Kempopeptin C, a Novel Marine-Derived Serine Protease Inhibitor Targeting Invasive Breast Cancer

Abstract

Kempopeptin C, a novel chlorinated analogue of kempopeptin B, was discovered from a marine cyanobacterium collected from Kemp Channel in Florida. The structure was elucidated using NMR spectroscopy and mass spectrometry (MS). The presence of the basic Lys residue adjacent to the N-terminus of the 3-amino-6-hydroxy-2-piperidone (Ahp) moiety contributed to its selectivity towards trypsin and related proteases. The antiproteolytic activity of kempopeptin C was evaluated against trypsin, plasmin and matriptase and found to inhibit these enzymes with IC₅₀ values of 0.19, 0.36 and 0.28 μM, respectively. Due to the significance of these proteases in cancer progression and metastasis, as well as their functional redundancy with respect to targeting overlapping substrates, we examined the effect of kempopeptin C on the downstream cellular substrates of matriptase: CDCP1 and desmoglein-2 (Dsg-2). Kempopeptin C was shown to inhibit the cleavage of both substrates in vitro. Additionally, kempopeptin C reduced the cleavage of CDCP1 in MDA-MB-231 cells up to 10 µM. The functional relevance of targeting matriptase and related proteases was investigated by assessing the effect of kempopeptin C on the migration of breast cancer cells. Kempopeptin C inhibited the migration of the invasive MDA-MB-231 cells by 37 and 60% at 10 and 20 µM, respectively.

Keywords: breast cancer; marine cyanobacteria; migration; serine proteases.

Figure 1

Kempopeptins A‒C (1‒3) and structurally-related 3-amino-6-hydroxy-2-piperidone (Ahp) containing cyclic depsipeptides bearing a basic residue (4‒6). The differences in the structures of 3‒6 compared to kempopeptin B (2) are highlighted.

Figure 2

Dose-response curves for kempopeptins B and C (2 and 3) against serine proteases. (A) Trypsin; (B) plasmin; and (C) matriptase. The dose-response is presented as % fold inhibition against solvent control (DMSO). Data are presented as the mean ± SD, n = 3.

Figure 3

Docked structures of kempopeptin B (pink) and kempopeptin C (green) in matriptase. (A) The atomic sizes of the Br (red) and Cl (green) are displayed as spheres; (B) the residues of the matriptase enzyme in close proximity to Br (Trp) and Cl (Phe and Thr) are displayed in stick representation (cyan).

Figure 4

The effect of kempopeptin C (3) on the in vitro cleavage of CDCP1 and desmoglein-2 (Dsg-2) by matriptase. (A,B) Incubation of recombinant human CDCP1 with recombinant human matriptase (enzyme to substrate ratio 1:5) in 50 mM Tris, 50 mM NaCl, 0.01% Tween 20 (pH 9.0) at 37 °C for 30 min and 2 h in the presence and absence of different concentrations (10‒0.01 µM) of kempopeptin C (3). (C) Incubation of recombinant human Dsg-2 with recombinant human matriptase (enzyme to substrate ratio 1:5) in 50 mM Tris, 50 mM NaCl, 0.01% tween 20 (pH 9.0) at 37 °C for 3 h in the presence and absence of different concentrations (10‒0.01 µM) of kempopeptin C (3). Fragments were separated on SDS-PAGE under reducing conditions and silver stained. (D) Densitometric analysis of Dsg-2 bands in the SDS-PAGE in Figure 3C.

Figure 5

The expression level of Dsg-2 and CDCP1 in protein lysates collected from different breast cancer cell lines. The expression levels were assessed by Western blot probed with anti-Dsg-2 and anti-CDCP1 antibodies. Actin was used as the loading control.

Figure 6

The effect of kempopeptins B and C (2 and 3) on CDCP1 cleavage in MDA-MB-231. (A) MDA-MB-231 cells were treated for 72 h with different concentrations of the test compounds. Following 72 h, the lysates were collected and analyzed by Western blot. (B) MDA-MB-231 cells were treated at different time points with 100 µM of kempopeptin C (3). Following 6 h, 12 h and 24 h, the lysates were collected and analyzed by Western blot. Dexamethasone (Dex) was used as a positive control. (C) MDA-MB-231 cells treated for 24 h with different concentrations of 3 in the presence of 10 nM recombinant human matriptase. Cell lysates were harvested and analyzed by Western blot. (D) Densitometric analysis of the full-length, LMW of CDCP1 and actin bands in (C).

Figure 7

The effect of kempopeptin C (3) on the migration of MDA-MB-231. MDA-MB-231 were incubated for 48 h in the presence of different concentrations (0, 10 or 20 µM) of 3 and the effect was compared to the solvent control. The graph represents the number of migrated cells in each treatment group. The asterisks denote the significance of p ≤ 0.01 relative to the solvent control using the two-tailed unpaired t-test. Data are presented as the mean ± SD, n = 3.