Developing P2X7 antagonists for the treatment of inflammatory eye disease using structure- and ligand-based virtual screening

Zuanon, Marika 2023. Developing P2X7 antagonists for the treatment of inflammatory eye disease using structure- and ligand-based virtual screening. PhD Thesis, Cardiff University. Item availability restricted.

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Abstract

P2X7 is a non-selective cation channel that is activated by high concentrations of extracellular ATP. Its activation leads to the release of proinflammatory cytokines and cell death, making P2X7 a promising drug target for chronic inflammatory diseases including Age-related Macular Degeneration (AMD), Glaucoma and Diabetic Retinopathy. To identify novel P2X7 antagonists, we prepared a molecular model of the human P2X7 receptor, and performed virtual screening, computationally analysing the binding of one million commercially available drug-like compounds at three potential binding pockets; the orthosteric pocket (ATP binding site), an allosteric pocket (adjacent to the orthosteric pocket) and the recently discovered GDP pocket in the cytoplasmic ‘ballast’ domain (thought to couple channel activation to downstream signalling). From our screen, 33 potential ‘hit’ compounds were selected and biologically evaluated on cells stably transfected with human P2X7 using two distinct functional assays; YO-PRO 1 dye uptake and Membrane Potential Red. Two molecules screened against the allosteric pocket (termed 7 and 26) inhibited P2X7 activation in both assays. The structure-activity relationship (SAR) of these two molecules, along with another hit compound previously identified (68), was studied, leading to the testing of a further 30 analogues and the identification of further two molecules (7g and 68d) with YO-PRO 1 IC50 values of 1.31± 0.2 µM and 204.0 ± 75.4 nM respectively. Our hit compounds may have potential for the treatment of inflammatory eye diseases, such as AMD. To test this, we used a human retinal pigment epithelial cell line which is a model system for AMD and has previously been reported to express P2X7 receptors (ARPE-19) but we were unable to detect P2X7 function or protein expression, even in conditions mimicking eye disease (high glucose, cobalt chloride-induced hypoxia or pre-treatment with ATP). We were, however, able to verify P2X7 protein expression in the posterior eye, using porcine and mouse ocular tissue, but functional assays were not attempted. To further investigate the GDP pocket, which has also been reported to be able to bind GTP, we used a combination of molecular docking and equilibrium molecular dynamics (MD) simulations on a model of human P2X7 embedded in a membrane. We found that although GDP and GTP present a similar binding mode, MD reveals a series of subtle conformational rearrangements in GDP-bound versus GTP-bound models, suggesting that GDP-GTP exchange may alter the structure of this important intracellular signalling domain.

Item Type:	Thesis (PhD)
Date Type:	Completion
Status:	Unpublished
Schools:	Biosciences
Subjects:	Q Science > Q Science (General)
Date of First Compliant Deposit:	4 June 2024
Last Modified:	04 Jun 2024 16:03
URI:	https://orca.cardiff.ac.uk/id/eprint/169464

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