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Molecular Mechanism of Inhibition of DNA Methylation by Zebularine Juan Aranda, Fedaa Attana, and Iñaki Tuñón ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.6b03381 • Publication Date (Web): 30 Jan 2017 Downloaded from http://pubs.acs.org on February 1, 2017
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Molecular Mechanism of Inhibition of DNA Methylation by Zebularine. Juan Aranda†, Fedaa Attana and Iñaki Tuñón* Departamento Química Física, Universitat de València, 46100 Burjassot. ABSTRACT: In this work we have analyzed the molecular mechanism of inhibition of a C5-DNA methyltransferase by zebularine using classical and QM/MM simulations. We found that the reaction proceeds with the addition of an unprotonated cysteine to the C6 position of the ring followed by methyl transfer to the C5 position. However, while the first step is reversible and presents a moderate free energy barrier, the second step presents a large free energy barrier, preventing the formation of the methylated complex. This mechanistic proposal agrees with recent experimental observations that point out to the formation of a reversible covalent complex between DNA containing zebularine and methyltransferases. The absence of the exocyclic amino group in zebularine as compared to the natural substrate cytosine, makes more difficult to reach an optimal orientation of the substrate both for cysteine addition and methyl transfer and decreases the nucleophilicity of the carbon atom to be methylated, making this step unaffordable at physiological conditions.
KEYWORDS: DNA Methylation, Inhibitors, Anticancer Drugs, QM/MM, Molecular Dynamics, zebularine
DNA methylation is one of the most important epigenetic modifying mechanisms, playing a fundamental role in numerous cell processes. It is directly related in the transcriptional level to the epigenetic mechanisms of silencing and expression of genes.1 DNA methylation also constitutes a mechanism to modulate the structure of the chromatin2 and regulates the binding of protein complexes to certain regions of it.3,4 In addition, DNA methylation is associated to the regulation of the genomic imprinting, X-chromosome inactivation, cell differentiation and undesired pathologies related to carcinogenic processes or aging.5-7 In cancer processes both hypomethylation and hypermethylation patterns are found, giving rise to an epigenetic deregulation and the silencing of tumor suppressor genes.8 In tumor tissues the percentage of CpG islands that are found to be methylated are significantly higher than in normal tissues.9 Likewise during the initiation of the tumoral process a high percentage of hypomethylation in the genome is produced. This hypomethylation results in an increase of the genomic instability and chromosomal anomalies.9 In mammals it was accepted that the only position that undergoes methylation is the C5 position of cytosine although it has recently been shown that a small percentage of adenines in vertebrates10 and in mouse stem cells are methylated in its N6 position11 opening new challenges to the epigenetic field. To date, four mammalian DNA methyltransferases enzymes (DNMTs) which methylate the C5 position of cytosine have been identified: the
Scheme 1. Proposed reaction mechanism for zebularine methylation by DNMTs. Cytosine presents an amino group at the H4 position. DNMT1 which acts as a maintenance DNMT, DNMT3a/b which act as de novo DNMTs and DNMT2, with a role still under debate.1 Cytosine DNA methylation is carried out by a DNMT enzyme that catalyzes the transfer of a methyl group from the well-known methyl donor Sadenosyl-L-methionine (SAM) cofactor to the C5 position of a cytosine base placed in a CpG sequence.1 According to our recent results,12 once the target base has been inserted into the enzymatic active site, the reaction mechanism of C5 methylation in cytosine consists firstly in the addition of a nucleophilic cysteine present in the active site to the C6 position of the cytosine ring, activating the cycle towards the methyl transfer which occurs in a se-
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cond step (see Scheme 1). Finally, a water molecule present in the active site abstracts the excess proton from the C5 position.12 Different strategies have been traced in order to synthetize DNMTs inhibitors. One strategy is to develop analogs of the cofactor SAM which are able to bind the enzyme and therefore inhibit the methylation process.13 However, the great variety of methyltransferases present in living organisms sharing SAM as a cofactor suppose a major impediment because of possible side-effects of the drug. In order to avoid this problem bisubstrate inhibitors have also been developed. These inhibitors have both features of the methyl donor and acceptor so this strategy of drug design could reach better selectivity.14 There are two main groups of substrate inhibitors under study: nucleoside analogs and non-analogs.15-17 The most important nucleoside analog inhibitors are zebularine, 5fluorodeoxycytidine, 5-Azacytidine and its 2’-deoxy analog, also known as Decitabine (see Figure 1). The last two inhibitors have been approved in 2006 by the FDA for the treatment of the myelodysplastic syndrome. In particular, zebularine has been shown to reactivate the genes that are involved in tumor suppression and that have been silenced by hypermethylation.18 These compounds are incorporated to DNA in its 2’-deoxy form and act presumably forming an adduct with DNMTs, which could result in a suicide complex.19 While aza-nucleosides possess high associated cytotoxicity,20 zebularine has been shown to have little cytotoxicity under in vitro and in vivo conditions and its stability in acidic and neutral conditions makes oral administration feasible.18 Moreover zebularine enhances the radiosensitivity both in vitro and in vivo through a mechanism that may involve the inhibition of DNA repair.21 In comparison to normal cells, zebularine is more selective and efficiently incorporated to cancerous cells.22
Figure 1. Most important nucleoside inhibitors of DNMTs.
The inhibition mechanism of DNMTs by zebularine at a molecular level has not yet been described and depending on the experiments carried out there have been different proposals. While some authors claim that zebularine forms a covalent complex by the addition of a cysteine to the C6 position of zebularine and the subsequent methyl transfer to the C5 position of the cycle,23,24 others state that the methyl transfer does not take place.25,26 Moreover it is not clear if the covalent complex formed between DNA containing zebularine and DNMTs is an irreversible covalent complex27 or a reversible one, as observed in the studies of van Bemmel et al.25 and Champion et al,26
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which could explain the different cytotoxicity between aza-nucleosides and zebularine. Here we present full atomistic hybrid Quantum Mechanics/Molecular Mechanics (QM/MM) and classical Molecular Dynamics (MD) simulations which shed light into the molecular mechanism of DNMTs inhibition by zebularine. Our QM/MM free energy calculations show that the reaction mechanism of zebularine consists first in the addition of cysteine to the C6 position of cytosine in a reversible step. The methyl transfer would take place in a subsequent step but it presents a too high activation free energy, resulting in a negligible reaction rate under physiological conditions and thus an irreversible complex is not formed. This molecular picture is in agreement with recent experimental studies.25,26 Our starting point was a previously equilibrated structure of the ternary complex formed by the DNMT M.HhaI, the cofactor SAM and a dodecamer of DNA, taken from our previous study for the cytosine containing system.12 Although M.HhaI is a bacterial DNMT it provides an excellent model system of mammalian DNMTs as they share the same catalytic domains. For this reason this enzyme has been often used in experimental and computational studies of DNMTs catalysis and inhibition.12,15,25,28-31 We then remodeled the target cytosine base flipped into the active site to zebularine by exchanging the exocyclic amino group for a proton in position C4 (see Scheme 1). Making use of the RED server32 we obtained the atomic charges for classical simulations of the nucleotide zebularine (provided as Suporting Information Table S1) keeping the same computational scheme followed by Cornell et al.33 Thus, these charges are fully compatible with the AMBER forcefield for DNA. For the rest of the system (DNA, protein, solvent molecules and counterions) we used the TIP3P34 and AMBER99SB force fields,35 including the parameters parmbsc0 for DNA,36 to carry out optimizations and molecular dynamics simulations with the NAMD program37 of the ternary complex formed by the protein, SAM cofactor and zebularine-containing DNA. For the details concerning the MD simulations we refer the reader to the Methods section in Supporting Information. Briefly, after equilibration, we performed 100 ns of a fully unrestrained MD simulation in the NVT ensemble at 300 K. The MD simulation shows that once incorporated into DNA and flipped out into the active site, zebularine is stabilized by means of several hydrogen bond interactions with active site residues. Arg163, Arg165 and Glu119 form hydrogen bond contacts with O2 and N3 respectively (see Figure S2). These hydrogen bond interactions are stable during the whole simulation, together with other DNAprotein contacts12 and contribute to the stability of the ternary complex. Obviously, the main difference with respect to the system containing cytosine as the target base for methylation is the absence of the hydrogen bond interactions formed by the exocyclic amino group in the latter with surrounding residues (see ref 12).
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ACS Catalysis scheme excluding a strong bias in our results due to the use of semiempirical geometries.
40 TS 2-3 30
Free Energy (kcalmol-1)
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2
0
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-10 Cys81 Addition
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TS1Cyt 2,5 kcal/mol TS1Zeb 4,7 kcal/mol
TS2Cyt 19.1 kcal/mol TS2Zeb 33.0 kcal/mol
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0
1
2 s (Å)
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Figure 2. AM1/MM PMFs, corrected at the M06-2X/6311+G** level, obtained for the Cys81 addition (1-2) and methyl transfer (2-3) for zebularine (blue) and cytosine (red) in M.HhaI as a function of the collective path coordinate s (see Supporting Information).
We selected a snapshot from the last 10 ns of the simulation to study the reactivity by means of the hybrid QM/MM methodology. In order to obtain the free energy profiles for the reaction mechanism we performed QM/MM-MD simulations with the fDynamo38 program to obtain minimum free energy paths (MFEPs) by means of the on-the-fly string method (consisting in 30 string nodes).39 Then a path collective variable40,41 was defined to obtain the potential of mean force (PMF) using 120 umbrella sampling windows.42 MFEPs were obtained at the AM1/MM level and the PMF free energies were corrected at the M06-2X/6-311+G** level using an interpolated scheme based in energy differences between both quantum methods.43 This methodology allows us to obtain the best reaction coordinate and characterize the preferred reaction mechanism in terms of the free energy landscape with no a priori mechanistic assumptions. We refer the reader to the Supporting Information section and to the Scheme S1. Our QM/MM-MD simulations show that the first step consists in the nucleophilic addition of the Cys81 residue to the C6 position of the zebularine ring (see Figure 2). This step has an activation free energy of 4.7 ± 0.3 kcal·mol-1 and the resulting intermediate state 2 is -1.9 kcal·mol-1 below the reactant state. Our simulations show that the nucleophilic addition to zebularine is a reversible and slightly exothermic step. Thus, the resulting adduct is reversible and heat labile, in agreement with experimental observations.25,26 Afterwards, methyl transfer to zebularine proceeds with a high activation free energy of 33.0 ± 0.2 kcal·mol-1. When cytosine is the target base the free energy barrier for the methylation of intermediate 2 is of only 19.1 ± 0.4 kcal·mol-1, and thus the reaction can proceed through an irreversible step (see Figure 2 and ref. 12). Additional explorations of the Potential Energy Surface at the M06-2X/6-31G**/MM level (see Figure S7 and S8 and Table S4) corroborate this stepwise mechanistic proposal where the nucleophilic addition precedes methylation. These direct DFT/MM calculations support our dual level
Table S2 provides the averaged distances involving the bonds to be formed or broken and the hydrogen bond interactions that zebularine establishes in the active site along the proposed mechanism. In the reactant state (1, before Cys81 addition) the sulfur atom of the Cys81 residue is found at 3.21 ± 0.07 Å from the C6 atom of zebularine, and the methyl group belonging to the cofactor SAM is 3.75 ± 0.21 Å away from the C5 atom of zebularine. These two distances are found to be larger in the reactant state of zebularine than when cytosine acts as substrate (3.15 ± 0.11 and 3.27 ± 0.09, respectively). The hydrogen bond interactions formed by the exocyclic amino group in the latter substrate assist the correct positioning of the target base in the active site for the nucleophilic addition step. For the first transition state (TS 1-2) where the Cys81 residue is added to the zebularine ring the distance between the sulfur atom of Cys81 and the C6 atom of zebularine is of 2.78 ± 0.06 Å. According to our explorations of the multidimensional free energy surface (see Supporting Information and Scheme S1) no proton transfer at the N3 position of the zebularine is needed to facilitate the addition of the Cys81 residue to the ring. In intermediate 2 the distance between the sulfur atom of the Cys81 and the C6 atom of zebularine is decreased to 2.08 ± 0.09 Å. The methyl group belonging to SAM is 3.25 ± 0.06 Å from the C5 carbon atom of zebularine which is longer than the distance observed for this intermediate in the cytosine system (2.93 ± 0.05 Å). Again, the presence of the exocyclic amino group in cytosine facilitates the optimal positioning of the ring for the methyl transfer. A more decisive influence of the exocyclic amino group is its effect on the nucleophilicity of the C5 atom. While in the cytosinecontaining intermediate 2 the CHELP44 derived charge for C5 is -1.27 a.u., for zebularine this charge is of only -0.95 a.u. Therefore the loss of the exocyclic amino group has a crucial impact on the reactivity of intermediate 2 towards methyl transfer. The free energy barrier for methylation of the zebularine adduct is simply too high to result in a measurable reaction rate at physiological conditions. This is mainly due to the lower nucleophilicity of the carbon atom to be methylated. If we compare the transition states (TS 2-3) associated to the methyl transfer from SAM to the C5 position in zebularine and cytosine rings (see Figure 3) we can observe that both the breaking and forming distances between the sulfur, the methyl carbon atom and the C5 carbon atom are slightly longer for zebularine (2.20 ± 0.06 Å and 2.29 ± 0.06 Å, respectively) than for cytosine (2.10 ± 0.05 and 2.21 ± 0.05 Å respectively). In addition the averaged S-CH3-C5 angle is closer to linearity for cytosine than for zebularine (170 ± 5 degrees and 165 ± 5 degrees, respectively). The decreased nucleophilicity of C5 in zebularine results in a less compact and linear transition state with a higher activation free energy. This is also reflected in the stabilizing interactions stablished between the methyl group halfway to be transferred and the carbonyl oxygen atoms of Asn304 and Gly74. These C-
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H···O hydrogen bonds for the zebularine containing system are lengthened by 0.12 and 0.17 Å respectively. Finally, as can be observed in Figure 2, the methylation step is not favored from the thermodynamical point of view for zebularine as much as for the natural substrate, cytosine, which exhibits a large exothermicity. The methylation step is irreversible for cytosine, as experimentally observed,45 while for zebularine the hypothetical reaction would be almost thermoneutral.
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in zebularine, which makes more difficult to reach an optimal orientation of the ring for both the cysteine addition and the methyl transfer and decreases the nucleophilicity of the carbon atom to be methylated. Our results provide a molecular picture to explain recent experimental observations and could be useful for future developments of DNMTs inhibitors.
AUTHOR INFORMATION Corresponding Author *E-mail:
[email protected] Present Addresses † Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology, Baldiri Reixac 10-12, 08028 Barcelona, Spain.
ASSOCIATED CONTENT Atomic charges for zebularine; extended computational methods regarding Molecular Dynamics simulations and QM/MM calculations; RMSD for the protein and DNA during classical simulations; distances between zebularine and M. HhaI active site residues during the MD simulation; QM subsystem employed and active space selected for the calculation of MFEP; string RMSD; representation of the converged MFEP; schemes, pictures and tables with the structural details of the different initial guesses considered in the string method; detailed table with averaged distances for reactants, TS, intermediate and product for the PMF; detailed table with averaged distances for reactants, TS, intermediate and product for the converged string; representation of representative structures for the converged string; Potential Energy Surface at the M06-2x/6-31G**/MM level for the reaction mechanism found; table showing the key distances of the PES and stationary structures found in the PES. This material is available free of charge via the Internet at http://pubs.acs.org.”
ACKNOWLEDGMENT
Figure 3. Representative TS structures for the natural occurring methylation of cytosine and the methylation step inhibited by zebularine. Hydrogen bonds are depicted in dotted blue lines, stabilizing interactions between the methyl group and carbonyl groups are indicated with dotted black lines and the red dotted lines show the distances involved in the methyl transfer. Numbers show the average values in Å.
Our classical and QM/MM simulations support an inhibition mechanism of DNMTs by zebularine where a covalent reversible adduct is formed after a nucleophilic attack by a cysteine residue to position C6 of the ring. However, this adduct cannot be methylated and thus no irreversible complex is formed. These mechanistic features are due to the absence of the exocyclic amino group
The authors gratefully acknowledge financial support from FEDER funds and the Ministerio de Economía y Competitividad (project CTQ2012-36253-C03-03) and Generalitat Valenciana (ACOMP/2015/239). J. A. thanks Ministerio de Economía y Competitividad for FPI fellowship. The authors also acknowledge the computational facilities of the Servei d’Informàtica de la Universitat de València in the “Tirant” Supercomputer.
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