Fig. 5: Structure-based design of lead compounds with affinity exceeding the native ligand.
From: Designing small molecules targeting a cryptic RNA binding site through base displacement
a, Modified click reaction25 to make Cbl derivatives from 58 using a variable azide. b, Simplified chemical structures of Cbls 63–65 that were made to systematically test our design hypotheses. c, Fluorescence displacement binding curve for CNCbl and 63–65 shown as the mean and s.e.m. from independent experiments (n = 4, except for data from CNCbl, where n = 3). KD values are reported as the mean and s.d. from independent experiments (n = 4, except for data from CNCbl, where n = 3). d, To verify the near-picomolar binding of 63 and 64, we used ITC with the env8 aptamer domain. Given that this RNA shows weaker ligand binding than the full-length env8 (ref. 19), it is in the perfect regime for ITC. Representative ITC binding isotherm for the env8 aptamer domain with CNCbl, 63 and 64. e, Cocrystal structure of env2–Cbl 63 showing only the binding-pocket nucleotides and the β-axial group. Proposed hydrogen bonds, which are weakly supported by the electron density, are shown as double arrows. All mesh representations correspond to a simulated annealing 2Fo − Fc map where A20 and the ligand were omitted from the model and are shown at 1σ contour. f, Same as e but for env2–Cbl 64. g, Fold repression for our reporter system in the presence of CNCbl and 63–65 and for cells with CNCbl and excess amounts of 63–65 added. The mean and s.d. from biological replicates (n = 4) are shown. For comparison, the mean fold repression of 10 nM CNCbl is shown as a dashed line and its s.d. is represented as a shaded box. h, Simplified chemical structure of the titratable pyridine Cbl 66. i, Representative ITC binding isotherms for env8 aptamer domain with CNCbl, 29 and 66 at pH 8 and pH 5. For all ITC data, KD values are reported as the mean and s.d. from independent experiments (n = 3).
