In HEK293T biosensor cells, GDC-0879 increased association of activated KRAS with BRAF. Its BRET EC50 was 30 ± 10 nM for WT BRAF and 1314 ± 522 nM for the dimerization-impaired R509H variant. These protein-association results show why target binding alone does not establish pathway suppression in every cellular context. R509H also weakens compound binding, which limits its use to isolate dimerization’s role. [1]
BRAF kinase-domain tracer displacement
Biochemical assay · Recombinant 6×His-tagged human BRAF kinase domain (residues 444–723), engineered with 16 solubilizing mutations; WT-background and R509H variants. [1]
GDC-0879 displaced a kinase-site fluorescent tracer. The R509H variant had a higher binding-assay IC50 than the WT-background construct. These are tracer-displacement endpoints, not catalytic enzyme inhibition IC50 values.
Literature-Reported Treatment Conditions
- Exposure duration
- 1 h at room temperature
- Method
- GDC-0879 concentration-response in recombinant-domain TR-FRET tracer displacement; signal normalized to DMSO-treated wells after blank subtraction.
- Vehicle
- DMSO
- Reported limits
- The full tested concentration series is not transcribed. Binding IC50 is an assay endpoint, not a treatment concentration or a catalytic-inhibition value.
Methods and source locations
Compound reported in the paper: GDC-0879; Compound label in the paper: GDC-0879
Results describe the paper’s GDC-0879 material and models; they do not validate an Adooq lot or establish salt-specific equivalence.
Figure 2d and caption; Methods, Plasmids and reagents and Drug-binding assay by TR-FRET; Supplementary Data 1, row 5; Supplementary Data 2, row 3
Tracer displacement from the WT-background engineered BRAF domain: TR-FRET competition assay; representative curves use technical duplicates. — Figure 2d, GDC-0879 panel and caption; Methods, Drug-binding assay by TR-FRET; Supplementary Data 1 A1:F5
Tracer displacement from engineered BRAF R509H domain: Same TR-FRET competition design as WT-background comparator. — Figure 2d, GDC-0879 panel and caption; Methods, Drug-binding assay by TR-FRET
GDC-0879 increases KRAS–BRAF association in cellular biosensors
Cell-based study · HEK293T cells expressing RlucII-tagged KRASG12V and GFP10-tagged full-length BRAF; BRAF WT, T529M gatekeeper and R509H comparator constructs. [1]
GDC-0879 concentration-dependently increased the KRASG12V–BRAF BRET signal. Gatekeeper T529M and dimerization-impaired R509H reduced compound potency. RAS activation-state and BRAF RAS-binding-domain controls supported probe specificity. This assay measures protein association, not tumor-cell viability or clinical benefit. Because R509H also reduces GDC-0879 binding affinity, this comparator cannot unambiguously isolate the contribution of dimerization.
Literature-Reported Treatment Conditions
- Treatment concentration
- Concentration-response; EC50 values are response endpoints, not administered fixed doses.
- Exposure duration
- 2 h
- Method
- Cellular BRET after GDC-0879 exposure at 37 °C. Equal GFP10 expression was monitored.
- Vehicle
- DMSO; shared methods specify final DMSO ≤0.5%.
- Reported limits
- No tumor-growth endpoint is measured by this biosensor experiment. Exact individual concentration steps are not transcribed.
Methods and source locations
Compound reported in the paper: GDC-0879; Compound label in the paper: GDC-0879
Results describe the paper’s GDC-0879 material and models; they do not validate an Adooq lot or establish salt-specific equivalence.
Figures 1a–c and 2c with captions; Results, RAF inhibitors stimulate RAS−RAF association; Methods, Plasmids and reagents and BRET titration curves and dose-response curves; Supplementary Data 2 row 3; Results, Compound-induced RAS−RAF binding correlates with dimerization, paragraph comparing R509H binding and BRET effects
Induction of KRASG12V–WT BRAF BRET: Cellular BRET dose-response; technical duplicate error bars on a representative biological replicate. — Figure 2c, GDC-0879 panel and caption; Methods, BRET titration curves and dose-response curves
Induction of KRASG12V–BRAF R509H BRET: Matched cellular BRET comparator; printed figure values. — Figure 2c, GDC-0879 panel and caption
