Fig. 4
Thermal stability, affinity determination, and ligand epitope mapping of hit compounds M23, M27, and M10. (A–B) Thermal unfolding of apo NHR2 and in complex with a 10-fold excess of the compounds were studied by nanoDSF ((A) Fluorescence intensity ratio at 350 nm / 330 nm; shown are representatives of unfolding events. (B) First derivative of the curves in (A)). Changes in the 350 nm / 330 nm fluorescence emission indicate blue- or redshifts. For apo NHR2 in 10% DMSO solvent, a Tm of 74.5 ± 0.16 °C was observed (the error was determined from fitting). Complexes showed reduced Tm (ΔTm of -1.6 ± 0.03 °C for M23, ΔTm of -1.0 ± 0.09 °C for M27, and ΔTm of -0.1 ± 0.04 °C for M10). (C–E) Hit compounds binding to NHR2 detected by MST assay. Titration of hit compounds to a constant concentration of Alexa488 dye-labeled NHR2 induces a change in thermophoresis. The data were fitted to a 1:1 binding model to obtain apparent KD values. The inflection point of the curve revealed a KD, app of 39 ± 21 µM for M23 (C), 114 ± 55 µM for M27 (D), and 89 ± 38 µM for M10 (E). (F–H) Binding of M23 (F), M27 (G), and M10 (H) to NHR2 studied by STD NMR (lower spectra) (see also Fig. 2B) and the corresponding reference 1D (STD-off) 1H-NMR (upper spectra). The assignment of the individual peaks is indicated in the chemical structures. The STD effects (Isat / I0) of each proton are indicated on top of the peaks and were mapped as a filled circle onto the structures. Darker red colors denote closer proximity to the protein. The STD effects reveal a particular ligand orientation at the protein.
