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Journal of Theoretical and Computational ChemistryVol. 8, No. 3 (2009) 491–506c© World Scientific Publishing Company
THEORETICAL STUDY ON POTENCY AND SELECTIVITYOF NOVEL NONPEPTIDE INHIBITORS OF MATRIX
METALLOPROTEINASES MMP-2 AND MMP-9
DAI-LIN LI∗, QING-CHUAN ZHENG∗, XUE-XUN FANG†,HAI-TAO JI†, JIN-GANG YANG† and HONG-XING ZHANG∗,‡
∗State Key Laboratory of Theoretical and Computational Chemistry,Institute of Theoretical Chemistry, Jilin University,
Changchun 130023, P. R. China
†Key Laboratory for Molecular Enzymology and Enzyme,Engineering of Ministry of Education, Jilin University,
Changchun 130023, P. R. China‡[email protected]
Received 14 May 2008Accepted 20 August 2008
Two novel matrix metalloproteinase (MMP) inhibitors, myricetin (m) and kaempferol(k), were found and the inhibitory activity is both in decreased order towards MMP-2and MMP-9. To understand the mechanism during the processes when inhibitors bind toMMP-2 and MMP-9, molecular modeling, docking, and density functional theory (DFT)calculations were performed. The calculated results indicated that the hydroxyls onbenzene ring of the inhibitors control the binding modes between inhibitors and MMPs,thus play an important role on the potency and selectivity. Besides coordinating with
the N atoms of three His residues, Zn also interacts with a hydroxyl group of inhibitorsby O–Zn distances of 2.66–2.78 A in all of the docked complexes, so that the hydroxylacts as a weak zinc binding group (ZBG). The DFT calculated results support theabove analysis. The binding affinity calculations between inhibitors and MMPs presentthe total interaction energies in the m-MMP < k-MMP order and the solvation energyof myricetin is less than that of kaempferol, which reflect the experimental inhibitoryactivity.
Keywords: MMP; inhibitors; docking; DFT.
1. Introduction
Matrix metalloproteinases (MMPs) are a large family of zinc-dependent enzymes,which show proteolytic activities against most components of the extracelluarmatrix, such as fibronectin, laminin, and interstitial collagens.1–3 The proteolyticactivity is partially regulated by endogenous tissue inhibitors of metalloproteinases
‡Corresponding author.
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(TIMPs).1 Imbalances in the expression of MMPs and their endogenous specificinhibitors may result in many diseases such as cancer, arthritis, and others.4,5
Therefore MMPs are important therapeutic targets for treatment of the diseasesmentioned above.
On the basis of substrate specificity and primary sequence similarity, MMPscan be classified into five subfamilies, i.e. collagenases (MMP-1, -8, and -13),stromelysins (MMP-3, -10, and -11), gelatinases (MMP-2 and -9), membrane-typeMMPs (MMP-14, -15, -16, -17, -24 and -25), a more heterogeneous subgroup con-taining matrilysin (MMP-7), macrophage metalloelastases (MMP-12 and -19), andenamelysin (MMP-20).6,7 Among the subfamilies of MMPs, gelatinase A (MMP-2)and gelatinase B (MMP-9) have been considered very promising for use in drugdevelopment, because they are thought to play an important role in triggering theprocesses of tumor growth, invasion, and metastasis by cleaving the vascular base-ment membrane, which consists of type IV collagen.8–13
General requirement for an inhibitor of MMPs is a zinc binding group (ZBG),which is capable of attaching to the catalytic zinc atom of MMPs.14 Inhibitors withseveral types of early found ZBG, such as hydroxamate,15–17 carboxylic acid,18–23
thiol,24–28 phosphonic acid and phosphinic acid,29,30 have been studied extensively.Inhibitors with the hydroxamic acid group were considered to be efficiently potent31
for the hydroxamic acid group binds to the catalytic ion in a bidentate way, block-ing substrate access to the active site and rendering the metal incapable of peptidehydrolysis. However, due to the release of hydroxylamine, a known carcinogenicagent, the hydroxamate inhibitors show poor pharmacokinetic properties and canentail toxicity in long-term therapy.32,33 The synthetic precursors to the hydroxa-mate inhibitors,1 carboxylic acids, were lately found to be a kind of inhibitors byacting as ZBG. Based on the examination of the O–Zn bond distances and geom-etry of the metal centers in the X-ray structures,18–23 the carboxylate ZBG wasrevealed to coordinate to the catalytic zinc ion in a monodentate way.34 As a mon-odentate inhibitor, carboxylates should bind to the zinc more weakly than hydrox-amates due to the loss of one O–Zn bond. Thus, most carboxylic acid inhibitorshave lower inhibitory activity than bidentate hydroxamate inhibitors.35 Thiol ZBGswere expected to be inhibitors due to the apparent thiophilicity of the zinc ion ina number of proteins. The crystal structures showed that thiol group binds to zincion in a monodentate way.36 Although thiols are almost as potent as hydroxamicacids,1 they are unstable in the body due to oxidation and formation of disulfidebonds with exposed Cys residues of various proteins.37 Inhibitors with phosphonicacid or phosphinic acid group also coordinate to zinc ion in a monodentate waybased on the O–Zn bond distances among the crystal structures.29,30,34 Therefore,this kind of inhibitors also has lower inhibitory activity than that of hydroxamates.In the last several years, the newly found pyrone-based inhibitors, which take theheterocyclic moiety as ZBG, were proposed to be more potent than the hydroxam-ates, nontoxic, and biocompatible, and showed promise to be a new type of MMPinhibitor.38–40
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(a) (b)
Fig. 1. Structures of inhibitors displayed in ball-and-stick model: (a) myricetin; (b) kaempferol.For consistency of the two molecules, we only labeled the atoms constituting the rings, the O,and H atoms are identified according to the labeled C atoms, i.e. labels of the O and H atomsin hydroxyl groups inherit the same number for the C atoms that the specific hydroxyl connectsto. For example, in the hydroxyl connecting to C1 atom, the O atom should be O1 and the H
atom H1.
So far, new types of inhibitors have continuously attracted the attention ofresearchers. Recently, we tested a class of novel nonpeptide inhibitors for MMPs,with the IC50 values toward MMPs in micromolar or submicromolar range, whichmay be useful in treating the diseases caused by MMPs.a These novel inhibitors,with hydroxyl as ZBG, include myricetin and kaempferol (Fig. 1). In the presentstudy, myricetin and kaempferol are taken as MMP inhibitors and simulatedto interact with MMP-2 and MMP-9 to investigate the binding modes, whichhave effects on the potency and selectivity of inhibitors. Our results may behelpful in structure-based design of MMP inhibitors with improved potency andselectivity.
2. Theory and Methods
The molecule simulations were performed on the SGI O3900 servers using InsightIIsoftware package developed by Accelrys.41 The extensible and systematic force field(ESFF) was used for energy minimization and docking simulations. Quantum chem-istry calculations were carried out using Gaussian 03.42
aFluorescence titration measurements were performed on a Shimadzu RF-5301PC (Tokyo, Japan)fluorescence spectrophotometer. The experiments were carried out at 25◦C in 50mM HEPESbuffer (PH = 7.5), including 0.2M NaCl, 10mM CaCl2, and 0.05% Brij-35. By means of thefluorescent substrate enzyme activity assay, we obtained the IC50 values as follows: myricetintowards MMP-2 and MMP-9 are 1.84 and 0.59 µM, respectively, and kaempferol toward MMP-2and MMP-9 are 312.26 and 255.23 µM, respectively.
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2.1. Modeling the initial structures of MMPs
The starting structures of MMP-2 (PDB code 1HOV) and MMP-9 (PDB code1GKC) were obtained from the Protein Data Bank (www.rcsb.org). The Buildermodule was used to modify the PDB files. Zinc binding ligands, hydroxamic acid andpeptidic reverse hydroxamate, were extracted from the proteinase complex in eachPDB file. Hydrogen atoms were added to the enzymes by setting PH=7.5, same asthe experimental condition in testing the IC50 values.a To rationalize the structuresof MMP-2 and MMP-9 in water solution from their crystal structures, we performedenergy minimizations of 300 steps of conjugate gradient with a convergence valueof 0.05 kcalmol−1A−1, where an explicit solvent model TIP3P water was used and5 A thickness of water layer was added to MMP-2 and MMP-9.43,44 In the energyminimizations, the relatively rigid structural units of α-helix and β-sheets in thesecondary structures were kept.
2.2. Docking inhibitors to MMPs
Affinity is a suite of programs for automatically docking a ligand to a receptor bya combination of Monte Carlo type and simulated annealing procedure.45,46 Afterthe energy minimizations, the potentials, in which the atom types were determinedby hybridization, formal charge, and symmetry rules, were calculated and assignedto the MMPs.47 Moreover, the partial charges, which were calculated by minimiz-ing the electrostatic energy under the constraint that the sum of the charges wasequal to the net charge on the molecule, were also assigned to the MMPs.47 Theformal charges were artificially assigned as +2.0 e to the catalytic zinc ion. Atomswithin the active sites of the receptors (MMPs) were totally released with the dif-ferent inhibitors during the docking process, while the structures of inactive regionswere held rigid. Six subsites around the catalytic zinc ion, named S1′–S3′ (primesite) and S1–S3 (nonprime site), which had been approved to interact with differenttypes of inhibitors, such as hydroxamic acid,15–17 carboxylic acid,18–23 thiol,24–28
etc. on experiments, were chosen as possible active sites. To clarify the positionof each subsite, we just take the structure of MMP-2 from the PDB file (code1HOV) (Fig. 2), for example, because the positions of each subsite in MMP-2 andMMP-9 are almost the same except for several different residues in constituents.The S1′ subsite, which was used to gain selectivity between MMP-2 and MMP-9 byintroducing large hydrophobic groups at P1′ into inhibitors,49 refers to the pocketconsisted of Leu116, Val117, Tyr144, and Thr145, extending into the inner of the“paper plane”. The S2′ subsite, which is partly solvent exposed, refers to the pocketmade up of Gly81, Pro140, and Ile141, pointing to the out of the “paper plane.”The S3′ pocket locates on the right of Asn111, Tyr112, and Tyr144 and is muchmore solvent exposed. The S1 pocket, which is formed of Ala86, Glu121, His124,and Ala125, extends into the inner of the “paper plane.” The S2 pocket involvesHis85, Ala86, His124, and His130 and points to the out of the “paper plane.” TheS3 pocket contains Phe87, Ala125, Met126, and Gly127 and locates on the left of
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Fig. 2. Representation of the active sites of MMP-2 chosen for docking calculations, consisted ofsix subsites named S1′–S3′ and S1–S3.
them. One point bearing in mind is that the residues of each subsite, we mentionedabove in Fig. 2, are used only for showing the positions of each subsite more clearly,and not the whole constituents of each subsite. Three-dimensional structures of theinhibitors, myricetin and kaempferol, were fully optimized through Gaussian 0342
and displayed in Fig. 1. The calculated structures of the docked complexes weretaken as the initiatives for further investigation of the interaction energy and thegeometrical matching quality. Moreover, the solvation energy composed of elec-trostatic energy and nonpolar energy, calculated by Delphi-based solvation modelwithin solvation module, was considered when comparing the inhibitory activity ofinhibitors.
2.3. Quantum chemistry calculations
The density functional theory (DFT) was used to characterize the interactionsbetween ZBG and the catalytic zinc ion more accurately. The Becke three para-meter functional and the Lee–Yang–Parr functional (B3LYP) method, a DFT typeof calculation approach based on a hybrid function has shown to be a favoritecandidate in calculation of geometries and energies for the first-row transition metalcomplexes.49 In the present work, the B3LYP calculations, under the basis of 6-31G*level, were chosen to optimize the docked complexes. Frequency calculations werethen carried out to verify the rationality of the optimized structures.
3. Results and Discussion
3.1. Potency
3.1.1. Potency of inhibitors on MMP-2
To investigate the potency of inhibitors on MMP-2, we theoretically studied thebinding characteristics between MMP-2 and the considered inhibitors, myricetin and
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kaempferol, when they form the inhibitor–acceptor complexes (Fig. 3) denoted bym-MMP2 and k-MMP2, respectively.
In m-MMP2 complex, myricetin, which owns three ortho-hydroxyls on the ben-zene ring, binds to the catalytic zinc ion with O1 atom, with the O1–Zn distance of2.71 A (Figs. 3(a) and 3(b)). The two-ring moiety at C5 atom acts as P1′ and occu-pies the S1′ subsite of MMP-2 partially. Three hydrogen bonds are formed, which
(a) (b)
(c) (d)
Fig. 3. Secondary structure representation of complexes, the zinc ions are shown as purple spheres:(a) m-MMP2; (d) k-MMP2. Detailed representation of inhibitors and MMP-2 active site, thedistance between binding atom and catalytic zinc is given by green line while the other linesare the hydrogen bonds: (b) m-MMP2; (e) k-MMP2. The meta-double-hydroxyl mode in thecomplexes: (c) m-MMP2; (f) k-MMP2. (Color online)
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(e) (f)
Fig. 3. (Continued)
are H3 with O of Gly81, O15 with H of Thr143, and H15 with O of Ala139. In orderto characterize the function mode of the meta-hydroxyl on benzene ring for clarity,the meta-double-hydroxyl mode is defined, which is when one hydroxyl binds tothe catalytic zinc, the other meta-hydroxyl will form at least one hydrogen bondwith MMP. For example, in m-MMP2, O1 binds to the catalytic zinc and H3 formsa hydrogen bond, which indicates that the meta-double-hydroxyl mode is formed(Fig. 3(c)).
In k-MMP2 complex, kaempferol, which owns just one hydroxyl on benzene ring,binds to the catalytic zinc ion with O13 atom, with the O13–Zn distance of 2.72 A(Figs. 3(d) and 3(e)). The benzene ring moiety at C7 atom acts as P2′ and pointsto S2′ subsite of MMP-2. Only one hydrogen bond is formed, which is H15 with Oof Ala84. The meta-double-hydroxyl mode is formed again, where O13 atom of onehydroxyl interacts with the catalytic zinc and H15 atom of the other meta-hydroxylforms a hydrogen bond with MMP-2 (Fig. 3(f)).
The interaction energy, a quantity reflecting the binding ability, can be used toillustrate the differences in potency and explain the calculated results mentionedabove more rationally. The interaction energies shown in Table 1 are −80.22 and−49.66 kcalmol−1 for m-MMP2 and k-MMP2, respectively, which indicates thatthe binding affinity is in the order of m-MMP2 > k-MMP2. In addition, the solva-tion energy, which has effects on the affinity between inhibitor and MMP, was alsoconsidered in evaluating the potency. It can be seen from Table 2 that the solva-tion energy of myricetin (−51.85 kcalmol−1) is much less than that of kaempferol(−37.32 kcalmol−1). This means that the solvation effect is more beneficial forthe combination between myricetin and MMP-2. From the analysis on interactionand solvation energy, it can be inferred that myricetin should be more potent thankaempferol. Table 3 shows that the IC50 values are 1.84 and 312.26µM for myricetinand kaempferol, respectively,a which is explicitly consistent with the above theo-retical deduction.
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Table 1. Solvation energies between inhibitors(myricetin and kaempferol) and MMPs (MMP-2 andMMP-9) (kcal mol−1).
Total interaction energy
Inhibitor MMP-2 MMP-9
Myricetin −80.22 −87.52Kaempferol −49.66 −50.45
Table 2. Solvation energies of inhibitors (myricetinand kaempferol) with MMPs (MMP-2 and MMP-9)(kcal mol−1).
Total interaction energy
Inhibitor MMP-2 MMP-9
Myricetin −51.85 −58.41Kaempferol −37.32 −37.84
Table 3. The IC50 values of myricetin and kaempferoltoward MMP-2 and MMP-9 (µM).
IC50
Inhibitor MMP-2 MMP-9
Myricetin 1.84 0.59Kaempferol 312.26 255.23
As a whole, kaempferol, short of two side hydroxyls on benzene ring comparedwith myricetin, binds to MMP-2 in a very different way from that of myricetin(Fig. 3). Therefore, we infer that the different numbers of hydroxyls on benzenering caused the completely binding modes, leading to the great loss of inhibitoryactivity of kaempferol, almost 170-fold less potent than myricetin (Table 3).a
3.1.2. Potency of inhibitors on MMP-9
To explore the potency when inhibitors, myricetin and kaempferol, interact withMMP-9 and form the complexes m-MMP9 and k-MMP9, we investigated the bind-ing characteristics between the considered inhibitors and MMP-9 (Fig. 4).
In m-MMP9 complex, myricetin binds to the catalytic zinc ion with O3 atom,with the O3–Zn distance of 2.66 A (Figs. 4(a) and 4(b)). The two-ring moiety at C5atom acts as P1′ and occupies the S1′ subsite of MMP-9 in degree. Four hydrogenbonds are formed, which are H2 with O of Ala189, H1 with O of Ala189, O1 with Hof Ala189, and H8 with O of Leu418. The meta-hydroxyls on benzene ring, of whichone (containing O3 and H3) binds to the catalytic zinc and the other (containingO1 and H1) forms two hydrogen bonds with MMP-9, function in the meta-double-hydroxyl mode.
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(a) (b)
(c) (d)
Fig. 4. Secondary structure representation of complexes, the zinc ions are shown as purple spheres:(a) m-MMP9; (c) k-MMP9. Detailed representation of inhibitors and MMP-9 active site, thedistance between binding atom and catalytic zinc is given by green line while the other lines arethe hydrogen bonds: (b) m-MMP9; (d) k-MMP9. (Color online)
In k-MMP9 complex, kaempferol binds to the catalytic zinc ion with O15 atom,with the O15–Zn distance of 2.78 A (Figs. 4(c) and 4(d)). The benzene ring moietyat C7 atom acts as P2′ and points to the S2′ subsite of MMP-9. Four hydrogenbonds are formed, which are H15 with O of Glu402, O13 with H of Ala189, O9with H of Leu188, and H8 with O of Gly186. The meta-hydroxyls, which containO15 atom binding to the catalytic zinc and O13 atom involved in a hydrogen bond,function in the meta-double-hydroxyl mode.
As a result, the binding mode of k-MMP9 is completely different from thatof m-MMP9 due to the lost of two side hydroxyls on benzene ring in kaempferol.
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The extreme differences in binding modes make that the total interaction energy ofk-MMP9 is 37.07 kcalmol−1 higher than that of m-MMP9 (Table 1). Furthermore,the solvation energy of myricetin (−58.41 kcalmol−1) is much less than that ofkaempferol (−37.84 kcalmol−1) (Table 2), indicating higher binding affinity betweenmyricetin and MMP-9. Our deduction is well consistent with the experimentalresulta that kaempferol is almost 430-fold less potent than myricetin on MMP-9(Table 3).
3.2. Selectivity
3.2.1. Selectivity of myricetin on MMP-2 and MMP-9
Selectivity, which is important in minimizing undesirable side effects during long-term medical treatment, has interested the field of drug design a lot.50 To investi-gate the differences in selectivity of myricetin on MMP-2 and MMP-9, we studiedthe binding characteristics when myricetin interacts with MMP-2 and MMP-9,respectively.
As the binding energies between myricetin and MMPs both partially result fromthe interactions between P1′ and S1′ in m-MMP2 and m-MMP9, we investigatethe interactions between P1′ and S1′ above all. Table 4 lists the energies betweenmyricetin and individual residues in S1′ subsite of MMPs, including the total, VDW,and electrostatic energies with total energies lower than −2 kcalmol−1. There arefive residues in m-MMP2 and m-MMP9, respectively, contributing more to theinteractions for lower total energies, which are Ala139, Pro140, Tyr142, Thr143,and Tyr144 in m-MMP2 and Leu397, Leu418, Met422, Tyr423, and Arg424 in
Table 4. The van der waals energies (Evdw), electro-static energies (Eele), and total energies (Etotal) betweenmyricetin and individual residues of S1′ subsite of MMP-2(a) and MMP-9 (b) (kcal mol−1).
Residues Evdw Eele Etotal
(a)Ala139 −1.91 −6.73 −8.64Pro140 −3.27 0.53 −2.74Tyr142 −6.38 −0.01 −6.39Thr143 −2.28 −3.20 −5.48Tyr144 −2.82 −0.61 −3.43
Sum −16.66 −10.02 −26.69
(b)Leu397 −2.49 0.20 −2.29Leu418 −3.16 −3.23 −6.39Met422 −4.68 −4.32 −9.00Tyr423 −5.51 −0.13 −5.64Arg424 −3.79 0.70 −3.09
Sum −19.63 −6.78 −26.41
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m-MMP9. The VDW energies −16.66 and −19.63 kcalmol−1 prevail in the sumin both m-MMP2 and m-MMP9 (Table 4), implying that the S1′ subsites ofMMP-2 and MMP-9 are hydrophobic, which is consistent with the results fromexperiments.50 The sum of total energies between P1′ and S1′ subsite, in m-MMP2and m-MMP9, are very similar, which are −26.69 and −26.41 kcalmol−1, respec-tively. This indicates that the interactions between P1′ and S1′ may not be the mainreason for the different selectivity of myricetin on MMP-2 and MMP-9. Therefore,we think that the reason may be the interactions between hydroxyls on benzene ringand MMPs. First, the bond distances of O1–Zn and O3–Zn are 2.71 and 2.66 A inm-MMP2 and m-MMP9, respectively. The shorter distance in m-MMP9 may indi-cate stronger binding affinity between hydroxyl O atom and catalytic zinc ion.Second, the hydroxyls on benzene ring form three hydrogen bonds with MMP-9while only one with MMP-2 (Figs. 3(b) and 4(b)). The contributions of hydrogenbonds to binding interactions are also more in m-MMP9 than that in m-MMP2.The two points above may cause the different selectivity.
The total interaction energies between myricetin and MMPs are −80.22 and−87.52 kcalmol−1 for m-MMP2 and m-MMP9, respectively (Table 1), which indi-cates that the differences in binding ability of myricetin with MMP-2 and MMP-9are relatively small and leads to a nonobvious selectivity on MMP-2 and MMP-9.This is consistent with the experimental results, which is myricetin that is almost3-fold selective on MMP-9 than MMP-2 (Table 3).a
3.2.2. Selectivity of kaempferol on MMP-2 and MMP-9
As being short of two hydroxyls on benzene ring, kaempferol binds to MMPs ina very different way from myricetin: it binds to MMPs with different atoms, O13atom in k-MMP2 and O15 atom in k-MMP9, and takes benzene ring moiety as P2′
group. In order to characterize the interactions between P2′ and S2′ subsite, ener-gies between kaempferol and each residue involved in S2′ subsite are calculated fork-MMP2 and k-MMP9, respectively. Table 5 lists the interaction energies includ-ing the total, VDW, and electrostatic energies with the total energies lower than−2 kcalmol−1.
It is evident from Table 5 that in k-MMP2, four residues of S2′ subsite, Gly81,Leu82, Pro140, and Ile141 contribute more to the binding affinity for lower totalenergies. The sum of VDW energy, −12.64 kcalmol−1, is much less than that of elec-trostatic energy, −2.95 kcalmol−1, indicating the VDW energy prevails between P2′
and S2′. The reason for this may be that though the P2′ is polar somewhat becauseof the hydroxyl (containing O2 and H2) on benzene ring, the hydroxyl has protrudedout of the S2′ subsite and been far away from the four residues mentioned above.Thus, the interactions mainly come from the nonpolar benzene ring and the fournonpolar residues, which make the VDW energy dominant. In k-MMP9, there arethree residues of S2′ subsite, Gly186, Leu187, and Pro421, which contribute more tothe interactions between P2′ and S2′. The sum of VDW energy, −9.70 kcalmol−1, is
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Table 5. The van der waals energies (Evdw), electro-static energies (Eele), and total energies (Etotal) betweenkaempferol and individual residues of S2′ subsite of MMP-2(a) and MMP-9 (b) (kcal mol−1).
Residues Evdw Eele Etotal
(a)Gly81 −3.22 −2.00 −5.22Leu82 −3.13 0.27 −2.86Pro140 −2.65 −0.62 −3.27Ile141 −3.64 −0.60 −4.24
Sum −12.64 −2.95 −15.59
(b)Gly186 −2.01 −2.08 −4.09Leu187 −4.85 −0.56 −5.41Pro421 −2.84 −0.40 −3.24
Sum −9.70 −3.04 −12.74
also much less than that of electrostatic energy, −3.04 kcalmol−1. This may be thesame reason as that for k-MMP2 because of similar binding modes of kaempferolin MMP-2 and MMP-9 in spite of different binding atoms.
The total binding energies between kaempferol and MMPs are −49.66 and−50.45 kcalmol−1 for k-MMP2 and k-MMP9, respectively (Table 1). The differ-ence is only 0.79 kcalmol−1, lower than that between m-MMP2 and m-MMP9,which indicates that kaempferol is more selective on MMP-9 than MMP-2, butwith even lower selectivity than that of myricetin. This is in agreement with theexperimental data, wherein the IC50 values are 312.26 and 255.23µM for kaempferoltoward MMP-2 and MMP-9, respectively, and the selectivity is less than 2-fold.a
Furthermore, we inferred above that the great decreased inhibitory activity ofkaempferol may arise from different binding modes. The main reason, we think,is the lost of stronger interactions between P1′ and S1′. The sum of total ener-gies between P1′ and S1′, −26.69 and −26.41 kcalmol−1 for m-MMP2 and m-MMP9, respectively, is much less than that between P2′ and S2′, −15.59 and−12.74 kcalmol−1 for k-MMP2 and k-MMP9, respectively (Tables 4 and 5). Thisindicates that interactions between P1′ and S1′ are indeed much stronger thanthat between P2′ and S2′. Thus, it can be deduced that the binding modes betweenkaempferol and MMPs, compared with that between myricetin and MMPs, in whichthe interactions between P1′ and S1′ are lost but with that between P2′ and S2′
instead, lead to the decreased potency of kaempferol on MMP-2 and MMP-9 in part.From all of the calculated complexes, m-MMP2, m-MMP9, k-MMP2, and k-
MMP9, it is evident that the new ZBG, hydroxyl, is a relatively weak ZBG for allof the distances between binding atom and the catalytic zinc ion are more than2.60 A while the general coordination bond distance is around from 1.90 to 2.20 A.Furthermore, the meta-double-hydroxyl mode may be conserved when inhibitors ofthis kind bind to MMPs.
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3.3. Quantum chemistry calculation
3.3.1. Interaction characteristics between ZBG and catalyticzinc ion of MMP
To investigate the interaction characteristics between ZBG and the catalytic zincion of MMP, we only take m-MMP2 for example, because myricetin owns morehydroxyls on benzene ring, which is more likely to result in alternative interactionways between ZBG and the zinc ion. Typical zinc coordination sphere in MMPs ismade up of three His residues and one exogenous ligand.51 Based on the dockingresult, the quantum chemistry calculation model is intercepted from the m-MMP2,which consists of the catalytic zinc ion, three imidazoles, and the whole of myricetin.The frequency calculation result of the optimized m-MMP2 shows that no imag-inary frequency exists, which indicates that the structure is in the state of trueminimum energy. As shown in Fig. 5, m-MMP2 adopts little distorted tetrahedralstructure with three angles of O1–Zn–N17, O1–Zn–N18, and O1–Zn–N19 105.3◦,98.3◦, and 107.3◦, respectively. These are close to that of standard tetrahedral con-figuration (109.5◦) and indicate that myricetin binds to MMP-2 with the ZBG inthe monodentate way.
3.3.2. Charge population analysis
The natural bond orbital (NBO) method, which uses natural atomic orbital tocalculate the atomic charge, is applied to the charge population analysis insteadof the Mulliken method, which is dependent on the basis set when calculating theatomic charge. We regard myricetin as a group for clarity and the group chargeis the summation of atomic charges of all of the atoms that compose myricetin.The calculation results show that the group charge is 0.0Q/e in free state while1.017Q/e in m-MMP2 complex. The group charge increase in m-MMP2 indicates
Fig. 5. Structure of m-MMP2 complex optimized through Gaussian 03.
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that the electrons have transferred from myricetin to MMP-2, which is a strongcertification that the ZBG, hydroxyl, can interact with the catalytic zinc ion.
4. Conclusions
Molecular modeling and docking simulations and quantum chemistry calcula-tions were carried out on the interactive systems when inhibitors, myricetin, andkaempferol, bind to MMP-2 and MMP-9, to investigate the binding modes andexplain the reasons for differences in potency and selectivity. Myricetin, with threeortho-hydroxyls on the benzene ring, owns the most potency on both MMP-2 andMMP-9. Kaempferol binds to MMPs in completely different way from myricetindue to the lost of two side hydroxyls on benzene ring, leading to the great decreasein potency on MMP-2 and MMP-9. The similar binding modes in each complexpair (m-MMP2 and m-MMP9, k-MMP2 and k-MMP9) make the nonobvious selec-tivities of inhibitors on MMP-2 and MMP-9. The results mentioned above areconsistent with the available inhibitory activity orders from experiments. All of thecomplex structures indicated that the new ZBG is a relatively weak ZBG, and themeta-double-hydroxyl mode may be conserved when inhibitors of this kind bind toMMPs. The quantum chemistry calculations show that the ZBG can interact withMMPs and inhibitors bind to MMPs in a monodentate way. We expect that thepresent theoretical investigations could provide a clue in structure-based design ofMMP inhibitors with improved potency and selectivity.
Acknowledgments
This work was supported by the Natural Science Foundation of China, KeyProjects in the National Science & Technology Pillar Program, and SpecializedResearch Fund for the Doctoral Program of Higher Education (Grant No. 20573042,2006BAE03B01, and 20070183046).
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