biostudies-literatureWang HYNCI NIH HHSNIGMS NIH HHS1443-1450https://www.ebi.ac.uk/biostudies/studies/S-EPMC10018702biostudies-literatureUnknown14(12)Ternatin-family cyclic peptides inhibit protein synthesis by targeting the eukaryotic elongation factor-1α. A potentially related cytotoxic natural product ('A3') was isolated from Aspergillus, but only 4 of its 11 stereocentres 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 an enhanced residence time and rebinding kinetics, as revealed by single-molecule fluorescence imaging of elongation reactions catalysed by eukaryotic elongation factor-1α in vitro. An increased residence time-stereospecifically conferred by the unique β-hydroxyl in SR-A3-was also observed in cells. Consistent with its prolonged duration of action, thrice-weekNature chemistrySynthesis and single-molecule imaging reveal stereospecific enhancement of binding kinetics by the antitumour eEF1A antagonist SR-A3.PMC10018702R35 CA242986T32 GM145460R01 GM079238R01 GM098859Yang HWeber JFAl-Khdhairawi AAQBlanchard SCOltion KTom HTaunton JRuggero DHolm MWang HYfalseSynthesis and single-molecule imaging reveal stereospecific enhancement of binding kinetics by the antitumour eEF1A antagonist SR-A3.Ternatin-family cyclic peptides inhibit protein synthesis by targeting the eukaryotic elongation factor-1α. A potentially related cytotoxic natural product ('A3') was isolated from Aspergillus, but only 4 of its 11 stereocentres 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 an enhanced residence time and rebinding kinetics, as revealed by single-molecule fluorescence imaging of elongation reactions catalysed by eukaryotic elongation factor-1α in vitro. An increased residence time-stereospecifically conferred by the unique β-hydroxyl in SR-A3-was also observed in cells. Consistent with its prolonged duration of action, thrice-week2022-01-01T00:00:00Z2022 Dec2025-05-29T22:11:49.114Z2025-05-29T22:11:49.114ZS-EPMC100187023612344910.1038/s41557-022-01039-3