Journal: bioRxiv

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

doi: 10.1101/2020.03.13.980946

Figure Lengend Snippet: ( 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 molecules 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. Significance designations derived from a permutation-based FDR estimation (q < 0.05) are provided in Supplementary Dataset 1. 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. ( f ) 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. ( g ) Immunoblot analysis of 293T WT or 293T VHL-/- FKBP12 F36V -KRAS G12V cells treated with DMSO or the indicated dTAG molecules for 24 h. Data in e - g are representative of n = 3 independent experiments. ( h ) 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. ( i ) Bioluminescence imaging to evaluate degradation of luciferase-FKBP12 F36V 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 value derived from a two-tailed Welch’s t -test (* P < 0.05, ** P < 0.01).

Article Snippet: The following cell lines were employed in this study: 293T (source: Thermo Fisher Scientific, media: DMEM with 10% FBS and 1% Penicillin-Streptomycin), 293FT (source: Thermo Fisher Scientific, media: DMEM with 10% FBS and 1% Penicillin-Streptomycin), PATU-8902 (source: DSMZ, media: DMEM with 10% FBS and 1% Penicillin-Streptomycin), MV4;11 (source: ATCC, media: RPMI with 10% FBS and 1% Penicillin-Streptomycin) and EWS502 (source: Dr. Stephen Lessnick, media: RPMI with 15% FBS and 1% Penicillin-Streptomycin-L-Glutamine).

Techniques: Derivative Assay, Western Blot, Clone Assay, Cell Culture, Imaging, Luciferase, Two Tailed Test