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Application Note


Real-time cellular assay quantitatively evaluates receptor-ligand binding


R


eceptor tyrosine kinases (RTK) are located at cell membranes where they transmit extracellular signals


into the cell. For example, the RTK vascular endothelial growth factor receptor 2 (VEGFR2) responds to stimulation by vas- cular endothelial growth factor (VEGF) by activating the MAPK pathway. On a cellu- lar level, this regulates proliferation and growth; on amulticellular level the receptor is implicated in angiogenesis. However, dis- turbance of the system in either the RTK, levels in VEGF or in the downstream path- way is related to increased tumour angio- genesis and growth. Therefore, the pathway is targeted by anti-cancer drugs such as Bevacizumab which inhibits VEGF1. This is why methods to study the interac-


tion of existing drugs and novel VEGFR2 modulators are needed.Due to its size,most assays work currently with parts of the receptor instead of studying the full length protein. Here, we show a method that anal-


Association kinetics of ligand binding to receptor tyrosine kinase With the NanoBRET assay binding of


yses kinetic binding of VEGF to VEGFR2 in live cells using the full length receptor.


Principle of the NanoBRET receptor-ligand binding assay The interaction assay is based on NanoBRET, a method employing energy transfer


from a donor


(Nanoluciferase-Nluc) to an acceptor fluo- rophore. The donor in the ligand binding assay is a fusion protein of VEGFR2 and Nluc stably expressed inHEK293 cells. This means the full length receptor is available for the studies. The VEGF isoform


VEGF165a coupled to the TMR fluorophore served as an acceptor. A novel labelling tech- nique allowed stoichiomet- ric labelling of the acceptor:


VEGF165a was expressed with a HaloTag that was proteolytically removed in order to allow conjugation with 6-TMR-PEG-CBT. As


a result, VEGF165a was conjugated with TMR at a single N-terminal cysteine. In presence of a luciferase substrate, the donor emits light (Nluc-VEGFR2), but only if


the acceptor


(VEGF165a-TMR) interacts with the receptor, luciferase


AAssay principle of NanoBRET assay to studyVEGF binding to


VEGFR2.Only if the ligand (VEGF165a-TMR) is bound to the receptor (Nluc-VEGFR2) energy transfer takes place and emission of fluorophore can be detected.B Ligand binding kinetics of


VEGF165a-TMR to NLuc-VEGFR2 in HEK293 cells. Furimazine was added to each well and after 5mins of equilibration the reaction was started by addingVEGF165a-TMR at the indicated concentration2


22


and fluorophore are close enough to transfer energy (Figure A) and thus for the fluorophore to emit light. The energy transfer is mea- sured by recording lumines- cence and fluorescence in a microplate reader and cal- culating their ratio after- wards.


VEGF165a to VEGFR2 was monitored in live cells. HEK293 cells expressing Nluc- VEGFR2 were exposed to increasing con- centrations of VEGF-TMR and both sig- nals (luminescence and fluorescence) were recorded on the PHERAstar microplate reader (BMG LABTECH) every 0.5s. The


luciferase


addition of 20nM VEGF165a-TMR result- ed in a steep increase in BRET ratio over the first six minutes after addition indica- tive of


ligand binding (Figure B).


Equilibrium at high level (approximately 0.012 BRET ratio) was reached after 10 minutes. As expected, lower concentrations displayed lower BRET ratios. The kinetic data allowed for calculation of the equilib-


rium dissociation constant KD (2.27nM), association rate kon (4.78 x 107 min-1M-1) and dissociation rate koff (0.11 min-1)2.


Conclusion The combination of an Nluc-fused full- length receptor with a stoichiometric labelled ligand allowed for recording of the ligand-binding event in real-time and live cells. However, in order to achieve the required temporal resolution, fast acquisi- tion of data is indispensable. The PHERAstar is specifically designed for fast, reliable and sensitivemeasurements. It does so by measuring two signals at the same time: luminescence and fluorescence. This does not only speed up the process but fur- ther provides highest quality of the data since both values are equally affected by possible interferences. The data prove the suitability of the novel NanoBRET assay combined with the PHERAstar microplate reader to study ligands binding to RTKs kinetically.


DDW


References 1 Shibuya,M (2011).Genes Cancer. 2011 Dec; 2(12): 1097-1105. doi: 10.1177/1947601911423031. 2 Kilpatrick, LE et al (2017). Biochem Pharmacol. Jul 15; 136:62-75. doi: 10.1016/j.bcp.2017.04.006.


Drug DiscoveryWorld Summer 2019


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