Synthesis and single-molecule imaging reveal stereospecific enhancement of binding kinetics by the antitumor eEF1A antagonist SR-A3

Ternatin-family cyclic peptides inhibit protein synthesis by targeting the eukaryotic elongation factor-1 (eEF1A). A potentially related cytotoxic natural product ("A3") was isolated from Aspergillus, but only 4 of its 11 stereocenters could be assigned. Here, we synthesized SR-A3 and SS-A3 - two out of 128 possible A3 epimers - and discovered that synthetic SR-A3 is indistinguishable from naturally derived A3. Relative to SS-A3, SR-A3 exhibits enhanced residence time and rebinding kinetics, as revealed by single-molecule fluorescence imaging of elongation reactions catalyzed by eEF1A in vitro. Increased residence time - stereospecifically conferred by the unique {beta}-hydroxyl in SR-A3 - was also observed in cells. Consistent with its prolonged duration of action, thrice-weekly dosing with SR-A3 led to dramatically increased survival in an aggressive Myc-driven mouse lymphoma model. Our results demonstrate the potential of SR-A3 as a cancer therapeutic and exemplify an evolutionary mechanism for enhancing cyclic peptide binding kinetics via stereospecific side-chain hydroxylation.