Fig. 1: Chemical synthesis of TMP-TACtz, genetic constructs and regulation of eDHFR in immortalized and primary cells.
From: Photocontrolled trimethoprim PROTACs targeting the eDHFR protein tag
A Synthesis of TMP-TACtz-1-4, (i) CuSO4, sodium ascorbate, 90 °C, 10–15 min; B Diagram of the eDHFR-YFP-T2A-Luciferase construct in which YFP is fused directly to the C-terminus of eDHFR; C Dose-dependent downregulation of YFP signal in Jurkat-eDHFR-YFP cells by TMP-TACtz-1-4 after 24 h incubation as assessed by flow cytometry; D Schematic representation of the anti-fibroblast activation protein (FAP) CAR construct involving directly fusion of the eDHFR protein to the C-terminus of the CD3zeta domain of the FAP CAR. This fusion allows for regulation with TMP-TACtz; T2A-BFP was also inserted downstream as a fluorescent marker; E Schematic representation of the FAP CAR construct without T2A-BFP (pTRPE FAP CAR-eDHFR); F Bar plots demonstrating dose-dependent regulation in primary human FAP CAR-eDHFR DF T cells by TMP-TACtz-3, mean fluorescence intensity (MFI) from the no-drug control was taken as the maximum CAR expression. For each treatment condition, CAR expression was quantified as the ratio of the sample MFI to the no-drug control MFI, expressed as a percentage: (Sample MFI / No-drug MFI) × 100, (n = 3), Error bars plotted mean with SD. G Regulation of surface CAR expression in primary human FAP CAR-eDHFR T cells by TMP-TACtz-3.
