Evaluation of pharmacological properties of Danshen compounds by computational and biochemical approaches
Creators: name: Zhou, Zhiwei
Publisher: name: RMIT University
Year: 2024

description: Endothelial dysfunction, caused by imbalance of responses to endothelium derived vasodilators and contracting factors, plays an important role in the pathogenesis of vascular diseases. Endothelial nitric oxide synthase (eNOS) uncoupling, occurred under oxidative stress, is a key driver to endothelial dysfunction. Functional eNOS requires adequate cofactors. Alterations of bioavailability of these cofactors, particularly, tetrahydrobiopterin (BH4) or other regulatory mechanisms such as heat shock protein (HSP) 90 can cause eNOS uncoupling. <br><br>Danshen has been used in traditional medicine to treat vascular diseases. Tanshinones, the active components of Danshen, have certain actions on the endothelium. However, their actions on eNOS uncoupling have not been investigated. Additionally, the relationship between chemical characteristics of Danshen compounds and their potential drug capability and the profile of absorption, distribution, metabolism, extraction and toxicity (ADME/Tox) are not fully understood. Thus, this project aims to evaluate the effects of Danshen compounds on eNOS uncoupling and the ADME/Tox profiles of natural compounds. <br><br>Firstly, the drug potential and toxicity risk of Danshen compounds were studied using established computational tools. Among 94 compounds evaluated, 76 compounds showed drug-like properties including major tanshinones-tanshinone I (Tan I), tanshinone IIA (Tan IIA) and cryptotanshinone (CT). Irritating effect is a potential toxicity risk of these compounds. Literature analysis indicates a number of commonly used natural compounds including Danshen compounds are the substrate or ligand of drug metabolizing enzymes, transporters, and nuclear receptors both in experimental and computational studies.<br><br>Secondly, eNOS uncoupling in human umbilical vein endothelial cell line (EA.hy926) was studied using a high glucose-induced eNOS uncoupling model. Cells were exhibited reduced nitric oxide (NO) and increased eNOS derived superoxide, reduced ratio of dimer/monomer of eNOS and BH4 bioavailability, and increased nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4) expression. Phosphoinositide 3-kinase (PI3K) signaling pathway was also enhanced and HSP90 expression was decreased. <br><br>Using this cell model, The effects of Tan I, Tan IIA and CT were tested on eNOS uncoupling. All three compounds significantly restored high glucose-induced eNOS uncoupling, with increased BH4 bioavailability and NO production, reduced superoxide generation and the expression of NOX4, up-regulated the expressions of guanosine triphosphate cyclohydrolase I (GTPCH1), dihydrofolate reductase (DHFR) and HSP90, down-regulated the expression of PI3K. In addition, two synthetic analogues of tanshinones were also found with similar effects on eNOS uncoupling. <br><br>The findings suggest that computational analysis of Danshen compounds is capable of predicting their drug potential and toxicity risk. The study has also demonstrated for the first time the mechanism of actions of tanshinones and two synthetic compounds on eNOS uncoupling via regulation of BH4 bioavailability and the expressions of GTPCH1 and DHFR, reduction of the expression of NOX4 and up-regulation of HSP90. The regulation of NOX4 by tanshinones may be related to PI3K pathway. These findings indicate that tanshinones may be used as a protype to develop new agents to treat eNOS uncoupling-mediated vascular diseases. Finally, an integration of computational approaches with well-established biochemical approaches make an important contribution in natural products research and drug discovery and development in the future.<br>