Grassystatins A-C from marine cyanobacteria, potent cathepsin E inhibitors that reduce antigen presentation

Abstract

In our efforts to explore marine cyanobacteria as a source of novel bioactive compounds, we discovered a statine unit-containing linear decadepsipeptide, grassystatin A (1), which we screened against a diverse set of 59 proteases. We describe the structure determination of 1 and two natural analogues, grassystatins B (2) and C (3), using NMR, MS, and chiral HPLC techniques. Compound 1 selectively inhibited cathepsins D and E with IC(50)s of 26.5 nM and 886 pM, respectively. Compound 2 showed similar potency and selectivity against cathepsins D and E (IC(50)s of 7.27 nM and 354 pM, respectively), whereas the truncated peptide analogue grassystatin C (3), which consists of two fewer residues than 1 and 2, was less potent against both but still selective for cathepsin E. The selectivity of compounds 1-3 for cathepsin E over D (20-38-fold) suggests that these natural products may be useful tools to probe cathepsin E function. We investigated the structural basis of this selectivity using molecular docking. We also show that 1 can reduce antigen presentation by dendritic cells, a process thought to rely on cathepsin E.

Figure 1

Structures of grassystatins A–C (1–3).

Figure 2

Structures of pepstatin A (including binding site nomenclature), tasiamide and tasiamide B.

Figure 3

ESIMS fragmentation pattern of grassystatins A (1) and B (2).

Figure 4

ESIMS fragmentation pattern for grassystatin C (3).

Figure 5

Protease screen treated with grassystatin A (1), 10 μM. Values represent % enzyme activity compared to solvent control, and additionally represented by a continuous color scale (0% red, 100% green).

Figure 6

Dose-response curves for grassystatin A (1) against A) cathepsins D and E, and B) ADAM9, ADAM 10 and TACE. % Activity figures are derived for initial reaction slope for A) and end-slope for B), due to apparent time-dependent inhibition (see Figure S1, Supporting Information).

Figure 7

Activities of grassystatin A (1) and pepstatin A against MCF7 cellular proteases as determined with a cathepsin D/E substrate (see text). A) MCF7 cell lysate was directly treated with grassystatin A (1) or pepstatin A. B) MCF7 cells were treated with grassystatin A (1) or pepstatin A for 1 h. Cells were lysed and the protease activity of the lysates assessed. *Denotes significance of P < 0.05 using a two-tailed t test. Data points are shown ± SD.

Figure 8

Downregulation of antigen presentation of T cells and T_H cells after treatment with grassystatin A (1) on activated PBMC and DC. A) Downregulation of the activation of CD3⁺ T cells on whole PBMC after treatment with different concentrations of 1. B) Downregulation of T_H activation (proliferation) by the addition of different concentrations of 1. C, D) Effect of 1 on the production of intracellular IFNγ (C) and IL-17 (D) by T_H cells induced by autologous activated DC. “Ctrl” refers to T cells that did not have DCs added to them. *Denotes significance of P < 0.05 using a two-tailed t test. Data points are shown ± SEM.

Figure 9

Effect of 1 on the production of A) intracellular IFNγ and B) IL-17 by T_H cells induced by allogeneic activated DC in an MLR. “Ctrl” refers to T cells that did not have DCs added to them. *Denotes significance of P < 0.05 using a two-tailed t test. Data points are shown ± SEM.

Figure 10

Docked structures of grassystatins A (1) and C (3) with cathepsins D and E. For each the protein is shown in green, and possible hydrogen bonds are shown as dotted yellow lines. A) Docked conformation of grassystatin A (1, yellow) with cathepsin D. B) Docked conformation of grassystatin A (1) with cathepsin E. C) Docked conformation of grassystatin C (3, pink) with cathepsin D. D) Docked conformation of grassystatin C (3) with cathepsin E. For docked conformations of pepstatin A, see Figure S2, Supporting Information.