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

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.