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Structure-based design of P2X receptor small molecule modulators

Pasqualetto, Gaia 2018. Structure-based design of P2X receptor small molecule modulators. PhD Thesis, Cardiff University.
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Abstract

P2X4 and P2X7 receptors have gained increasing significance as drug targets for their involvement in neurotransmission, pain, cancer, inflammation and immunity. To date, numerous P2X7 antagonists have been developed, yet relatively few P2X4 antagonists have been reported, and no data is available regarding their binding sites on the receptor. Using in silico techniques, we attempted to identify novel P2X4 and P2X7 modulators, and combined experimental and structural data to explore allosteric pockets with future potential for drug design. Two virtual screenings of commercially available drug-like compounds performed in the human P2X4 homology model led to the selection, purchase and biological evaluation via calcium influx assay of 42 compounds. While no compound with significant antagonist activity at human P2X4 was found, multiple compounds abolished ATP-induced YO-PRO dye uptake at human P2X7, including Compound 25 (IC50 value of 8 μM). Further 27 structural analogues to Compound 25 were purchased and assayed, expanding the number of active antagonists and offering an insight in the structure-activity relationship. In parallel, a mutagenesis study of human and rat P2X4 receptors based on (i) species-specific pharmacology and (ii) the recently published panda P2X7 crystal structures led us to identify the allosteric binding site for the P2X4-selective antagonist BX-430. The pocket was then used to perform a new virtual screen, identifying 20 potential ‘hit’ candidates for future biological evaluation. Finally, we performed docking simulations of the positive P2X4 allosteric modulator ivermectin and the partially selective P2X7 agonist 2’(3’)-4-O-benzoylbenzoyl A TP (BzA TP), with good correlation between binding conformations and previously published pharmacological data. In conclusion, although no novel human P2X4 antagonist has been identified, this work led to (i) the discovery of an allosteric site not previously described in human P2X4, (ii) the identification of a novel P2X7 antagonist with micromolar potency and (iii) and increased understanding of the molecular basis for subtype-specific modulator potency at P2X4 and P2X7 receptors.

Item Type: Thesis (PhD)
Date Type: Completion
Status: Unpublished
Schools: Biosciences
Pharmacy
Subjects: R Medicine > RM Therapeutics. Pharmacology
Funders: Life Science Research Network Wales
Date of First Compliant Deposit: 21 May 2019
Last Modified: 21 May 2019 10:00
URI: http://orca-mwe.cf.ac.uk/id/eprint/122715

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