Developing P2X7 antagonists for the treatment of inflammatory eye disease

Abstract

Purpose : Age-related Macular Degeneration (AMD), glaucoma and Diabetic Retinopathy (DR) are the most common cause of blindness worldwide, with few effective treatments available. All are characterized by a state of chronic inflammation, thought to be at least in part mediated by the P2X7 receptor, an ion channel widely expressed in the eye, which is activated by high concentrations of extracellular ATP present during inflammation. P2X7 activation promotes the release of pro-inflammatory cytokines and cell death, contributing to disease progression and leading to loss of cells critical for vision. Blocking P2X7 activation may present a valid therapeutic strategy to treat chronic inflammation in eye disease.
This study has two main aims; (1) to confirm the involvement of P2X7 activation and signalling in the progression of AMD using retinal pigment epithelial (RPE) cells, and (2) to use structure-based virtual screening methods to develop novel-small molecule P2X7 antagonists.

Methods : For Aim 1, P2X7 expression and function have been evaluated in a human RPE cell line (ARPE-19) using Western blotting and ATP-stimulated fluorescent dye uptake (YO-PRO1). For Aim 2, structure-based screening was performed on human P2X7 molecular models to identify potential hit molecules (from commercially available libraries) likely to bind to orthosteric and allosteric pockets. Potential hits were purchased and tested on 1321N1 astrocytoma cells stably transfected with human P2X7 using ATP-stimulated YO-PRO 1 uptake and the Membrane Potential Red assay (MPR), to assess their ability to inhibit P2X7 activation.

Results : ATP-stimulated YO-PRO1 uptake was observed in non-polarised ARPE-19 cells (EC₅₀ in the low millimolar range), consistent with P2X7 expression, but Western blot analysis failed to detect protein, suggesting low expression levels. A total of 30 potential hit molecules were selected and their activity was assessed by YO-PRO 1 and MPR assays. A number of compounds showed a reduction in the response to ATP compared to control when pre-applied at either 10 µM or 30 µM.

Conclusions : This work demonstrates that ATP-stimulated activity consistent with that of P2X7 is observed in ARPE-19 cells, despite no protein being detected by Western blot. In addition, this study demonstrates the identification of P2X7 antagonist hit compounds, which have the potential to be developed into novel anti-inflammatory therapeutics.

This is a 2021 ARVO Annual Meeting abstract.