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A fluorescent approach for identifying P2X1 ligands.
Marc-David Ruepp1, James A Brozik2, Iwan J P de Esch3
1Department of Chemistry and Biochemistry, University of Bern, Freiestrasse 3, CH-3012 Bern, Switzerland.
Neuropharmacology
|June 1, 2015
Summary
Researchers identified novel P2X1 receptor ligands using voltage-sensitive dyes and fragment-based drug discovery. This cost-effective method screened 1443 fragments, yielding 46 promising leads for P2X1 receptor pharmacology.
Area of Science:
- Pharmacology
- Neuroscience
- Drug Discovery
Background:
- Limited availability of small, receptor-specific P2X1 ligands hinders research.
- Existing antagonists like NF449 are potent but complex; many ligands lack selectivity.
- Rapid receptor desensitization complicates P2X1 screening assays.
Purpose of the Study:
- To develop a flexible and cost-effective method for identifying novel P2X1 receptor ligands.
- To overcome screening challenges posed by P2X1 receptor desensitization.
- To utilize fragment-based drug discovery in conjunction with functional assays.
Main Methods:
- Employed fluorescent voltage-sensitive dyes to monitor P2X1 channel activity via membrane potential changes.
- Screened a library of 1443 fragments at a single concentration (300 μM).
- Validated initial hits by measuring concentration-dependence and using confocal microscopy with a fluorescent ATP competitor.
Main Results:
- Identified 46 novel ligand leads from 1443 fragments, achieving a 3.2% hit rate.
- Validated hits demonstrated promising structural diversity.
- Determined kinetic parameters for Alexa-647-ATP (kon = 1.142 × 10^6 M^-1 s^-1, koff = 0.136 s^-1, Kd = 119 nM).
Conclusions:
- Functional screening using voltage-sensitive dyes is a versatile and cost-effective approach for P2X1 ligand discovery.
- The method is applicable to various ion channels, including other P2X family members.
- This technique allows probing of both agonist and antagonist activities in a single assay.
