Rapid and direct control of target protein levels with VHL-recruiting dTAG molecules

Abstract

Chemical biology strategies for directly perturbing protein homeostasis including the degradation tag (dTAG) system provide temporal advantages over genetic approaches and improved selectivity over small molecule inhibitors. We describe dTAG^V-1, an exclusively selective VHL-recruiting dTAG molecule, to rapidly degrade FKBP12^F36V-tagged proteins. dTAG^V-1 overcomes a limitation of previously reported CRBN-recruiting dTAG molecules to degrade recalcitrant oncogenes, supports combination degrader studies and facilitates investigations of protein function in cells and mice.

Fig. 1. dTAG^V-1 is an exclusively selective degrader of FKBP12^F36V-tagged proteins.

a Schematic depiction of the dTAG system using VHL-recruiting dTAG molecules. VHL-recruiting dTAG molecules promote ternary complex formation between the FKBP12^F36V-tagged target protein and E3 ubiquitin ligase complex, inducing target protein ubiquitination and degradation. b Chemical structures of dTAG^V-1 and dTAG^V-1-NEG. c DMSO-normalized ratio of Nluc/Fluc signal of 293FT FKBP12^WT-Nluc or FKBP12^F36V-Nluc cells treated with dTAG^V-1 or dTAG^V-1-NEG for 24 h. Data are presented as mean ± s.d. of n = 4 biologically independent samples and are representative of n = 3 independent experiments. d Protein abundance after treatment of PATU-8902 LACZ-FKBP12^F36V clone with 500 nM dTAG^V-1 for 4 h compared to DMSO treatment. Volcano plots depict fold change abundance relative to DMSO versus P value derived from a two-tailed Student’s t-test. Fold change values and significance designations derived from a two-tailed Student’s t-test and a permutation-based FDR estimation are provided in Supplementary Data 2. Data are from n = 3 biologically independent samples. e Immunoblot analysis of PATU-8902 LACZ-FKBP12^F36V clone co-treated with DMSO, THAL-SNS-032, and/or dTAG^V-1 as indicated for 24 h. Data is representative of n = 3 independent experiments. Source data for c and e are provided as a Source Data file.

Fig. 2. dTAG^V-1 is an in vivo-compatible degrader of FKBP12^F36V-tagged proteins.

a Immunoblot analysis of PATU-8902 FKBP12^F36V-KRAS^G12V; KRAS^−/− clone treated with DMSO, dTAG^V-1, or dTAG^V-1-NEG for the indicated time-course. b Immunoblot analysis of 293T^WT FKBP12^F36V-KRAS^G12V or 293T^VHL-/- FKBP12^F36V-KRAS^G12V cells treated with DMSO or the indicated dTAG molecules for 24 h. Data in a, b are representative of n = 3 independent experiments. c DMSO-normalized antiproliferation of PATU-8902 LACZ-FKBP12^F36V or FKBP12^F36V-KRAS^G12V; KRAS^-/- clones treated with dTAG^V-1 or dTAG^V-1-NEG for 120 h. Cells were cultured as ultra-low adherent 3D-spheroid suspensions. Data are presented as mean ± s.d. of n = 4 biologically independent samples and are representative of n = 3 independent experiments. d Bioluminescent imaging to evaluate degradation of luciferase-FKBP12^F36V in mice was performed daily as follows: day 0 to establish baseline signal, day 1–3 to monitor luciferase-FKBP12^F36V signal 4 h after vehicle or dTAG molecule treatment (T), day 4 to monitor duration of luciferase-FKBP12^F36V signal 28 h after third and final vehicle or dTAG molecule treatment. Total flux for each mouse is depicted. Data are presented from vehicle (n = 5 biologically independent mice at day 0–4), dTAG-13 (n = 5 biologically independent mice at day 0–3; n = 4 biologically independent mice at day 4) or dTAG^V-1 (n = 5 biologically independent mice at day 0–4) treated mice. P values are derived from a two-tailed Welch’s t-test (*P < 0.05, **P < 0.01) and are provided as a Source Data file. Source data for a–d are provided as a Source Data file.

Fig. 3. EWS/FLI degradation reverses abnormal proteomic signaling and proliferation.

a, b Immunoblot analysis of EWS502 FKBP12^F36V-GFP or FKBP12^F36V-EWS/FLI; EWS/FLI^−/− cells treated with DMSO (a, b), dTAG-13 (a), or dTAG^V-1 (b) for 24 h. Data in a, b are representative of n = 3 independent experiments. c Protein abundance after treatment of EWS502 FKBP12^F36V-EWS/FLI; EWS/FLI^−/− cells with 1 μM dTAG^V-1 for 6 h compared to DMSO treatment. Volcano plots depict fold change abundance relative to DMSO versus P value derived from a two-tailed Student’s t-test. Fold change values and significance designations derived from a two-tailed Student’s t-test and a permutation-based FDR estimation are provided in Supplementary Data 2. Data are from n = 2 for DMSO and n = 3 for dTAG^V-1 biologically independent samples. d Immunoblot analysis of EWS502 FKBP12^F36V-GFP or FKBP12^F36V-EWS/FLI; EWS/FLI^−/− cells treated with DMSO, 1 μM dTAG^V-1 or 1 μM dTAG^V-1-NEG for 24 h. Data are representative of n = 3 independent experiments. e GSEA signatures upon assessment of significantly differentially expressed target proteins (FDR < 0.05) after treatment of EWS502 FKBP12^F36V-EWS/FLI; EWS/FLI^−/− cells as described in c. Data are from n = 2 for DMSO and n = 3 for dTAG^V-1 biologically independent samples. f Relative growth of EWS502 FKBP12^F36V-EWS/FLI; EWS/FLI^−/− cells treated with DMSO, dTAG^V-1, or dTAG^V-1-NEG. Y-axis represent luminescence values relative to day 0. Data are presented as mean ± s.d. of n = 8 technical replicates and are representative of n = 3 independent experiments. Source data for a, b, d, and f are provided as a Source Data file.