Grassystatins D-F, Potent Aspartic Protease Inhibitors from Marine Cyanobacteria as Potential Antimetastatic Agents Targeting Invasive Breast Cancer

Abstract

Three new modified peptides named grassystatins D-F (1-3) were discovered from a marine cyanobacterium from Guam. Their structures were elucidated using NMR spectroscopy and mass spectrometry. The hallmark structural feature in the peptides is a statine unit, which contributes to their aspartic protease inhibitory activity preferentially targeting cathepsins D and E. Grassystatin F (3) was the most potent analogue, with IC₅₀ values of 50 and 0.5 nM against cathepsins D and E, respectively. The acidic tumor microenvironment is known to increase the activation of some of the lysosomal proteases associated with tumor metastasis such as cathepsins. Because cathepsin D is a biomarker in aggressive forms of breast cancer and linked to poor prognosis, the effects of cathepsin D inhibition by 1 and 3 on the downstream cellular substrates cystatin C and PAI-1 were investigated. Furthermore, the functional relevance of targeting cathepsin D substrates was evaluated by examining the effect of 1 and 3 on the migration of MDA-MD-231 cells. Grassystatin F (3) inhibited the cleavage of cystatin C and PAI-1, the activities of their downstream targets cysteine cathepsins and tPA, and the migration of the highly aggressive triple negative breast cancer cells, phenocopying the effect of siRNA-mediated knockdown of cathepsin D.

Figure 1

Grassystatins D–F (1–3) isolated from the marine cyanobacterium VPG 14–61. The differences in structures 2 and 3 compared to 1 are highlighted.

Figure 2

Pepstatin A (4) with binding site nomenclature and structurally related cyanobacterial compounds. The structures are color coded to highlight the similarities and differences relative to grassystatins D–F (1–3).

Figure 3

Dose response curves for grassystatins D–F (1–3) and pepstatin A (4) against A) cathepsin D and B) cathepsin E. The dose-response is presented as % fold inhibition against solvent control (DMSO) and normalized IC₅₀ values are given.

Figure 4

The cellular inhibitory activities of grassystatins D and F (1 and 3) and pepstatin A (4) against cathepsin D/E proteases using cell lysates and intact live cells. A) MDA-MB-231 lysates were treated with grassystatins (1 and 3) or pepstatin A (4) in the presence of cathepsin D/E fluorogenic substrate. B) MDA-MB-231 cells were treated with pepstatin A (4), grassystatin D (1) or F (3) for 4 h, cells were lysed, and the protease inhibitory activity was analyzed. C) MDA-MB-231 cells were treated with pepstatin A (4) or grassystatin F (3) for 4 h, cells were trypsinized, collected by centrifugation then lysed, and the protease inhibitory activity was analyzed. D) MDA-MB-231 cells were seeded in 12-well plates in duplicate, after 24 h the medium was replaced with serum free medium buffered with 50 mM HEPES, pH 6.6 and incubated with either DMSO, pepstatin A (4), grassystatins D or F (1 or 3) for 3 days at 37 ºC. The activity of secreted cathepsins D/E was quantified by incubating the 3-day conditioned media (CM) in assay buffer (37 °C, pH 3.5) in the presence of fluorogenic substrate. The asterisks denote significance of P < 0.05 relative to solvent control using two-tailed unpaired t test (* denotes P ≤ 0.05, ** denotes P ≤ 0.01, *** denotes P ≤ 0.001, and **** denotes P ≤ 0.0001).

Figure 5

Docked structures of A) grassystatin F (3) and B) grassystatin D (1) in cathepsin D (1LYB) using Autodock Vina.

Figure 6

The secretion level of cathepsin D, PAI-1, and cystatin C in conditioned media (CM) prepared from breast cancer cells. 24 h serum free CM (pH 7.4) collected from four breast cancer cell lines (MCF7, MDA-MB-231, MDA-MB-436, and BT474) were centrifuged to remove detached cells, concentrated and analyzed by A) Western blot, B) PAI-1 ELISA, and C) Cys-C ELISA.

Figure 7

Cleavage of Cys-C and PAI-1 by cathepsin D in vitro. A) Incubation of recombinant human Cys-C with cathepsin D (enzyme to substrate ratio 1:5) in 100 mM NaCl, 100 mM sodium acetate (pH 4.0) at 37 °C for 3 h in the presence and absence of cathepsin D inhibitors (1 and 5 μM). B) Incubation of active PAI-1 with cathepsin D (enzyme to substrate ratio 1:5) in 100 mM NaCl, 100 mM sodium acetate (pH 4.0) at 37 °C for 3 h in the presence and absence of cathepsin D inhibitors (1 μM). Fragments were separated on SDS-PAGE under reducing conditions and silver stained.

Figure 8

The effect of cathepsin D inhibition on the degradation and activity of extracellular Cys-C and cysteine cathepsins, respectively. MDA-MB-231 cells were seeded in 12-well plates in duplicate, after 24 h the medium was replaced with serum free medium buffered with 50 mM HEPES, pH 7.4 or pH 6.6 and incubated with either DMSO or pepstatin A (4), grassystatins D and F (1 and 3) for 3 days at 37 ºC. A, B) The concentration of Cys-C in the 3-day CM was quantified by human Cys-C ELISA. C, D) The activity of cysteine cathepsins was quantified by incubating the 3-day CM in assay buffer (37 °C, pH 5.5) in the presence of fluorogenic substrate. The asterisks denote significance of P < 0.05 relative to solvent control using two-tailed unpaired t test (* denotes P ≤ 0.05, ** denotes P ≤ 0.01, *** denotes P ≤ 0.001, and **** denotes P ≤ 0.0001).

Figure 9

The effect of cathepsin D inhibition on the degradation and activity of extracellular PAI-1 and tPA respectively. MDA-MB-231 cells were seeded in 12-well plates in duplicate, after 24 h the medium was replaced with serum free medium buffered with 50 mM HEPES, pH 7.4 or pH 6.6 and incubated with either DMSO or pepstatin A (4), grassystatins D and F (1 and 3) for 3 days at 37 ºC. A, B) The concentration of PAI-1 in the 3-day CM was quantified by PAI-1 human ELISA kit. C, D) The activity of tPA was quantified by tPA human chromogenic activity assay. The asterisks denote significance of P < 0.05 relative to solvent control using two-tailed unpaired t test (* denotes P ≤ 0.05, ** denotes P ≤ 0.01, *** denotes P ≤ 0.001, and **** denotes P ≤ 0.0001).

Figure 10

The effect of cathepsin D inhibitors and siCTSD on the migration of MDA-MB-231 cells. A) MDA-MB-231 cells were incubated for 48 h in the presence of 5 μM cathepsin D inhibitors (grassystatins D and F (1 and 3) and pepstatin A (4)) and the effect was compared to the solvent control. B) MDA-MB-231 cells were incubated in the presence of 20 nM siCTSD and the effect was compared to the negative control. The graph represents number of migrated cells in each treatment group. The asterisks denote significance of P < 0.05 relative to solvent control using two-tailed unpaired t test (* denotes P ≤ 0.05, ** denotes P ≤ 0.01, *** denotes P ≤ 0.001, and **** denotes P ≤ 0.0001).

Figure 11

Proposed mechanisms by which cathepsin D contributes to tumor aggressiveness and metastasis via its effect on PAI-1 and Cys-C.