Description
Study Cellular E3 Ligase Target Engagement
Protein degradation via small molecules such as PROTACs or molecular glues is governed by multiple steps. The initial steps involve entry of the small molecule degrader compound into the cell, followed by binding to the E3 ubiquitin ligase and protein target. The NanoBRET® Target Engagement (TE) Intracellular E3 Ligase Assays allow live-cell measurement of compound binding or affinity for an E3 ubiquitin ligase protein, Cereblon (CRBN) or von Hippel-Lindau disease tumor suppressor (VHL). In addition, the assays can be used to quantify intracellular availability of the compound. These assays are based on the NanoBRET® technology, an energy transfer technique designed to measure molecular proximity in living cells.
The NanoLuc® E3 ligase fusion vectors are designed to be the energy donor in the NanoBRET® TE Intracellular E3 Ligase Assay. The energy acceptor in these assays is a fluorescent cell-permeable NanoBRET® Tracer that binds to the NanoLuc® E3 ligase fusion protein. The close proximity of the tracer to NanoLuc® in the E3 ligase fusion results in a NanoBRET® signal. Addition of compounds to cells that bind the E3 ligase results in a dose-dependent decrease in NanoBRET® signal. This data allows quantitation of the intracellular affinity of degrader compounds for a target E3 ligase protein.
