Research Article Investigation of Potential Pyruvate ...downloads.hindawi.com/journals/bmri/2014/189495.pdfIn Silico Investigation of Potential Pyruvate Kinase M2 Regulators from Traditional

Research ArticleIn Silico Investigation of Potential Pyruvate Kinase M2Regulators from Traditional Chinese Medicine against Cancers

Kuan-Chung Chen,1 Kuen-Bao Chen,2,3,4 Hsin-Yi Chen,3 and Calvin Yu-Chian Chen2,3,5,6

1 School of Pharmacy, China Medical University, Taichung 40402, Taiwan2 School of Medicine, College of Medicine, China Medical University, Taichung 40402, Taiwan3Department of Biomedical Informatics, Asia University, Taichung 41354, Taiwan4Department of Anesthesiology, China Medical University Hospital, Taichung 40447, Taiwan5Human Genetic Center, Department of Medical Research, China Medical University Hospital, Taichung, Taiwan6Research Center for Chinese Medicine & Acupuncture, China Medical University, Taichung 40402, Taiwan

Correspondence should be addressed to Calvin Yu-Chian Chen; [email protected]

Received 22 February 2014; Revised 5 March 2014; Accepted 5 March 2014; Published 25 June 2014

Academic Editor: Chung Y. Hsu

Copyright © 2014 Kuan-Chung Chen et al. This is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properlycited.

A recent research in cancer research demonstrates that tumor-specific pyruvate kinase M2 (PKM2) plays an important role inchromosome segregation and mitosis progression of tumor cells. To improve the drug development of TCM compounds, we aimto identify potent TCM compounds as lead compounds of PKM2 regulators. PONDR-Fit protocol was utilized to predict thedisordered disposition in the binding domain of PKM2 protein before virtual screening as the disordered structure in the proteinmay cause the side effect and downregulation of the possibility of ligand to bind with target protein. MD simulation was performedto validate the stability of interactions between PKM2 proteins and each ligand after virtual screening. The top TCM compounds,saussureamine C and precatorine, extracted from Lycium chinenseMill. and Abrus precatorius L., respectively, have higher bindingaffinities with target protein in docking simulation than control. They have stable H-bonds with residues A:Lys311 and some otherresidues in both chains of PKM2 protein. Hence, we propose the TCM compounds, saussureamine C and precatorine, as potentialcandidates as lead compounds for further study in drug development process with the PKM2 protein against cancer.

1. Introduction

Recently, more and more pathogenesis of diseases has beenidentified [1, 2] to reveal potential target proteins for drugdesign [3–5]. A recent research in cancer research demon-strates that tumor-specific pyruvate kinase M2 (PKM2) playsan important role in chromosome segregation and mitosisprogression of tumor cells [6, 7]. PKM2 proteins can betreated as drug target proteins against cancers [8, 9].

The computer-aided drug design hadwildly been used forvirtual drug screening in the drug design [10, 11]. In previousstudy, many compounds from traditional Chinese medicine

(TCM) have been identified as potential lead compoundsin computer-aided drug design for the treatment of cancers[12–14], metabolic syndrome [15], diabetes [16], stroke [17,18], inflammation [19], and some other diseases [20]. Toimprove the drug development of TCM compounds, weemployed TCM compounds from TCM Database@Taiwan[21] to virtual screening of the potent lead compounds ofPKM2 regulators. As the disordered structure in the proteinmay cause the side effect and downregulation of the possi-bility of ligand to bind with target protein [22], PONDR-Fitprotocol was performed to predict the disordered dispositionin binding domain of PKM2 protein before virtual screening.

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014, Article ID 189495, 14 pageshttp://dx.doi.org/10.1155/2014/189495

2 BioMed Research International

1.0

0.5

0.0

Diso

rder

disp

ositi

on

0 50 100 150 200 250 300 350 400 450 500 550

Residue index

P-Fit

Figure 1: Disordered disposition predicted by PONDR-Fit.

RMSD: 0.4873

CrystalRedock

Chain AChain BBinding site

Figure 2: Binding site of PKM2 protein defined as the volume of 𝑁-(4-((4-(pyrazin-2-yl)piperazin-1-yl)carbonyl)phenyl)quinoline-8-sulfonamide and root-mean-square deviation value between crystallized structure (orange) and docking pose (violet).

TheMD simulation was performed after virtual screening, tovalidate the stability of interactions between PKM2 proteinsand each ligand in each docking pose.

2. Materials and Methods

2.1. Data Collection. The X-ray crystallography structure ofthe human pyruvate kinase M2 (PKM2) was downloaded

from RCSB Protein Data Bank with PDB ID: 4G1N [9].To predict the disordered disposition in PKM2 protein,PONDR-Fit [23] protocol was employed with the sequenceof PKM2 protein from Swiss-Prot (UniProtKB: P14618). Inpreparation section, the final structure of PKM2 proteinwas protonated with Chemistry at HARvardMacromolecularMechanics (CHARMM) force field [24] and removed watermolecules in the X-ray crystallography structure by Prepare

BioMed Research International 3

O

O

+H2N O

NH2

O

O−

(a)

N+

O

O

OH

OOH

O−

(b)

N SO

OHN

ONNN

N

(c)

Figure 3: Chemical scaffold of controls and top two TCM candi-dates with their scoring function and sources. (a) Saussureamine C,(b) precatorine, and (c) NZT.

Protein module in Discovery Studio 2.5 (DS2.5). The TCMcompounds from TCM Database@Taiwan [21] were filteredby Lipinski’s Rule of Five [25], and their final structurewas protonated using Prepare Ligand module in DS2.5. Thebinding site was defined by the volume of the cocrystallizedPKM2 activator for virtual screening.

2.2. Docking Simulation. TheTCMcompoundswere dockinginto the binding site defined above by LigandFit protocol[26] in DS 2.5 using a shape filter and Monte-Carlo ligandconformation generation. CHARMM force field [24] wasemployed to optionallyminimize the docking poses, and thenthe clustering algorithmwas employed to filter out the similar

Table 1: Scoring functions of top candidates from TCM databasescreening.

Name Source Dock ScoreSaussureamine C Lycium chinenseMill. 166.382Precatorine Abrus precatorius L. 161.002NZT∗ 73.062∗Control: N-(4-((4-(pyrazin-2-yl)piperazin-1-yl)carbonyl)phenyl)quinoline-8-sulfonamide.

poses. Each docking pose was evaluated by Dock Score usingthe following equation:

Dock Score = − (ligand receptor interaction energy

+ligand internal energy) .(1)

2.3. Molecular Dynamics (MD) Simulation. The moleculardynamics (MD) simulation was employed with classicalmolecular dynamics theory using Gromacs 4.5.5 [27] tosimulate the variation of each protein-ligand complex underdynamic conditions. In preparation section, the PKM2 pro-teins were prepared by pdb2gmx protocol of Gromacs toprovide topology and parameters with CHARMM27 forcefield, and each ligand was prepared by SwissParam program[28] to provide topology and parameters with CHARMM.A cubic box solvated using TIP3P water model and 0.145MNaCl model was defined based upon the edge approx. 12 Afrom the protein complexes periphery. In the minimizationsection, we employed steepest descents [29] minimizationwith a maximum of 5,000 steps to remove bad van der Waalscontacts. In the equilibration section, we perform position-restrained molecular dynamics with the linear constraintalgorithm for all bonds by Gromacs program with NVTequilibration, Particle Mesh Ewald method, and Berendsenweak thermal coupling method. In the production section,we perform 10,000 ps production simulation by Gromacsprogram with time step in unit of 2 fs under NPT ensemblesand particle mesh Ewald (PME) option. A series of protocolsin Gromacs program was employed to analyze the MDtrajectories of 5000 ps.

3. Results and Discussion

3.1. Disordered Protein Prediction. The disordered disposi-tion predicted by PONDR-Fit protocol with the sequenceof PKM2 protein from Swiss-Prot (UniProtKB: P14618) wasdisplayed in Figure 1. The key residues in the binding site donot locate in disordered domain (>0.5), which indicates thatPKM2 protein expresses a stable binding domain in proteinfolding. We employed the crystallography structure of PKM2protein for docking simulation as the residues in the bindingsite of target protein have no significant variation.

3.2. Docking Simulation. To validate the accuracy of Lig-andFit protocol in DS2.5, the cocrystallized PKM2 proteinactivator was redocked into the binding site of PKM2


A:ASN350

A:LEU353

A:ASP354

A:MET30

B:ASN350

B:LYS311

B:ASP354

B:GLY355

A:LEU27

B:ASN318

B:LEU394

B:GLU397

B:GLN393

B:TYR390

Pi

+

B:PHE26

A:LYS311 A:PHE

26

B:LEU353

(a)

B:ASP354

A:MET30

B:ASN350

A:ASP354

B:ILE389

B:ASN318

A:ASN350

B:LEU394

B:GLY355

B:GLN393

A:PHE26

B:LYS311

B:PHE26

B:LEU353

A:LYS311

(b)

Figure 4: Continued.


A:GLU397

A:GLY355

B:LEU27 A:ASP

354

A:GLY315 B:MET

30 B:LEU353

B:LEU394

B:HIS391

B:ILE389

B:ASP354

A:GLN393

A:TYR390

B:PHESigma

i

26A:LEU394

A:LEU353

B:TYR390

A:ALA388

B:GLN393

A:PHE26 A:ILE

389

A:MET30

B:LYS311

B:LEU353

B:LEU394

B:TYR390

(c)

Figure 4: Docking pose of PKM2 protein complex with (a) saussureamine C, (b) precatorine, and (c) NZT.

protein. The root-mean-square deviation (RMSD) valuebetween crystallized structure and docking pose of controlis 0.4873 (Figure 2), which shows a good accuracy in thedocking simulation by LigandFit protocol. So we employLigandFit protocol as suitable for virtual screening withPKM2 protein. The top TCM compounds ranked by DockScore [26] and control, 𝑁-(4-((4-(pyrazin-2-yl)piperazin-1-yl)carbonyl)phenyl)quinoline-8-sulfonamide (NZT), areshown in Table 1. For the top two TCM compounds, saus-sureamine C and precatorine were extracted from LyciumchinenseMill. and Abrus precatorius L. The chemical scaffoldof saussureamine C, precatorine, and control is illustrated inFigure 3. According to the docking poses in Figures 4 and5, the top two candidate compounds have hydrogen bonds(H-bonds) with common residue B:Lys311 and an interactionwith residues in both chains of PKM2 protein as control.

3.3. Molecular Dynamics Simulation. LigandFit protocol per-forms a docking simulation with a rigid body of PKM2proteins. The docking poses with PKM2 protein may modifyunder dynamic conditions. We employed the MD simula-tion to validate the stability of interactions of each ligandwith PKM2 proteins. Root-mean-square deviations (RMSDs)

illustrated the atomic fluctuations during MD simulation.Figure 6 displays the atomic fluctuations of PKM2 proteinsand ligands in complexes with saussureamine C, precatorine,and control during 10,000 ps MD simulation. It shows thatPKM2 proteins tend to be stable after a short period of MDsimulation. In addition, there is no significant variance forthe total energies of each PKM2 protein complex duringMD simulation (Figure 7). The variation and distribution ofradii of gyration for protein and ligand over 10,000 ps MDsimulation in Figure 8 indicate that the radii of gyrationof PKM2 protein complexes with ligands, saussureamineC, precatorine, and control were stabilized under dynamicscondition after 5,000 ps MD simulation. The variation ofmean-square displacement (MSD) and total solvent accessi-ble surface area (SASA) for each protein and ligand displayedin Figure 9. They indicate that the SASA of PKM2 proteinin complexes with precatorine was decreased after MDsimulation, which implies that precatorine may cause twochains of PKM2 protein more compact. Root-mean-squarefluctuations (RMSFs) for each residue over 10,000 ps MDsimulation are shown in Figure 10. They indicate that PKM2proteins dockingwith saussureamineC andprecatorine causeflexibility for PKM2 proteins as control.


2.99

C1

C2C3

C4C5

C6C7

C8C9

C10

O11

C12

C13O14

C15C16

C19

N20

C21

C22

O23

O24 C25

C26 N27

O28

N CA

CB

CG

CD1

CD2

CO

Leu353

Phe26

Phe26Leu353

Asp354

Lys311

Leu394

Asp354

Tyr390

(a)

2.722.48

2.69

C1 C2

N3

C4

C5

C6

C7

O8O9

C10

C11C12

C13

C14C15

O16

C17O18

O19

O20

C21

NCA

CBCG

OD1

OD2

CO

Asp354

NCA

CB

CG

CD

CE

NZ

C

O

Lys311

Met30

N

CA

CB

CG

CD

CE

NZ

C

O

Lys311

Phe26

Phe26

Asn350

Leu353

Leu394

Asp354

(b)

2.66

2.69

2.71

C1

C2 C3

C4 C5

C6

C7N8

C9C10

S11O12

O13N14

C15C16

C17

C18

C19

C20C21

O22N23

C24

C25

C26C27

N28

C29

N30C31

C32

N33

C34

NCA

CB

CG

CD1

CD2

C

O Leu353

N

CA

CBCG

CD1CE1

CD2 CE2

CZ OH

C

O

Tyr390 Leu394

N

CA CB

CGCD

CE

NZCO

Lys311

Phe26

Ile389

Tyr390

Gln393

Phe26

Leu353

Met30

Asp354

Leu394Met30

Asp354

Glu397

Leu27

Ligand bondNonligand bondHydrogen bond and its length Nonligand residues involved in hydrophobic

Corresponding atoms involved in hydrophobic contact(s)

3.0

(c)

Figure 5: Docking pose of PKM2 protein complex with (a) saussureamine C, (b) precatorine, and (c) NZT drawn by LIGPLOT program.


0 2000 4000 6000 8000 10000

Time (ps)Li

gand

RMSD

_2Li

gand

RMSD

_1C

ompl

exRM

SD

0.50.6

0.41.30.20.10.0

0.3

0.2

0.1

0.00.3

0.2

0.1

0.0

Saussureamine CPrecatorineNZT

Figure 6: Root-mean-square deviations in units of nm for protein and ligand for PKM2protein complexes with saussureamineC, precatorine,and NZT. Ligand RMSD 1 and Ligand RMSD 2 are calculated with least squares fit by protein and ligand, respectively.

−2406000

−2408000

−2410000

−2412000

−2414000

−2416000

−2418000

−2420000

−2422000

−2424000

Tota

l ene

rgy

(KJ/m

ol)

0 2000 4000 6000 8000 10000

Time (ps)


(a)

12

10

8

6

4

2

0−2410000 −2415000 −2420000 −2425000 −2430000

Dist

ribut

ion

(%)

Total energy (KJ/mol)

(b)

Figure 7: (a) Distribution and (b) variation of total energy for PKM2 protein complexes with saussureamine C, precatorine, and NZT over5000 ps of MD simulation.


0 2000 4000 6000 8000 10000

Time (ps)

3.20

3.15

3.10

3.05

3.00

0.55

0.50

0.45

0.40

0.35

0.30

Liga

nd g

yrat

e (nm

)Pr

otei

n gy

rate

(nm

)


(a)

70

60

50

40

30

20

10

0

Dist

ribut

ion

(%)

3.00 3.04 3.08 3.12

Protein gyrate (nm)

(b)

0.35 0.40 0.45 0.50 0.55

Ligand gyrate (nm)

70

60

50

40

30

20

10

0

Dist

ribut

ion

(%)

(c)

Figure 8: (a) Variation of radii of gyration for protein and ligands in PKM2 complexes with saussureamine C, precatorine, and NZT over5000 ps of MD simulation. Distribution of radii of gyration for (b) protein and (c) ligands in PKM2 complexes with saussureamine C,precatorine, and NZT over 10,000 ps of MD simulation.

After MD simulation, we decide the representative struc-tures of PKM2 protein complexes by the RMSD valuesand graphical depiction of the clusters analysis with cutoffof 0.1 nm (Figure 11). To compare with the interactions indocking simulation and in representative structures of PKM2protein complexes after MD simulation, the snapshots of

each docking pose were displayed in Figure 12. They indicatethat two TCM candidates remain the H-bonds with residuesLys311 in chain A of PKM2 protein. Table 2 and Figure 13display the H-bond occupancy and distance variation foreach ligand with PKM2 proteins. They indicate that bothTCMcompounds have stableH-bondswith residuesA:Lys311


0 2000 4000 6000 8000 10000

Time (ps)

0.08

0.06

0.04

0.02

0.00

Prot

ein

MSD

(nm

S-2

N-1

)

(a)

0 2000 4000 6000 8000 10000

Time (ps)

Liga

nd M

SD (n

mS-2

N-1

)

0.08

0.06

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(b)

0 2000 4000 6000 8000 10000

Time (ps)

Prot

ein

SASA

(nm

S-2

N-1

)

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(c)

Protein SASA (nmS-2N-1)170165160155150

50

40

30

20

10

0

Dist

ribut

ion

(%)

(d)


0 2000 4000 6000 8000 10000

Time (ps)

Liga

nd S

ASA

(nm

S-2

N-1

)

4.5

4.0

3.5

3.0

2.5

2.0

1.5

1.0

0.5

(e)


Ligand SASA (nmS-2N-1)0.8 1.2 1.6 2.0 2.4 2.8 3.2 3.6 4.0 4.4

20

10

40

30

60

50

80

90

70

0

Dist

ribut

ion

(%)

(f)

Figure 9: Variation of (a) mean square displacement (MSD) of protein, (b) ligand MSD, (c) total solvent accessible surface area (SASA)of protein, and (e) ligand SASA and distribution of (d) protein SASA and (f) ligand SASA for PKM2 complexes with saussureamine C,precatorine, and NZT over 10,000 ps of MD simulation.


0.5

0.4

0.3

0.2

0.1

0.0

RMSF

(nm

)

Saussureamine C

0 50 100 150 200 250 300 350 400 450 500 550

Residue

(a)

RMSF

(nm

)

Precatorine

0 50 100 150 200 250 300 350 400 450 500 550

Residue

0.4

0.5

0.3

0.2

0.1

0.0

(b)

0 50 100 150 200 250 300 350 400 450 500 550

Residue

0.4

0.5

0.3

0.2

0.1

0.0RMSF

(nm

)

NZT

(c)

Figure 10: Root-mean-square fluctuation (RMSF) for residues in PKM2 complexes with saussureamine C, precatorine, and NZT.

Time (ps)

Tim

e (ps

)

5000 6000 7000 8000 9000 100005000

6000

7000

8000

9000

10000 0.256

0

RMSD

(nm

)

(a)

Time (ps)

Tim

e (ps

)

5000 6000 7000 8000 9000 100005000

6000

7000

8000

9000

10000

0

0.273

RMSD

(nm

)

(b)

Time (ps)

Tim

e (ps

)

5000 6000 7000 8000 9000 100005000

6000

7000

8000

9000

10000 0.257

0

RMSD

(nm

)

(c)

Figure 11: Root-mean-square deviation value (upper left half) and graphical depiction of the clusters with cutoff 0.1 nm (lower right half) forPKM2 complexes with (a) saussureamine C, (b) precatorine, and (c) NZT.


(a) (b)

(c) (d)

(e) (f)

Figure 12: Docking poses in docking simulation (left) andmiddle RMSD structure in the major cluster (right) for PKM2 complexes with (a),(b) saussureamine C, (c), (d) precatorine, and (e), (f) NZT.


B: N27/Lys311: HZ3 B: H50/Asn350: OD1B: H50/Asp354: OD1B: H50/Asp354: OD2

B: O28/Lys311: HZ3A: H50/Lys311: NZ

Dist

ance

(nm

)

1.5

1.2

0.9

0.6

0.3

0.0

0 2000 4000 6000 8000 100000 2000 4000 6000 8000

Time (ps) Time (ps)

(a)

B: O19/His29: HE2B: H29/Leu353: O

A: O18/Lys311: HZ3B: O20/Tyr390: HN

Dist

ance

(nm

)

0.8

1.0

0.6

0.4

0.2

0.00 2000 4000 6000 8000 100000 2000 4000 6000 8000

Time (ps) Time (ps)

(b)

B: O22/Lys311: HZ3A: O12/Tyr390: HNA: O13/Tyr390: HN

A: H41/Leu353: OB: N28/Gln393: HE22B: N30/Gln393: HE22

Dist

ance

(nm

)

1.5

1.8

1.2

0.9

0.6

0.3

0.0

0 2000 4000 6000 8000 100000 2000 4000 6000 8000

Time (ps) Time (ps)

(c)

Figure 13: Distance variation of H-bonds with PKM2 protein during MD simulation. (a) Saussureamine C, (b) precatorine, and (c) NZT.


Table 2: H-bond occupancy for key residues of PKM2 protein with top TCMcompounds overall 10,000 ps ofmolecular dynamics simulation.

Ligand H-bond Ligand atoms Amino acid Distance (nm) Occupancy (%)Max. Min. Average

Saussureamine C

1 O23 A:Lys311:HZ3 0.553 0.259 0.438 15.80%2 O24 A:Lys311:HZ3 0.57 0.146 0.294 94.80%3 N27 A:Lys311:HZ3 0.593 0.234 0.408 30.00%4 O28 B:Lys311:HZ3 0.726 0.161 0.519 10.40%5 O28 B:Lys311:HZ3 0.63 0.15 0.31 73.20%6 H50 B:Lys311:NZ 0.76 0.24 0.43 46.20%7 H50 A:Asn350:OD1 0.64 0.16 0.36 44.80%8 H52 B:Asp354:OD1 0.54 0.27 0.31 96.20%9 H50 B:Asp354:OD1 0.66 0.19 0.49 20.40%10 H50 B:Asp354:OD2 0.79 0.15 0.51 23.20%

Precatorine

1 O18 A:Lys311:HZ3 0.39 0.14 0.29 98%2 O19 B:His29:HE2 0.41 0.15 0.19 97%3 O20 B:Tyr390:HN 0.58 0.17 0.24 91%4 H29 B:Leu353:O 0.52 0.16 0.37 25%

NZT∗

1 O12 A:Tyr390:HN 0.32 0.16 0.19 100%2 O13 A:Tyr390:HN 0.46 0.17 0.37 15%3 O22 B:Lys311:HZ3 0.78 0.15 0.42 53%4 N28 B:Gln393:HE22 1.03 0.23 0.56 18%5 N30 B:Gln393:HE22 0.99 0.19 0.49 40%6 H41 A:Leu353:O 0.30 0.16 0.20 100%

H-bond occupancy cutoff: 0.35 nm.∗Control: N-(4-((4-(pyrazin-2-yl)piperazin-1-yl)carbonyl)phenyl)quinoline-8-sulfonamide.

and some other residues in both chains of PKM2 protein,which can stabilize the docking poses in the binding domain.

4. Conclusion

This study aims to investigate the potent lead drug fromTCMcompounds for PKM2 protein inhibitors against cancers.The top TCM compounds, saussureamine C and precatorine,have higher binding affinities with PKM2 proteins in dockingsimulation than control. They have H-bonds with residuesA:Lys311 and some other residues in both chains of PKM2protein. After MD simulation, the top TCM compoundsmaintain the similar docking poses under dynamic con-ditions. In addition, the top two TCM compounds, saus-sureamine C and precatorine, were extracted from Lyciumchinense Mill. and Abrus precatorius L., respectively. Hence,we propose the TCM compounds, saussureamine C andprecatorine, as potential candidates as lead compounds forfurther study in drug development process with the PKM2protein against cancer.

Conflict of Interests

The authors declare that there is no conflict of interests.

Authors’ Contribution

Kuan-Chung Chen, Kuen-Bao Chen, and Hsin-Yi Chencontributed equally to this work.

Acknowledgments

The research was supported by Grants from the NationalScience Council of Taiwan (NSC102-2325-B039-001 andNSC102-2221-E-468-027-), Asia University (ASIA100-CMU-2, ASIA101-CMU-2, and 102-ASIA-07), and China Medi-cal University Hospital (DMR-103-058, DMR-103-001, andDMR-103-096).This study is also supported in part byTaiwanDepartment of Health Clinical Trial and Research Center ofExcellence (DOH102-TD-B-111-004), Taiwan Department ofHealth Cancer Research Center of Excellence (MOHW103-TD-B-111-03), and CMU under the Aim for Top UniversityPlan of the Ministry of Education, Taiwan.

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