Kinase inhibitor potency and selectivity is routinely measured using biochemical assays, which typically measure binding to the enzyme or inhibition of enzymatic activity in vitro. However, many compounds show a much lower potency for their inhibition of kinase activity in cells. This disconnect may arise from multiple factors, including the use of isolated kinase domains rather than full length protein, the absence of important partner proteins, or the use of ATP at concentrations well below what is found in living cells. For this reason, the SGC-UNC in collaboration with the Promega Corporation has applied NanoBRET technology to the development of kinase target engagement assays in live cells.

The development of nanoluciferase (Nluc) by Keith Wood and colleagues has accelerated interest in the use of bioluminescence to monitor target engagement of small molecules with kinases and other proteins. Nluc is an extremely bright luciferase derived from the deep-sea shrimp Oplophorus gacsilorstis that was generated by a combination of protein engineering and a chemical optimization of the enzyme substrate to be both significantly smaller and 150-fold brighter than firefly or Renilla luciferases. When employed with an efficient red-shifted fluorophore acceptor, the combination of greater light intensity and wide spectral resolution gives improved detection sensitivity and dynamic range over conventional BRET technologies.  The high intensity requires the protein-Nluc fusion to be transiently expressed at only low levels in the assay. NanoBRET assays involve real-time monitoring of interactions within the cell and are able to assess binding kinetics.

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Figure. A NanoBRET target engagement assay

The key principle behind the NanoBRET assay is bioluminescence resonance energy transfer (BRET). This energy transfer occurs when a bioluminescent energy donor (e.g., the light-producing Nluc) is in close proximity to an energy acceptor (see Figure above). The acceptor is commonly a BODIPY dye incorporated into a tracer molecule that is derived from a promiscuous kinase inhibitor. When the BODIPY dye and the Nluc tagged kinase are in close proximity to each other a BRET signal is generated: energy is transferred between the donor and acceptor and, after thermal relaxation, a longer wavelength light is emitted as the observable BRET signal. As small molecule ATP-competitive inhibitors are titrated into the system, the tracer undergoes competitive binding displacement with a concomitant loss of BRET signal.

Working closely with Matt Robers and his team at Promega, we have configured NanoBRET assays for the majority of the human kinases. These assays are being used routinely at the SGC-UNC to support the characterization of cell active inhibitors for the dark kinases and for the optimization of kinase chemical probes.