Molecular docking study on the molecular mechanism of rhaponticin for treatment of chronic myelocytic leukemia

Li-Juan Liu,Chao Zhou,Fu-Cheng Song,Sen-Sen Zhang,Wen-Jing Teng,Jing Zhuang,Chang-Gang Sun*,Hong-Zong Si

Introduction

Chronic myelocytic leukemia(CML)is a kind of chronic myeloproliferative disorders of primitive pluripotent hematopoietic stem cells.It accounts for 20%of all adult leukemias.Its symptoms may include lethargy,weight loss,unusual bleeding,sweats,anemia,and splenomegaly.The cytogenetic hallmark of CML is the Philadelphia chromosome(Ph),created by a reciprocal translocation between chromosomes 9 and 22 (t[9;22][q34;q11]).The conjugation of the breakpoint cluster region gene on chromosome 22 and the Abelson kinase gene on chromosome 9 creates oncogene BCR/ABL,which codes for a deregulated tyrosine kinase,activates multiple signal transduction pathways,including Ras/Raf/mitogen-activated protein kinase (MAPK), phosphatidylinositol 3 kinase,STAT5/Janus kinase,and Myc.The activity of BCR-ABL leads to uncontrolled cell proliferation and reduced apoptosis, resulting in the malignant expansion of pluripotent stem cells in bone marrow.Now it is generally accepted that the occurrence of CML involves many abnormal gene regulation,multiple signaling pathways,such as the change of tumor suppressor genes,the activation of telomerase,chromosome abnormalities,cancer gene abnormalities and abnormal repair function[1-2].

Rhubarb is one of the four traditional Chinese medicine(TCM)in China.It has many kinds of active ingredients, such as anthraquinones, styrene,anthracene ketone,tannic acid and polysaccharide.Its pharmacological effects mainly include regulating gastrointestinal function,anti-inflammatory,anti-tumor,cardiovascular protection, protecting liver and anti-aging,etc [3-5].The styrene,an important component of rhubarb,has significant effect on resistance to free radicals,anti-aging,anti-tumor.Its main ingredients include rhaponticin,piceatannol 3’-O-Glc,rhapontigenin,resveratrol-4’-O-β-D-glucopy-ranoside,resveratrol 4’-O-β-D-(6’’-O-galloyl)-glucop-yranoside and so on.

Rhaponticin(RT)widely exists in the leaves of Polygonaceae plants and is one of the main active components of Rhubarb.It is the derivatives of styrene(structural formula in Figure 1)and is a kind of stilbene glucoside.Rhaponticin has a role of improving microcirculation,reducing blood fat,blood sugar suppression,antitumor,allergic reactions,regulating the body's immune defenses,antithrombotic and antioxidant[6].But details of its antitumor mechanism have not been specific explained.Based on molecular docking approach,we research the interaction between rhaponticin and CML to detect treatment mechnism of rhaponticin on CML.

Materialsand Methods

1.1 Ligand preparation

Fig 1 The structure of rhaponticin

Firstly,we built the molecular structure of rhaponticin by software Chem Draw Ultra 8.0[7],then import the molecular structure to SYBYL 7.3[8],hydrogenation,give charge by using Gasteiger-HUckel method,make structure optimization through 1000 steps molecular mechanics by Tripos force field of calculation module,Powell as iterative algorithm, energy gradient 0.209KJ·mol-1·nm-1 as the terminating condition,1.0nm as the key interaction cutoff value,1.0 as dielectric constant,get the optimal configuration,store as MOL2 file,small molecules as the ligand for docking.

1.2 Dataset

We developed a protein-protein interaction network of CML based on genetic genes in our previous work[10].Through the Network topology attribute analysis,we regarded the nodes which the connectivity was greater than/equal to 30 as the key nodes(hub).Key nodes included TP53,MAPK8,STAT3,EPHB2,MYC,TNFSFL13,PIK3CA,JUN,JAK2,MAPK3,SRC,MAPK14,FRAP1,BCL2L1,BCR,CD4,CDKN1A,PARP1,ABL1,regard as the receptor of this study.The crystal structure data (4WA9.pdb, 3ZLN.pdb,3IK3.pdb, 4KA2.pdb, 4NJD.pdb, 1JPA.pdb,3OAW.pdb, 5AEP.pdb, 2GO1.pdb, 2ZOQ.pdb,3PZE.pdb, 2YIX.pdb, 4Y7R.pdb, PARP1.pdb,4WAF.pdb,SRC.pdb,4ZIA.pdb,1U5Y.pdb,3DCY.pdb)were taken from the protein data bank database[9].

1.3 Molecular docking

Molecular simulation was run based on Surflex-Dock,a part of software SYBYL 7.3[8].We import 19 protein crystals in SYBYL,then input small molecules file,respectively for docking.Score ligand-receptor affinity by consistency score function,and observe hydrogen bonding and combining site.

Results and Analysis

We got the docking scores and the numbers of hydrogen bonds of rhaponticin with 19 receptors based on molecular docking approach(Table 1)

In table 1,we can see that rhaponticin with JUN(2G01)receptor has the highest docking score,promting that the two combined the best.We can infer that JUN (2G01)is the preference receptors for rhaponticin.Secondly,SRC(SRC),JAK2(5AEP),

MAPK14(2YIX),FRAP1(3OAW),MAPK8(3PZE),PARP1(1U5Y)have high scores(Figure 2-6).

Figure2 JUN

Figure3 SRC

Figure4 JAK2

Figure5 MPKK14

Figure6 FRAP1

Discussion

TCM is a composite of many complicated chemical components and has the characteristic of multiple targets.Its effectiveness is often a result of the combined action.that is why TCM reflects the whole modulation,small side effects,and high tolerance in the process of prevention and control of complicated disease.However,the characteristics of multiple components and multiple targets,also cause the complexity of the research on TCM,and make it hard to own clear targets and accurate concentration-response relationship. Molecular docking is a kind of process that simulate molecular geometric structure and molecular inter-atomic forces,research molecular interactions and explore the low-energy combination model of ligand and receptor through chemometrics methods by computer technology[11].Virtual screening TCM chemical composition database for one or more target protein associated with disease by molecular docking,getting the candidate compounds which has a specific role with its target protein,combining with biology,spectroscopy and other experiments, eventually making chemical essence of TCM for treatment of the disease clear.

In latest Pharmacological research, RT has antitumor activity.Xiaoyan Shi[12]etc found that rhaponticin inhibited human liver cancer cell(SMMC-7721 cells)proliferating and promoted the differentiation in vitro with concentration and time dependence.Surong Zhao[13-14]etc found that rhaponticin can induce apoptosis of breast cancer cell.The mechanism may be related to the expression of protein involved in regulating apoptosis such as Bcl-2,Bax and the activation of caspase-3.And it can inhibit cell proliferation,which may be associated with inhibition of p-Ak expression.But there are ralely reports about rhaponticin on treatment of CML.

The result of molecular docking indicate that JUN,SRC,JAK2,MAPK14,FRAP1,MAPK8,PARP1 have more higher scores.Jun is a critical component of transcription factor AP1,composed of Jun family members(c-Jun,JunB and JunD)and plays an important role in pathogenesis of CML.C-Jun,a major downstream factor of JNK,is transcriptionally actived upon phosphorylation at Ser63 and Ser73 within its N-terminal transactivation domain by c-jun N-terminal kinase(JNK)[15].It plays a crucial role in proliferation,apoptosis,survival and differentiation of cell.Reports showed that it could activate the JNK pathway and the expression of c-Jun of Bcr/Abl mutant cells associated with JNK pathway.

SRC,the product of Proto-oncogene c-SRC,is the earliest discovered SRC kinase(SFKs)family member.Its relative molecular weight is about 60000 and is a kind of receptor tyrosine protein kinase.The disorder of expression of C-SRC in time and space make the quality and quantity of protein products change,that is one of the important reasons for CML.Src family play an important role in regulating cell proliferation and differentiation and cell signal transduction.When combined with other kinases,highly activated c-Src protein can induce cell canceration by changing the signal transduction of normal cell[16].In tumor metastasis,FAK closely associated with protein c-Src.FAK is the downstream molecular targets of Src protein and it can pass signals which from the integrin receptors,growth factor receptor,and the Src protein to the downstream mediators(PI-3k,ERK)[17].

The mutations of JAK2 gene in the Ph-MPD(PV,ET and IMF)is common,but rarely express in CML/AML[17-19].CML patients with JAK2 V617F mutation have longer survival and good prognosis.Amanta[20]found that Jak2 inhibition activated Lyn kinase in CML and induced apoptosis in imatinib resistant cells through SET-PP2A-SHP1 pathway.Jak2 inhibition also induced apoptosis in CML cells from blast crisis patients but not in normal hematopoietic cells.It will be an excellent strategy to imatinib drug-resistant CML,and also to MPN with jak2 mutation.

Studies have confirmed that the occurrence of tumor are relevant to abnormal changes of mitogen-activated protein kinase(MAPK)signal transduction pathways.MAPKs are highly conserved serine/threonine protein kinases functioning in various fundamental cellular processes,such as growth/proliferation,differentiation,motility and apoptosis/survival,as well as stress response[21].

According to the existing literature[22-25]:BCR is a crucial gene associated with the pathogenesis of CML.Number-growing evidence suggests the role of Stat protein family in the development of leukemia,and the activation of the Stat proteins are associated with poor prognosis and shortened survival in patients.CD4's immune inhibitory makes cancer gene escape immune surveillance.IL-6 is a pleiotropic cytokine that can regulate a variety of cellular functions including cell proliferation, cell differentiation,immune defense mechanisms and blood cell production.The abnormalities of tumor suppressor gene p53 and oncogene myc is associated with rapid change of CML.The above points are all related to the pathogenesis of CML.We constructed a genes-protein interaction network and got some key nodes involving those above genes or proteins,and proved a certain authenticity and representation on describing the pathogenesis of CML in molecules.In view of that,our result can provide some new methods and targets for our further research on CML treatment,but further experiment should be needed.

Conclusion

This study explored the interaction between rhaponticin and genes associated with CML.The result showed that rhaponticin and JUN(2G01)had the highest docking score,promting that the two combined the best and SRC(SRC),JAK2(5AEP),MAPK14(2YIX),FRAP1(3OAW),MAPK8(3PZE),PARP1(1U5Y)had high scores.The treatment of rhaponticin for CML is multiple targets and multiple ways.

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