Study on Structure Activity Relationship of Natural Flavonoids Against Thrombin by Molecular Docking Virtual Screening Combined with Activity Evaluation in Vitro
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molecules Article Study on Structure Activity Relationship of Natural Flavonoids against Thrombin by Molecular Docking Virtual Screening Combined with Activity Evaluation In Vitro 1,2, 3,4, 3,4 1 1,2 Xiaoyan Wang y, Zhen Yang y, Feifei Su , Jin Li , Evans Owusu Boadi , Yan-xu Chang 1,2,* and Hui Wang 3,4,* 1 Tianjin State Key Laboratory of Modern Chinese Medicine, Tianjin University of Traditional Chinese Medicine, Tianjin 300193, China 2 Tianjin Key Laboratory of Phytochemistry and Pharmaceutical Analysis, Tianjin University of Traditional Chinese Medicine, Tianjin 300193, China 3 Tianjin Key Laboratory of Chinese Medicine Pharmacology, Tianjin University of Traditional Chinese Medicine, Tianjin 300193, China 4 College of Chinese Materia Medica, Tianjin University of Traditional Chinese Medicine, Tianjin 300193, China * Correspondence: [email protected] (Y.-x.C.); [email protected] (H.W.); Tel./Fax: +86-22-59596163 (Y.-x.C.) These authors contributed equally to this work. y Received: 20 December 2019; Accepted: 18 January 2020; Published: 20 January 2020 Abstract: Thrombin, a key enzyme of the serine protease superfamily, plays an integral role in the blood coagulation cascade and thrombotic diseases. In view of this, it is worthwhile to establish a method to screen thrombin inhibitors (such as natural flavonoid-type inhibitors) as well as investigate their structure activity relationships. Virtual screening using molecular docking technique was used to screen 103 flavonoids. Out of this number, 42 target compounds were selected, and their inhibitory effects on thrombin assayed by chromogenic substrate method. The results indicated that the carbon-carbon double bond group at the C2, C3 sites and the carbonyl group at the C4 sites of flavones were essential for thrombin inhibition, whereas the methoxy and O-glycosyl groups reduced thrombin inhibition. Noteworthy, introduction of OH groups at different positions on flavonoids either decreased or increased anti-thrombin potential. Myricetin exhibited the highest inhibitory potential against thrombin with an IC50 value of 56 µM. Purposively, the established molecular docking virtual screening method is not limited to exploring flavonoid structure activity relationships to anti-thrombin activity but also usefully discovering other natural active constituents. Keywords: flavonoids; molecular docking; structure activity relationship; thrombin 1. Introduction In recent years, thrombotic disease has been a major threat to human health worldwide. As mentioned earlier, thrombin, a principal serine protease, plays a critical role in blood coagulation cascade response by activating blood platelet and catalyzing the conversion of soluble plasma-protein fibrinogen to insoluble fibrin [1,2]. This implies that thrombin metabolic disorder is a major triggering factor for thrombotic diseases. In clinical practice, there are some effective anti-thrombin inhibitors such as bivalirudin, argatroban, heparin among others. However, the cost of treatment with these drugs is expensive and also associated with serious side effects [2–4]. These reasons underscore the need to seek and discover safer, affordable and effective natural thrombin inhibitors for the treatment of thrombotic disease. Molecules 2020, 25, 422; doi:10.3390/molecules25020422 www.mdpi.com/journal/molecules Molecules 2020, 25, 422 2 of 14 In recent research, plant-derived flavonoids, namely, kaempferol, quercetin and its glycosylated derivatives, (+)-catechin, (-)-epicatechin, cyanidin, cyanidin-3-glucoside, eupatilin-7-O-β-d- glucopyranoside and 5,6,20,40-tetrahydroxy-7,50-dimethoxyflavone have been proven to have inhibitory effect on thrombin [5–8]. Notably, Traditional Chinese Medicines (TCMs) such as Typha pollen, Salvia miltiorrhizae, Radix et rhizoma, Chuanxiong rhizoma and Platycladi cacumen are enriched with natural flavonoids and have been used for treating thrombotic diseases for centuries [9,10]. Structurally, 2-phenylchromanone and C6-C3-C6 form the backbone of flavonoids. Some well-known flavonoids include but are not limited to flavonols, isoflavones, flavones, flavanonols, anthocyanidins, chalcones, dihydrochalcone and aurones. [11,12]. These compounds elicit diverse pharmacological activity such as antioxidant, anti-inflammation, antibacterial, antithrombus and antineoplastic due to the difference in structural diversities in basic parent structure or substituent group [13]. Structure activity relationship (SAR) of flavonoids based on the above biological activities has gained lots of attention in recent years [14–16]. To the best of our knowledge, there is only one literature report on the SAR of flavonoids inhibiting thrombin [17]. Noticeably, it did not investigate anti-thrombin activity for all the substituent groups on flavonoid. In view of this, it was imperative to study the structural characteristics of more natural flavonoids for unraveling the mechanism of thrombin inhibition. Molecular docking technology, an algorithm that predicts the putative geometry of a protein–ligand complex has been successfully applied in the studies of SAR of compounds and TCM as well as predicting binding affinities of ligands [18–22]. Irrespective of these gains, there also exist disadvantages such as low accuracy and high false positive rate. A chromogenic substrate assay (CSA), which is a rapid and very effective technique is another in vitro method for examining thrombin inhibition activity [4,19,23]. This assay determines the activity of enzymes in samples by measuring the absorbance of colored p-nitroaniline produced by the reaction of blood coagulation factor (such as FX, thrombin and prothrombin) and chromogenic substrate [24]. Clinically, CSA is widely used in diagnosing and monitoring of hemophilia A/B, investigating anticoagulant effects and screening enzyme inhibitors [5,25,26]. Therefore, the combination of the chromogenic substrate method and molecular docking could greatly improve the screening accuracy. In this study, the SAR of 103 flavonoids was studied using molecular docking combined with chromogenic substrate assay. The findings of this research aim to provide a basis for the study of mechanism of thrombin inhibition and also serve as a model for studying new flavonoid-type thrombin inhibitors. 2. Results and Discussion 2.1. The Feasibility of the Established Docking Protocol To verify the feasibility of the established docking protocol, the original ligand molecule was extracted from the co-crystallized thrombin complex (2GDE and the original ligand SN3) and re-docked into 2GDE using CDOCKER. The conformation of the re-docked co-crystallized thrombin complex (2GDE and SN3) is shown in Figure1. The value of low root mean-square deviation (RMSD) between re-docked conformation and the crystal conformation was 0.7457 Å. In general, if RMSD 2.0 Å, the ≤ molecular docking model should be considered as feasible [27]. Molecules 2020, 25, 422 3 of 14 Molecules 2019, 24, x FOR PEER REVIEW 3 of 14 Figure 1. Conformation of re-docked co-crystallized thrombin complex (2GDE and SN3). Figure 1. Conformation of re-docked co-crystallized thrombin complex (2GDE and SN3). 2.2. In Silico Screening for Thrombin Inhibitors 2.2. In Silico Screening for Thrombin Inhibitors One hundred and three flavonoids compounds were docked into the active site of thrombin model and identifiedOne hundred by CDOCKER and three moduleflavonoids in discovery compounds studio were 4.5 docked software into (Accelrys, the active Biovia, site of Boston, thrombin MA, modelUSA). Theand results identified showed by 62CDOCKER hit compounds module with in -CDOCKER_energydiscovery studio 4.5 above software 20.0 kcal(Accelrys,/mol and Biovia, 54 hit Boston,compounds MA, with USA). -CDOCKER_INTERACTION The results showed 62 hit energy compounds above 40.0 with kcal -CDOCKER_energy/mol (see Table S1 in Supportingabove 20.0 kcal/molMaterial). and The 54 docking hit compounds effect was with good -CDOCKER_INTERACTION since the -CDOCKER_energy energy was higher above than 40.0 15 kcal/mol kcal/mol (see [24]. TableBased S1 in onSupporting the above Material). results, the The anti-thrombin docking effect activities was good of 42 since target the flavonoids -CDOCKER_energy were selected was for higherfurther than research 15 kcal/mol using a [24]. chromogenic substrate in vitro method. Selection of flavonoids was done accordingBased toon threethe above principles. results, Thethe anti-thrombin first rule was activi the valueties of of 42 -CDOCKER_energy target flavonoids were should selected exceed for further25 kcal/ molresearch or -CDOCKER_INTERACTION using a chromogenic substrate energy in shouldvitro method. exceed 45Selection kcal/mol of in flavonoids order to improve was done the accordingaccuracy of to measurement three principles. results. The Secondly, first rule flavonoidswas the value including of -CDOCKER_energy flavonols, flavones, should flavanonols exceed and 25 kcal/molcatechins or with -CDOCKER_INTERACTION comparable structural features energy were should given exceed preference. 45 kcal/mol Lastly, in naturally order to improve derived andthe accuracyeasily obtained of measurement compounds results. were chosen. Secondly, The flavonoids chemicalstructures includingof flavonols, the 42 flavonoids flavones, are flavanonols shown in andFigure catechins S1 (in Supporting with comparable Material). structural features were given preference. Lastly, naturally derived and easily obtained compounds were chosen. The chemical structures of the 42 flavonoids are shown in2.3. Figure In Vitro