Discovery and Characterization of Panaxatriol as a Novel Thrombin Inhibitor from Panax notoginseng Using a Combination of Computational and Experimental Approaches

Xing Wang; Yuqing Ma; Chunfang Zuo; Zixi Zhao; Ruonan Ma; Lele Wang; Yuzhen Fang; Yuxin Zhang; Xia Wu

doi:10.1055/a-2339-2720

Planta Medica, Inhaltsverzeichnis

Planta Med 2024; 90(10): 801-809
DOI: 10.1055/a-2339-2720

Biological and Pharmacological Activity

Original Papers

Discovery and Characterization of Panaxatriol as a Novel Thrombin Inhibitor from Panax notoginseng Using a Combination of Computational and Experimental Approaches

Xing Wang ‡

¹Beijing Key Lab of Traditional Chinese Medicine Collateral Disease Theory Research, School of Traditional Chinese Medicine, Capital Medical University, Beijing, China

,

Yuqing Ma‡

¹Beijing Key Lab of Traditional Chinese Medicine Collateral Disease Theory Research, School of Traditional Chinese Medicine, Capital Medical University, Beijing, China

,

Chunfang Zuo

²Pharmacy Department, The 989th Hospital of the Joint Logistics Support Force of the Chinese Peopleʼs Liberation Army, Luoyang, China

,

Zixi Zhao

¹Beijing Key Lab of Traditional Chinese Medicine Collateral Disease Theory Research, School of Traditional Chinese Medicine, Capital Medical University, Beijing, China

,

Ruonan Ma

¹Beijing Key Lab of Traditional Chinese Medicine Collateral Disease Theory Research, School of Traditional Chinese Medicine, Capital Medical University, Beijing, China

,

Lele Wang

³Key Laboratory of Ethnomedicine (Ministry of Education), School of Pharmacy, Minzu University of China, Beijing, China

,

Yuzhen Fang

³Key Laboratory of Ethnomedicine (Ministry of Education), School of Pharmacy, Minzu University of China, Beijing, China

,

Yuxin Zhang

³Key Laboratory of Ethnomedicine (Ministry of Education), School of Pharmacy, Minzu University of China, Beijing, China

,

Xia Wu

¹Beijing Key Lab of Traditional Chinese Medicine Collateral Disease Theory Research, School of Traditional Chinese Medicine, Capital Medical University, Beijing, China

› Institutsangaben

Abstract

Thrombin is a crucial enzyme in the coagulation cascade, and inhibitors of thrombin have been extensively studied as potential antithrombotic agents. The objective of this study was to identify natural inhibitors of thrombin from Panax notoginseng and evaluate their biological activity in vitro and binding characteristics. A combined approach involving molecular docking, thrombin inhibition assays, surface plasmon resonance, and molecular dynamics simulation was utilized to identify natural thrombin inhibitors. The results demonstrated that panaxatriol directly inhibits thrombin, with an IC₅₀ of 10.3 µM. Binding studies using surface plasmon resonance revealed that panaxatriol interacts with thrombin, with a K_D value of 7.8 µM. Molecular dynamics analysis indicated that the thrombin-panaxatriol system reached equilibrium rapidly with minimal fluctuations, and the calculated binding free energy was − 23.8 kcal/mol. The interaction between panaxatriol and thrombin involves the amino acid residues Glu146, Glu192, Gly216, Gly219, Tyr60A, and Trp60D. This interaction provides a mechanistic basis for further optimizing panaxatriol as a thrombin inhibitor. Our study has shown that panaxatriol serves as a direct thrombin inhibitor, laying the groundwork for further research and development of novel thrombin inhibitors.

Keywords

thrombin inhibitor - panaxatriol - Panax notoginseng - molecular dynamics - surface plasmon resonance

Volltext

Referenzen

References
1 Yu J, Wang T, Zhang X, Chen Q, Hu Y, Liang Q, Shi Y, Wang Y, Zhang Y, Yu M, Zhao B. Anti-thrombotic effects mediated by a novel dual-target peptide inhibiting both platelet aggregation and thrombin activity without causing bleeding. Thromb Haemost 2024; 124: 108-121
2 Melzig MF, Henke K. Inhibition of thrombin activity by selected natural products in comparison to neutrophil elastase. Planta Med 2005; 71: 787-789
3 Al-Amer OM. The role of thrombin in haemostasis. Blood Coagul Fibrinolysis 2022; 33: 145-148
4 Wenzel P. Thromboinflammation in cardiovascular disease: Lessons learned for the management of hypertension, heart failure, and aortic valve stenosis. Eur Heart J 2022; 43: 4611-4613
5 Sun W, Han D, Awad MA, Leibowitz JL, Griffith BP, Wu ZJ. Role of thrombin to non-physiological shear stress induced platelet activation and function alternation. Thromb Res 2022; 219: 141-149
6 Cheng S, Wu D, Yuan L, Liu H, Ei-Seedi HR, Du M. Crassostrea gigas-based bioactive peptide protected thrombin-treated endothelial cells against thrombosis and cell barrier dysfunction. J Agric Food Chem 2022; 70: 9664-9673
7 Laura BC, Morales-Maldonado MA, Rodgers A, Kitt LA, Humphry M, Figg N, Bennett MR, Clarke M. Thrombin activated Interleukin-1α drives atherogenesis, but also promotes VSMC proliferation and collagen production. Cardiovasc Res 2023; 119: 2179-2189
8 Hansen FC, Nadeem A, Browning KL, Campana M, Schmidtchen A, van der Plas M. Differential internalization of thrombin-derived host defense peptides into monocytes and macrophages. J Innate Immun 2022; 14: 418-432
9 Huang Y, Li X, Zhu L, Huang C, Chen W, Ling Z, Zhu S, Feng X, Yi C, Gu W, Yan C, Wang J, Ma L, Su X, Dai R, Shi G, Sun B, Zhang Y. Thrombin cleaves IL-33 and modulates IL-33-activated allergic lung inflammation. Allergy 2022; 77: 2104-2120
10 Johnson C, Quach HQ, Lau C, Ekholt K, Espevik T, Woodruff TM, Pischke SE, Mollnes TE, Nilsson PH. Thrombin differentially modulates the acute inflammatory response to Escherichia coli and Staphylococcus aureus in human whole blood. J Immunol 2022; 208: 2771-2778
11 Alexander ET, Gilmour SK. Immunomodulatory role of thrombin in cancer progression. Mol Carcinog 2022; 61: 527-536
12 Yang T, Jiang H, Luo X, Hou Y, Li A, He B, Zhang X, Hao H, Song H, Cai R, Wang X, Wang Y, Yao C, Qi L, Wang Y. Thrombin acts as inducer of proinflammatory macrophage migration inhibitory factor in astrocytes following rat spinal cord injury. J Neuroinflammation 2022; 19: 120
13 Park MJ, Won JH, Kim DK. Thrombin induced apoptosis through calcium-mediated activation of cytosolic phospholipase A₂ in intestinal myofibroblasts. Biomol Ther (Seoul) 2023; 31: 59-67
14 Gupta R, Lin M, Mehta A, Aedma SK, Shah R, Ranchal P, Vyas AV, Singh S, Kluck B, Combs WG, Patel NC. Protease-activated receptor antagonist for reducing cardiovascular events – A review on Vorapaxar. Curr Probl Cardiol 2023; 48: 101035
15 van Paridon P, Panova-Noeva M, van Oerle R, Schulz A, Prochaska JH, Arnold N, Schmidtmann I, Beutel M, Pfeiffer N, Munzel T, Lackner KJ, Ten CH, Wild PS, Spronk H. Relationships between coagulation factors and thrombin generation in a general population with arterial and venous disease background. Thromb J 2022; 20: 32
16 French JK, Edmond JJ, Gao W, White HD, Eikelboom JW. Adjunctive use of direct thrombin inhibitors in patients receiving fibrinolytic therapy for acute myocardial infarction. Am J Cardiovasc Drugs 2004; 4: 107-115
17 Markovitz RC, Healey JF, Parker ET, Meeks SL, Lollar P. The diversity of the immune response to the A2 domain of human factor VIII. Blood 2013; 121: 2785-2795
18 Asanuma K, Yoshikawa T, Yoshida K, Okamoto T, Asanuma Y, Hayashi T, Akita N, Oi T, Nishimura A, Hasegawa M, Sudo A. Argatroban more effectively inhibits the thrombin activity in synovial fluid than naturally occurring thrombin inhibitors. Cell Mol Biol 2016; 62: 27-32
19 Shimizu M, Natori T, Tsuda K, Yoshida M, Kamada A, Oi K, Ishigaku Y, Oura K, Narumi S, Yamamoto M, Terayama Y. Thrombin-induced platelet aggregation-effect of dabigatran using automated platelet aggregometry. Platelets 2020; 31: 360-364
20 Warkentin TE. Bivalent direct thrombin inhibitors: Hirudin and bivalirudin. Best Pract Res Clin Haematol 2004; 17: 105-125
21 Schmidt DE, Chaireti R, Bruzelius M, Holmstrom M, Antovic J, Agren A. Correlation of thromboelastography and thrombin generation assays in warfarin-treated patients. Thromb Res 2019; 178: 34-40
22 Zhang Y, Zhang M, Tan L, Pan N, Zhang L. The clinical use of Fondaparinux: A synthetic heparin pentasaccharide. Prog Mol Biol Transl Sci 2019; 163: 41-53
23 Warkentin TE, Greinacher A, Koster A. Bivalirudin. Thromb Haemost 2008; 99: 830-839
24 Doepker B, Mount KL, Ryder LJ, Gerlach AT, Murphy CV, Philips GS. Bleeding risk factors associated with argatroban therapy in the critically ill. J Thromb Thrombolysis 2012; 34: 491-498
25 Gong JH, Liu GJ, Zhou F, Sun ZQ. Dabigatran-induced chronic progressive immune hemolytic anemia: A case report. J Stroke Cerebrovasc Dis 2020; 29: 104924
26 Mendes TC, Dos RLF, de Souza P, Gebara KS, Junior AG. Cellular and molecular mechanisms of antithrombogenic plants: A narrative review. Curr Pharm Des 2020; 26: 176-190
27 Mazumder T, Salam MA, Mitra S, Hossain S, Hussain MS. Current antithrombotic therapies and prospects of natural compounds in the management of the thrombotic disorder. Nat Resour Hum Health 2022; 3: 134-175
28 Fan W, Liao Q, Fan L, Li Q, Liu L, Wang Z, Mei Y, Li L, Yang L, Wang Z. An innovative processing driven efficient transformation of rare ginsenosides enhances anti-platelet aggregation potency of notoginseng by integrated analyses of processing-(chemical) profiling-pharmacodynamics. J Ethnopharmacol 2023; 319: 117126
29 Huang LF, Shi HL, Gao B, Wu H, Yang L, Wu XJ, Wang ZT. Decichine enhances hemostasis of activated platelets via AMPA receptors. Thromb Res 2014; 133: 848-854
30 Yang X, Xiong X, Wang H, Wang J. Protective effects of Panax notoginseng saponins on cardiovascular diseases: A comprehensive overview of experimental studies. Evid Based Complement Alternat Med 2014; 2014: 204840
31 Cao Y, Li Q, Yang Y, Ke Z, Chen S, Li M, Fan W, Wu H, Yuan J, Wang Z, Wu X. Cardioprotective effect of stem-leaf saponins from Panax notoginseng on mice with sleep derivation by inhibiting abnormal autophagy through PI3K/Akt/mTOR pathway. Front Cardiovasc Med 2021; 8: 694219
32 He X, Deng FJ, Ge JW, Yan XX, Pan AH, Li ZY. Effects of total saponins of Panax notoginseng on immature neuroblasts in the adult olfactory bulb following global cerebral ischemia/reperfusion. Neural Regen Res 2015; 10: 1450-1456
33 Salomon-Ferrer R, Götz AW, Poole D, Le Grand S, Walker RC. Routine microsecond molecular dynamics simulations with AMBER on GPUs. 2. Explicit solvent particle mesh Ewald. J Chem Theory Comput 2013; 9: 3878-3888
34 Hui Z, Sha DJ, Wang SL, Li CS, Qian J, Wang JQ, Zhao Y, Zhang JH, Cheng HY, Yang H, Yu LJ, Xu Y. Panaxatriol saponins promotes angiogenesis and enhances cerebral perfusion after ischemic stroke in rats. BMC Complement Altern Med 2017; 17: 70
35 Wu Q, Wang R, Shi Y, Li W, Li M, Chen P, Pan B, Wang Q, Li C, Wang J, Sun G, Sun X, Fu H. Synthesis and biological evaluation of panaxatriol derivatives against myocardial ischemia/reperfusion injury in the rat. Eur J Med Chem 2020; 185: 111729
36 Ruhmann EH, Rupp M, Betz M, Heine A, Klebe G. Boosting affinity by correct ligand preorganization for the S2 Pocket of thrombin: A study by isothermal titration calorimetry, molecular dynamics, and high-resolution crystal structures. ChemMedChem 2016; 11: 309-319
37 Li Y, Zhang Y, Wu X, Gao Y, Guo J, Tian Y, Lin Z, Wang X. Discovery of natural 15-LOX small molecule inhibitors from Chinese herbal medicine using virtual Screening, biological evaluation and molecular dynamics studies. Bioorg Chem 2021; 115: 105197
38 Zhao C, Zhou T, Li M, Liu J, Zhao X, Pang Y, Liu X, Zhang J, Ma L, Li W, Yao X, Feng S. Argatroban promotes recovery of spinal cord injury by inhibiting the PAR1/JAK2/STAT3 signaling pathway. Neural Regen Res 2024; 19: 434-439
39 Koromili M, Kapourani A, Valkanioti V, Manioudaki AE, Assimopoulou AN, Barmpalexis P. In depth analysis of pilocarpine-carbomer molecular interactions via molecular dynamics simulations. Planta Med 2022; 88: 1576-1577
40 Frisch MJ, Trucks GW, Schlegel HB, Scuseria GE, Robb MA, Cheeseman JR, Scalmani G, Barone V, Petersson GA, Nakatsuji H. Gaussian 16 Revision C. 01. Wallingford CT: Gaussian, Inc.; 2016. Available online (accessed on 16. October 2023): https://gaussian.com/
41 Case DA, Belfon K, Ben-Shalom I, Brozell SR, Cerutti D, Cheatham T, Cruzeiro VWD, Darden T, Duke RE, Giambasu G. Amber 2020. 2020 Available online: https://ambermd.org/CiteAmber.php accessed on 16 October 2023
42 Jorgensen WL, Chandrasekhar J, Madura JD, Impey RW, Klein ML. Comparison of simple potential functions for simulating liquid water. J Chem Phys 1983; 79: 926-935
43 Maier JA, Martinez C, Kasavajhala K, Wickstrom L, Hauser KE, Simmerling C. ff14SB: Improving the accuracy of protein side chain and backbone parameters from ff99SB. J Chem Theory Comput 2015; 11: 3696-3713
44 Darden T, York D, Pedersen L. Particle mesh Ewald: An N⋅log (N) method for Ewald sums in large systems. J Chem Phys 1993; 98: 10089-10092
45 Miller 3rd BR, McGee jr. TD, Swails JM, Homeyer N, Gohlke H, Roitberg AE. MMPBSA.py: An efficient program for end-state free energy calculations. J Chem Theory Comput 2012; 8: 3314-3321

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