Journal: Cell Death & Disease

Article Title: VHL regulates the sensitivity of clear cell renal cell carcinoma to SIRT4-mediated metabolic stress via HIF-1α/HO-1 pathway

doi: 10.1038/s41419-021-03901-7

Figure Lengend Snippet: A Analyzed the correlation between HIF-1α and SIRT4 in mutated VHL and nonmutated VHL group from a TCGA cohort of ccRCC patients. B HIF-1α protein in SIRT4 overexpressed 786-O and Caki-2 cells were displayed by western blot. The quantification analysis is shown on the right panel. C Caki-2 cells were treated with VHL inhibitor, VH-298 (50 µM), for 24 h followed by western blot. Densitometric analyses of HIF-1α/HO-1 are shown on the right panel. D Molecular docking of SIRT4 and HIF-1α was realized by zdock. E The immunoprecipitation was used to analysis the interaction of exogenous SIRT4 with endogenous HIF-1α.

Article Snippet: VH-298 (HY-100947) was purchased from MCE.

Techniques: Western Blot, Immunoprecipitation

Journal: bioRxiv

Article Title: Targeting cancer with small molecule pan-KRAS degraders

doi: 10.1101/2023.10.24.563163

Figure Lengend Snippet: a , KRAS and GAPDH levels by Western blot of GP5d cells treated with compounds 2 or - 3 (24 h), numbers indicate normalised KRAS levels vs controls ( N =3). b, Dose-dependent degradation of KRAS G12V in SW620 cells (24 h, N =3, SD). c, Dose-dependent degradation of Hibit-KRAS G12D in GP5d cells for compounds 2 and - 3 (24 h, N =3, SD). d, KRAS G12D levels in GP5d cells treated with compound 2 in presence or absence of MLN4924 or VH298 by capillary electrophoresis (24 h, N =3, SD). e, Detection of compound 2 and - 3 induced ternary complex formation for KRAS G12D in Gp5d cells (4 h, N =3, SD). f, KRAS G12D ubiquitination in Gp5d cells treated with compounds 2 or - 3 (4 h, N =3, SD). g, SPR characterization of ternary complex for VCB, KRAS G12D :GDP and compound 3 . h, Data as in with protein abundance stated as molecules per cell.

Article Snippet: Immediately after, VH298 (Selleckchem Cat. No. S8449), MG132 (Selleckchem Cat. No. S2619) and MLN4924 (Tocris Cat. No. 6499) were added to the wells at 10 μM final concentration and incubated for 4h.

Techniques: Western Blot, Electrophoresis, Ubiquitin Proteomics, Quantitative Proteomics

Journal: bioRxiv

Article Title: Targeting cancer with small molecule pan-KRAS degraders

doi: 10.1101/2023.10.24.563163

Figure Lengend Snippet: a , VHL engagement by nanoBRET in live or permeabilised HEK293 cells for ACBI3 ( N =3, SD). b, Detection of VCB:ACBI3:KRAS G12D complexes in Gp5d cells (4 h, N =3, SD, one-way ANOVA, Dunnet correction). c, KRAS G12D ubiquitination in Gp5d cells treated with ACBI3 (4 h, N =3, SD, one-way ANOVA, Dunnet correction). d, KRAS and GAPDH levels by Western blot of Gp5d cells treated with ACBI3 or e, inactive stereoisomer compound 8 (24 h), numbers indicate normalised KRAS levels vs controls ( N =3). f, KRAS levels in GP5d cells treated with ACBI3 in presence or absence of MLN4924 or VH298 by Western blot (24 h), numbers indicate normalised KRAS levels vs controls ( N =3). g, Degradation of KRAS G12D or KRAS G12V in GP5d, GP2d or SW620 cells by ACBI3, respectively (24h – 18 h for GP2d, N =3, SD). h, Live cell degradation time course of Hibit-KRAS G12D in presence of varying concentrations of ACBI3 ( N =6). i, Degradation rate by live cell degradation vs concentration for ACBI3 (mean and 95% CI). j, Degradation timecourse of endogenous KRAS G12D in GP2d cells at varying concentrations of ACBI3 by capillary electrophoresis ( N =3). k, Degradation rate for endogenous KRAS G12D vs concentration for ACBI3 (mean and 95% CI). l, Degradation of indicated retrovirally transduced HiBit-KRAS mutants by ACBI3 (24 h, N =3). m, Proliferation inhibition data for 240 cancer cell lines for ACBI3, bars represent IC 50 per cell line (5 days, N =3). n, Proliferation inhibition data for 240 cancer cell lines for ACBI3 in presence of 2.5 µM Zosuquidar, bars represent IC 50 per cell line (5 days, N =3). o, Cumulative proliferation data for ACBI3 in 240 cell line panel in presence or absence of 2.5 µM Zosuquidar for KRAS WT vs KRAS mutant cell lines (geometric mean, geometric SD). p, Effect of ACBI3 or vehicle treatment on body weight of GP2d tumour bearing mice ( N =10, mean and SD).

Article Snippet: Immediately after, VH298 (Selleckchem Cat. No. S8449), MG132 (Selleckchem Cat. No. S2619) and MLN4924 (Tocris Cat. No. 6499) were added to the wells at 10 μM final concentration and incubated for 4h.

Techniques: Ubiquitin Proteomics, Western Blot, Concentration Assay, Electrophoresis, Inhibition, Mutagenesis

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: The VHL ligands included VH032 ( 1 ), VH298 ( 2 ), VH032 amine , Me-VH032 amine ( 7 ), BOC-VH032 ( 8 ), VH032 phenol ( 9 ), VH032-PEG4-amine ( 10 ), and the dual VHL and BRD PROTAC ligand MZ1 ( 3 ).

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: The VHL ligands included VH032 ( 1 ), VH298 ( 2 ), VH032 amine , Me-VH032 amine ( 7 ), BOC-VH032 ( 8 ), VH032 phenol ( 9 ), VH032-PEG4-amine ( 10 ), and the dual VHL and BRD PROTAC ligand MZ1 ( 3 ).

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: The VHL ligands included VH032 ( 1 ), VH298 ( 2 ), VH032 amine , Me-VH032 amine ( 7 ), BOC-VH032 ( 8 ), VH032 phenol ( 9 ), VH032-PEG4-amine ( 10 ), and the dual VHL and BRD PROTAC ligand MZ1 ( 3 ).

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: 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: The VHL ligands included VH032 ( 1 ), VH298 ( 2 ), VH032 amine , Me-VH032 amine ( 7 ), BOC-VH032 ( 8 ), VH032 phenol ( 9 ), VH032-PEG4-amine ( 10 ), and the dual VHL and BRD PROTAC ligand MZ1 ( 3 ).

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: The VHL ligands included VH032 ( 1 ), VH298 ( 2 ), VH032 amine , Me-VH032 amine ( 7 ), BOC-VH032 ( 8 ), VH032 phenol ( 9 ), VH032-PEG4-amine ( 10 ), and the dual VHL and BRD PROTAC ligand MZ1 ( 3 ).

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: The VHL ligands included VH032 ( 1 ), VH298 ( 2 ), VH032 amine , Me-VH032 amine ( 7 ), BOC-VH032 ( 8 ), VH032 phenol ( 9 ), VH032-PEG4-amine ( 10 ), and the dual VHL and BRD PROTAC ligand MZ1 ( 3 ).

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: The VHL ligands included VH032 ( 1 ), VH298 ( 2 ), VH032 amine , Me-VH032 amine ( 7 ), BOC-VH032 ( 8 ), VH032 phenol ( 9 ), VH032-PEG4-amine ( 10 ), and the dual VHL and BRD PROTAC ligand MZ1 ( 3 ).

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: The VHL ligands included VH032 ( 1 ), VH298 ( 2 ), VH032 amine , Me-VH032 amine ( 7 ), BOC-VH032 ( 8 ), VH032 phenol ( 9 ), VH032-PEG4-amine ( 10 ), and the dual VHL and BRD PROTAC ligand MZ1 ( 3 ).

Techniques: FP Assay, Concentration Assay

Journal: Scientific Reports

Article Title: SOCS domain targets ECM assembly in lung fibroblasts and experimental lung fibrosis

doi: 10.1038/s41598-024-83187-9

Figure Lengend Snippet: VHL protein inhibition does not decrease SMA intensity in myofibroblasts. ( A ) IMR90 lung fibroblasts were untreated or differentiated with 5 ng/mL TGFβ for 48 h. Differentiated myofibroblasts were then treated with 100 µM VH298 for 1 h. Cells were then lysed in SDS buffer and immunoblotted for HIF1-⍺. β-Tubulin was used as the loading control. ( B ) Fibroblasts, myofibroblasts and myofibroblasts treated as in ( A ) were immunostained for FN (red) or SMA (red) and nuclear stain, DAPI (blue). Scale bar = 10 µm.

Article Snippet: VH298 (Axon MedChem, #2810) was used at working concentrations of 100 μM for 1 h in serum media.

Techniques: Inhibition, Control, Staining