1. Biflavones from Ginkgo biloba as inhibitors of human thrombin
Tian-Ran Chen, et al. Bioorg Chem. 2019 Nov;92:103199. doi: 10.1016/j.bioorg.2019.103199. Epub 2019 Aug 16.
Ginkgo Biloba leaf extract has been widely used for the prevention and treatment of thrombosis and cardiovascular disease in both eastern and western countries, but the bioactive constituents and the underlying mechanism of anti-thrombosis have not been fully characterized. The purpose of this study was to investigate the inhibitory effects of major constituents in Ginkgo biloba on human thrombin, a key serine protease regulating the blood coagulation cascade and the processes of thrombosis. To this end, a fluorescence-based biochemical assay was used to assay the inhibitory effects of sixteen major constituents from Ginkgo biloba on human thrombin. Among all tested natural compounds, four biflavones (ginkgetin, isoginkgetin, bilobetin and amentoflavone), and five flavonoids (luteolin, apigenin, quercetin, kaempferol and isorhamnetin) were found with thrombin inhibition activity, with the IC50 values ranging from 8.05 μM to 82.08 μM. Inhibition kinetic analyses demonstrated that four biflavones were mixed inhibitors against thrombin-mediated Z-GGRAMC acetate hydrolysis, with the Ki values ranging from 4.12 μM to 11.01 μM. Molecular docking method showed that the four biflavones could occupy the active cavity with strong interactions of salt bridges and hydrogen bonds. In addition, mass spectrometry-based lysine labeling reactivity assay suggested that the biflavones could bind on human thrombin at exosite I rather than exosite II. All these findings suggested that the biflavones in Ginkgo biloba were naturally occurring inhibitors of human thrombin, and these compounds could be used as lead compounds for the development of novel thrombin inhibitors with improved efficacy and high safety profiles.
2. Natural constituents of St. John's Wort inhibit the proteolytic activity of human thrombin
Ling-Hua Wei, Tian-Ran Chen, Hong-Bo Fang, Qiang Jin, Shui-Jun Zhang, Jie Hou, Yang Yu, Tong-Yi Dou, Yun-Feng Cao, Wen-Zhi Guo, Guang-Bo Ge Int J Biol Macromol. 2019 Aug 1;134:622-630. doi: 10.1016/j.ijbiomac.2019.04.181. Epub 2019 Apr 29.
Thrombin, a multifunctional serine protease responsible for the proteolytic hydrolysis of soluble fibrinogen, plays a pivotal role in the blood coagulation cascade. Currently, thrombin inhibitor therapy has been recognized as an effective therapeutic strategy for the prevention and treatment of thrombotic diseases. In this study, the inhibitory effects of natural constituents in St. John's Wort against human thrombin are carefully investigated by a fluorescence-based biochemical assay. The results clearly demonstrate that most of naphthodianthrones, flavonoids and biflavones exhibit strong to moderate inhibition on human thrombin. Among all tested compounds, hypericin shows the most potent inhibitory capability against thrombin, with the IC50 value of 3.00 μM. Further investigation on inhibition kinetics demonstrates that hypericin is a potent and reversible inhibitor against thrombin-mediated Z-GGRAMC acetate hydrolysis, with the Ki value of 2.58 μM. Inhibition kinetic analyses demonstrate that hypericin inhibits thrombin-mediated Z-GGRAMC acetate hydrolysis in a mixed manner, which agrees well with the results from docking simulations that hypericin can bind on both catalytic cavity and anion binding exosites. All these findings suggest that hypericin is a natural thrombin inhibitor with a unique dianthrone skeleton, which can be used as a good candidate to develop novel thrombin inhibitors with improved properties.
3. Discovery of hCES2A inhibitors from Glycyrrhiza inflata via combination of docking-based virtual screening and fluorescence-based inhibition assays
Yun-Qing Song, Xiao-Qing Guan, Zi-Miao Weng, Jun-Ling Liu, Jing Chen, Lu Wang, Long-Tao Cui, Sheng-Quan Fang, Jie Hou, Guang-Bo Ge Food Funct. 2021 Jan 7;12(1):162-176. doi: 10.1039/d0fo02140g. Epub 2020 Dec 8.
Human carboxylesterase 2 (hCES2A) is a key target to ameliorate the intestinal toxicity triggered by irinotecan that causes severe diarrhea in 50%-80% of patients receiving this anticancer agent. Herbal medicines are frequently used for the prevention and treatment of the intestinal toxicity of irinotecan, but it is very hard to find strong hCES2A inhibitors from herbal medicines in an efficient way. Herein, an integrated strategy via combination of chemical profiling, docking-based virtual screening and fluorescence-based high-throughput inhibitor screening assays was utilized. Following the screening of a total of 73 herbal products, licorice (the dried root of Glycyrrhiza species) was found with the most potent hCES2A inhibition activity. Further investigation revealed that the chalcones and several flavonols in licorice displayed strong hCES2A inhibition activities, while isoliquiritigenin, echinatin, naringenin, gancaonin I and glycycoumarin exhibited moderate inhibition of hCES2A. Inhibition kinetic analysis demonstrated that licochalcone A, licochalcone C, licochalcone D and isolicoflavonol potently inhibited hCES2A-mediated fluorescein diacetate hydrolysis in a reversible and mixed inhibition manner, with Ki values less than 1.0 μM. Further investigations demonstrated that licochalcone C, the most potent hCES2A inhibitor identified from licorice, dose-dependently inhibited intracellular hCES2A in living HepG2 cells. In summary, this study proposed an integrated strategy to find hCES2A inhibitors from herbal medicines, and our findings suggested that the chalcones and isolicoflavonol in licorice were the key ingredients responsible for hCES2A inhibition, which would be very helpful to develop new herbal remedies or drugs for ameliorating hCES2A-associated drug toxicity.