Weinhausen Stephanie, Nagel Jessica, Namasivayam Vigneshwaran, Spanier Claudia, Abdelrahman Aliaa, Hanck Theodor, Hausmann Ralf, Müller Christa E
University of Bonn, PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical & Medicinal Chemistry, An der Immenburg 4, 53121 Bonn, Germany.
RWTH Aachen University, Institute of Clinical Pharmacology, Wendlingweg 2, 52074 Aachen, Germany.
Life Sci. 2022 Dec 15;311(Pt A):121143. doi: 10.1016/j.lfs.2022.121143. Epub 2022 Nov 1.
P2X receptors are ATP-gated ion channels which play a role in many pathophysiological conditions. They are considered as novel drug targets, particularly in the fields of pain, (neuro) inflammation, and cancer. Due to difficulties in developing drug-like orthosteric ligands that bind to the highly polar ATP binding site, the design of positive and negative allosteric modulators (PAMs and NAMs) is a promising strategy. The P2X4 receptor was proposed as a novel target for neuropathic and inflammatory pain (antagonists), and for the treatment of alcoholism (PAMs). So far, little is known about the allosteric binding site(s) of P2X4 receptors. The aim of this study was to identify the binding site(s) of the macrocyclic natural product ivermectin, the urea derivative BX430, and the antidepressant drug paroxetine that act as allosteric modulators of P2X4 receptors.
We generated chimeric receptors in which extracellular sequences of the human P2X4 receptor were exchanged for corresponding residues of the human P2X2 receptor, complemented by specific single amino acid residue mutants. Chimeric and mutated receptors were stably expressed in 1321N1 astrocytoma cells, and characterized by fluorimetric measurement of ATP-induced Ca-influx. In addition, docking studies utilizing a homology model of the human P2X4 receptor were performed.
Our results suggest a common binding site for ivermectin and BX430 in an extracellular receptor domain, while paroxetine might bind to the cation pore.
The obtained results provide a basis for the development of positive and negative allosteric P2X4 modulators with improved properties and will support future drug development efforts.
P2X受体是ATP门控离子通道，在许多病理生理状况中发挥作用。它们被视为新型药物靶点，尤其在疼痛、（神经）炎症和癌症领域。由于难以开发与高度极性的ATP结合位点结合的类药物正构配体，设计正变构调节剂（PAMs）和负变构调节剂（NAMs）是一种有前景的策略。P2X4受体被认为是神经性和炎性疼痛（拮抗剂）以及酒精中毒治疗（PAMs）的新型靶点。到目前为止，关于P2X4受体的变构结合位点知之甚少。本研究的目的是确定大环天然产物伊维菌素、尿素衍生物BX430和抗抑郁药帕罗西汀作为P2X4受体变构调节剂的结合位点。
我们构建了嵌合受体，其中人P2X4受体的细胞外序列被替换为人P2X2受体的相应残基，并辅以特定的单氨基酸残基突变体。嵌合受体和突变受体在1321N1星形细胞瘤细胞中稳定表达，并通过荧光法测量ATP诱导的Ca2+内流进行表征。此外，利用人P2X4受体的同源模型进行了对接研究。
我们的结果表明伊维菌素和BX430在细胞外受体结构域有一个共同的结合位点，而帕罗西汀可能结合到阳离子孔。
所得结果为开发具有改进特性的正变构和负变构P2X4调节剂提供了基础，并将支持未来的药物开发工作。
