Structure-Based Drug Design of Quercetin and Its Derivatives against HMGB1

High mobility group box 1 (HMGB1) is known as non-histone nuclear protein which has many biological functions, and it plays a significant role in many diseases inhibition, such as inflammatory and cancer diseases. HMGB1 in cancer cells could induce cell proliferation, cell differentiation, carcinogenesis, and tumorigenesis. In addition, HMGB1 function and location highly depends on the redox states. Our work focuses on molecular interaction studies of quercetin and its derivatives with HGMB1 protein target. Early reports showed that quercetin could inhibit tumors and cancers in different experimental examinations. However, clinical studies of quercetin showed low performance in its bioavailability. Therefore, structural modification of quercetin is needed to enhance its pharmacological properties. Here, we found 9 molecules of quercetin derivatives (QD) which have favorable interaction with HGMB1 and improved pharmacological properties. These findings indicate that QD might be the potential candidates against HMGB1 as therapeutic target for anticancer.


INTRODUCTION
High mobility group box 1 (HMGB1) is a non-histone chromosomal and highly conserved protein that serves various roles in intracellular and extracellular systems. This group of proteins was appointed as the "high mobility group," due to their rapid mobility properties in polyacrylamide gel electrophoresis. HMGB1 is discovered firstly as a chromatin-associated protein, extensive studies of this group have directed to the identification of many critical functions of HMGB1. In eukaryotic organisms, HMGB1 protein is mainly found in the nucleus, where its function is chaperoning DNA molecules This is an Open Access article licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License (https://creativecommons.org/licenses/by-nc-sa/4.0/ ), which permits unrestricted Non Commercial use, distribution and reproduction in any medium, provided the original work is properly cited.
Published by Oriental Scientific Publishing Company © 2017 alleviating replication, recombination, transcription, DNA repair, and nucleosome formation stabilization by forming the DNA helical structure and promoting the binding of regulatory complexes to DNA. HMGB1 takes part in regulating autophagy and maintaining balance between autophagy and apoptosis within the cytoplasm compartment 1,2 .
HMGB1 is also implicated as an important mediator in cancer. HMGB1 expression is high in many cancer cells, especially in colon cancer, breast cancer, cervical cancer, lung cancer, prostate cancer, and pancreatic cancer [3][4][5][6] . Previous studies exhibited that there is a correlation between HMGB1 and various malignant cancer mechanisms. The principle behind this connection may be due to the role of extracellular HMGB1 in angiogenesis, cell migration, and macrophages recruitment, which could contribute to tumor growth and metastasis. HMGB1 has an elaborated role in carcinogenesis which could suppress tumorigenesis by interacting with tumor suppressor genes such as p53, p73 and RB. Moreover, HMGB1 has mixed effects on the superiorities of cancer including unlimited replicative potential, ability to blood vessels development (angiogenesis), deflection of programmed cell death (apoptosis), self-sufficiency in growth signals, insensitivity to inhibitors of growth, tissue invasion and metastasis which cause inflammation 7,8 . This exhibits that HMGB1 is a potential target for cancer treatment.
Suppression of HMGB1 is important and it becomes prospective strategy for treating various cancers. Recently, various synthetic and natural products have been used to inhibit HMGB1 expression. Quercetin is a secondary metabolite compound which has polyphenol groups, and it belongs to the flavonol class of flavonoids. The broad range of biochemical and pharmacological properties of quercetin and its metabolites are due to the relative substitution of various functional groups on the flavonol molecule 9 . Phytochemical investigations of various plant extracts have revealed that quercetin can exist in a free state as an aglycone, or as its derivative by conjugating with: carbohydrates as quercetin glycosides 10 ; alkyls as quercetin methyl or ethyl 11,12 ; hydroxyl group as quercetin ethers [13][14][15] ; and a sulfate group as quercetin derived-sulfates 16 . Our research aim is to investigate the interaction binding between quercetin derivatives (QD) and HMGB1 through molecular docking analysis. Furthermore, the best docking hit of QD were analysed the ADMET (absorption, distribution, metabolism, excretion and toxicity) prediction 17,18 to evaluate its pharmacological properties of QD in different mechanisms in the body. Here, we report the highlight molecular nature of QD action mechanims with various pharmacological properties as HGMB1 inhibitors.

Protein Preparation
The protein sequence of human High  19 . The coordinates of hHGMB1 only consists of amino acid residues in .pdb format file.

Ligand Preparation
The structure of the ligands were obtained from PubChem 20, 21 in .sdf format files (Fig. 1). Ligands were converted into .pdb format using GUI OpenBabel 22 . Some of ligand structures were drawn and the 3D structures were optimized by using ChemDraw 23 . Pharmacokinetic properties of all the ligands were analysed by using SwissADME 17 .

ADMET Prediction
Absorption, distribution, metabolism, excretion and toxicity (ADMET) prediction for the top docking of quercetin derivatives were predicted using ACD/I-Lab 18 . This platform predicts physicochemical descriptors as well as to predict ADMET parameters. ACD/I-Lab provides particular molecule's properties with 95% of known drugs. Evaluation of ADMET properties, were based on absorption involved maximum passive absorption through intracellular and paracellular pathways, Volume distribution (Vd) was also involved in this work. Plasma Protein Binding percentage (%PPB) and P-gp specificity were used to evaluate metabolism and excretion profiles, respectively. Additionally, LD50 mouse and probability of health effects predictions of QD were calculated using ACD/I-Lab which is a web-based service that provides instant access to spectral and chemical databases, ADME predictions, and toxicity characteristics. A comparative analysis was performed for mouse LD50 (intraperitoneal, oral, intravenous, subcutaneous) and probability of health effect of blood, cardiovascular system, gastrointestinal system, kidney, liver and lung by QD's biological activities.  Quercetin, 3'-ethyl ether 560 570 270 120

RESULTS
The derivatives of Quercetin have been successfully built for further in silico analysis. The structures of Quercetin derivatives are based on the variation of their side group, as depicted in Fig. 1.  (Table 1).
Molecular docking analysis showed that Quercetin, 3 sodium sulfate showed the best interaction with HMGB1, base don its affinity energy (-11.2 kcal/mol) and Ki value (6.04x10 -9 M). This interaction does not involve hydrogen bond, but it involves hydrophobic interaction with the residues in HMGB1 binding site, including: Ala69, Glu72, Arg73, and Lys76 (   (Table 3 and Table  4). These analyses were conducted by using ACD/I-LAB program.

DISCUSSION
Quercetin is categorized by a benzo-(g)pyrone skeletal structure with C6-C3-C6 carbon form, consisting of two benzene rings, A and B, linked by three carbons pyrone ring C as shown in Fig. 1. Quercetin is referred to pentahydroxy flavonol due to the presence of five hydroxyl groups on its flavonol skeletal framework at 3, 30, 40, 5, and 7 carbons [29][30][31] . The broad range of biochemical and pharmacological properties of quercetin and its derivatives are due to functional group substitutions of flavonol molecule 32 . However, glycosylation of other hydroxyl groups has also been found from plants 33,34 . The sugar moieties could be in the form of monosaccharides, disaccharides, or polysaccharides. The most common monosaccharides substituents are glucose, galactose, rhamnose, and xylose. Isoquercetin glycosides also has been reported having radical scavenging properties 35,36 .
In addition, the conformational analysis of quercetin showed the presence of 12 conformations of this molecule having Gibbs energies in the range of 0 to 5.33 kcal/mol 37,38 . Furthermore, quercetin exhibits strong intramolecular Hydrogen bond interactions, which displaying its biological multifunctional activities and renders its ability to form strong complex interactions, including with metals, affecting its bioavailability and transport system in the cells (39)(40)(41) . Among these H-bonds, two bonds are with carbonyl groups and third one is between hydroxyl groups (42,43) . Moreover, the derivatives of quercetin glycosides may also contain acyl and sulfur substituents becomes sugar moieties. In the case of quercetin derivatives, the hydroxyl groups of quercetin are attached with alcohols via ether bonds. Although quercetin is lipophilic, the glycosylation of quercetin derivatives can increase the hydrophilicity and enable the molecules to transport through all parts of the plant (44,45) .
Pharmacophore properties of quercetin and its derivatives showed various properties according to Lipinski's rule of five (Ro5). Ro5 consist of HBA/ HBD value up to 10 and 5, respectively; MW less than 500, Log P value less than 5 and total polar surface area (TPSA) value less than 140 Å (46) . From the results showed that there was no violation of Ro5 for quercetin; Quercetin, 3-sulfate; Quercetin, 3-sodium sulphate; Quercetin, 3'-methyl ether; and Quercetin, 3'-ethyl ether, while the 4 other derivatives showed 3 violations of Ro5 as shown on Table 1.
Analysis of molecular docking was performed by using Autodock Vina. The best docking is Quercetin, 3-sodium sulfate with Affinity binding -11.2 kcal/mol. This favorable binding was contributed by hydrophobic interactions from four amino acid residues from HGMB1 protein including Ala69, Glu72, Arg73, and Lys76 (Fig.2). The inhibition constant (Ki) of Quercetin, 3-sodium sulfate is 6.04x10-9 M. The result of docking interaction, Ki value, and HGMB1 residues involved in hydrophobic interaction is shown on Tabel 2.
Prediction of ADME properties of quercetin and its derivatives were done by using ACD/I-Lab platform as shown on Table 3. Maximum passive absorptions of Quercetin, Quercetin, 3'-methyl ether and Quercetin, 3'-methyl ether were found 100%. Meanwhile, other quercetin derivatives were found less than 15%. Volume distribution rates of quercetin and its derivatives we found within 0.25 -0.65 L/kg. Plasma binding protein ability rate in metabolisms prediction from quercetin and its derivatives were within 85. 36 -99.82. Excretion probabilities by involving P-gp specificity (AB/logP) from quercetin and its derivatives had value between 2.5-5 were possibly inhibitor, meanwhile P-gp specificity value less than 2.5 indicated were not inhibitors as well.
Moreover, toxicity analyses of quercetin and its derivatives were done by using ACD/I-Lab platform as shown on Table 4 and health effects prediction on Table 5. Quercetin, 3-sodium sulfate was found as acceptable ADME value, meanwhile toxicity of Quercetin, 3-sodium sulfate seems high on oral system with LD 50 = 1600 mg/kg, whatsoever other derivatives gave toxicity prediction values in various deposition in the body tissues. To evaluate health effect in the body system responses, we also analysed the health effect prediction in blood, cardiovascular, gastrointestinal, kidney, lung, and liver. Quercetin, 3-sodium sulfate might give high effect to gastrointestinal which causes dysfunction of gastrointestinal.

CONCLUSION
The molecular docking studies with quercetin and its derivatives into the binding cavity of human HGMB1 inducible showed the derivatives having more favorable interaction than quercetin with docking score involving hydrogen bond and ligandprotein interaction energy compared to quercetin. As earlier reported in literature, quercetin is known for having anticancer property and inhibiting the HGMB1 protein, the derivatives were docked at the binding cavity could have also possess some sort of anticancer property as it is 95% similar to quercetin retrieved form the NCBI PubChem database. The docked compounds used in the present study do not violate the Lipinski rule of five parameters. Also, from the ADME-Toxicity prediction using ACD/I-Lab revealed the docked compounds are in the acceptable range of various pharmacological parameters and they have similar behaviour of health effects and LD50 compared to quercetin. Therefore, we conclude that quercetin derivative compounds could be potential candidate molecules and could support for experimental testing against HGMB1 protein as anticancer agents.