Journal: NAR Cancer

Article Title: Polycomb group proteins in cancer: multifaceted functions and strategies for modulation

doi: 10.1093/narcan/zcab039

Figure Lengend Snippet: Mechanism of action and structure of Polycomb modulators

Article Snippet: Simultaneously to UNC6852, a team at AstraZeneca reported different EED-based PROTAC molecules that utilize VHL recruitment to degrade PRC2 subunits and reduce PRC2 activity ( ).

Techniques: Activity Assay, Mutagenesis, Methylation, In Vitro, Concentration Assay, Inhibition, Mouse Assay, Expressing, Binding Assay, Derivative Assay, Functional Assay

Journal: NAR Cancer

Article Title: Polycomb group proteins in cancer: multifaceted functions and strategies for modulation

doi: 10.1093/narcan/zcab039

Figure Lengend Snippet: Mechanism of action and structure of Polycomb modulators

Article Snippet: Simultaneously to UNC6852, a team at AstraZeneca reported different EED-based PROTAC molecules that utilize VHL recruitment to degrade PRC2 subunits and reduce PRC2 activity ( ).

Techniques: Activity Assay, Mutagenesis, Methylation, In Vitro, Concentration Assay, Inhibition, Mouse Assay, Expressing, Binding Assay, Derivative Assay, Functional Assay

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: Design of BODIPY FL VH032 ( 5 ).

Article Snippet: We then use BODIPY FL VH032 ( 5 )-based TR-FRET and FP assays to test a panel of reported VHL ligands ( Figure ), including VH032 , VH298 , MZ1 ( 3 ), VH032 amine ( 6 ), Me-VH032 amine ( 7 ), BOC-VH032 ( 8 ), VH032 phenol ( 9 ), and VH032-PEG4-amine ( 10 )., , − We also test non-VHL ligands ( Figure ), including (+)-JQ1 ( 4 ), thalidomide-4′-oxyacetamido-alkylC4-amine ( 11 , cereblon E3 ligase ligand), and dBET1 ( , bivalent BRD-CRBN PROTAC).

Techniques:

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: TR-FRET binding affinity of BODIPY FL VH032 ( 5 , 1 to 2 dilutions, at an optimized concentration range of 0.06–500 nM) to GST–VCB. (A) Binding interaction of BODIPY FL VH032 ( 5 ) to 2 nM Tb-anti-GST in the presence of 2 nM GST–VCB or in the absence of GST–VCB at the designated incubation times. “+” and “–” (after each time point) represent “with GST–VCB” and “without GST–VCB”, respectively. The TR-FRET signals were expressed as relative TR-FRET units (RTU), which were calculated using 10,000 × 520 nm/490 nm. (B) Binding interaction of BODIPY FL VH032 ( 5 ) and 2 nM Tb-anti-GST, with 2 nM GST–VCB, 2 nM GST–VCB + VH298 ( 2 , 30 μM), or without GST–VCB + DMSO at the 90-min incubation time. (C) Fold changes in the TR-FRET signals of BODIPY FL VH032 ( 5 ) with (2 nM GST–VCB + DMSO) to (2 nM GST–VCB + VH298) ( 2 , 30 μM) (blue curve) or to (without GST–VCB + DMSO) (red curve).

Article Snippet: We then use BODIPY FL VH032 ( 5 )-based TR-FRET and FP assays to test a panel of reported VHL ligands ( Figure ), including VH032 , VH298 , MZ1 ( 3 ), VH032 amine ( 6 ), Me-VH032 amine ( 7 ), BOC-VH032 ( 8 ), VH032 phenol ( 9 ), and VH032-PEG4-amine ( 10 )., , − We also test non-VHL ligands ( Figure ), including (+)-JQ1 ( 4 ), thalidomide-4′-oxyacetamido-alkylC4-amine ( 11 , cereblon E3 ligase ligand), and dBET1 ( , bivalent BRD-CRBN PROTAC).

Techniques: Binding Assay, Concentration Assay, Incubation

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: Signal stability of the BODIPY FL VH032 ( 5 )-based VHL TR-FRET assay. (A) TR-FRET interaction of 4 nM BODIPY FL VH032 ( 5 ) and 2 nM Tb-anti-GST with 2 nM GST–VCB + DMSO (negative control), 2 nM GST–VCB + VH298 ( 2 , 30 μM) (positive control), or without GST–VCB + DMSO (background control) at specified incubation time points. (B) TR-FRET signal-fold change to 2 nM GST–VCB + VH298 ( 2 , 30 μM) of 2 nM GST–VCB + DMSO (negative control), 2 nM GST–VCB + VH298 ( 2 , 30 μM) (positive control), or without GST–VCB + DMSO (background control) in the presence of 4 nM BODIPY FL VH032 ( 5 ) and 2 nM Tb-anti-GST at specified incubation time points. (C) Dose–response inhibition curves of VH298 ( 2 , 1–3 dilutions, in the concentration range of 2.1 pM to 30 μM) at specified incubation time points in the presence of 4 nM BODIPY FL VH032 ( 5 ), 2 nM GST–VCB, and 2 nM Tb-anti-GST.

Article Snippet: We then use BODIPY FL VH032 ( 5 )-based TR-FRET and FP assays to test a panel of reported VHL ligands ( Figure ), including VH032 , VH298 , MZ1 ( 3 ), VH032 amine ( 6 ), Me-VH032 amine ( 7 ), BOC-VH032 ( 8 ), VH032 phenol ( 9 ), and VH032-PEG4-amine ( 10 )., , − We also test non-VHL ligands ( Figure ), including (+)-JQ1 ( 4 ), thalidomide-4′-oxyacetamido-alkylC4-amine ( 11 , cereblon E3 ligase ligand), and dBET1 ( , bivalent BRD-CRBN PROTAC).

Techniques: Negative Control, Positive Control, Incubation, Inhibition, Concentration Assay

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: We then use BODIPY FL VH032 ( 5 )-based TR-FRET and FP assays to test a panel of reported VHL ligands ( Figure ), including VH032 , VH298 , MZ1 ( 3 ), VH032 amine ( 6 ), Me-VH032 amine ( 7 ), BOC-VH032 ( 8 ), VH032 phenol ( 9 ), and VH032-PEG4-amine ( 10 )., , − We also test non-VHL ligands ( Figure ), including (+)-JQ1 ( 4 ), thalidomide-4′-oxyacetamido-alkylC4-amine ( 11 , cereblon E3 ligase ligand), and dBET1 ( , bivalent BRD-CRBN PROTAC).

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: BODIPY FL VH032 ( 5 ) concentration optimization in a VHL FP assay with GST–VCB (1 to 2 dilutions; in an optimized concentration range of 0.03–1000 nM) and a 90-min incubation time. (A) VHL FP assay performance with BODIPY FL VH032 ( 5 ) concentrations of 70, 60, 50, 40, 30, 20, and 10 nM. (B) VHL FP assay performance with BODIPY FL VH032 ( 5 ) concentrations of 10, 5, 2, and 1 nM.

Article Snippet: We then use BODIPY FL VH032 ( 5 )-based TR-FRET and FP assays to test a panel of reported VHL ligands ( Figure ), including VH032 , VH298 , MZ1 ( 3 ), VH032 amine ( 6 ), Me-VH032 amine ( 7 ), BOC-VH032 ( 8 ), VH032 phenol ( 9 ), and VH032-PEG4-amine ( 10 )., , − We also test non-VHL ligands ( Figure ), including (+)-JQ1 ( 4 ), thalidomide-4′-oxyacetamido-alkylC4-amine ( 11 , cereblon E3 ligase ligand), and dBET1 ( , bivalent BRD-CRBN PROTAC).

Techniques: Concentration Assay, FP Assay, Incubation

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: Determination of the binding affinity of BODIPY FL VH032 ( 5 , 10 nM) to GST–VCB in an FP assay with GST–VCB (1 to 2 dilutions; in the optimal concentration range of 0.03–1000 nM) + DMSO (total interactions), GST–VCB (1 to 2 dilutions; in the optimal concentration range of 0.03–1000 nM) + VH298 ( 2 , 30 μM) (i.e., GST–VCB-mediated nonspecific interactions), or DMSO without GST–VCB (i.e., background interactions) at a 90-min incubation time.

Article Snippet: We then use BODIPY FL VH032 ( 5 )-based TR-FRET and FP assays to test a panel of reported VHL ligands ( Figure ), including VH032 , VH298 , MZ1 ( 3 ), VH032 amine ( 6 ), Me-VH032 amine ( 7 ), BOC-VH032 ( 8 ), VH032 phenol ( 9 ), and VH032-PEG4-amine ( 10 )., , − We also test non-VHL ligands ( Figure ), including (+)-JQ1 ( 4 ), thalidomide-4′-oxyacetamido-alkylC4-amine ( 11 , cereblon E3 ligase ligand), and dBET1 ( , bivalent BRD-CRBN PROTAC).

Techniques: Binding Assay, FP Assay, Concentration Assay, Incubation

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: We then use BODIPY FL VH032 ( 5 )-based TR-FRET and FP assays to test a panel of reported VHL ligands ( Figure ), including VH032 , VH298 , MZ1 ( 3 ), VH032 amine ( 6 ), Me-VH032 amine ( 7 ), BOC-VH032 ( 8 ), VH032 phenol ( 9 ), and VH032-PEG4-amine ( 10 )., , − We also test non-VHL ligands ( Figure ), including (+)-JQ1 ( 4 ), thalidomide-4′-oxyacetamido-alkylC4-amine ( 11 , cereblon E3 ligase ligand), and dBET1 ( , bivalent BRD-CRBN PROTAC).

Techniques: FP Assay, Incubation

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: DMSO tolerance of the BODIPY FL VH032 ( 5 )-mediated VHL TR-FRET assay in the presence of 4 nM BODIPY FL VH032 ( 5 ), 2 nM GST–VCB, and 2 nM Tb-anti-GST at the 90-min incubation time point. (A) RTU of the negative control DMSO or the positive control VH298 ( 2 , 30 μM) in the presence of 0.2, 0.5, 1, 2, 5, or 10% DMSO. (B) % RTU (% RTU of 0.2% DMSO was set as 100%) of the negative control DMSO or the positive control VH298 ( 2 , 30 μM) in the presence of 0.2, 0.5, 1, 2, 5, or 10% DMSO. (C) Dose–response inhibition curves of VH298 ( 2 , 1–3 dilutions; in the concentration range of 2.1 pM to 30 μM) in the presence of indicated DMSO concentrations.

Article Snippet: We then use BODIPY FL VH032 ( 5 )-based TR-FRET and FP assays to test a panel of reported VHL ligands ( Figure ), including VH032 , VH298 , MZ1 ( 3 ), VH032 amine ( 6 ), Me-VH032 amine ( 7 ), BOC-VH032 ( 8 ), VH032 phenol ( 9 ), and VH032-PEG4-amine ( 10 )., , − We also test non-VHL ligands ( Figure ), including (+)-JQ1 ( 4 ), thalidomide-4′-oxyacetamido-alkylC4-amine ( 11 , cereblon E3 ligase ligand), and dBET1 ( , bivalent BRD-CRBN PROTAC).

Techniques: Incubation, Negative Control, Positive Control, Inhibition, Concentration Assay

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: Pilot screening using the BODIPY FL VH032 (5)-mediated VHL TR-FRET binding assay in the presence of 4 nM BODIPY FL VH032 ( 5 ), 2 nM GST–VCB, and 2 nM Tb-anti-GST at the 90-min incubation time point. (A) Plate-by-plate negative (DMSO) and positive (VH298, 2 , 30 μM) control performance. (B) Screening scatterplot of plate Z -prime. (C) Screening scatterplot of activity values, where the positive control is VH298 ( 2 , 30 μM, green dots, 100% inhibition), the negative control is DMSO (red dots, 0% inhibition), active compounds (blue dots) are chemicals with % inhibition ≥30% with the 30% activity cutoff defined by the black dotted line, and inactive compounds (black dots) are chemicals with % inhibition <30%. (D) Chemical structures and VHL binding activities of compounds SJ000994241-1 ( 14 ), SJ000994129-1 ( 15 ), SJ000994509-1 ( 16 ), and SJ000994244-1 ( 17 ).

Article Snippet: We then use BODIPY FL VH032 ( 5 )-based TR-FRET and FP assays to test a panel of reported VHL ligands ( Figure ), including VH032 , VH298 , MZ1 ( 3 ), VH032 amine ( 6 ), Me-VH032 amine ( 7 ), BOC-VH032 ( 8 ), VH032 phenol ( 9 ), and VH032-PEG4-amine ( 10 )., , − We also test non-VHL ligands ( Figure ), including (+)-JQ1 ( 4 ), thalidomide-4′-oxyacetamido-alkylC4-amine ( 11 , cereblon E3 ligase ligand), and dBET1 ( , bivalent BRD-CRBN PROTAC).

Techniques: Binding Assay, Incubation, Activity Assay, 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: Comparison of a Previously Reported VHL FP Assay and Our Newly Developed VHL TR-FRET and FP Assays

Article Snippet: We then use BODIPY FL VH032 ( 5 )-based TR-FRET and FP assays to test a panel of reported VHL ligands ( Figure ), including VH032 , VH298 , MZ1 ( 3 ), VH032 amine ( 6 ), Me-VH032 amine ( 7 ), BOC-VH032 ( 8 ), VH032 phenol ( 9 ), and VH032-PEG4-amine ( 10 )., , − We also test non-VHL ligands ( Figure ), including (+)-JQ1 ( 4 ), thalidomide-4′-oxyacetamido-alkylC4-amine ( 11 , cereblon E3 ligase ligand), and dBET1 ( , bivalent BRD-CRBN PROTAC).

Techniques: FP Assay, Concentration Assay