcutaneous injection from 10-13.5 weeks of age. D5W: n=18 mice, VBY-825: n=21 mice. (B) The cumulative tumor volume at the 13.5 week trial endpoint was decreased by 52% in VBY-825 treated RT2 mice compared to the D5W vehicle control-treated group, ** P<0.01.

Figure 3. Effects of VBY-825 treatment on the balance between cell proliferation and apoptosis in RT2 tumors

(A) Proliferating cells were detected by Ki67 staining (brown) and representative images are shown for D5W-treated tumors (upper panel) and VBY-825 treated tumors (lower panel) at the 13.5 week trial endpoint. Scale bar = 50 μm. Graph on right shows the percentage of proliferating cells (Ki67 positive) over the total number of hematoxylin-positive cells in the tumor. VBY-825 treatment resulted in a 25% decrease in cell proliferation. (B) Apoptotic cells were detected by cleaved caspase 3 staining (red) and representative images are shown for D5W-treated tumors (upper panel) and VBY-825 treated tumors (lower panel) at the 13.5 week trial endpoint. Scale bar = 50 μm. Graph on right shows the percentage of apoptotic cells (cleaved caspase 3 positive) over the total number of DAPI-positive cells in the tumor. VBY-825 treatment resulted in a 42% increase in apoptosis. The means and SEM are indicated in both graphs, n=5 mice per group.

Figure 4. VBY-825 treatment does not affect RT2 tumor invasion or angiogenesis at the 13.5 week trial endpoint

(A) Graph showing the relative proportions of encapsulated, microinvasive, and highly invasive carcinomas in 13.5 week D5W controls compared to VBY-825 treated RT2 mice. VBY-825 treatment did not significantly alter the distribution of tumor grades compared to D5W-treated mice, n=15 mice per group. (B, C) The tumor vasculature was analyzed by perfusion of mice with Texas Red-dextran and by staining with an endothelial cell specific antibody (CD31) (n=2-8 mice per group). The percentage of CD31-positive blood vessels over the total tumor area (DAPI-positive) is graphed in (B), showing no difference in vessel coverage between the D5W-and VBY-825 treated tumors. The percentage of dextran-covered area over the total tumor area (DAPI-positive) is graphed in (C), showing no difference in vessel leakiness (as assessed by dextran diffusion from blood vessels) between the D5W-and VBY-825 treated tumors.