Fig. 6: Structure-informed screen identifies cryptic binders of env8 divorced from Cbl.
From: Designing small molecules targeting a cryptic RNA binding site through base displacement
a, Chemical structure of the eight hits that emerged from our structure-informed docking, with their biphenyl-like scaffold highlighted in gray. b, TO-based displacement assay to identify env8-binding compounds that induce fluorescence attenuation. This was used as an orthogonal binding experiment because we observed ligand-induced fluorescence induction of CNCbl–5×PEG-ATTO590. c, MST trace of fluorescently labeled env8 alone and in the presence of CNCbl, 68 and 71. All ligands induced an observable change to the MST trace of env8, suggestive of binding. d, TO displacement binding curve for CNCbl, 68 and 71 shown as the mean and s.d. from independent experiments (n = 3). KD values are reported as the mean and s.d. from independent experiments (n = 3). e, Native polyacrylamide gel showing a gradual shift from a slower-migrating, extended conformation to a faster-migrating, kissing-loop conformation of env8 upon titration of CNCbl (0.01–10 µM). Data are shown from an individual experiment. f, Same as e but after addition of CNCbl, 68 and/or 71 to demonstrate competitive binding of 68 and 71. Similar results were obtained from repeated experiments (n = 8 for CNCbl, n = 3 for 68 and n = 2 for 71). g, MST trace from a competition experiment where env8–CNCbl was monitored with and without the addition of excess amounts of 68 or 71. Both ligands induced an observable change to the MST trace of env8–CNCbl, suggestive of competitive binding. All MST data are shown as representative traces from multiple independent experiments (n = 4). h, Top-ranked docking poses of 68 and 71 to env2 in the A20-out and A20-in states.
