Extended Data Fig. 2: Negative controls for OI-RD measurements and validation of the compounds’ interaction with HTT and LC3 by MST. | Nature

Extended Data Fig. 2: Negative controls for OI-RD measurements and validation of the compounds’ interaction with HTT and LC3 by MST.

From: Allele-selective lowering of mutant HTT protein by HTT–LC3 linker compounds

Extended Data Fig. 2

a, Similar to Fig. 1c–e, but for negative control proteins MBP–His8 (MBP), sfGFP and Rpn10 (Rpn10). Association–dissociation curves of surface immobilized compounds 8F20 and 10O5 with these proteins were measured by OI-RD, and no compound–protein interactions were detected. For all association–dissociation curves, vertical dashed lines mark the starts of association and dissociation phases of the binding event. b, Binding of 10O5 and 8F20 to full-length HTT(Q73) (flHTT(Q73), black dots) or LC3B (red dots) in standard treated capillaries measured by MST. The compound-bound protein fractions (bound/total) were calculated from the MST signals (Fnorm) at each compound concentration, as well as the bound (Fnorm_bound, set as 100%) and the unbound (Fnorm_unbound, set as 0%) MST signals: bound/total = (Fnorm – Fnorm_unbound)/(Fnorm_bound – Fnorm_unbound) × 100%. The fitted curves and Kd values calculated by Nanotemper analysis software (v.1.5.41) for flHTT(Q73) and LC3B are indicated in each panel. Consistent with the OI-RD measurements (Fig. 1e), no binding was observed for the flHTT(Q23) protein (blue dots). The MST experiments were repeated more than three times and showed consistent results. c, Similar to b, except using the compounds indicated on the x axis. MST measurements of the binding of indicated compounds to full-length HTT(Q73) (flHTT-Q73), full-length HTT(Q23) (flHTT-Q23) and LC3B in standard treated capillaries. The proteins tested are indicated in the legends. d, Similar to Fig. 1c–e, but plotting the association–dissociation curves of surface immobilized compound AN2 with full-length HTT(Q73) (Q73), or full-length HTT(Q23) (Q23), LC3B or the negative-control proteins MBP–His8 (MBP), sfGFP and Rpn10. For all association–dissociation curves, vertical dashed lines mark the starts of association and dissociation phases of the binding event. The red dashed lines are global fits to a Langmuir reaction model with the global fitting parameters listed at the bottom of each plot. No binding signals were observed for full-length HTT(Q23) proteins, and thus the parameters were not presented. e, Cell viability measurement of cultured HD neurons measured by the CellTiter-glo assay. No toxicity was observed within the concentration range presented in Fig. 2, although the compound 8F20 became toxic to the cells when the concentration reached 300 nM.

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