Structure-activity-relationship study of semi-synthetically modified fusicoccins on their stabilization effect for 14-3-3-phospholigand interactions.

The diterpene glucoside fusicoccin-A (FC-A) is a fungal phytotoxin that stabilizes the interaction of plant 14-3-3 protein and plasma membrane H+-ATPase by forming a stable ternary complex. Previous studies demonstrated that structurally modified FC-A derivatives exhibit significant antitumor activities but their synthesis involves an explosive reagent, limiting their utility and opportunities for further structure-activity-relationship studies. In this study, we synthesized a series of FC derivatives by introducing various substituents on the fusicoccan scaffold and on the glucoside moiety, and evaluated their stabilization effects on the binding of 14-3-3 to fluorescently labeled mode-1 and mode-3 phosphopeptides. The results showed that introducing an amino group at the 6'-position of the glucoside moiety improves stabilization. Furthermore, cell-based evaluation demonstrated that 6'-amino benzyl 21b exhibits higher antiproliferative activity than previously developed FC agents.

Copper-Free Huisgen Cycloaddition for the 14-3-3-Templated Synthesis of Fusicoccin-Peptide Conjugates.

Mid-sized molecules have emerged as an attractive chemical space and potentially provide a robust basis for the development of synthetic agents to control intracellular protein interactions. However, the limited cell permeability and chemical tractability of such agents remain to be addressed. We envisioned that target-templated synthesis of such mid-sized molecules might provide a solution. Here, we exploited a copper-free Huisgen cycloaddition for template synthesis using a peptide fragment containing a 4,8-diazacyclononyne (DACN) moiety and an azide-containing fusicoccin derivative in the presence or absence of recombinant 14-3-3ζ protein in vitro. Time-course changes in the yield of products demonstrated that the reaction was accelerated in the presence of 14-3-3 and one of the regioisomers was generated predominantly, supporting the template effect.

Structural Effects of Fusicoccin upon Upregulation of 14-3-3-Phospholigand Interaction and Cytotoxic Activity.

Fusicoccins (FCs) exhibit various cellular activities in mammalian cells, but details of the mechanism of action are not fully understood. In this study, we synthesized two pairs of model derivatives of FCs differing only in the presence and absence of a 12-hydroxyl group and evaluated their binding to a 14-3-3 protein together with various mode 1 and mode 3 phosphopeptide ligands. Our results demonstrate that the 12-hydroxyl group hampers binding to 14-3-3 with mode 1 phospholigands, presumably due to steric repulsion with the i+2 residue. Furthermore, cell-based evaluations showed that only non-substituted FCs exhibit significant cytotoxicity and all 12-hydroxyl derivatives were inactive, demonstrating a clear correlation with their ability to form ternary complexes with 14-3-3 and a mode 1 ligand. These results suggest that binding to 14-3-3 and a partner protein(s) possessing a mode 1 sequence plays a role in the mechanism of action of 12-non-substituted FCs.