Journal: ACS Omega

Article Title: Development of BODIPY FL VH032 as a High-Affinity and Selective von Hippel–Lindau E3 Ligase Fluorescent Probe and Its Application in a Time-Resolved Fluorescence Resonance Energy-Transfer Assay

doi: 10.1021/acsomega.0c05221

Figure Lengend Snippet: Dose–response curves of a panel of VHL ligands and non-VHL ligands in the presence of BODIPY FL VH032 ( 5 , 4 nM), 2 nM GST–VCB, and 2 nM Tb-anti-GST at a 90-min incubation time. Ligand-relative TR-FRET units (RTUs) at their individual concentrations were normalized to that of VH298 ( 2 , 30 μM, positive control, 100% inhibition) and DMSO (negative control, 0% inhibition) and fitted to a sigmoidal equation with GraphPad PRISM to derive the IC 50 values, if applicable. The K i values were calculated with the Cheng–Prusoff equation. (A) Dose–response curves of VHL ligands VH032 ( 1 ), VH298 ( 2 ), VH032 amine ( 6 ), Me-VH032 amine ( 7 ), BOC-VH032 ( 8 ), and VH032 phenol ( 9 ). (B) Dose–response curves of VHL ligands VH298 ( 2 ), MZ1 ( 3 ), and VH032-PEG4-amine ( 10 ) and of non-VHL ligands (+)-JQ1 ( 4 ), thalidomide-4′-oxyacetamido-alkylC4-amine ( 11 ), and dBET1 ( 12 ).

Article Snippet: The dose–response curves of MZ1 ( 3 ), dBET1 ( 12 ), (+)-JQ1 ( 4 ), VH032-PEG4-amine ( 10 ), thalidomide-4′-oxyacetamido-alkylC4-amine ( 11 ), and VH298 ( 2 ) positive control are depicted in Figure B. VH032 ( 1 ), VH298 ( 2 ), VH032 amine , Me-VH032 amine ( 7 ), BOC-VH032 ( 8 ), VH032 phenol ( 9 ), VH032-PEG4-amine ( 10 ), and MZ1 ( 3 ) exhibited IC 50 values of 77.8 nM, 44.0 nM, 13.3 μM, 7.9 μM, 4.9 μM, 34.0 nM, 5.9 nM, and 14.7 nM, respectively, and K i values of 33.4 nM, 18.9 nM, 5.7 μM, 3.4 μM, 2.1 μM, 14.6 nM, 6.8 nM, and 6.3 nM, respectively.

Techniques: Incubation, Positive Control, Inhibition, Negative Control

Journal: ACS Omega

Article Title: Development of BODIPY FL VH032 as a High-Affinity and Selective von Hippel–Lindau E3 Ligase Fluorescent Probe and Its Application in a Time-Resolved Fluorescence Resonance Energy-Transfer Assay

doi: 10.1021/acsomega.0c05221

Figure Lengend Snippet: Activities of controls and selected VHL or non-VHL ligands in the BODIPY FL VH032 ( 5 )-mediated VHL FP assay with 10 nM BODIPY FL VH032 ( 5 ) and 100 nM GST–VCB at a 90-min incubation time. (A) FP assay signals of DMSO and VH298 ( 2 , 30 μM). (B) FP signal fold change from VH298 ( 2 , 30 μM) of DMSO and VH298 ( 2 , 30 μM). (C) FP dose–response curves of the VHL ligands VH032 ( 1 ), VH298 ( 2 ), VH032 amine ( 6 ), Me-VH032 amine ( 7 ), BOC-VH032 ( 8 ), and VH032 phenol ( 9 ). (D) FP dose–response curves of VH298 ( 2 ), MZ1 ( 3 ), and VH032-PEG4-amine ( 10 ) and of the non-VHL ligands (+)-JQ1 ( 4 ), thalidomide-4′-oxyacetamido-alkylC4-amine ( 11 ), and dBET1 ( 12 ).

Article Snippet: The dose–response curves of MZ1 ( 3 ), dBET1 ( 12 ), (+)-JQ1 ( 4 ), VH032-PEG4-amine ( 10 ), thalidomide-4′-oxyacetamido-alkylC4-amine ( 11 ), and VH298 ( 2 ) positive control are depicted in Figure B. VH032 ( 1 ), VH298 ( 2 ), VH032 amine , Me-VH032 amine ( 7 ), BOC-VH032 ( 8 ), VH032 phenol ( 9 ), VH032-PEG4-amine ( 10 ), and MZ1 ( 3 ) exhibited IC 50 values of 77.8 nM, 44.0 nM, 13.3 μM, 7.9 μM, 4.9 μM, 34.0 nM, 5.9 nM, and 14.7 nM, respectively, and K i values of 33.4 nM, 18.9 nM, 5.7 μM, 3.4 μM, 2.1 μM, 14.6 nM, 6.8 nM, and 6.3 nM, respectively.

Techniques: FP Assay, Incubation