Spider venoms and chronic pain – developing novel pharmacological tools from the spider venoms to target P2X4 in microglia

Bibic, Lucka (2020) Spider venoms and chronic pain – developing novel pharmacological tools from the spider venoms to target P2X4 in microglia. Doctoral thesis, University of East Anglia.

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Today, one in five adults experience chronic pain and this figure increases for those over 65 years old. However, frustration is mounting over the inadequate treatment for chronic neuropathic pain since its symptoms are challenging to treat and often resistant to opioids. Processing of pain signals relies on the activities of ion channels with the microglial P2X4 receptor being an important player. Animal venoms play an essential role in drug discovery as they contain a rich source of bioactive molecules evolutionarily fine-tuned to target ion channels such as P2X receptors. First, we have established and validated several fluorescent-based high throughput screening assays for assessing the activity of venom toxins at P2X receptors. Second, a diverse selection of 180 crude venoms has been screened against human P2X4 in HEK293 and 1321N21 cells, resulting in several venoms containing inhibitors against hP2X4. Two of them, LK-601 and LK-729, were confirmed to be structurally uncharacterized acylpolyamines, which potently inhibited hP2X4 with the apparent IC50 values between 1.1 – 4.5 μM, however only LK-601 showed a relatively high level of selectivity over hP2X3, hP2X7 and NMDA 1a/2a. Species differences were evident with no effect at rat P2X4, however, blocking the mouse P2X4. Using LK-601 as a structural guide, the fragment-based screening was carried out and five smaller toxin analogues chemically synthesized. One of them, LA-3, was found to block the hP2X4 (IC50 of 9.7 – 18.6 μM) and showed selectivity to hP2X4 over hP2X3, hP2X7 and rP2X4 with a modest inhibition at mP2X4. Due to the differential sensitivity of LA-3 to block P2X4 orthologues, the potential binding site were identified, and the validation showed that two crucial amino acid residues, D220 and N238, might be involved in LA-3 binding to hP2X4; however, more experiments are needed to confirm that effect fully. In summary, we discovered a novel toxin from a spider venom with inhibitory activity at human P2X4 ion channels that shows selectivity at hP2X4 over other P2X receptors. Further characterization and validation are required to understand whether these novel compounds could be useful as analgesics.

Item Type: Thesis (Doctoral)
Faculty \ School: Faculty of Science > School of Pharmacy
Depositing User: Chris White
Date Deposited: 14 Jul 2020 14:24
Last Modified: 01 Jan 2021 01:38
URI: https://ueaeprints.uea.ac.uk/id/eprint/76039


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