Molecular Structure and Pronounced Conformational Flexibility of

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Molecular Structure and Pronounced Conformational Flexibility of Doxorubicin in Free and Conjugated State within a Drug-Peptide Compound Yana Tsoneva, Hendrik R. A. Jonker, Manfred Wagner, Alia V. Tadjer, Marco Lelle, Kalina Peneva, and Anela Ivanova J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/jp509320q • Publication Date (Web): 20 Jan 2015 Downloaded from http://pubs.acs.org on January 24, 2015

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The Journal of Physical Chemistry

1

Molecular Structure and Pronounced Conformational Flexibility of Doxorubicin in Free and Conjugated State within a Drug-Peptide Compound

Yana Tsoneva,[a] Hendrik R.A. Jonker,[b] Manfred Wagner,[c] Alia Tadjer,[a] Marco Lelle,[c] Kalina Peneva,[c] Anela Ivanova[a],*

[a]

University of Sofia, Faculty of Chemistry and Pharmacy, Department of Physical Chemistry, 1 James Bourchier Ave., 1164 Sofia, Bulgaria

[b]

Goethe University Frankfurt, Institute for Organic Chemistry and Chemical Biology, Center for Biomolecular Magnetic Resonance, Max von Laue Strasse 7, 60438 Frankfurt am Main, Germany [c]

*

*

Max Planck Institute for Polymer Research, Ackermannweg 10, 55128 Mainz, Germany

Corresponding author: e-mail: [email protected]; Tel.: ++35928161520; FAX: ++35929625438

Corresponding author: e-mail: [email protected]; Tel.: ++35928161520; FAX: ++35929625438

ACS Paragon Plus Environment


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2

Abstract The search of targeted drug delivery systems requires the design of drug-carrier complexes, which could both reach the malignant cells and preserve the therapeutic substance activity. A promising strategy aimed at enhancing the uptake and reducing the systemic toxicity is to bind covalently the drug to a cell-penetrating peptide. For understanding the structure-activity relation in such preparations, the chemotherapeutic drug doxorubicin was investigated by unrestrained molecular dynamics simulations, supported by NMR, which yielded its molecular geometry in aqueous environment. Furthermore, the structure and dynamics of a conjugate of the drug with a cell-penetrating peptide was obtained from molecular dynamics simulations in aqueous solution. The geometries of the unbound compounds were characterized at different temperatures, the extent to which they change after covalent binding and whether/how they influence each other into the drug-peptide conjugate. The main structural fragments that affect the conformational ensemble of every molecule were found. The results show that the transitions between different substructures of the three compounds require modest amount of energy. At increased temperature either more conformations become populated as a result of the thermal fluctuations, or the relative shares of the various conformers equalize at the nanosecond scale. These frequent structural interconversions suggest expressed conformational freedom of the molecules. Conjugation into the drug-peptide compound partially immobilizes the molecules of the parent compounds. Nevertheless, flexibility still exists as well as an effective intra- and intermolecular hydrogen bonding that stabilizes the structures. We observe compact packing of the drug within the peptide that is also based on stacking interactions. All this outlines the drug-peptide conjugate as a prospective building block of a more complex drug-carrier system.

Keywords: Doxorubicin, drug delivery, drug-peptide conjugate, molecular dynamics, octaarginine


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3

Introduction

A class of the most widely used chemotherapeutic agents is that of the anthracycline antibiotics, among which doxorubicin is the most effective and the one with the most diverse activity.1-3 This drug is a natural product and is originally derived from Streptomyces. Its mechanism of action is extensively studied and there are a lot of possible expressions of bioactivity: inhibition of DNA and RNA synthesis by intercalation between the bases of the macromolecules,4-8 blocking transcription and replication by inhibition of the enzyme Topoisomerase II, thus inducing apoptosis;9 covalent binding to DNA and alkylation; free oxygen radical generation, which damages the DNA and the cell membranes; or crosslinking of the DNA helices.3 Historically, the role of DOX as a DNA intercalator was considered the most important. However, there is also a number of works, which report evidence for covalent binding of the drug to DNA responsible for its cytotoxicity. 10-15 Overall, the mechanism of bioactivity of DOX is still debated. Clarification of the mode of action of doxorubicin was assisted by several theoretical reports. For characterizing the structure of doxorubicin Zhu et al.16 carried out a Density functional theory study and determined the most stable geometry in vacuum. The authors concluded that: (i) this geometry is stabilized by intramolecular hydrogen bonds between the quinone and hydroquinone, (ii) the side chain has perpendicular alignment with respect to the anthracycline system, and (iii) the most flexible part is the sugar residue. However, the behavior of the drug in solution, i.e., in its natural environment, has not been described. The main problems with the usage of doxorubicin as a chemotherapeutic agent are its toxicity to healthy tissues17 and the multidrug resistance of tumor cells. That is why it is a subject of many investigations associated with its more directed targeting and has thus undergone different modifications, which can decrease its side effects.1,2 These modifications can affect either the structure of the drug itself or can consist of binding it to other molecules18 resulting in more complex constructs.



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4 Liposomes, nanoparticles, different polymeric and dendritic formations have been used as targeting carriers.19,20,21,2223 In a theoretical study of a structural model of doxorubicin interacting with oligomeric polyalkyl cyanoacrylate in nanoparticles, Poupaert et al.24 found that the multidrug resistance was reversed due to adsorption of the particles onto the cell membrane, which created high drug gradient close to the cell surface. The interaction of doxorubicin with other target biomolecules was modeled by molecular dynamics simulations as well.25-29 It has also been demonstrated that conjugation of drugs to peptides in general30,31 or more specifically of doxorubicin to a cell-penetrating peptide (CPP) can overcome multidrug resistance in vitro.32 CPPs are short peptide sequences (usually built of less than 30 residues) that have a proven capacity to penetrate the cell membrane.33 This class of peptides is efficient34 and numerous studies confirm that among all arginine-containing CPPs, especially those with eight or nine consecutive arginine residues are the most effective and less cytotoxic35-39 The translocation of arginine (as polyarginine or included into other peptide sequences) through a bilayer was addressed theoretically as well and the energy barriers were estimated40-44 confirming the importance of the presence of this amino acid. The attachment of small molecules like fluorophores or the addition of hydrophobic components can significantly influence not only the cellular uptake and distribution of CPPs, but also the overall toxicity. Therefore, one important aspect that should be considered when designing novel drug-peptide conjugates is the mutual influence of the cargo and the CPP on their inherent structure and properties in unbound state because usually the native structure of such molecules is essential for their bioactivity. In spite of the number of questions already clarified with respect to the bioactivity of doxorubicin and CPPs, there are no theoretical studies tackling the molecular structure and properties of CPP-drug conjugates. A powerful approach for elucidating the molecular structure and dynamics of drugs and for getting access to their interactions with biomolecules is the combination of NMR spectroscopy and molecular dynamics (usually with imposed NMR-derived restraints) simulations. Such an ACS Paragon Plus Environment

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5 interdisciplinary scheme has been applied successfully to various drug-containing systems45-47 but there are just a few reports closely related to the objects of the current work. NMR measurements on anthracycline antibiotics were used predominantly to assess their aggregation behavior in solution 1b or when attached to phospholipid vesicles.48 Depending on the experimental conditions and on the particular representative, the drugs were found to form dimers (by stacking of the -conjugated fragments) in aqueous solution2 or larger stacked assemblies when bound to a peptide.49 NMR spectra of three anthracycline derivatives (4’-epiadriamycin, DOX, daunorubicin) in D2O at room temperature were recorded and structural parameters were extracted thereof.50 The molecular geometry was obtained by Density functional theory (B3LYP with basis sets STO-3G, 6-31G, or 6-31G**) calculations in the gas phase and restrained molecular dynamics (employing the NMR-derived distance restraints) simulations in solution. Three conformers with respect to the gycosidic bond were proposed out of which one was outlined as the biologically relevant structure. In all instances the importance of the complementarity between NMR and molecular simulations was outlined. In view of the above, one of the aims of the current study is to characterize the molecular structure of doxorubicin in aqueous solution both experimentally (by NMR spectroscopy) and theoretically (by classical molecular dynamics) in order to extract in a concerted fashion the most essential structural features of the drug. The second objective of this work is to characterize the structure of the doxorubicin-octaarginine conjugate, coupled via a cleavable linker, and to assess the perturbation of the structure of each molecule due to the presence of the other. In the conjugate DOX is bound covalently to the CPP using a novel heterobifunctional linker. This structure of the molecule complies with several important requirements: (i) secure attachment of the drug to the peptide for safe passage along the transduction path in vivo; (ii) enhanced capacity for penetration of cell membranes; (iii) two linker bonds, which are easily cleavable in physiological conditions. These labile bonds are an oxime fragment with sufficient hydrolytic stability to prevent premature release of the drug in the bloodstream



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6 and a disulfide bond to the bioactive cargo, which can be reduced in the cytosol to ensure the proper biological function of doxorubicin.

Computational protocol and experimental setup

MD simulations Separate atomistic molecular dynamics (MD) simulations of one molecule doxorubicin (DOX) in its protonated bioactive form, one molecule of a cell-penetrating peptide formed by one cysteine and eight arginine residues, and the product (DOX-peptide) of their covalent binding (Scheme 1), were carried out. The organic molecules were described by the Amber0351 force field as implemented within the Gromacs 4.5.252 program package. Geometry optimizations were carried out in order to obtain the atomic charges of DOX, the end-cap groups of the peptide (Scheme 1, Figures S1, S2 of the Supporting information) and the acetate counter ions (the arginine residues and DOX are charged at the pH at which experiments with this system are done); then the electrostatic potentials and the restrained electrostaticpotential-derived (RESP) atomic charges53,54 were generated (Tables S1, S2 and Figure S3 of the Supporting information). The latter were used for evaluation of the electrostatic interactions of these residues in all MD simulations. All compounds were solvated in explicit TIP3P55,56 water and periodic boundary conditions were applied. Three simulations were performed for each system with the pressure always fixed at 1 atm but at different temperatures: 4  , 57 25  or 37  (NPT ensemble), maintained with Berendsen58 barostat and thermostat. Chloride anions were used to neutralize the charge of unconjugated DOX, while acetate anions were the counterions of the peptide and of the DOX-peptide conjugate, which corresponded to the setup of the experimental studies of the same systems.59 Information about the size and composition of all models is given in Table S3 of the Supporting Information.


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7 O

O

O

O H 2N O

1

OH

O NH 2

O

O O

H 2N

NH 2

NH

O

H 2N

NH 2

NH

H2 N

NH 2

NH

NH

OH O O

OH

O

O

OH

O

O

O

H N

N H

H N

N H

O

2

O

H N

N H

H O

O

H N

HN

N H

O

NH 2

O

O

HS OH

Cl

NH

NH

NH

NH

NH3 O H 2N

NH 2

O

H 2N

O

NH 2

O

O

H 2N

NH

O H2 N

O O

NH 2

H2N

NH

NH 2

O

O O

NH 2

NH 2

O

O O

H 2N

O

H2 N

O NH 2

H2 N

NH

NH 2 NH

O O

O

S

O

H N

N H O

O

H N

HN

H N

N H

O

O

H N

N H

N H

O

O

NH 2 O

S

N H

NH

NH

NH

NH

O O

N

OH

OH

O H 2N O

OH

NH 2

O

H 2N

NH 2

O

O

H2 N O

NH 2

O H2 N

NH 2

O

H O

O

OH

O

O

O

NH3

O OH

Scheme 1: Chemical formulae of doxorubicin with notations of the hydroquinone hydroxyl groups (top left), the studied peptide (top right) and the drug-peptide conjugate (bottom)

During the simulations all hydrogen-containing bonds were restrained with SETTLE60 (for water) and LINCS61 (for the rest of the molecules). The potential for non-bonded interactions was Lennard-Jones with cutoff of 12 Å and switch functions activated at 10 Å. The electrostatic interactions were assessed in monopole approximation with cutoff of 14 Å turning on a switch function at 12 Å. The long-range electrostatic contributions were evaluated with the particle mesh Ewald (PME) method.62 The time step was 2 fs and integration of the equations of motion was done with the leap-frog algorithm.63 The systems were simulated following a standard protocol,51,64 including energy minimization, MD equilibration (0.8 ns for DOX, 5.8 ns for the peptide, and between 5.8 ns and 20.8 ns



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8 for the conjugate) and production (10 ns for DOX, 25 ns for the peptide, and 30 ns for the conjugate) stages.65 10 ns fragments of the production trajectory of DOX and of the peptide and 25 ns of that of the conjugate were used for the statistical analysis with frames extracted at intervals of 0.2 ps, i.e., the data presented below encompass 50 000 structures for DOX and the peptide and 125 000 for DOX-peptide unless otherwise specified.66 The cluster analysis was performed with the method of Jarvis-Patrick67 on 10 000 structures for DOX and the peptide and 25 000 for DOX-peptide; the subsequent frames were separated by 1.0 ps. Only the clusters with population of more than 200 structures were considered significant. The cutoff was 0.04 nm for DOX, 0.11 nm for octaarginine, and 0.10 nm to 0.12 nm for the conjugate at the different temperatures.68 All MD simulations were carried out with the program package Gromacs 4.5.252 and VMD69 was used for visualization. Gaussian 0970 was employed for geometry optimization and calculation of electrostatic potentials. The utilities of Amber 871 were used for generation of the RESP charges.

Quantum calculations The energy change upon dissociation of the oxime and disulfide bonds of the linker fragment of the DOX-peptide conjugate was quantified by performing rigid potential energy surface (PES) scans on geometries taken from the MD trajectories. Unrestricted MP2 with 6-31G** atomic basis set was employed for all single point calculations. The presence of the aqueous environment was accounted for by implicit (PCM72,73) solvation. The wavefunctions minima with respect to variation of the coefficients were verified by stability analysis. The length of each of the two bonds (S—S or N—O ) was used as a reaction coordinate to construct separate PES profiles for a set of probable conformers. The bond lengths were varied in the following intervals: from 1.7 Å to 5.0 Å for S—S and from 1.1 Å to 5.0 Å for N—O. The increments were 0.05 Å in the proximity of the minima and 0.5 Å at the larger distances. The dissociation energy was determined as energy difference between the maximum and the minimum of each profile. ACS Paragon Plus Environment

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9

NMR NMR spectra of 3mM DOX were measured in H2O/D2O (9:1) at 298.3 K and pH 7.4 on a Bruker 700 MHz Avance III spectrometer with a 5 mm BBI 1H/BB probe equipped with a z-gradient. Spectra were processed using Topspin 3.1 (Bruker Biospin) and analysed using Sparky (T.D. Goddard and D.G. Kneller, University of California, San Francisco). Several homo- and heteronuclear 2D NMR spectra (HSQC, HMBC, COSY, TOCSY and NOESY) were recorded for the assignment of the 1H and

13

C

resonances at natural isotope abundance.

Results and discussion The first stage of the study encompasses unrestrained MD simulations of the aqueous solutions of the drug molecule and the peptide separately in order to assess their unperturbed geometry. Then, the combined system is characterized and its structure compared to that of the constituents. Based on that, the overall structural effect due to the presence of the drug is estimated and probable consequences on the bioactivity of the conjugate are discussed.

1. Characteristics of the structure of doxorubicin and changes after conjugation with the peptide

To understand the general interaction between DOX and octaarginine we characterized the molecules in terms of root-mean-square deviation (RMSD) of the atomic coordinates along the MD trajectory (Figure 1). This allows assessment of the structural changes after covalent binding.



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10

Figure 1: RMSD of the DOX coordinates during the simulation with respect to those of the minimized structure (Min); an experimental X-ray geometry74 (ID number 1D12 in the Protein Data Bank) is shown for comparison

The reference structure for all RMSD calculations is obtained after quantum-chemical geometry optimization (B3LYP/6-31G*) of DOX in the gas phase started from the X-ray structure,74 followed by its energy minimization with molecular mechanics in aqueous solution.75 The graphs at all temperatures reveal two different structures: one with RMSD in the range 0.08  0.16 nm (substructure 1), the second – with coordinates deviating from 0.16 nm to 0.24 nm (substructure 2). We have assigned these to two different conformers, which were further verified by cluster analysis. The outcome of the cluster analysis confirms that there are two preferred conformations at all temperatures (Figure 2A). The main difference between the two most populated clusters is the orientation of the side chain and partially of the heterocycle (Figure 2B). The changes in the positioning of these two residues define the dynamics of the molecule at each temperature (see below). The superposition of the central structures of the most populated clusters at the highest temperature (Figure 2B) is an excellent illustration of the extreme side chain mobility – it can change its


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11 orientation from perpendicular to parallel with respect to the anthracycline system. The methoxy group does not demonstrate any pronounced flexibility but it rotates in a relatively narrow range and is positioned only on one side of the conjugated system. The steady existence of two more substantially populated, i.e. with population exceeding 5 %, clusters at each temperature is in accordance with the RMSD data as well. The observed clusters have been verified with experimental results from solution state NMR. In 2D 1H1H-NOESY spectra of DOX in water, close contacts (NOE crosspeaks) are observed between specific protons from the heterocycle and the anthracycline system (Figure 3A, C). The NOE crosspeak intensities from NOESY spectra at high mixing time are prone to spin-diffusion, but globally they correlate well with the distances observed in the MD simulations (Figure 3B).

Figure 2: (A) Population of the clusters and (B) superposition of the central structures of the populated clusters of unbound DOX obtained by cluster analysis of the MD trajectories at the three temperatures

The NOESY crosspeaks between 7 and 26/27 are the strongest, indeed corresponding to the shortest distances (Figure 3D). The crosspeaks between 7 and 58/59 are very weak (only observed in NOESY spectra at higher mixing times) and the observed average distances in the MD (about 5 to 6.5



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12 Å) in fact clearly indicate the NOE detection limit. The resonances of 27* and (19/20) overlap, but the corresponding 26* resonance is well separated. Furthermore, the crosspeaks intensities from 11 and (2/3/4) resonances are a bit hard to estimate due to relaxation T1-noise (11 overlaps with (40/41/42)); nevertheless, their contacts match well the MD observations.

Figure 3: (A) 2D 1H1H-NOESY spectra of DOX in water with proton resonance assignment (* denotes ambiguous assignment for CH2 groups); (B) the distances between the characteristic NOESY proton pairs plotted for the structures from the most populated MD cluster of DOX (cluster 2 in Figure 2A) at room temperature and (D) their average values together with NOE category of the corresponding experimental signals (** shows that there is signal overlap); (C) atom numbering used for assignment of the NOESY signals


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13 Comparing the central structures of the most populated clusters with the X-ray geometry (Figure 1) would indicate whether the molecule preserves its solution structure also in the solid state. In the case of DOX the preferred solution structures have certain similarities to the X-ray one but some differences can be outlined as well. The resemblance is in the orientation of the two hydroxyl groups (1 and 2 in Scheme 1) attached to the aromatic ring – both in solution (at room and body temperature) and in the crystal they are likely to form intramolecular H-bonds. The alignment of the heterocycle, the methoxy group and of the side chain, however, differs in solution and in the crystal. As could be expected, the crystal structure is characterized by much more compact geometry. Thus, it can be concluded that the Xray structure is not fully representative of the drug geometry in solution even at the lowest temperature. Since the molecular geometry may have impact on the biological activity of the compound, it would be more relevant to base bioactivity discussions on solution structural data.

H O

O

H

O

O

O

O

OH

OH

OH

O

O

H 3C

O

1

OH

H

O

H O

O

H

O

H3 C

O H

O

O

2

OH

OH

NH 3 H O

3 O

O

NH 3 H O

O

OH

4'

O OH OH

OH H O

O

H 3C

O

O

O

O

O

H 3C

H

4 ''O

H

O H

O

OH

OH NH3

NH3 H O

O

H

O

O

O

OH

6

OH

O H 3C

O

O

5

OH OH

H O

O

H O

O

H

H 3C

O

O

O

O

H

OH NH3

OH NH 3

Scheme 2: Chemical formula of doxorubicin with illustration and notations of the six dihedral angles analyzed (marked in red) ACS Paragon Plus Environment


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14 The two different conformers are verified by tracking the evolution of several dihedral angles (Scheme 2), which describe the mobility of the sugar residue (1, 2 and 5; Figure S4, S5 and S6 of the Supporting information) and the side chain (3 and 6; Figures S7 and S8 of the Supporting information). The angles 4 and 4 (Figure S9 of the Supporting information) are used for assessment of the intramolecular hydrogen bonds. The angles 1, 2 and 6 can be outlined as responsible for the transformation of one substructure of doxorubicin into another (Table 1). At low temperature DOX has limited mobility of the sugar residue and more significant flexibility of the side chain (Figures S4, S5 and S8 of the Supporting information).

Table 1: Range of dihedral angles values characteristic for the substructures of the unconjugated and conjugated drug at different temperatures

4

Temperature Angle, o

1

2

25  6

1

37 

2

6

1

2

6

RMSD , nm Unconjugated drug 0.08 - 0.16

60-140

45-120

40-100

45-140

45-120

35-100

35-140

35-120

30-105

0.16 - 0.24

135-180

120-180

140-180

135-180

120-180

135-180

135-180

120-180

130-180

Conjugated drug 0.03 - 0.08

135-180

70-180

---

135-180

100-180

45-100

125-180

80-180

---

0.08 - 0.22

40-160

45-120

---

30-150

30-160

50-120

60-150

50-120

---

Upon temperature increase the drug molecule becomes more flexible and the enhanced dynamics stems mainly from the heterocycle. The latter retains predominant gauche-conformation with respect to


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15 the -conjugated part at all temperatures. Moreover, the positive values of 5 correspond to positioning of the sugar in all structures on that side of the anthracycline system, which corresponds to the bioactive form of the drug. At low temperature the sugar is primarily trans-oriented along the ether bond but upon heating some gauche-conformers become populated as well, giving rise even to a third probable conformer at 37  (Figure S4). Due to the steric hindrance, only one of the two possible gauche-ranges is accessible. The more populated conformation along 5 coincides with the one detected in a previous NMR/rMD study of the related antibiotics epiadriamycin, adriamycin, and daunomycin,2 which is the biologically active conformer. At low temperature the side chain is gauche-positioned with respect to the conjugated fragment while at the two higher temperatures more coplanar conformations become accessible. At body temperature the two side chain orientations are equiprobable. In all simulations the DOX molecule is stabilized by the formation of two intramolecular hydrogen bonds between the hydroxyl groups 1 and 2 in Scheme 1 and the proximate keto-groups, which is in accordance with previous findings.16-15 In the drug-peptide conjugate none of the dihedral angles that describe the transitions between the substructures of DOX remains correlated to the overall RMSD of the conjugate (Figure S10 of the Supporting information, middle panel). This is expected because the total RMSD variations correspond to structural changes in the entire molecule as can be seen by the much larger RMSD magnitude relative to that of unbound doxorubicin (Figure 1). The angles 1, 2, 3, 5 and 6, however, remain correlated to the partial RMSD variation of DOX into the conjugate (Figure S10 of the Supporting information, bottom panel). The populated ranges of the sugar dihedrals (Figures S4-S6 of the Supporting information) are the same as in the unbound DOX, just the relative shares are different in some cases. The only exception is the angle 3, for which the two gauche and the eclipsed conformation become preferential in DOX-peptide. This is related to the



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16 formation of a hydrogen bond between the hydroxyl group in the side chain and the oxygen atom of the oxime bond (see Section 5).

Figure 4: (A) Population of the clusters and (B) central structures of the clusters with population probability larger than 10 % as obtained from cluster analysis of the MD trajectories of DOX-peptide at the three temperatures


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17 The distribution of the torsion angles indicates that the conformational flexibility of the heterocycle is not substantially perturbed after covalent binding to the peptide but that preference is given to different among the found conformers. More specific, the formation of the conjugate immobilizes only the side chain of DOX and solely the gauche-orientations remain accessible. The intramolecular quinone-hydroquinone hydrogen bonds are present in the conjugate but become slightly more dynamic at the two higher temperatures (Figure S9 of the Supporting information). The cluster analysis of the trajectories of DOX-peptide at 4 C shows that more conformations than in the unbound molecules become populated (Figure 4A). In the most abundant geometries at the higher temperatures (Figure 4B) there are various alignments of DOX with respect to the peptide backbone. A common feature of all the populated conformations is compact packing of the anthracycline part of the drug into the side chains of the peptide, which is probably stabilized by hydrophobic interactions between DOX and the guanidinium residues.

2. Characteristics of the structure of the peptide and changes after conjugation with the drug

Figure 5 presents the results of the cluster analysis of the conformations of the unconjugated peptide. At the lowest temperature the peptide geometry is relatively fixed with increasing mobility upon temperature increase. Although at body temperature there are three most populated structures, the sum of the frames belonging to them represents only 13 % of the whole set of conformations. This illustrates the extreme flexibility of the peptide geometry at this temperature. The structure of the peptide is partially immobilized after conjugation with the drug due to hydrogen bonds and stacking interactions. This can affect the bioactivity if the peptide flexibility is responsible for the cell-penetrating ability of the peptide. However, the effect on bioactivity might not be strong because at body temperature the peptide retains its mobility to a large extent (Figure 6). This was confirmed experimentally by the fact that no legible NOESY NMR spectra could be recorded for the ACS Paragon Plus Environment


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18 drug-peptide conjugate due to very fast dynamics of the molecule. On the other hand, the presence of the drug might facilitate the membrane translocation of the conjugate through the hydrophobic core of the lipid bilayer because of the substantial hydrophobic fragment (the anthracycline system) of DOX.

Figure 5: (A) Population of the clusters and (B) superposition of the central structures of the most populated clusters of the unbound peptide obtained by cluster analysis of the MD trajectories at the three temperatures

Two alternative approaches were used to quantify the peptide secondary structure – DSSP analysis76 and Ramachandran plots. The former identifies characteristic hydrogen bonding patterns of the peptide backbone, whereas the latter is a correlation plot of two characteristic peptide backbone dihedral angles ( and ). The DSSP analysis of the secondary structure of the peptide yields random coil in all cases, which is in agreement with our experimental data from circular dichroism spectra.59 The Ramachandran plots of the peptide in bound and unbound state show heaping of most of the average values of the two characteristic dihedrals in the region of the -sheet (Figure S11 of the Supporting information). The standard deviations (Tables S4 and S5 of the Supporting information), however, are significant and as a result no certain secondary structure is stabilized.


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19 These substantial fluctuations are in line with the result for root-mean-square fluctuations (RMSF) of the

atoms (Figure 6). It can be concluded from the graphs that all of the amino acids are

very mobile and this is the reason why there is no definite secondary structure. It is also noteworthy that there is no regular temperature dependence of the RMSFs in any state of the peptide.

Figure 6: RMSF of

during the simulations of the unbound peptide (left) and of DOX-peptide (right)

3. Analysis of hydrogen bonds

Both intra- and intermolecular hydrogen bonds were determined for all studied systems. A cutoff length of 0.35 nm and a hydrogen-donor-acceptor angle ≤ 30° were used to distinguish the H-bonds. For the unbound doxorubicin there are 1.60.8 hydrogen bonds formed on average at the lowest temperature and 1.80.9 and 1.70.9 at 25 oC and 37 oC, respectively. In the initial structure one of the intramolecular hydrogen bonds between the quinone and hydroquinone does not exist, but it has already taken place in the production stage of the simulations at the three temperatures (Figure S9 of the Supporting information). However, there are competitive interactions of the hydroquinone OH2-group (see Figure 1 for notation) with the ammonium cation from the sugar residue and with the ether oxygen atom. As a result, this quinone-hydroquinone hydrogen bond breaks occasionally, which leads to



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20 average values lower than 2. The length of the intramolecular hydrogen bonds at 4 oC varies in a relatively narrow range (from 1.5 Å to 2.0 Å) with a maximum population at 1.73 Å (Figure S12 of the Supporting information), representative of strong intramolecular hydrogen bonds. The most populated bonds at higher temperature have the same most probable length, which confirms the hypothesis of their key role for stabilization of the molecular structure. For the peptide the average number of intramolecular hydrogen bonds at 4 oC is 1.00.4.77 The formed bonds are between the guanidinium groups of the arginine side chains and oxygen or nitrogen atoms of the backbone. The length of these hydrogen bonds is relatively large (Figure S12 of the Supporting information) and perhaps this is one of the reasons why they do not last long. At the higher temperatures the profiles of length distribution look identical and the average numbers are 0.80.3 for 25 and 1.40.5 for body temperature. The formation of a drug-peptide conjugate substantially affects the intramolecular hydrogen bonding since the average numbers for 4 oC, 25 oC and 37 oC increase to 5.51.8, 8.11.9, 6.11.5, respectively. Two of these bonds are the ones between the quinone and the hydroquinone (denoted in Scheme 3). The other ones correspond to H-bonds within the peptide, within DOX and a small number within the linker and from it to DOX or the peptide. Most important, a substantial part of the various bonds found (22-28 % of all types) are between DOX and the peptide, which is one of the driving forces for placement of the drug in between the arginine side chains and a major cause for the somewhat reduced flexibility of the peptide. As could be expected, the profiles for length distribution are superposition of those of the unconjugated drug and peptide and do not differ upon temperature changes (Figure S12 of the Supporting information). With the water molecules the unbound DOX forms 15.42.4 H-bonds on average at 4

,

15.22.4 at RT and 14.82.5 at body temperature. At the two higher temperatures the most populated lengths are short with maxima ~1.9 Å (Figure S13 of the Supporting information), which corresponds to


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21 strong hydrogen bonds between DOX and water that do not depend on changes in the temperature. The peptide forms 61.55.0 hydrogen bonds with water on average at 4

, 59.45.4 at room and 59.15.2 at

body temperature (Figure S13 of the Supporting information). This effective hydrogen bonding does not change after the drug-peptide conjugation, since the mean numbers of formed bonds are 79.85.8, 75.05.9 and 77.65.9 at the three different temperatures: 4 C, 25 C, and 37 C, respectively, which is roughly the sum of the bonds formed by the two unbound compounds. Thus, formation of the drugpeptide conjugate does not influence the effective hydrogen bonding of the drug and the peptide to the water molecules. The length distribution is additive, too (Figure S13 of the Supporting information). The intermolecular hydrogen bonding is not largely affected by temperature increase.

4. Mutual orientation in the drug-peptide conjugate For studying the mutual orientation of the two components in the conjugated compound, the evolution of two additional dihedral angles (denoted in Scheme 3) was analyzed. The populated values of 7 (Figure S14 of the Supporting information) vary from 90 to 180 and those of 8 – from 60 to 120. This corresponds to location of the drug inside a peptide ‘pocket’ formed by the side chains. The smaller angles describe deeper penetration of DOX into the pocket. The most preferred combinations of the two dihedrals depict structures where the anthracycline system faces the guanidinium groups (smaller 8) or where the drug is outside the peptide but in close proximity to two or three guanidinium groups. Overall, the junction of the molecule is very mobile and does not restrict the mobility of the drug.

Scheme 3: Chemical structure of DOX-peptide with the two analyzed dihedrals (7 and 8) marked in red ACS Paragon Plus Environment


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22 In addition, radial pair distribution functions (RDF) were estimated. From the profiles of these functions for the carbon atoms (Cg) from the guanidinium groups (Figure 7), it can be concluded that in the solution of the unbound peptide there is no long-range order of the arginine side chains but in DOXpeptide there is more pronounced ordering at every temperature, which can be judged from the three well-defined peaks in the RDF. The second peak at 25 oC is very well expressed, which means that the interactions there are stronger.

Figure 7: Radial distribution function of Cg-Cg (top) for the peptide (left) and for DOX-peptide (right) and of CgDOX (bottom)

The second analyzed distribution function is between the Cg atoms and the center of mass of the anthracycline system. There the structuring at the two lower temperatures is also evident from the multiple sharp peaks. The first maximum at all temperatures is centered at 0.38 nm, which corresponds


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23 to a distance ensuring stacking interactions between the guanidinium groups and the drug cyclic fragment.

5. Linker bonds dissociation energy

As pointed out in the Introduction, an important prerequisite for the linker fragment is that it should contain easily cleavable bonds, so that the drug can be released from the peptide after the conjugate reaches the cytosol of the target cells. Since the linker contains two relatively labile covalent bonds – an oxime and a disulfide (scheme 1), it is of significance to characterize their energy of dissociation. For the purpose, the energy variation upon elongation of the two bonds was estimated by electron-correlated quantum calculations. Separate PES scans were performed to describe the dissociation of the two bonds.

Figure 8: Geometries of the linker fragment for which UMP2/6-31G** PES scans were performed; LIN25-1 and LIN25-2 are the respective structures of the linker from clusters 1 and 5 at 25 C, LIN37-1 and LIN25-1 were extracted from clusters 14 and 7 at 37 C (Figure 4)



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24 The linker is a linear molecule with flexible geometry. Therefore, it is essential to take into account its various possible structures. The conformations from the two most populated clusters at room and body temperature were selected (Figure 8) as representative for the linker fragment. Several atoms from the side chain of DOX were included in the model, too, because they participate in a hydrogen bond with atoms from the linker. Inclusion of this H-bond is important for proper reproduction of the dissociation of the oxime bond. The obtained PES profiles are shown in Figure 9. In all of them well shaped minima are observed with a preferred bond length of 1.45 Å for N—O and between 2.05 and 2.1 Å for S—S. As expected, the potential well for the S—S bond is wider than that for the oxime. The correct asymptotic behavior is reproduced in all cases. There is some conformational dependence of the energy profiles, as the minimum energy of the different conformers varies.

Figure 9: UMP2/6-31G** PES profiles of N—O and S—S bonds dissociation for the most probable linker structures at 25 C and 37 C; relative energies with respect to a reference completely dissociated structure (with bond length of 20 Å) are plotted


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25 The estimates for the dissociation energy of the studied linker conformers are summarized in Table 2. It should be noted that these are upper bounds of the actual energies because of the rigid nature of the scans. Nevertheless, this semi-quantitative assessment is valuable since it singles out the pure contribution of the scanned degrees of freedom, i.e. the energetics of the two bonds alone in their correct atomic surrounding.

Table 2: Dissociation energy (Ediss) estimates for the most probable linker conformers at room and body temperature

Structure Ediss, kJ/mol N-O bond LIN25-1 191 LIN25-2 186 LIN37-1 199 LIN37-2 229

Structure Ediss, kJ/mol S-S bond LIN25-1 217 LIN25-2 228 LIN37-1 226 LIN37-2 184

The energy required for dissociation of each bond spans a similar range: from 185 to 230 kJ/mol. This corresponds to medium bond strength, which is comparable for the two bonds. The relative strength of the two bonds turns out to depend on the local atomic environment. This is illustrated by the reversal of the dissociation energies for structure LIN37-2 where the dissociation of the oxime is more difficult than that of the disulfide, whereas in the other three conformers the relation is reverse. The energy barriers for the two bonds seem to be roughly complementary – the order in which the dissociation energy grows for the N-O bond is opposite for the S-S one at each temperature. There is no definite influence of the temperature on the strength of the two bonds, the local conformation is more important. Since there is no energy preference for dissociation of the two bonds at molecular level, the specificity for cleavage of the S—S bond, registered in the experimental study,59 can be attributed to the influence of environmental factors such as the pH or to the nature of the bioactive reagent – glutathione in the particular case.



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26

Conclusions

Identifying agents or delivery systems that improve drug efficacy, while reducing toxicity to healthy tissues is a major goal in drug delivery research. Computational studies that can predict structural changes and can directly be applied for design and optimization of novel delivery systems are highly desirable. To this end, molecular dynamics simulations of three compounds: the chemotherapeutic drug doxorubicin, a cell-penetrating peptide and a conjugated compound formed by them (DOX-peptide) in aqueous solution were carried out and analyzed. A simultaneous NMR study of DOX allowed explicit identification and confirmation of its molecular structure. From the evolution and the distribution of the monitored drug dihedral angles it can be concluded that doxorubicin at low temperature has low flexibility except for the side chain. With increase of the temperature the structure becomes much more mobile mainly because of the dynamics of the sugar residue. Up to three definite substructures can be discriminated. This mobility is retained after binding to the peptide, but it is restrained in the side chain because of the better opportunity for stacking interactions of the anthracycline part of the drug with the side chains of the peptide. The DOX molecules are stabilized by intramolecular hydrogen bonds between the quinone and hydroquinone residue of the anthracycline system. Apart from these bonds, effective temperature-independent hydrogen bonding between the organic system and the surrounding water molecules is observed. The stability of the formed conjugate is aided by the enhanced affinity for hydrogen bonding, too. The results from the root-mean-square deviation for every simulation show that upon temperature increase more geometry configurations of the three systems become accessible. This is a proof that the thermal energy suffices for the transitions between different conformations both for the DOX and peptide alone as well as within the conjugated compound. The results for the spatial


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27 orientation of the peptide coincide with the experimental data, namely, lack of a certain rigid secondary structure. These observations remain valid in the molecule of the drug-peptide conjugate, but the flexibility of the peptide backbone is slightly decreased because of hydrogen bonding and stacking interactions with the drug. Overall, conjugation of doxorubicin to octaarginine does not change the essential structural characteristics of the two molecules. The fact that the peptide backbone remains extremely flexible, combined with the increased hydrophobicity due to the drug, should facilitate the drug delivery. We believe that the flexibility of the peptide-drug compound is important during the stage of cell membrane translocation. The fact that the geometry of the molecule can rearrange without loss of energy during the unrestrained MD simulations implies that the conjugate will be able to easily adjust its structure to the local membrane surrounding, which could facilitate the translocation. This assumption is confirmed by the preliminary results of our ongoing bioactivity tests where elevated levels of the conjugate inside two lines of cancer cells are registered during in vitro studies.59 The hypothesis will be verified also by future cell membrane penetration studies. The experimental results have shown that the disulfide bond of the linker is efficiently cleaved by glutathione in conditions mimicking those in the living organism. Theoretical findings confirm that both the disulfide and the oxime bond require moderate energy for cleaving and show that the relative strength of the two bonds depends on the local atomic environment at body temperature. This analysis of the dynamics of the doxorubicin-peptide compound in aqueous solution should help clarifying the experimentally observed behavior and is part of a more complex approach for developing a system that is suitable for targeted drug transport and increased uptake of the highly efficient chemotherapeutic agent.



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28

Acknowledgement The authors are grateful to the FP7 project BeyondEverest.

Supporting Information The following material is available as Supporting information: atom numbering and RESP atomic charges of doxorubicin (Figure S1, Table S1) and peptide end-capping groups (Figure S2, Table S2); RESP atomic charges of the acetate anion (Figure S3); time evolution and probability distribution of dihedral angles 1 to 6 (Figures S4-S9); evolution of the RMSD during the production MD part of the atomic coordinates of the peptide, the whole conjugate, DOX within the conjugate, and cross-correlation functions of the latter with several characteristic dihedral angles (Figure S10); details of the models size and composition (Table S3); Ramachandran plot (Figure S11) and average values of the backbone angles  and  (Tables S4, S5) of the unbound and bound peptide; length distributions of the intramolecular (Figure S12) and intermolecular (Figures S13) hydrogen bonds; time evolution and probability distribution of dihedral angles 7 and 8 (Figure S14). This material is available free of charge via the Internet at http://pubs.acs.org


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29

TOC Graphic



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30

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32 (42) Dorairaj, S.; Allen, T. W. On the Thermodynamic Stability of a Charged Arginine Side Chain in a Transmembrane Helix. Proc. Natl. Acad. Sci. USA, 2007, 104, 4943-4948 (43) Li, L.; Vorobyov, I.; Allen, T. W. Potential of Mean Force and pK(a) Profile Calculation for a Lipid Membrane-exposed Arginine Side Chain. J. Phys. Chem. B 2008, 112, 9574-9587 (44) Freites, J. A.; Tobias, D.J.; Heijne, G.; White, S. H. Interface Connections of a Transmembrane Voltage Sensor. Proc. Natl. Acad. Sci. USA 2005, 102, 15059-15064 (45) Molinari, H.; Pastore, A.; Lian, L.-Y.; Hawkes,II, G. E.; Sales, K. Structure of Vancomycin and a Vancomycin/D-Ala-D-Ala Complex in Solution. Biochemistry 1990, 29, 2271-2277 (46) Juretschke, H.-P.; Lapidot, A. Intramolecular Interactions, Mesomerism and Dynamics in Actinomycin D Studied by 15N NMR Spectroscopy. Eur. J. Biochem. 1985, 147, 313-324 (47) Yongye, A. B.; Calle, L.; Ardá, A.; Jiḿnez-Barbero, J.; Andŕ, S.; Gabius, H.-J.; Martínez-Mayorga, K.; Cudic, M. Molecular Recognition of the Thomsen-Friedenreich Antigen–Threonine Conjugate by Adhesion/Growth Regulatory Galectin-3: Nuclear Magnetic Resonance Studies and Molecular Dynamics Simulations. Biochemistry 2012, 51, 7278−7289 (48) de Wolf, F. A.; Maliepaard, M.; van Dorsten, F.; Berghuis, I.; Nicolay, K.; de Kruijff, B. Comparable Interaction of Doxorubicin with Various Acidic Phospholipids Results in Changes of Lipid Order and Dynamics. Biochim. Biophys. Acta 1991, 1096, 67-80 (49) Karki, Sh. B.; Ostovic, D. Assessing Aggregation of Peptide Conjugate of Doxorubicin Using Quasi-elastic Light Scattering and 600 MHz NMR. Int. J. Pharmac. 2004, 271, 181-187 (50) Barthwal, R., Agrawal, P., Tripathi, A. N., Sharma, U., Jagannathan, N. R., Govil, G. Structural Elucidation of 4 -Epiadriamycin by Nuclear Magnetic Resonance Spectroscopy and Comparison with Adriamycin and Daunomycin Using Quantum Mechanical and Restrained Molecular Dynamics Approach Arch. Biochem. Biophys. 2008, 474, 48–64 (51) Duan, Y.; Wu, C.; Chowdhury, S.; Lee, M. C.; Xiong, G.; Zhang, W.; Yang, R.; Cieplak, P.; Luo, R.; Lee, T. et al. A Point-charge Force Field for Molecular Mechanics Simulations of Proteins Based on Condensedphase Quantum Mechanical Calculations. J. Comput. Chem. 2003, 24, 1999-2012 (Full citation is given in the SI) (52) van der Spoel, D.; Lindahl, E.; Hess, B.; Groenhof, G.; Mark, A. E.; Berendsen, H. J. C. GROMACS: Fast, Flexible, and Free. J. Comput. Chem. 2005, 26, 1701-1718 (53) Bayly, C. I.; Cieplak, P.; Cornell, W. D.; Kollman, P. A. A Well-behaved Electrostatic Potential Based Method Using Charge Restraints for Deriving Atomic Charges – the RESP Model. J. Phys. Chem. 1993, 97, 10269-10280 (54) Cieplak, P.; Cornell, W. D.; Bayly, C.; Kollman, P. A. Application of the Multimolecule and Multiconformational RESP Methodology to Biopolymers – Charge Derivation for DNA, RNA, and Proteins. J. Comput. Chem. 1995, 16, 1357-1377 (55) (a) Jorgensen, W. L.; Chandrasekhar, J.; Madura, J. D.; Impey, R. W.; Klein, M. L. Comparison of Simple Potential Functions for Simulating Liquid Water. J. Chem. Phys. 1983, 79, 926-935 (56) Jorgensen, W. L.; Madura, J. D. Temperature and Size Dependence for Monte Carlo Simulations of TIP4P Water. Mol. Phys. 1985, 56, 1381-1392 (57) This low temperature was chosen to mimic the conditions of in vitro cell experiments aimed at investigating the mechanism of drug uptake/release. (58) Berendsen, H. J. C.; Postma, J. P. M.; DiNola, A.; Haak, J. R. Molecular Dynamics with Coupling to an External Bath. J. Chem. Phys. 1984, 81, 3684-3691 (59) Lelle, M.; Frick, S. U.; Steinbrink, K.; Peneva, K. Novel cleavable cell-penetrating peptide–drug conjugates: synthesis and characterization. J. Peptide Sci. 2014, 20, 323-333 (60) Miyamoto, S.; Kollman, P. A. A. SETTLE – An Analytical Version of the SHAKE and RATTLE Algorithm for Rigid Water Models. J. Comput. Chem. 1992, 13, 952-962 (61) Ryckaert, J-P.; Ciccotti, G.; Berendsen H. J. C. Numerical Integration of the Cartesian Equations of Motion of a System with Constraints: Molecular Dynamics of n-Alkanes. J. Comput. Phys. 1977, 23, 327-341 (62) Darden, T.; York, D.; Pedersen, L.The Particle-Mesh Ewald – an NlogN Method for Ewald Sums in Large Systems. J. Chem. Phys. 1993, 98, 10089-10093 (63) Allen, M. P.; Tildesley, D. J. Computer Simulation of Liquids, Clarendon Press, Oxford, 2009 ACS Paragon Plus Environment

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33 (64) Ivanova, A.; Jezierski, G.; Vladimirov, E.; Rösch, N. Structure of Rhodamine 6G-DNA Complexes from Molecular Dynamics Simulations. Biomacromolecules 2007, 8, 3429-3438 (65) Even though it might seem that such run times are short for MD simulations of drug-peptide complexes, there are no sampling issues in this particular case for two reasons: (i) the peptide is short and very flexible and can sample fast its conformational space as evident from its RMSD and Ramachandran analysis; (ii) the drug is composed of a rigid part (the anthracycline cyclic system) with no expressed dynamics needing longer sampling and of a very mobile sugar residue, which is shown to sample freely its conformational space in the results section. Thus, the reported length of the simulations is considered sufficient. (66) It was necessary to analyze larger part of the trajectory of DOX-peptide because of the slower and more expressed structural dynamics of the molecule, i.e., various substructures with existence times of the order of 10 ns were witnessed in the RMSD profile and needed to be characterized. (67) Jarvis, R. A.; Patrick, E. A. Clustering Using a Similarity Measure Based on Shared Near Neighbors. IEEE Trans. Computers C 1973, 22, 1025-1034 (68) The values are different because of the dissimilar size of the molecules and of the variations in mobility upon temperature increase. (69) Humphrey, W.; Dalke, A.; Schulten, K. VMD - Visual Molecular Dynamics. J. Molec. Graphics 1996, 14, 33-38. (70) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci, B.; Petersson, G. A et al. Gaussian 09 Gaussian, Inc.: Wallingford CT, 2009. (Full citation is given in the Supporting Information) (71) Case, D. A.; Darden, T. A.; Cheatham, T. E.; Simmerling, C. L.; Wang, J.; Duke, R. E.; Luo, R.; Merz, K. M.; Wang, B.; Pearlman, D. A. et al. AMBER 8, University of California, San Francisco, 2004. (Full citation is given in the Supporting Information) (72) (a) Tomasi, J.; Mennucci, B.; Cammi, R. Quantum Mechanical Continuum Solvation Models. Chem. Rev. 2005, 105, 2999-3094 (73) Miertus, S.; Scrocco, E.; Tomasi, J. Electrostatic Interaction of a Solute with a Continuum. A Direct Utilization of Ab initio Molecular Potentials for the Prevision of Solvent Effects. Chem. Phys. 1981, 55, 117129 (74) Frederick, C. A.; Williams, L. D.; Ughetto, G.; van der Marel, G. A.; van Boom, J. H.; Rich, A.; Wang, A. H. Structural Comparison of Anticancer Drug DNA Complexes – Adriamycin and Daunomycin. Biochemistry 1990, 29, 2538-2549 (75) This initial preparation of the molecular structure was chosen to correspond as closely as possible to the protocol used for implementation of the peptide residues into the force field. (76) Kabsch, W.; Sander, C. Dictionary of Protein Secondary Structure: Pattern Recognition of Hydrogen-bonded and Geometrical Features. Biopolymers 1983, 22, 2577-2637 (77) For average values below 2.0 the data are not normally distributed and, hence, the standard deviations are estimated as for Boltzmann distributions.



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Figure 1: RMSD of the DOX coordinates during the simulation with respect to those of the minimized structure (Min); an experimental X-ray geometry53 (ID number 1D12 in the Protein Data Bank) is shown for comparison 150x108mm (300 x 300 DPI)


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Figure 2: (A) Population of the clusters and (B) superposition of the central structures of the populated clusters of unbound DOX obtained by cluster analysis of the MD trajectories at the three temperatures 94x48mm (300 x 300 DPI)



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Figure 3: (A) 2D

1H1H-NOESY spectra of DOX in water with proton resonance assignment (* denotes ambiguous assignment for CH2 groups); (B) the

distances between the characteristic NOESY proton pairs plotted for the structures from the most populated MD cluster of DOX (cluster 2 in Figure 2A) at room temperature and (D) their average values together with NOE category of the corresponding experimental signals (** shows that there is signal overlap); (C) atom numbering used for assignment of the NOESY signals 151x134mm (300 x 300 DPI)


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Figure 4: (A) Population of the clusters and (B) central structures of the clusters with population probability larger than 10 % as obtained from cluster analysis of the MD trajectories of DOX-peptide at the three temperatures 209x238mm (300 x 300 DPI)



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Figure 5: (A) Population of the clusters and (B) superposition of the central structures of the most populated clusters of the unbound peptide obtained by cluster analysis of the MD trajectories at the three temperatures 160x76mm (300 x 300 DPI)


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Figure 6: RMSF of Сα during the simulations of the unbound peptide (left) and of DOX-peptide (right) 153x57mm (300 x 300 DPI)



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Figure 7: Radial distribution function of Cg-Cg (top) for the peptide (left) and for DOX-peptide (right) and of Cg-DOX (bottom) 150x122mm (300 x 300 DPI)


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Figure 8: Geometries of the linker fragment for which UMP2/6-31G** PES scans were performed; LIN25-1 and LIN25-2 are the respective structures of the linker from clusters 1 and 5 at 25 °C, LIN37-1 and LIN25-1 were extracted from clusters 14 and 7 at 37 °C (Figure 4) 129x100mm (300 x 300 DPI)



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Figure 9: UMP2/6-31G** PES profiles of N—O and S—S bonds dissociation for the most probable linker structures at 25 °C and 37 °C; relative energies with respect to a reference completely dissociated structure (with bond length of 20 Å) are plotted 99x83mm (300 x 300 DPI)


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Scheme 1: Chemical formulae of doxorubicin with notations of the hydroquinone hydroxyl groups (top left), the studied peptide (top right) and the drug-peptide complex (bottom) 21x20mm (300 x 300 DPI)



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Scheme 2: Chemical formula of doxorubicin with illustration and notations of the six dihedral angles analyzed (marked in red) 34x37mm (300 x 300 DPI)


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Scheme 3: Chemical structure of DOX-peptide with the two analyzed dihedrals (Θ7 and Θ8) marked in red 159x98mm (300 x 300 DPI)



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TOC Graphic 50x50mm (300 x 300 DPI)


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Molecular Structure and Pronounced Conformational Flexibility of

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