Pasqualetto Gaia, Zuanon Marika, Brancale Andrea, Young Mark T
School of Biosciences, Cardiff University, Cardiff, United Kingdom.
School of Pharmacy and Pharmaceutical Sciences, Cardiff University, Cardiff, United Kingdom.
Front Pharmacol. 2023 Jan 12;13:1094607. doi: 10.3389/fphar.2022.1094607. eCollection 2022.
P2X4 and P2X7 receptors are ATP-gated ion channels, which play important roles in neuropathic and inflammatory pain, and as such they are important drug targets in diseases of inflammatory origin. While several compounds targeting P2X4 and P2X7 receptors have been developed using traditional high-throughput screening approaches, relatively few compounds have been developed using structure-based design. We initially set out to develop compounds targeting human P2X4, by performing virtual screening on the orthosteric (ATP-binding) pocket of a molecular model of human P2X4 based on the crystal structure of the receptor. The screening of a library of approximately 300,000 commercially available drug-like compounds led to the initial selection of 17 compounds; however, none of these compounds displayed a significant antagonist effect at P2X4 in a Fluo-4 ATP-induced calcium influx assay. When the same set of compounds was tested against human P2X7 in an ATP-stimulated Yo-Pro1 dye uptake assay, one compound (an indeno(1,2-b)pyridine derivative; GP-25) reduced the response by greater than 50% when applied at a concentration of 30 µM. GP-25 displayed an IC value of 8.7 μM at human P2X7 and 24.4 μM at rat P2X7, and was confirmed to be active using whole-cell patch clamp electrophysiology and not cytotoxic. Schild analysis suggested that mode of action of GP-25 was orthosteric. Screening of a further 16 commercially available analogues of GP-25 led to the discovery of five additional compounds with antagonist activity at human P2X7, enabling us to investigate the structure-activity relationship. Finally, docking of the R- and S-enantiomers of GP-25 into the orthosteric pocket of molecular models of human P2X4 and human P2X7 revealed that, while both enantiomers were able to make multiple interactions between their carboxyl moieties and conserved positively charged amino-acids in human P2X7, only the S-enantiomer of GP-25 was able to do this in human P2X4, potentially explaining the lack of activity of GP-25 at this receptor.
P2X4和P2X7受体是ATP门控离子通道，在神经性疼痛和炎性疼痛中发挥重要作用，因此它们是炎症性疾病的重要药物靶点。虽然已经使用传统的高通量筛选方法开发了几种靶向P2X4和P2X7受体的化合物，但使用基于结构的设计开发的化合物相对较少。我们最初着手开发靶向人P2X4的化合物，通过基于该受体的晶体结构对人P2X4分子模型的正构（ATP结合）口袋进行虚拟筛选。对大约300,000种市售类药物化合物库的筛选导致初步选择了17种化合物；然而，在Fluo-4 ATP诱导的钙内流试验中，这些化合物在P2X4上均未显示出显著的拮抗作用。当在ATP刺激的Yo-Pro1染料摄取试验中针对人P2X7测试同一组化合物时，一种化合物（茚并(1,2-b)吡啶衍生物；GP-25）在浓度为30 μM时应用，使反应降低了50%以上。GP-25在人P2X7上的IC值为8.7 μM，在大鼠P2X7上为24.4 μM，并通过全细胞膜片钳电生理学证实具有活性且无细胞毒性。Schild分析表明GP-25的作用模式是正构的。对另外16种市售的GP-25类似物进行筛选，发现了另外5种对人P2X7具有拮抗活性的化合物，使我们能够研究构效关系。最后，将GP-25的R和S对映体对接至人P2X4和人P2X7分子模型的正构口袋中，结果显示，虽然两种对映体都能够在其羧基部分与人P2X7中保守的带正电荷氨基酸之间进行多次相互作用，但只有GP-25的S对映体能够在人P2X4中做到这一点，这可能解释了GP-25在该受体上缺乏活性
