NanoBRET assay measures apparent affinity of small molecules by competitive displacement of a target specific tracer, reversibly bound to target of interest fused to a NanoLuc tag. It has found extensive use in drug discovery for screening and in characterizing compounds to study signal transduction pathways, protein dynamics, and protein-small molecule interactions in living cells.
o2h biologist have extensive experience in establishing NanoBRET assay for different targets and have screened >1000 in-house synthesized small molecules, supporting hit to lead identification with an average turn-around-time of 3-4 days.
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Figure: NanoBRET assay validation and compound screening. A) Tool compound showing a dose-dependent displacement of the tracer with the optimal inhibition at tracer concentrations at 0.5 μM and 1 μM. B) An example illustrating inter-day and inter-person variability with tool compound CTx-0294885 performed as a 5-point DRC as indicated above. C) Representative dose-response curves of compounds with different potencies along with the tool/control compound. D) Time kinetics of a potent compound showing displacement of the tracer in comparison with an inactive compound.
At o2h discovery, we quantitatively access targeted protein degradation utilizing HiBiT-LgBiT complementation system which are high affinity tags and produces a very bright, sensitive kinetic readout. Stable cell lines are generated using LgBiT, and the HiBiT tag is introduced at the endogenous target locus using CRISPR gene editing. This approach allows rank ordering PROTACs based on DC50 determination and degradation rates, also suitable for high-throughput screening.
In order to detect protein-protein interactions (PPI) in live cells, we can customize BRET-based target complementation with different effector or transducer proteins. This involves a two-subunit system comprising of small BiT (SmBiT) and large BiT (LgBiT) subunits fused to targets of interest. When expressed, enables PPI and in forming a functional enzyme thereby generating a bright luminescent signal.
To know more about our biology services offering or to request our brochure, please reach out to us at discovery@o2h.com.
