Structure-activity relation of ivermectin: a Positive Allosteric Modulator of the human P2X4 receptor

Meades, Jessica (2023) Structure-activity relation of ivermectin: a Positive Allosteric Modulator of the human P2X4 receptor. Doctoral thesis, University of East Anglia.

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Ivermectin (IVM) is as a positive allosteric modulator of the P2X4 receptor, a ligand-gated ion channel activated by ATP. Various features restrict IVM as a viable pharmacological tool for P2X4 receptor modulation, including its ability to bind and modulate other mammalian ion channels. Moreover, IVM's binding site and mode of action at the P2X4 receptor has yet to be fully elucidated. Lack of selective modulators for P2X4 channels has hindered research advancement, despite the growing evidence implicating P2X4 as a feasible therapeutic target in several cardiovascular and neurological disorders.
In this study, a library of IVM-analogues was investigated, and the structure-activity relationship was examined by calcium influx assays in 1321N1 cells stably expressing the human P2X4 receptor. Pharmacologically characterising these compounds has allowed ranking of potency and efficacy, and the chemical features of IVM that produce effects at P2X4 to be determined. Such features include redundancy of the large disaccharide moiety and positioning of carbon chains in the spiroketal group. The novel chemical information determined will be useful in the development of more efficacious and potent positive allosteric modulators of P2X4. Another objective of this study was to perform a selectivity screen of the IVM analogues against the GABA(A) receptor. Each compound was screened as an agonist of the GABA(A) receptor at two fixed concentrations (1 µM and 10 µM) using the FLIPR membrane potential assay in L(tk-) cells stably expressing the human recombinant GABA(A) (α1β3γ2) receptor. Two of the 33 compounds screened were identified as having selectivity towards the human P2X4 receptor over the GABA(A) receptor. The information gained from this screen will aid in the discovery and design of novel compounds for selective targeting of P2X4 receptors for future medications.
The P2X4 receptor is highly expressed in vascular endothelial cells. Multiple studies have linked activation of this receptor to the regulation of vascular tone by promoting flow-dependent vasodilation in blood vessels. To test this hypothesis, pressure myography studies were performed on the isolated second-order mesenteric arteries of male mice. This provides an ex vivo model to study the effect of IVM in the vasculature at physiological pressures and rates of luminal flow. In this study, IVM was found to significantly potentiate flow-mediated vasodilation in pre-constricted mouse mesenteric arteries. Since IVM is known to enhance P2X4 function, this data suggests that P2X4 receptors are involved in the flow-sensitive mechanism that regulates blood pressure. This demonstrates the therapeutic potential of positive modulation of P2X4 in the treatment of cardiovascular diseases such as hypertension or ischemia.

Item Type: Thesis (Doctoral)
Faculty \ School: Faculty of Science > School of Biological Sciences
Depositing User: Kitty Laine
Date Deposited: 27 Jun 2023 11:35
Last Modified: 27 Jun 2023 11:35

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