| Assay Method Information | |
| | Binding Affinity of Example 1 Compounds to Cbl-b Protein |
| Description: | Compounds were assessed for binding activity to Cbl-b by their ability to compete with and displace a probe comprised of BODIPY-FL fluorophore conjugated to a Cbl-b inhibitor (see Example 54 in WO2020264398). In the assay, a 36-427 aa truncated form of Cbl-b with an N-terminus Avi-tag was incubated 20 mM HEPES pH 7.5, 150 mM NaCl, 0.01% Triton X-100, 0.1% BSA, 0.5 mM TCEP buffer with streptavidin-terbium (Cisbio). After one hour of incubation at room temperature, 1 uM each of fluorescent probe and compound were combined with the reaction mixture. Compounds with Cbl-b binding activity competitively displace the fluorophore tagged inhibitor thereby disrupting the FRET signal from the terbium-BODIPY-FL complex. The reaction mixtures were incubated for one additional hour at room temperature to allow for competition between inhibitor and candidate compounds to occur. The time resolved FRET (TR-FRET) signal was then read at 520/620 nM (excitation/emission) using a Spectramax M5e plate reader (Molecular Devices) to measure probe displacement. Compounds with Cbl-b binding activity had decreased FRET signal owing to disruption of the terbium-BODIPY-FL FRET complex. Standard methods in PRISM were used to calculate the compound IC50 values from the experimental data. |
| Affinity data for this assay | |
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