KI04 是一种氨基糖苷类衍生分子，可作为人源连接蛋白 46 半通道在 HeLa 细胞中表达的抑制剂。

KI04 an Aminoglycosides-Derived Molecule Acts as an Inhibitor of Human Connexin46 Hemichannels Expressed in HeLa Cells.

机构信息

Department of Chemistry and Biochemistry, Utah State University, Logan, UT 84322-0300, USA.

Laboratorio de Neurobiología, Facultad de Medicina y Facultad de Ciencias de la Vida, Instituto de Ciencias Biomédicas, Universidad Andres Bello, Santiago 7780272, Chile.

出版信息

Biomolecules. 2023 Feb 22;13(3):411. doi: 10.3390/biom13030411.

DOI:10.3390/biom13030411

PMID:36979346

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC10046693/

Abstract

BACKGROUND

Connexins (Cxs) are proteins that help cells to communicate with the extracellular media and with the cytoplasm of neighboring cells. Despite their importance in several human physiological and pathological conditions, their pharmacology is very poor. In the last decade, some molecules derived from aminoglycosides have been developed as inhibitors of Cxs hemichannels. However, these studies have been performed in , which is a very simple model. Therefore, our main goal is to test whether these molecules have similar effects in mammalian cells.

METHODS

We transfected HeLa cells with the human Cx46tGFP and characterized the effect of a kanamycin-derived molecule (KI04) on Cx46 hemichannel activity by time-lapse recordings, changes in phosphorylation by Western blot, localization by epifluorescence, and possible binding sites by molecular dynamics (MD).

RESULTS

We observed that kanamycin and KI04 were the most potent inhibitors of Cx46 hemichannels among several aminoglycosides, presenting an IC close to 10 μM. The inhibitory effect was not associated with changes in Cx46 electrophoretic mobility or its intracellular localization. Interestingly, 5 mM DTT did not reverse KI04 inhibition, but the KI04 effect completely disappeared after washing out KI04 from the recording media. MD analysis revealed two putative binding sites of KI04 in the Cx46 hemichannel.

RESULTS

These results demonstrate that KI04 could be used as a Cx46 inhibitor and could help to develop future selective Cx46 inhibitors.

摘要

背景

连接蛋白（Cxs）是帮助细胞与细胞外介质和相邻细胞的细胞质进行通讯的蛋白质。尽管它们在人类多种生理和病理条件中都具有重要作用，但它们的药理学性质却很差。在过去的十年中，一些源自氨基糖苷类的分子已被开发为 Cx 半通道的抑制剂。然而，这些研究都是在中进行的，这是一种非常简单的模型。因此，我们的主要目标是测试这些分子在哺乳动物细胞中是否具有相似的作用。

方法

我们用 Cx46tGFP 转染 HeLa 细胞，并通过时差记录、Western blot 检测磷酸化变化、荧光显微镜检测定位以及分子动力学（MD）检测可能的结合位点，来表征来源于卡那霉素的分子（KI04）对 Cx46 半通道活性的影响。

结果

我们观察到，在几种氨基糖苷类中，卡那霉素和 KI04 是对 Cx46 半通道抑制作用最强的抑制剂，其 IC 接近 10 μM。抑制作用与 Cx46 的电泳迁移率或其细胞内定位的变化无关。有趣的是，5 mM DTT 不能逆转 KI04 的抑制作用，但在从记录介质中冲洗出 KI04 后，KI04 的作用完全消失。MD 分析揭示了 KI04 在 Cx46 半通道中的两个可能的结合位点。