Fig. 2: iDegs interact with IDO1.
From: Monovalent pseudo-natural products supercharge degradation of IDO1 by its native E3 KLHDC3
a,b, CETSA in intact SKOV-3 cells treated with 50 µM iDeg-1 or DMSO for 1 h followed by heat treatment and immunoblotting. a, Representative immunoblot for IDO1. b, Quantification of band intensities from a. Data are presented as mean values ± s.d.; n = 3. c, Structures of iDeg-2 and iDeg-3 and IC50 values in the Kyn assay in BxPC3 cells. Data are presented as mean values ± s.d.; n = 3 biological replicates. d,e, Influence of iDeg-1, iDeg-2 and iDeg-3 on IDO1 protein levels in BxPC3 cells. Cells were treated with IFN-γ and the compounds (3.33 µM) for 24 h before immunoblotting (d). Quantification of the band intensities is shown in e. Data are presented as mean values ± s.d.; n = 3 biological replicates. Dashed lines indicate where lanes of the same blot were spliced together. f, Influence on in vitro rhIDO1 activity. rhIDO1 was pre-incubated with the compounds at 37 °C for 90 min before the detection of Kyn levels using p-DMAB. Data are presented as mean values ± s.d.; n = 3 independent experiments. g, rhIDO1 thermal stability in the presence of 50 µM iDeg-1, iDeg-2 or iDeg-3 or DMSO and the apo-IDO1 inhibitor linrodostat (50 µM) using nanoDSF. rhIDO1 and the compounds were pre-incubated for 3 h at 37 °C before measurement. Representative results are shown (n = 3 independent experiments). h, Detection of haem-bound IDO1 by means of UV–vis spectroscopy in the presence of iDeg-1, iDeg-2 or iDeg-3 (100 µM), DMSO or linrodostat (100 µM). Incubation was performed at 37 °C for 3 h. Representative data are presented for n = 3 independent experiments.
