Journal: Nature Communications
Article Title: Development of a RIPK1 degrader to enhance antitumor immunity
doi: 10.1038/s41467-024-55006-2
Figure Lengend Snippet: A Chemical structures of RIPK1 PROTAC LD4172 and its negative control, LD4172-NC. LD4172-NC shares the same warhead and linker as LD4172 but contains an inactive VHL ligand, making it unable to recruit VHL for ubiquitination. B Quantification of RIPK1 levels in Jurkat and B16F10 cells treated with various concentrations of LD4172 for 24 h, analyzed by Western blot. Results are representative of three independent experiments. Source data are provided as a file. C The DC 50 and D max values of LD4172 were determined in various human and mouse cell lines. The screening of LD4172 across different cell types was conducted in a single experiment. DC 50 represents the concentration at which 50% of RIPK1 is degraded, while Dmax indicates the maximum level of degradation achieved. D Kinetics of RIPK1 degradation induced by LD4172 (1 µM) and resynthesis upon LD4172 washout in Jurkat and B16F10 cells. Representative Western blots ( n = 3) show that the degradation half-life of RIPK1 is less than 2 h in both cell lines. RIPK1 resynthesis begins 4 h post-washout, with half-lives of ~48 h in Jurkat cells and ~24 h in B16F10 cells. Source data are provided as a file. E Quantification of RIPK1 degradation and resynthesis from ( D ). F NanoBRET-based in-cell RIPK1 target engagement assay. HEK293 cells transfected with nLuc-RIPK1 were incubated with a RIPK1 NanoBRET tracer (500 nM) and different concentrations of LD4172 or LD4172-NC ( n = 3 biological independent replicates, three independent experiments). G Time-resolved fluorescence resonance energy transfer (TR-FRET) biochemical binding assay for RIPK1. GST-tagged human RIPK1 (1 nM), Tb-labeled anti-GST antibody (0.3 nM), a RIPK1 TR-FRET tracer (350 nM), and various concentrations of LD4172 were incubated for 2 h ( n = 3 biological independent replicates, three independent experiments), followed by TR-FRET measurements at excitation 340 nm and emission 495/520 nm. H NanoBRET-based ternary complex formation assay. HEK293T cells co-transfected with nLuc-RIPK1 and VHL-HaloTag were treated with different concentrations of LD4172 or LD4172-NC ( n = 3 biological replicates, three independent experiments). Graph bars represent mean ± SD, and statistical significance was determined using a two-tailed unpaired t -test with P values indicated. I LD4172-induced RIPK1 degradation depends on ternary complex formation, neddylation, and proteasome activity. Representative Western blots (three independent experiments) of RIPK1 in Jurkat and B16F10 cells treated with T2I, a VHL ligand, MLN4924 (neddylation inhibitor), or Carfilzomib (proteasome inhibitor) for 4 h, followed by LD4172 treatment. Source data are provided as a file. J Proteomic profiling of LD4172-induced degradation. MDA-MB-231 cells were treated with LD4172 or LD4172-NC (200 nM) for 6 h ( n = 3 biological independent replicates from the single experiment). Proteins were ranked in a volcano plot based on their P value (−log10) and fold change (log 2 FC) between LD4172 and LD4172-NC treatments. RIPK1 (red dot) showed >50% degradation with P < 0.01, while blue dots represent kinases inhibited by the LD4172 warhead but not degraded. Source data are provided as a file.
Article Snippet: Human RIPK1 cDNA insert was cloned into pLenti6.2-ccdB-nLuc plasmid (87075, Addgene, a kind gift from Taipale Lab) using gateway cloning kit (11791020, Thermo) and standard protocol to obtain pLenti6.2-RIPK1-nLuc fusion vector.
Techniques: Negative Control, Ubiquitin Proteomics, Western Blot, Concentration Assay, Drug discovery, Transfection, Incubation, Fluorescence, Förster Resonance Energy Transfer, Binding Assay, Labeling, Tube Formation Assay, Two Tailed Test, Activity Assay
