Fig. 2: Design and mechanism of bioPROTACs, based on genetic fusion of DARPins and CHIP E3 ligase.
From: Molecular features defining the efficiency of bioPROTACs
Asterisks (*) indicate dye label. a, b Mean degradation rate constants of DARPin-CHIPΔTPR bioPROTACS, labeled with TMR dye (red bars), either alone (b) or in a one-to-one complex with GS-eGFP (green bars, a), determined by microinjection into HEK293 cells. Error bars represent standard deviations. Numbers of analyzed cells per analyte are shown in Supplementary Table ST1. Ineffective bioPROTACs are indicated by a blue circle. c Graphical representation of labeled DARPin-CHIPΔTPR bioPROTAC dimer in complex with GS-eGFP used for degradation determination. d–f Plots comparing the indicated degradation rate constants shown in (a, b), each axis shows the underlined compound. g–i Western blot images of ubiquitination reactions. Samples were detected with either anti-eGFP antibody or anti-DARPin polyclonal antibody to show GS-eGFP or bioPROTAC ubiquitination. Shown are images from single experiments. To image marker bands and antibody-stained bands, the same membranes were imaged in two separate channels. Uncropped membranes are shown in Supplementary Fig. S17. g Western blot images resulting from the LysateUb assay, based on HEK293 lysates and involving GS-eGFP/bioPROTAC complexes. h, i Western blot images of InVitroUb assays, an in vitro ubiquitination assay with purified E1, E2 and ubiquitin with bioPROTACs (i) or GS-eGFP/bioPROTAC complexes (h). Black triangle (▲) shows controls without added ubiquitination reaction components. j Graph comparing DARPin affinities determined by SPR with indicated degradation rate constant of GS-eGFP. k–m Comparison of DARPin denaturation midpoints, determined by guanidinium-induced unfolding, and degradation rate constants. Pearson’s correlation coefficients (r) are 0.86 for (d) when ignoring the two outliers in blue, 0.58 for (e), and 0.70 for (f) when ignoring the two outliers in blue. The r values for j–m are between 0.21 and −0.41, therefore not supporting a correlation. For a and b, the number of analyzed cells are shown in Supplementary Table ST1.
