A Fluorescent Approach for Identifying P2X1 Ligands
Brozik, James A
de, Esch Iwan JP
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Ruepp, M., Brozik, J. A., de, E. I. J., Farndale, R., Murrell-Lagnado, R., & Thompson, A. (2015). A Fluorescent Approach for Identifying P2X1 Ligands. Neuropharmacology, 98 13-21. https://doi.org/10.1016/j.neuropharm.2015.05.016
There are no commercially available, small, receptor-specific P2X1 ligands. The natural agonist is ATP, of which there are several synthetic derivatives and there are also several structurally-complex antagonists such as PPADS, NTP-ATP, suramin and its derivatives such as NF449. NF449 is the most potent and selective P2X1 receptor ligand, but the potencies of many of the others are not particularly high and can also have actions at other P2X, P2Y and non-purinergic receptors. While there is clearly scope for further work on the pharmacology of P2X1 receptors it can be difficult so screen for novel ligands owing to the rapid desensitisation of the P2X1 receptor agonist response. To remove this rapid desensitisation, substitutions can be made within the N-terminus of the P2X1 receptor and ligands tested on the more slowly desensitising mutant, although there is a danger that such changes could also affect ligand properties. Another approach is the use of fluorescent voltage-sensitive dyes that respond to the changes in membrane potential that result from channel opening. Here we utilised this second approach in conjunction with fragment-based drug-discovery. Using a single concentration (300 µM) of each fragment we identified 46 novel leads from a fragment library of 1443 compounds, giving an overall hit rate of 3.2 %. These hits were independently validated by measuring the concentration-dependence of the fluorescent response using the same voltage-sensitive dye, and by visualising the competition of identified compounds with an Alexa-647-ATP fluorophore using confocal microscopy; confocal measurements of kon (1.142 x 106 M-1 s-1) and koff (0.136 s-1) yielded a Kd (119 nM) for Alexa-647-ATP. The identified hit fragments showed a promising structural diversity. In summary, the measurement of functional responses using voltage-sensitive dyes provided a flexible and cost-effective approach because labelled competitive ligands were not needed, effects were not dependent upon competition at a known binding site, and both agonist and antagonist actions were probed in a single assay. As the method uses voltage-sensitive dyes it can be applied to all P2X family members and other receptor types, including voltage-gated and ligand-gated ion channels.
Our thanks are given to Richard Evans for the P2X1 cDNA, and to Prof. Oliver Mühlemann for kindly providing lab space for M-DR. M-DR was supported by the HOLCIM Stiftung zur Förderung der wissenschaftlichen Fortbildung. The British Heart Foundation supported AJT (grant; PG/13/39/30293) and RWF (grant; RG/09/003/27122).
British Heart Foundation (PG/13/39/30293)
British Heart Foundation (RG/09/003/27122)
External DOI: https://doi.org/10.1016/j.neuropharm.2015.05.016
This record's URL: https://www.repository.cam.ac.uk/handle/1810/248447
Attribution-NonCommercial-NoDerivs 2.0 UK: England & Wales
Licence URL: http://creativecommons.org/licenses/by-nc-nd/2.0/uk/
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