ORIGINAL RESEARCH

Quercetin and taxifolin completely break MDM2–p53 association:molecular dynamics simulation study

Sharad Verma • Amit Singh • Abha Mishra

Received: 18 March 2012 / Accepted: 9 October 2012

� Springer Science+Business Media New York 2012

Abstract Inhibition of the MDM2–p53 interaction has been

becomes a new therapeutic strategy to activate wild-type p53

in tumors. Molecular dynamics (MD) simulations were used

to study the effects of quercetin and taxifolin on MDM2–p53

complex. We found that binding of ligands (quercetin and

taxifolin) led to the dissociation of MDM2–p53 complex.

Analyses of the hydrophobic contacts between the inhibitors

and MDM2–p53 were performed, and the results suggested

that these ligands form stable hydrophobic interactions with

MDM2 which led to complete disruption of MDM2–p53

hydrophobic interactions and dissociation of p53 from the

complex. Our study suggests that the pi–pi stacking between

Tyr 51 of MDM2 and aromatic rings of ligands is the critical

event in MDM2–p53 dissociation.

Keywords MDM2–p53 � Taxifolin � Quercetin �Molecular dynamics simulation

Introduction

The p53 (tumor suppressor protein) is one of the key players

which regulate the cell cycle, apoptosis, and DNA repair to

protect cells from malignant transformation (Vassilev et al.,

2004; Vogelstein et al., 2000; Levine, 1997). However,

activity of p53 is regulated by MDM2, a protein that inhibits

the ability of p53 to bind to DNA and activate transcription.

Tumors have over-expressed MDM2 which led to down-reg-

ulated tumor suppressor activity of p53 (Dastidar et al., 2009).

The p53 interacts with MDM2 by inserting its hydrophobic

residues (Phe19, Trp23, and Leu26) into a deep groove in

MDM2 (Allen et al., 2009). Many peptide inhibitors that

mimic the MDM2–p53 interaction have been reported, but

these inhibitors display only modest potency because they

have poor membrane permeability (Bautista et al., 2010; Fasan

et al., 2004; Kritzer et al., 2004; Stoll et al., 2001; Zhao et al.,

2002). Several small-molecule inhibitors have been designed

by structure-based methods to interrupt the binding of p53 to

MDM2 which mainly include Nutlins (based on cis-imidaz-

olidine) (Vassilev et al., 2004; Chene 2003), benzodiazepin-

edione derivatives (Popowicz et al., 2010; Koblish et al., 2006)

and spirooxindole (Shangary and Wang, 2008, 2009). These

studies are mainly concentrated on the binding of inhibitor at

hydrophobic cleft of MDM2 to interrupt the p53 binding. In

recent years, naturally occurring polyphenolic phytochemicals

(such as taxifolin and quercetin) have remained completely

ignored in this regard. The tremendous potentials of taxifolin

and quercetin (Fig. 1) to inhibit the cancer are well known (Lee

et al., 2007; Seufi et al., 2009). In this study, we tried to elu-

cidate the effects of these polyphenols on MDM2–p53 com-

plex with the help of molecular docking and molecular

dynamic simulation along with the possible mode of action.

Computational methods

Molecular docking

AutoDock 4.0 suite was used as molecular-docking tool to

carry out the docking simulations. The crystal structure of

Electronic supplementary material The online version of thisarticle (doi:10.1007/s00044-012-0274-9) contains supplementarymaterial, which is available to authorized users.

S. Verma � A. Mishra (&)

School of Biochemical Engineering, Indian Institute

of Technology, Banaras Hindu University,

Varanasi 221005, India

e-mail: abham.bce@itbhu.ac.in

A. Singh

Department of Pharmacology, Institute of Medical Sciences,

Banaras Hindu University, Varanasi 221005, India

123

Med Chem Res

DOI 10.1007/s00044-012-0274-9

MEDICINALCHEMISTRYRESEARCH

MDM2–p53 complex (pdb id. 1ycq) was obtained from the

RCSB protein data bank. The structures of ligands, taxif-

olin and quercetin were generated from smile strings fol-

lowed by energy minimization. All the heteroatoms were

removed during the preparation of protein coordinate file.

Hydrogen atoms were added to protein crystal structures

using AutoDock program, while all the nonpolar hydrogen

atoms were merged. Six bonds were made ‘‘active’’ or

rotatable for the taxifolin and quercetin (Fig. 1). Lamarkian

genetic algorithm was used as a search parameter, which is

based on adaptive local search. Short range van der Waals

and electrostatic interactions, hydrogen bonding, entropy

losses were included for energy-based AutoDock scoring

function (Morris et al., 1998; Morris et al., 2009). The

Lamarckian GA parameters used in the study were the

number of runs, 30; population size, 150; the maximum

number of eval, 250,00,000; the number of generations,

27,000; rate of gene mutation, 0.02; and the rate of cross

over, 0.8. Blind docking is carried out using grid sizes of

126, 126, and 126 along the X, Y, and Z axes with 0.375 A

spacing. RMS cluster tolerance was set to 2.0 A. Semi-

flexible docking was performed which includes a flexible

ligand and a rigid receptor.

Molecular dynamic simulation

MD simulation of the complex was carried out by means of

the GROMACS4.5.4 package using the GROMOS96 43a1

force field (Berendsen et al., 1995; Lindah et al., 2001).

The lowest binding energy (most negative) docking con-

formation of MDM2–p53–ligand complex generated by

Autodock was taken as initial conformation for MD sim-

ulation. The topology parameters of proteins were created

by means of the Gromacs program. The topology param-

eters of taxifolin were built using the Dundee PRODRG

server (Schuttelkopf and van Aalten, 2004). The complex

Fig. 1 Structures of quercetin

and taxifolin

Fig. 2 a Plot of RMSD of

backbone of MDM2–p53

complexed with taxifolin

(black) and MDM2–p53

complexed with quercetin

(grey). RMSDs were calculated

using the initial structures as

templates. The trajectories were

captured at every 0.5 ps until

the simulation time reached

12,000 ps, b Plot of RMSD of

taxifolin (black) and quercetin

(grey)

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was immersed in an octahedron box of extended simple

point charge water molecules (Van Gunsteren et al., 1996;

Van Gunsteren et al., 1998). To release conflicting con-

tacts, energy minimization was performed using the

steepest descent method of 10,000 steps followed by the

conjugate gradient method for 10,000 steps. MD simulation

studies consist of equilibration and production phases.

Position-restrained dynamics simulation (NVT and NPT)

of the system was done at 300 K for 200 ps. Finally, the

full system was subjected to MD production run at 300 K

temperature and 1 bar pressure for 12,000 ps. For the

purpose of analysis, the atom coordinates were recorded

every 0.5 ps during the MD simulation. All the structural

images were generated using PYMOL.

Result and discussion

Molecular docking

Taxifolin and quercetin were found to bind at the interface of

MDM2–p53 complex with the highest negative binding ener-

gies of -8.22 and -8.29 kcal/Mol, respect ively. Free energy

of binding is calculated as the sum of four energy terms of

intermolecular energy (van der Waals, hydrogen bond,

desolvation energy, and electrostatic energy), total internal

energy, torsional free energy, and unbound system energy.

Judging from the values of mean binding energy, the rank of

conformations of top five docking poses is detailed in Supple-

mentary table 1. These results clearly indicate that both ligands

have high affinity for the MDM2–p53 interface. The major

interactions shown in the MDM2–p53 interface are the

important H-bonds with residues Lys 24 and Leu 26 of p53, and

Tyr 51 and Gln 55 of MDM2 (Supplementary table 1). The

groups involved in H-bonding were hydroxyl (hydrogen donor)

and carbonyl (hydrogen acceptor) groups of taxifolin and quercetin.

Molecular dynamic simulation

The MDM2–p53–taxifolin and MDM2–p53–quercetin

complexes, with the binding energies of -8.22 kcal/mol

and -8.29 kcal/mol, respectively, and obtained using

Autodock, were used for carrying out MD simulation. All-

atom MD simulations represent a convenient method for

investigating the differences in motions of residues/atoms

that are subjected to structural and chemical changes. We

have analyzed the time-dependent behaviors of MD tra-

jectories for MDM2–p53 ligand including root mean

square deviation (RMSD) for all backbone atoms and

ligands along with the average residue fluctuations of the

Fig. 3 a Number of H-bonds

formed between taxifolin and

MDM2–p53 interface residues,

b Number of H-bonds formed

between quercetin and MDM2–

p53 interface residues during

12,000-ps MD simulation

Fig. 4 RMSFs of MDM2–p53 residues backbone in taxifolin-bound

(black), quercetin-bound (grey) and ligand-unbound form (light grey)

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residues (RMSF). The RMSD of backbone atoms with

respect to the initial conformation was calculated as a

function of time to assess the effect of ligand binding on

conformational stability of MDM2–p53 complex during

the simulations. Figure 2a shows that the RMSD trajectory

was always less than 0.25 nm up to *11,500 ps for

Fig. 5 Snap shots at different time intervals of MDM2–p53 complexed with taxifolin for 12,000 ps MD simulation showing dissociation of

complex

Fig. 6 Snap shots at different time intervals of MDM2–p53 complexed with quercetin for 12,000 ps MD simulation showing dissociation of

complex

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taxifolin-bound form except at *7,000 ps which was

found to be associated with partial disruption of complex.

A huge rise in the RMSD was observed at *11,500 ps, and

subsequently, a constant profile was observed with up and

down movements for very small time intervals. This

increase in RMSD was found to be in good agreement with

the snapshots recorded at and after 11,000 ps (described

later) which revealed the separation of p53 segment from

MDM2. Analysis of taxifolin RMSD indicates that taxif-

olin showed remarkable stability at the interface of

MDM2–p53 complex during MD simulation. However,

some high fluctuations were observed during the simulation

(Fig. 2b). In quercetin-bound form, the RMSD trajectories

of backbone showed high increase from the value of

0.25 nm after *10,000 ps, and subsequently, a near-con-

stant profile was observed (Fig. 2a). Similar to taxifolin,

this increase of RMSD was found to be associated with the

dissociation of MDM2–p53 complex (described later).

Analysis of quercetin RMSD indicated that quercetin

showed stability at interface of MDM2–p53 complex dur-

ing MD simulation. However, an increase in RMSD was

observed after *9,000 ps (Fig. 2b). This increase in

RMSD indicates the dissociation of quercetin along with

p53. Previously, simulation studies on MDM2–p53 com-

plex in the absence of ligands showed stable RMSD profile

which confirmed high increase in RMSD associated

with disruption of MDM2–p53 complex and the complex

disruption effect of taxifolin and quercetin during MD

simulation (Espinoza-FonsecaJose and Trujillo-Ferrara,

2006, Espinoza-FonsecaJose and Garcıa-Machorro, 2008;

Bharatham et al., 2011). The number of H-bonds (cut off

0.35 nm) which was formed during MD simulation

between ligands and MDM2–p53 was also calculated. A

variable profile was observed which fluctuates from 0 to 3

and from 0 to 4 with corresponding average values of 0.15

and 0.25 for taxifolin and quercetin, respectively (Fig. 3).

Furthermore, to identify the flexible residues of the protein,

root mean square fluctuation (RMSF) of backbone atoms

from its time-averaged position was analyzed. All the

residues of both MDM2 and p53 showed markedly higher

fluctuation in taxifolin- and quercetin-bound forms as

compared with unbound form. The p53 residues showed

Fig. 7 Interaction of taxifolin with p53 residues at different time intervals during 12,000-ps MD simulation (cyan-taxifolin) (Color figure online)

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Fig. 8 Interaction of taxifolin with MDM2 residues at different time intervals during 12,000-ps MD simulation (green-taxifolin) (Color figure

online)

Fig. 9 2D plots of interaction between taxifolin and MDM2–p53 at different time intervals of 12,000-ps MD simulation

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fluctuation increased by *1 nm compared with ligand-

unbound form (Fig. 4). This profile confirmed that the

ligandbinding induced a movement in the residues of protein

complex.

Coordinates of the MDM2–p53–taxifolin system recor-

ded at different time intervals of simulation revealed

that binding of the MDM2–p53 remained intact for

*11,000 ps; thereafter, P53 segment detached from the

MDM2 cleft as observed during the 11,500-ps and final

12,000-ps snapshots (Fig. 5). These results were supported

by the RMSD and RMSF profiles described earlier. In

the case of MDM2–p53–quercetin, major changes were

observed at *10 ns. p53 segment along with quercetin

leaves the MDM2 binding site as observed in the 11-ns

snapshot, while at 12 ns, quercetin was found to be bound

to MDM2 (Fig. 6). Analysis of the 12-ns snapshot of

MDM2–p53 complex (without ligand) recorded during

simulation revealed that only few residues contributed to

the interaction of these two proteins. Phe 19, Trp 23, and

Leu 26 of P53 were the residues which oriented toward the

MDM2 binding cleft. The importance of these residues was

previously described several times (Allen et al., 2009; Moll

and Petrenko, 2003; Chen et al., 2011). In the case of

MDM2, Lys 47, Tyr 51, Gln 55, and Met 58 were found as

critical residues involved in interaction with p53. The

interaction of these two proteins is mainly dependent on the

hydrophobic interactions (Allen et al., 2009; Moll and

Petrenko, 2003; Chen et al., 2011). To find the effect of

ligands on these residues, we analyzed snapshots of sim-

ulations at different time intervals.

Interaction of taxifolin with MDM2–p53

Phe 19 side chain of p53 showed movement toward the p53

backbone in taxifolin-bound form. This bending might be

due to the hydrophobic stacking with B–C rings of taxif-

olin. Trp 23 side chain showed higher bending. The side

chain indole ring was switching its interaction from Tyr 51

of MDM2 to taxifolin B and C rings. However, the ori-

entation of Leu 26 remained nearly the same (Fig. 7). Ring

A of taxifolin oriented itself parallel to the side chain ring

Fig. 10 Interaction of quercetin with p53 residues at different time intervals during 12,000-ps MD simulation (green-quercetin) (Color figure online)

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of Tyr 51 of MDM2 which led to strong pi–pi stacking

(Fig. 8). Possibly, this strong interaction suppressed the

interaction between Trp 23 of p53 and Tyr 51 of MDM2.

The bonding between Tyr 51 and A ring was found to be

the main reason behind the stability of taxifolin at MDM2

cleft. The 2D plot of taxifolin and MDM2–p53 interaction

generated by Discovery studio 3.1 (Accelrys Software Inc.,

2011) at different time intervals of MD simulation showed

and confirmed that the hydrophobic interactions were

dominating during simulation. Initially, taxifolin showed

pi–pi interaction with both MDM2 and p53, which finally,

completely, switched to pi–pi and cation-pi interactions

between A-ring and Tyr 51, and between A-ring and Lys

47, respectively (Fig. 9). In this way, interactions of Trp 23

of p53 and Tyr 51 of MDM2 transformed into Trp 23–B–C

and Tyr 51–A interactions, and finally into A-ring of

taxifolin and Tyr 51, and into A-ring–Lys 47 of MDM2. On

the basis of these results, the strength of hydrophobic

interactions can be arranged in the order as follows:

MDM2�p53� p53�taxifolin\MDM2�taxifolin

Interaction of quercetin with MDM2–p53

Although p53 residues side chains did not show very high

difference in their orientations during simulation, the B and

C rings of quercetin showed movement toward Phe 19 and

Trp 23 (Fig. 10). These interactions were solely responsi-

ble for the masking of the interaction between MDM2 and

p53 residues and the separation of P53–quercetin complex.

The snapshot recorded at 12 ns revealed that B and C rings

were in hydrophobic contact to the side chain aromatic ring

of Tyr 51 of MDM2 (Fig. 11). The possible reason might

be the lack of more favorable orientation between the B–C

rings and p53 residues which led to weak hydrophobic

interaction as compared with Tyr 51 of MDM2 and B and,

C rings so that quercetin dissociated from p53 and bound to

MDM2. The 2D plot of quercetin and MDM2–p53 inter-

action also showed that initially, quercetin involved in

pi–pi interaction with MDM2 by C-ring. At 12,000 ps, B

and C rings of quercetin were found in pi–pi interaction

with Tyr 51(Fig. 12). On the basis of these results, the

order of hydrophobic interaction strength was found as

follows:

MDM2�p53\p53�quercetin\MDM2�quercetin

It is well established that interaction between MDM2

and p53 is governed by hydrophobic groups of residues.

The presence of aromatic rings in the ligands was found as

the main reason behind the masking of the interaction

between these two proteins. Further, Tyr 51 residue was

found to play a leading role in its interaction with p53 as

Fig. 11 Interaction of quercetin with MDM2 residues at different time intervals during 12,000-ps MD simulation (green-quercetin) (Color figure

online)

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the masking of Tyr 51 hydrophobic side chain by ligands

led to the separation of p53. However, there are several

studies regarding the inhibition of interaction of MDM2–

p53, but no previous study has reported the separation of

MDM2 and p53 during simulation.

Conclusion

Taxifolin and quercetin are naturally occurring polyphenolic

compounds. Both compounds were found to inhibit the

MDM2 and p53 interaction as evidenced by molecular

dynamic simulation. The interaction of MDM2 and p53 is

dominantly governed by the hydrophobic residues. Taxifolin

and quercetin efficiently mask these interactions, leading to

the separation of p53. The hydrophobic aromatic ring system

of ligands mainly contributed to this action. These phyto-

chemicals being the natural compounds and considering

their bioavailability in natural food products, they can be

used for targeting MDM2 and p53 interaction and develop-

ing more efficient inhibitors for cancer chemoprevention.

Acknowledgments One of the authors (Sharad Verma) is thankful

to the Council of Scientific and Industrial Research (CSIR), India for

providing Senior Research Fellowship.

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