Drug Discovery
disrupting the cell membrane. It would also be desirable to evaluate other kinases in a similar man- ner to generate a profile of kon and koff for com- pounds to determine residency time selectivity and add value to the decision process. With recent advances in assay technology it is
now possible to study target engagement and resi- dency time in live cells in a kinetic manner2. By using a bioluminescence resonance energy transfer technology (NanoBRET) designed to generate light (photons), coupled with a cell-permeable energy-transfer probe (fluorescence tracer) that reversibly binds the active site of the kinase, the interaction of the tracer with the binding site of the kinase can be examined in real time in live cells. Cells are transiently tansfected with a con- struct composed of the full-length kinase cDNA coupled to a small (19KD) Nanoluc® luciferase cDNA. When the cells are treated with the Nanoluc substrate, photons are generated and detected at 460nm. On administration of the fluo- rescence tracer to the cells, this molecule binds the kinase, bringing the tracer into close proximity with the Nanoluc generated photons and resulting in BRET. The fluorescence signal is detectable at 600+nm and the result can be expressed as a ratio of the 460 to 600+nm signal. Binding of test com- pounds to the kinase can be examined by incubat- ing the tracer at a fixed concentration with the cells, followed by various concentrations of a test compound, to observe the decrease in the BRET signal as the compound competes the tracer from the kinase. Residency time can also be studied by first incubating a fixed concentration of test com- pound with the cells, then washing the cells to remove the compound from the well followed by the addition of a fixed concentration of tracer. The rate of dissociation of the compound can be detected as an increase in BRET signal over time as the tracer competes for binding to the kinase. By using this approach both binding and residency time of test compounds within the native environ- ment of the cell can be determined in a kinetic manner. Furthermore, the addition of the same concentration of compound to other residency time experiments using other Nanoluc kinase con- structs allows for the development of a ‘picture’ of both binding and residency time of the compound in cells and may add value to the selectivity versus non-selectivity question for many of the 243 clini- cal kinase inhibitors.
Comments… I applaud the authors of this paper for shedding some light on drug discovery within the human
Drug Discovery World Spring 2018
kinome. There is overwhelming evidence to suggest that mutations in kinase proteins result in modifi- cations of phosphorylation that in a clinical setting manifest themselves as cancer and inflammatory disease. With 37 kinase inhibitors in the clinic and more than 243 being developed, it is clear that the scientific community recognises the potential to alleviate these diseases with both small molecules and biologics to target kinases. New and innova- tive technologies, when combined with past and current technologies, can help to add value to his- toric data and shed light on our understanding of how compounds can be developed in the future. In addition, it is also possible to repurpose past clini- cal candidates that have failed by revisiting their potential using some of these new technologies, such as residency time. It is possible that focusing on residency time in addition to binding affinity and selectivity may lead to a better understanding of kinase biology and better clinical candidates. So let’s ‘boldly go’ into kinase space using all the tech- nologies available to us.
DDW
References 1 Klaeger, et al. Science 358, eaan4368 (2017). 2 Huwiler, et al. Genetic Engineering and Biotechnology News, Vol. 38, No 2 (2018).
Dr Gary Allenby is the Chief Scientific Officer of Aurelia Bioscience Ltd, a Contract Research Organisation located in Nottingham, UK. Gary is a card-carrying cell biologist and pharmacologist having spent more than 30 years in pre-clinical drug discovery working at Hoffmann La Roche, GSK and AstraZeneca. He has worked within numerous disease areas developing innovative technology assays for screening small molecules and biologics.
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