Identification and characterization of novel ligands for purinergic P2X receptors

Abstract

P2X receptors are ligand-gated ion channels activated by adenosine-5’-triphosphate (ATP). Seven different subtypes exist termed P2X1 - P2X7. P2X receptors exist as homo- or hetero-trimers. They show a ubiquitous expression, but particularly high P2X receptor expression levels are found in the central and peripheral nervous system. P2X receptors are pathophysiologically associated with pain, inflammation and cancer. The aim of this study was the identification and characterization of novel P2X receptor antagonists, with a special focus on the homomeric P2X3 receptor subtype.<br /> The first of three sub-projects dealt with the establishment of a fluorescence-based calcium influx assay for the homomeric S15V mutant of the rat P2X3 receptor. A cell line stably expressing the receptor mutant was kindly provided by PD Dr. Ralf Hausmann of RWTH Aachen. The assay system was established and validated. Since the Z’-factor was higher than 0.5 (0.64 ± 0.02), the assay system was found suitable for high-throughput screening. Subsequent screening of a compound library of anthraquinone derivatives identified several P2X3 receptor antagonists with micromolar potency and a competitive mode of action. The most potent compound was YB120 with an IC50 value of 2.39 ± 1.00 μM. Furthermore, several compounds were identified as positive allosteric modulators of P2X3 receptor activity. Phenylethylamino-substitution at the position 4 of the anthraquinone core could be identified as a structural element that leads to positive allosteric modulation of the P2X3 receptor.<br /> The second sub-project resulted in the identification of polyoxometalates (POMs) as P2X receptor antagonists with low nanomolar potency. The compounds available for testing contained tungsten or rhenium as the main transition metal. For one of the potent compounds, the influence of PEGylation with varying chain lengths on the inhibitory potency was determined and shown to be well tolerated. One of the most potent compounds was the P2X2 receptor antagonist K-POM, a polyoxotungstate containing phosphorus and vanadium, which displayed an IC50 value of 0.00660 ± 0.00117 μM. The mechanism of action was determined to be competitive for several of the investigated structures.<br /> By assessing the inhibitory potency of a library of 440 approved drugs with the aim of repurposing for new targets or to identify new molecular mechanism of action, several compounds were identified as potent antagonists of P2X receptor subtypes. The two most interesting hits, bisacodyl at the P2X7 receptor (IC50 0.0962 ± 0.0324 μM) and niclosamide at P2X3 and P2X7 receptors (IC50 0.0134 ± 0.0029 μM and 0.322 ± 0.129 μM) were selected for further evaluation. A library of nine bisacodyl derivatives was synthesized. The cleavage of the ester residues and further etherification of the phenol residues of bisacodyl shifted the inhibitory potency from the P2X7 to the P2X3 receptor, yielding compounds with nanomolar potency. The most potent compound was the phenol derivative THV-B01 with an IC50 value of 0.0194 ± 0.0036 μM at the P2X3 receptor. The high potency suggests that P2X3 receptor inhibition might contribute to the laxative mechanism of bisacodyl, since the free phenol derivative is its active metabolite. Furthermore, a library of hydroxybenzamide derivatives with high structural similarity to niclosamide led to the identification of compounds with enhanced potency at P2X7, but not at P2X3 receptors. The most potent compound was MT175 with IC50 values of 0.0109 ± 0.0052 μM at P2X3 and 0.0513 ± 0.0025 μM at P2X7 receptors. Further experiments indicated that bisacodyl, niclosamide and the related MT175 act as allosteric antagonists of P2X3 and P2X7 receptors, respectively.<br /> The newly identified P2X receptor ligands constitute a basis for the development of future drugs acting on P2X receptors. Subsequent studies should focus on improving the compounds’ potency, selectivity and pharmacokinetic properties by medicinal chemistry approaches.