Grassystatin G, a new cathepsin D inhibitor from marine cyanobacteria: discovery, synthesis, and biological characterization.

Abstract

Through ongoing investigations of marine cyanobacteria, a prolific source of structurally diverse secondary metabolites, we isolated grassystatin G (1), a new statine-containing linear peptide, closely related to the cathepsin E (CatE) inhibitors grassystatins A-F, some of which may function as CatE probes. The planar structure of 1 was determined by analysis of 1D, 2D NMR and MS/MS fragmentation data, and is structurally distinct from its analogs by being shorter and containing a hydrophobic residue (Val) adjacent to the statine unit instead of a polar residue (Asn, Gln). We employed chiral HPLC analysis and modified Marfey's method to assign the absolute configuration of constituent amino acids, suggesting the presence of N-Me-l-Phe instead of N-Me-d-Phe in other grassystatins. To prove the structure and overcome the lack of material for further biological studies and mechanistic characterization, we developed a 3 + 3 convergent synthesis and have accessed the peptide with an overall yield of 19% using standard peptide coupling. As the statine moiety is a known pharmacophore with an inhibitory effect against aspartic proteases, we screened grassystatin G against a panel of human and virus aspartic proteases. In contrast to grassystatins A-F, preferentially targeting CatE over CatD with 18-66-fold selectivity, grassystatin G displayed 2-fold selectivity for CatD over CatE, suggesting that the key structural differences may be exploited for CatD probe design. Docking and molecular dynamics provided insights into the structural features responsible for the selectivity towards CatD. CatD is well-documented to play a role in cancer proliferation and metastasis, particularly in the context of breast cancer. We tested grassystatin G against MDA-MB-231 triple-negative breast cancer cells and demonstrated its cooperative effects with TRAIL. RNA-seq highlighted the potential pathways and molecular mechanisms governed by grassystatin G alone and in combination with TRAIL.