Translational Neurodegeneration Section "Albrecht-Kossel", Department of Neurology, University Medical Center Rostock, 18147 Rostock, Germany.
Leibniz Institute for Catalysis, University of Rostock, 18059 Rostock, Germany.
Int J Mol Sci. 2022 Mar 24;23(7):3536. doi: 10.3390/ijms23073536.
Ambroxol (ABX) is a mucolytic agent used for the treatment of respiratory diseases. Bioactivity has been demonstrated as an enhancement effect on lysosomal acid β-glucosidase (β-Glu) activity in Gaucher disease (GD). The positive effects observed have been attributed to a mechanism of action similar to pharmacological chaperones (PCs), but an exact mechanistic description is still pending. The current study uses cell culture and in vitro assays to study the effects of ABX on β-Glu activity, processing, and stability upon ligand binding. Structural analogues bromohexine, 4-hydroxybromohexine, and norbromohexine were screened for chaperone efficacy, and in silico docking was performed. The sugar mimetic isofagomine (IFG) strongly inhibits β-Glu, while ABX exerts its inhibitory effect in the micromolar range. In GD patient fibroblasts, IFG and ABX increase mutant β-Glu activity to identical levels. However, the characteristics of the banding patterns of Endoglycosidase-H (Endo-H)-digested enzyme and a substantially lower half-life of ABX-treated β-Glu suggest different intracellular processing. In line with this observation, IFG efficiently stabilizes recombinant β-Glu against thermal denaturation in vitro, whereas ABX exerts no significant effect. Additional β-Glu enzyme activity testing using Bromohexine (BHX) and two related structures unexpectedly revealed that ABX alone can refunctionalize β-Glu in cellula. Taken together, our data indicate that ABX has little in vitro ability to act as PC, so the mode of action requires further clarification.
氨溴索（ABX）是一种黏液溶解剂，用于治疗呼吸系统疾病。在戈谢病（GD）中，已证明其具有增强溶酶体酸性β-葡萄糖苷酶（β-Glu）活性的生物活性。观察到的积极作用归因于一种作用机制类似于药理学伴侣（PCs），但确切的机制描述仍有待确定。本研究使用细胞培养和体外测定来研究 ABX 对β-Glu 活性、加工和稳定性的影响，以及配体结合。结构类似物溴己新、4-羟基溴己新和去甲溴己新被筛选用于伴侣功效，并且进行了计算机对接。糖类似物异法沙明（IFG）强烈抑制β-Glu，而 ABX 则在微摩尔范围内发挥抑制作用。在 GD 患者成纤维细胞中，IFG 和 ABX 将突变型β-Glu 活性增加到相同水平。然而，Endoglycosidase-H（Endo-H）消化酶的带型特征和 ABX 处理的β-Glu 半衰期明显降低表明了不同的细胞内加工。与这一观察结果一致，IFG 有效地稳定了体外热变性的重组β-Glu，而 ABX 则没有显著影响。使用溴己新（BHX）和两种相关结构进行的额外β-Glu 酶活性测试出人意料地表明，ABX 本身可以在细胞内使β-Glu 重新发挥功能。总之，我们的数据表明 ABX 在体外几乎没有作为 PC 的能力，因此作用模式需要进一步阐明。
