Identification and structure activity relationship of small molecule antagonists of the human P2X4 receptor

Ahmad, Izzuddin (2018) Identification and structure activity relationship of small molecule antagonists of the human P2X4 receptor. Doctoral thesis, University of East Anglia.

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P2X4 is a purinergic receptor distributed all over the body with various roles. Among them, it was reportedly overexpressed in several neuronal and immune cell types following peripheral nerve injury and its activation leads to neuropathic pain. Several compounds were found to block P2X4 but none has gone into a clinical stage, probably due to insufficient information about the compound itself and P2X4 in general. One of such compounds, 5-(3-Bromophenyl)-1,3-dihydro-2H-Benzofuro[3,2-e]-1,4-diazepin-2-one (5-BDBD) is known to be widely used in P2X4-related studies despite its limited information. Therefore, this study aimed to (i) find a novel compound that can block the activation of P2X4 through high throughput screening and characterise it, and (ii) study structural-activity relationship between 5-BDBD and P2X4. Human P2X4 receptor was stably expressed in human 1321N1 astrocytoma cells and 1710 compounds from National Cancer Institute were screened for their activity at P2X4. Extensive tests led to identifying a natural product (thaspine) as the most potent inhibitor at reducing P2X4 activation. Further characterisation experiments revealed that thaspine had an IC50 value of 3.8 ± 0.2 μM and showed an allosteric mode, time-dependent and irreversible inhibition. Thaspine was similarly potent at mouse P2X4, but not effective at human P2X2, P2X2/3 and P2X7. It was also inactive at human P2Y2 and P2Y6 at concentrations below 10 μM and 30 μM respectively. In primary microglial cell model (BV2), it inhibited ivermectin-potentiated responses but not normal ATP-evoked responses. Meanwhile, 5-BDBD was found to be inhibiting P2X4 receptor competitively and diazepinone was a pivotal group of the structure to cause inhibition. The binding pocket of 5-BDBD at P2X4 was also predicted using molecular docking. In this study, a novel compound thaspine, has been shown to be effective at inhibiting human P2X4 and thus may have potential therapeutic applications while novel information about 5-BDBD was also acquired.

Item Type: Thesis (Doctoral)
Faculty \ School: Faculty of Science > School of Biological Sciences
Depositing User: Users 9280 not found.
Date Deposited: 07 Sep 2018 12:40
Last Modified: 26 Mar 2020 01:38


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