On the preferred structure of dicoumarol and implications for enzyme binding: A quantum chemical analysis

Chemical Physics Letters 602 (2014) 45–51

Contents lists available at ScienceDirect

Chemical Physics Letters journal homepage: www.elsevier.com/locate/cplett

On the preferred structure of dicoumarol and implications for enzyme binding: A quantum chemical analysis Bilal M. Hussain a, Kassam Hassam a, Qing-Xi Ooi a,b, Richard A. Bryce a,⇑ a b

Manchester Pharmacy School, University of Manchester, Oxford Road, Manchester M13 9PT, UK Clinical Research Centre Perak, Raja Permaisuri Bainun Hospital, Jalan Hospital, 30990 Ipoh, Malaysia

a r t i c l e

i n f o

Article history: Received 19 November 2013 In final form 7 April 2014 Available online 13 April 2014

a b s t r a c t Dicoumarol and related coumarin compounds are potent inhibitors of oxidoreductase NQO1, an enzyme overexpressed in several types of solid tumour. Using density function theory, we study dicoumarol conformation in various tautomeric and ionisation states, in the gas phase and low and high dielectric environments. In aqueous solution, where the monoanionic form of dicoumarol is predominant, we predict a syn rotamer as favoured, which is the conformation crystallographically observed bound to NQO1. Comparison of internal distortion energies and protein docking calculations rationalise why only the syn form is found bound to NQO1. Ó 2014 Elsevier B.V. All rights reserved.

1. Introduction Coumarin-based compounds have found a wide range of applications, from dyes and sensors [1] to potent enzyme inhibitors. Recently, for example, coumarin derivatives have been identified as potential dual inhibitors of acetyl and butyrylcholinesterase [2]. Interestingly, coumarin was chosen as a probe druglike molecule for computational studies of transport across a lipid membrane using molecular dynamics simulations [3]. Recently, we have sought to identify and develop inhibitors of two oxidoreductases linked to cancer, NAD(P)H:quinone oxidoreductase 1 (NQO1) and NRH:quinone oxidoreductase 2 (NQO2) [4–9]. For NQO1, which is overexpressed in some solid tumours [10], we have identified coumarin scaffolds as having inhibitory activity [6,7]. Indeed, dicoumarol [3,30 -methylenebis(4-hydroxycoumarin)] has previously been shown to be one of the most potent competitive inhibitors of NQO1, with an inhibition constant Ki of 1–10 nM [11]. The structure of human NQO1/dicoumarol complex has been solved by X-ray crystallography at 2.75 Å (PDB code 2F1O) [12]. This structure reveals that dicoumarol adopts a V-shaped conformation, complementary to the active site shape, and hydrogen bonds via its O2, O200 , O4 and O400 with NQO1 residues Tyr1280 and His161 (Figure 1). Indeed, the structure suggests that dicoumarol takes on a syn conformation, where the two ring oxygens, O1 and O100 , orient on approximately the same side of the molecule (Figure 1b). However, an alternative anti orientation of the ligand, with the ring oxygens on opposite sides, was also ⇑ Corresponding author. Fax: +44 (0)161 275 2481. E-mail address: [email protected] (R.A. Bryce). http://dx.doi.org/10.1016/j.cplett.2014.04.009 0009-2614/Ó 2014 Elsevier B.V. All rights reserved.

proposed by our subsequent docking calculations (Figure 1c) [7]. Interestingly, an anti conformation is observed in small molecule crystal structures of dibromodicoumarol, 3,30 -methylene (bis-6-bromo-4-hydroxycoumarin) [13], and a linker-subsititued dicoumarol, (phenyl)bis(4-hydroxybenzo-2H-pyran-2-one-3-yl) methane [14]. Furthermore, dicoumarol, a member of the 4-hydroxycoumarin family, is theoretically capable of existing in a range of keto-enol tautomeric forms, which include dicoumarin, mixed coumarin–chromone and dichromone forms [15]. As a first step to understanding the NQO1-bound form of dicoumarol, an accurate description of its preferred unbound tautomeric and conformational behaviour in solution is desirable. To date, conformational analyses of dicoumarol have focused on its neutral tautomer [16–18], of which only trace exists in solution. Here, we analyse conformations and tautomers of dicoumarol at the B3LYP/6-311++G(2d,2p) level of theory. We consider dicoumarol in its neutral, monoanionic and dianionic ionisation states in vacuo and in polar and nonpolar solvents. In this way, we seek to model in a straightforward fashion the dielectric effects of an aqueous solution and more hydrophobic protein-bound environment on the ligand structure [19–21]. 2. Computational methods Dicoumarol can potentially exist as six different neutral tautomers [19]. Three of these forms are enol tautomers, N1, N2 and N3 (Figure 2), which can convert into three keto tautomers through rearrangement of the carbon–carbon double bond; this however has a profound impact on stability due to loss of resonance. For this reason, we focus here on the more stable enol

46

B.M. Hussain et al. / Chemical Physics Letters 602 (2014) 45–51

Figure 1. (a) Bound conformation of dicoumarol in active site of human NQO1; hydrophobic protein surface in yellow. Overlay (b) of crystal pose of dicoumarol (blue) and docked syn structure; (c) of crystal pose of dicoumarol (blue) and docked anti structure. Interactions of O2/O200 and O4/O400 oxygens of dicoumarol (d) in NQO1 crystallographic complex and (e) docked anti pose. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Figure 2. Structure of neutral enol tautomers (N1, N2 and N3), monoanionic tautomers (M1 and M2) and dianionic form (D1) of dicoumarol. Linker torsion angles also shown.

47

B.M. Hussain et al. / Chemical Physics Letters 602 (2014) 45–51

tautomers. Deprotonation of N1, N2 and N3 produces monoanionic tautomers, M1 and M2 (Figure 2); subsequential deprotonation of M1 and M2 gives rise to dianionic form, D1 (Figure 2). Prior molecular dynamics simulations of the NQO1/dicoumarol complex has suggested six possible conformations at the linkage between the coumarin rings [6]. These conformations are described by torsion angles U and W, defined as C4–C3–C11–C300 and C400 –C300 –C11–C3 respectively (Figure 3a). The three potential syn rotamers have a negative W torsional angle and are labelled a, b and c (Table 1, Figure 3a); anti rotamers A, B and C have a positive W torsional angle (Table 1, Figure 3b). Conformations were constructed for each of the six dicoumarol tautomer/charge states (N1, N2, N3, M1, M2, D1), leading to 36 structures. A further three structures were obtained by modelling alternative OH orientations in N2A, N2B and N2C; these are labelled mN2A, mN2B and mN2C respectively. We also define a as C3–C11–C300 for the CH2 linker bond angle (Figure 3a). All 39 structures were built using the molecular modelling programme Sybyl 8.0 [22]. Subsequent geometry optimisations were performed using the GAUSSIAN 03 quantum chemistry suite [23]. A preliminary conformational analysis was performed at the HF/631G⁄ level (data not shown). Geometry optimisation in the gas phase at this level led to convergence of structures to a core of 12 conformations: six neutral tautomers (N1a, N1A, N2a, mN2A, N3b, N3B), four monoanionic structures (M1b, M1A, M2a, M2A) and two dianionic conformers (D1b and D1A). The geometries of the 12 tautomers were further optimised using the hybrid B3LYP density functional theory method [24] with the 6-311++G(2d,2p) basis set. These calculations were performed in vacuo, in aqueous solvent and chloroform solvent. The conductor-like polarisable model (COSMO) implicit solvent model [25] was used with a dielectric of e = 4.8 for chloroform and e = 80.1 for water. Non-polar contributions were estimated using the universal force field (UFF) model [26]. Thermochemical contributions at 298.15 K and 1 atmosphere were estimated from gas-phase second derivative calculations at the B3LYP/6-311++G(2d,2p) level. Proton affinities (PA) were calculated as the difference in gas phase enthalpies for the protonated and dissociated systems:

PA ¼ DH0g ðA Þ þ DH0g ðHþ Þ  DH0g ðHAÞ

ð1Þ

where DH0g ðHþ Þ is taken as 2.5RT, i.e. 1.48 kcal/mol. 3. Results 3.1. Neutral dicoumarol We consider first the minimum energy structures and energetics of the six neutral structures of dicoumarol at the B3LYP/6311++G(2d,2p) level. We note that, here, N1 labels conformations in the dichromone form, protonated at both O2 and O200 oxygens (Figure 2); for N2, a mixed coumarin–chromone form is explored, protonated at O2 and O400 ; and for N3, the dicoumarin form is modelled, protonated at both O4 and O400 oxygens. At the B3LYP/

Table 1 Initial linker conformations of neutral, monoanionic and dianionic dicoumarol structures. Torsion angle

U W

syn

anti

a

b

c

A

B

C

80.0 100.0

60.0 130.0

30.0 110.0

80.0 80.0

60.0 50.0

30.0 70.0

6-311++G(2d,2p) level, we predict that the gas phase relative stabilities of these neutral tautomers as, N3B > N2a > N1A  N3b > mN2A > N1a (Table 2). Thus, dicoumarin form N3B is found to be the lowest in energy in the gas phase. This structure is in an anti conformation (Figure 4), forming two hydrogen bonds, both of O  O distance 2.68 Å (Table 3, Figure 4). N2a and N1A also form two hydrogen bonds (Table 3, Figure 4) but these structures, protonated at O2/O400 and O2/O200 atoms respectively, lie 5.1 and 9.5 kcal/mol higher in free energy than N3B (Table 2). This suggests that the O4 site of protonation is preferred to O2 by 4–5 kcal/mol. In comparison, a study of neutral tautomers of dicoumarol at the PM3 level by Kumar et al. [19] also found the dicoumarin form to be the lowest in energy, 3.3 kcal/mol more stable than the mixed coumarin–chromone N2 form and 19 kcal/ mol more stable than the dichromone N1. Although not explicitly stated, it appears that their work explored only anti structures, following pre-existing experimental small molecule X-ray [13,14] and NMR data [15] which support an anti conformation of dicoumarol and its derivatives in the solid state and deuterochloroform solution. N1a, mN2A and N3b structures are considerably higher in energy relative to the lowest energy geometry N3B, at 18.5– 32.1 kcal/mol (Table 3). Correspondingly, these structures form only one hydrogen bond of interoxygen distance less than 3 Å (Table 3). For example, syn N3b forms one good hydrogen bond in vacuo, of O  O distance 2.87 Å (Table 3); the other inter-oxygen distance is 3.43 Å, and lies 18.5 kcal/mol above N3B (Table 2). Interestingly, these three higher energy structures have considerably larger dipole moments (6.8–8.6 D, Table 4) compared to 1.4–2.4 D for the three lower energy structures. For these dicoumarol tautomers, the higher dipoles reflect their lower symmetry and less optimal intramolecular interactions. In an aqueous environment, as represented by the COSMO solvent model, the relative stabilities of dicoumarol are predicted as, N3B > N2a > N3b  N1A > mN2A > N1a (Table 2). This is the pattern exhibited in the gas phase, with the exception that N1A and N3b are now approximately degenerate. Interestingly, in water N1A remains about the same stability with respect to N3B as in vacuo, but the relative stability of N3b improves markedly, by 8.5 kcal/mol (Table 2). Similar changes in stability on solvation are also found for N1a and mN2A. This is not unexpected, given the larger gas-phase dipoles of N1a, mN2A and N3b noted earlier, which now result in greater solvation of these species relative to the remaining three conformers. For these three higher energy structures, the existing hydrogen bonding tightens. Likely due to relief of inter-oxygen repulsion by solvent screening, all hydrogen

Figure 3. Conformation of (a) syn and (b) anti tautomers of dicoumarol. Linker bond and torsion angles also shown.

48

B.M. Hussain et al. / Chemical Physics Letters 602 (2014) 45–51

Table 2 Relative Gibbs free energy, DG (kcal/mol) of optimal dicoumarol tautomers in gas phase (gas), water (aq) and chloroform (CHCl3).

a

Tautomer

Conf

Neutral N1a N1A N2a mN2A N3b N3B

Protonation

DGgas

DGaq

DGCHCl3

syn anti syn anti syn anti

O2 O2 O2 O2 O4 O4

O200 O200 O400 O400 O400 O400

32.1 9.5 5.1 26.2 18.5 0.0

25.4 10.1 5.5 18.1 10.0 0.0

27.7 10.0 5.4 20.6 12.7 0.0

Monoanion M1b M1A M2a M2A

syn anti syn anti

– – – –

O2 O2 O400 O400

Dianion D1b D1A

syn anti

– –

– –

11.0 a

9.7 a

9.9 a

0.0 4.2

0.0 3.3

0.0 3.6

0.0 0.4

0.0 0.0

0.0 0.1

Converts to M2A.

site. As expected, the calculated relative stabilities of neutral tautomers are intermediate between gas and aqueous phase values. The predicted ordering of stability is, N3B > N2a > N1A  N3b > mN2A > N1a (Table 2), following the gas phase trend. As for gas phase and aqueous solution, none of the DG values are close enough to suggest any structure other than anti N3B as accessible. 3.2. Monoanionic dicoumarol In the monoanionic form of dicoumarol, we again consider both syn (M1b, M2a) and anti structures (M1A and M2A). At the B3LYP/ 6-311++G(2d,2p) level, M1A was not found to be a stable minimum in gas phase or solvent, but converted to M2A by transfer of a proton across rings, from the O2 to O400 position (Figure 5). This further highlights that the O400 position is the more favourable site of protonation (and that the transfer here is barrierless). In the gas phase, the three remaining structures have the stability M2a > M2A > M1b (Table 2). Unlike the neutral minimum energy structure N3B, the most stable monoanionic tautomer, M2a, is a syn conformation (Figure 5). The equivalent anti structure to M2a is M2A, which is 4.2 kcal/mol higher in energy than M2a. M2a is protonated at the O400 position and is 11.0 kcal/mol higher than its O2-protonated counterpart, M1b (Table 2). All three monoanionic structures form hydrogen bonds between the coumarin rings, with interoxygen distances of 2.45 Å for M2a, 2.52 Å for M2A and 2.46 Å for M1b (Table 3). On solvation, M2A and M1b are stabilised in relation to M2a by 0.9 and 1.3 kcal/mol respectively in water; and 0.6 and 1.1 kcal/ mol respectively in chloroform (Table 2). All structures are favoured by decreased interoxygen repulsion, reflected in decreased interoxygen distances, by up to 0.15 Å (M2a), 0.17 Å (M1b) and 0.17 Å in water (M2A) (Table 3). Accompanying this, the linker torsion angle W exhibit changes on aqueous solvation by 3.3° (M2a), 5.9° (M1b) and 6.0° (M2A) (Table 3). Similar but smaller effects on gas phase energetics and geometry are observed on solvation by chloroform, reflecting the smaller dielectric of the medium (Tables 2 and 3). 3.3. Dianionic dicoumarol

Figure 4. Optimal conformations of N1A, N2a and N3B in gas phase. Mulliken charges (in e) given for oxygen and selected hydrogen atoms.

bond distances for N1a, mN2A and N3b decrease by 0.03–0.09 Å (Table 3); by comparison, the N3B, N2a and N1A structures experience increases in distance by up to 0.02 Å (Table 3). We also compute relative stabilities in chloroform solvent, as an approximation of the nonpolar environment of the NQO1 active

Here, we consider the dianionic form of dicoumarol; these structures differ only in conformation, as anti conformer D1A and syn conformer D1b. From our B3LYP/6-311++G(2d,2p) calculations, no clear energetic preference emerges for the syn or anti conformation (Table 2). In gas phase, the anti conformer D1A is 0.4 kcal/mol less stable than syn conformer D1b (Table 2). The difference in DG values for D1b and D1A in both aqueous and chloroform solvents are effectively zero (Table 2), and suggests that facile interconversion between syn and anti conformations is possible in all three environments. For D1A and D1b, no intramolecular hydrogen bond is possible. For both structures, significant charge resides on both coumarin rings, with Mulliken charges on O2/O200 atoms of between 0.65 and 0.68 e and on O4/O400 atoms of between 0.72 and 0.75 e (Figure 6). The gas phase D1A conformation possesses generally shorter interoxygen interactions than D1b, by 1.66, 0.26 and 0.09 Å for O2  O400 , O4  O200 and O4  O400 distances respectively (Table 3) and may experience greater electrostatic repulsion; accordingly, D1A is energetically stabilised on aqueous solvation by 0.4 kcal/mol relative D1b (Table 2). 4. Discussion and conclusions From conformational analysis of neutral, monoanionic and dianionic conformations of dicoumarol, we observe the following:

49

B.M. Hussain et al. / Chemical Physics Letters 602 (2014) 45–51

Table 3 Torsion angles U and W (°), bond angle a (°) and distances between key oxygen atoms (Å) in optimal dicoumarol tautomers, in gas (g) water (w) and chloroform (ch) phases. Note that M1A converts to M2A.

W

a

O2–O200

O2–O400

O4–O200

O4–O400

80.1 82.6 81.9

121.3 119.3 120.5

115.9 116.3 116.1

2.89 2.82 2.83

5.70 5.68 5.69

4.99 5.08 5.03

3.60 3.57 3.60

g aq ch

87.3 87.8 87.7

87.3 87.8 87.7

114.8 114.7 114.8

5.49 5.46 5.47

2.59 2.59 2.59

2.59 2.59 2.59

5.27 5.29 5.28

N2a

g aq ch

88.1 88.9 88.5

89.6 89.3 89.7

115.2 115.2 115.3

2.64 2.65 2.66

5.52 5.50 5.52

5.30 5.33 5.32

2.64 2.64 2.65

mN2A

g aq ch

79.6 82.6 81.9

66.4 69.2 68.6

115.9 116.4 116.2

5.84 5.84 5.84

2.82 2.77 2.78

3.47 3.42 3.43

4.87 4.97 4.95

N3b

g aq ch

65.8 71.4 68.1

105.5 102.7 103.8

116.0 116.5 116.2

3.43 3.34 3.39

5.84 5.84 5.84

4.81 4.99 4.90

2.87 2.80 2.82

N3B

g aq ch

90.0 88.8 89.9

90.0 88.8 90.0

115.7 115.7 115.7

5.33 5.39 5.36

2.68 2.70 2.69

2.68 2.70 2.70

5.54 5.49 5.52

M1b

g aq ch

100.1 99.4 98.3

120.1 114.2 117.9

118.9 118.6 118.6

2.46 2.48 2.48

5.66 5.64 5.65

5.41 5.46 5.40

3.78 3.61 3.69

M2a

g aq ch

86.0 87.3 87.6

70.6 73.9 71.7

118.2 117.9 117.9

3.66 3.51 3.57

5.44 5.44 5.41

5.64 5.63 5.64

2.45 2.48 2.48

M2A

g aq ch

96.9 95.9 94.5

65.8 71.8 68.4

118.2 117.9 117.8

5.67 5.66 5.67

2.52 2.54 2.55

3.74 3.55 3.62

5.34 5.39 5.32

D1b

g aq ch

73.3 64.0 64.5

151.3 129.6 132.1

119.8 117.4 117.5

3.73 3.59 3.62

5.56 5.75 5.74

4.16 4.43 4.38

4.46 3.82 3.91

D1A

g aq ch

57.9 57.9 60.7

58.0 60.6 58.1

118.1 117.1 116.8

5.75 5.76 5.78

3.90 3.76 3.69

3.90 3.69 3.75

4.37 4.39 4.42

Tautomer

Phase

N1a

g aq ch

N1A

U

Table 4 Dipole moment (D) of optimal dicoumarol tautomers in the gas phase, water and chloroform.

a

Tautomer

Gas

Water

Chloroform

N1a N1A N2a mN2A N3b N3B

6.77 1.55 2.44 7.35 8.57 1.38

11.17 2.17 3.29 12.57 14.43 2.05

9.73 2.11 3.03 10.92 12.57 1.76

M1b M1A M2a M2A

4.09

D1b D1A

6.87

6.05

a

a

a

6.30 5.13

10.82 8.72

9.45 7.68

4.53 6.05

10.15 11.90

8.81 10.43

Converts to M2A.

in gas phase, the lowest energy forms are N3B, M2a and D1b. These preferences are maintained in aqueous and chloroform solvents. We predict that in all three states, the coumarin tautomeric form is favoured for both rings, i.e. that both O4 and O400 is protonated in preference to O2 or O200 atoms. This is reflected by the corresponding computed proton affinities involving compounds N3B, M2a and D1b (Table 5): protonation of lowest energy monoanionic structure M2a at the O2 atom to form the mixed coumarin– chromene structure N2a liberates 313.8 kcal/mol (Table 5).

Alternatively protonating O4 of M2a to form N3B releases 319.0 kcal/mol, 5.2 kcal/mol more favourable than at O2. Similarly, protonation of D1b at O2 to form M1b is 11.0 kcal/mol less favourable than pronation of O400 to produce M2a (Table 5). Clearly, protonation of O2, proximal to O1, has a more disruptive effect on the aromatic stability of the conjugated coumarin system as compared to protonation at O4. Secondly, lowest energy structures N3B, M2a and D1b correspond to an anti neutral form, syn monoanionic form and syn dianionic form of dicoumarol respectively (although for the latter, we note that the anti form lies very close in energy to the syn form). NMR and IR supports a neutral form of dicoumarol in deuterochloroform, where two intramolecularly hydrogen bonded protons were observed [15]. However, the experimentally measured pKa values of dicoumarol are 3.9 ± 0.2 and 8.0 ± 0.1 [27]. This suggests that at physiological pH, less than 0.1% of dicoumarol will exist in the neutral form, whereas approximately 80% will be monoanion and approximately 20% dianion. Thus, the form predicted to bind to enzyme NQO1 would be mainly monoanion. Our lowest energy monoanionic structure is M2a, a syn conformation; the syn conformation of dicoumarol is also observed bound to NQO1 in the crystal structure [12]. We note that the crystal structure was determined at pH 8.1, which would have a balance of monoanion and dianion in solution. Indeed, molecular dynamics simulation of the dicoumarol/NQO1 crystal structure showed closer correspondence to the crystallographic conformation when the ligand was modelled as monoanion rather than dianion [6]. Indeed, in

50

B.M. Hussain et al. / Chemical Physics Letters 602 (2014) 45–51 Table 5 Gas phase proton affinity, PA (kcal/mol).

Figure 5. Optimal conformations of M2a and M2A in gas phase. Mulliken charges (in e) given for oxygen and selected hydrogen atoms.

Figure 6. Optimal conformations of D1b and D1A in chloroform. Mulliken charges (in e) given for oxygen and selected hydrogen atoms.

the latter case, the ligand flipped during the course of the trajectory into an anti conformation [6]. The linker conformation (U,W) of dicoumarol in the crystal structure is (78.1°, 96.7°), as

A

HA

Protonation site

PA

M2a M2a D1b D1b

N2a N3B M1b M2a

O2 O4 O2 or O200 O4 or O400

313.8 319.0 395.1 406.1

measured in complex with a representative NQO1 subunit formed by polypeptide chains G and H [12]. This is not dissimilar to the low dielectric chloroform structures of M2a (87.6°,71.7°) and D1b (64.5°, 132.1°) that we observe here (Table 3). The calculated bond angles a of M2a (117.9°) and D1b (117.5°) compares well with the crystal structure (117.8°). Our previous docking calculations to the NQO1 active site predicted that an anti dicoumarol-bound structure scored equally to a syn structure: the computed ChemScore value [28] was 10.8 kcal/mol for both syn and anti forms [7]; indeed, the putative anti conformation superimposes closely onto the crystallographic syn conformation in the active site (Figure 1c; also compare Figure 1d and e). We find here that at the B3LYP/6-311++G(2d,2p) level, the computed syn M2a and anti M2A geometries possess a strong intramolecular hydrogen bond, promoting a V-shape suited to the dicoumarol cavity [12]. This V-shape enables p stacking with the flavin adenine dinucleotide (FAD) and along the access pocket. We note that different active site constraints can influence the dicoumarol into alternative structures: a pose where the coumarin rings are orthogonal in plane to one another is found for the nitroreductase, NTR [29]. Interestingly, for the more superficially located binding site in azoreductase, AzoR, a syn V-shaped bound pose of dicoumarol is observed [30], similar to that found in NQO1. A quantum chemical analysis of neutral dicoumarol in the anti conformation at the B3LYP/6-31G⁄ level [16] suggested that the O2  O4 intramolecular hydrogen bond is unusually strong, approximately 12.2 kcal/mol in strength. The geometry of the intramolecular hydrogen bond in monoanionic dicoumarol is likely to be stronger than that of the neutral form, with shorter O  O hydrogen bond distances calculated here for M2a (2.5 Å) compared to N3B (2.7 Å) (Table 3). Both the crystallographic syn and predicted anti poses of dicoumarol appear to maintain an intramolecular hydrogen bond (Figure 1d and e). However, from our B3LYP/6-311++G(2d,2p) calculations here, the energy required for dicoumarol to adopt an anti geometry in aqueous solution from the minimum energy syn pose is predicted to be 3.3 kcal/mol (comparing syn M2a with the nearest anti geometry, M2A) (Table 2). This energetic penalty would be offset by favourable interactions of dicoumarol with NQO1. Inspection of the (syn) dicoumarol/NQO1 crystal structure indicates that dicoumarol forms key polar interactions with Tyr1280 and His161: ligand O2 and O200 atoms interact with Og-H Tyr1280 (Figure 1d), with O  O distances of 2.71 and 3.95 Å respectively; and ligand O4 and O400 atoms with His161, with O  N distances of 3.15 and 3.42 Å respectively (as before, taken from the G/H subunit of NQO1) [12]. Interestingly, the docked bound pose of anti dicoumarol (Figure 1e) situates its oxygen atoms in a proximity to Tyr1280 and His161 very similar to the syn form. Here, dicoumarol O4 and O200 atoms interact with Og-H Tyr1280 (Figure 1d), with O  O distances of 2.88 and 4.18 Å respectively; and dicoumarol O2 and O400 atoms with His161, with O  H–Ne distances of 2.39 and 2.98 Å respectively. One can therefore surmise that the hydrogen bond interactions with NQO1 are likely to be similar for bound syn and anti forms. However, the computed distortion penalty of >3 kcal/ mol, to adopt the anti M2A conformation from the syn M2a structure in water (Table 2), disfavours NQO1 binding of the anti

B.M. Hussain et al. / Chemical Physics Letters 602 (2014) 45–51

form. This energetic cost is somewhat larger than the internal energy penalty estimated from the ChemScore function, of 0.5 kcal/mol favouring the syn form. This combines with a protein clash term of 0.5 kcal/mol favouring the syn form as the better fitting ligand in the active site. These terms are counterbalanced by a kilocalorie stronger protein-ligand hydrogen bonding component of the anti form, leading to the degenerate ChemScores for syn and anti conformations. We note here that His161 is modelled as protonated at Ne. For this form of NQO1, both docked syn and anti poses form good hydrogen bonds between dicoumarol O400 and His161. Alternatively protonating the Nd atom of His161 in docking calculations, such that the N–H bond points away from the ligand, led to loss of a His161 NAH  O hydrogen bond with the ligand in the docked pose and an associated drop in docking score of 0.8 kcal/mol for both predicted syn and anti poses of dicoumarol. There was no evidence from docking to either His161 tautomer of NQO1 that the dicoumarol OH group could adopt the appropriate geometry to act as a hydrogen bond donor to the His161 Ne atom. For docking to NQO1 with the cationic form of His161 (i.e. protonation at both Nd and Ne), syn and anti poses were again predicted as degenerate and to have scores similar to that of the Ne form. Overall, these calculations suggest that His161 acts as a hydrogen bond donor to dicoumarol. In summary, using density functional theory calculations, we have explored the preferred conformations of dicoumarol in neutral, monoanionic and dianionic forms. In the neutral form, dicoumarol prefers an anti conformation of its coumarin rings; this is in agreement with IR, NMR and X-ray data. As monoanion, dicoumarol prefers a syn conformation. As dianion, syn and anti conformations are effectively degenerate. Incorporating the effect of high and low dielectric solvent, to represent water and protein environments respectively, does not change these preferences. In all cases, dicoumarol is predicted to prefer the coumarin tautomer as opposed to forms involving chromene. Comparing with our calculations of dicoumarol in solution, monoanionic dicoumarol is crystallographically observed in the lowest energy syn form when bound to the protein NQO1. Although the anti form is predicted from docking calculations to make similar polar interactions with NQO1 as syn dicoumarol, incorporation of a penalty of distortion

51

of 3.3 kcal/mol may explain why this bound form is not observed experimentally. Acknowledgements We thank Siobhan McClair, Diana Chan Leong, Craig Marsh, David Riley, Phillip Edwards, David Frenkel, Karen Nolan and Ian Stratford for their contributions to this work. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30]

H.E. Katerinopoulos, Curr. Pharm. Des. 10 (2004) 3835. M. Alipour et al., Arch. Pharm. Chem. Life Sci. 346 (2013) 577. M. Paloncyova, K. Berka, M.J. Otyepka, Chem. Theory Comput. 8 (2012) 1200. M.S. Dunstan et al., J. Med. Chem. 54 (2011) 6597. K.A. Nolan, D.J. Timson, I.J. Stratford, R.A. Bryce, Bioorg. Med. Chem. Lett. 16 (2006) 6246. K.A. Nolan et al., J. Med. Chem. 50 (2007) 6316. K.A. Nolan et al., J. Med. Chem. 52 (2009) 7142. K.A. Nolan, M.P. Humphries, R.A. Bryce, I.J. Stratford, Bioorg. Med. Chem. Lett. 20 (2010) 2832. K.A. Nolan, M.C. Caraher, M.P. Humphries, H.A.-A. Bettley, R.A. Bryce, I.J. Stratford, Bioorg. Med. Chem. Lett. 20 (2010) 7331. M. Faig et al., Structure 9 (2001) 659. S. Hosoda, W. Nakamura, K. Hayashi, J. Biol. Chem. 249 (1974) 6416. G. Asher, O. Dym, P. Tsvetkov, J. Adler, Y. Shaul, Biochemistry 45 (2006) 6372. N.W. Alcock, E. Hough, Acta Crystallogr. B 28 (1972) 1957. E.J. Valente, D.S. Eggleston, Acta Crystallogr. C 45 (1989) 785. D.W. Hutchinson, J.A. Tomlinson, Tetrahedron 25 (1969) 2531. N. Trendafilova, G. Bauer, T. Mihaylov, Chem. Phys. 302 (2004) 95. T. Mihaylov, N. Trendafilova, I. Georgieva, J. Mol. Model. 14 (2008) 353. T. Mihaylov, I. Georgieva, G. Bauer, I. Kostova, I. Manolov, N. Trendafilova, Int. J. Quantum Chem. 106 (2006) 1304. A. Kumar, S.K. Jain, R.C. Rastogi, J. Mol. Struct. (Theochem) 678 (2004) 55. E. Knobloch, Z. Prochazka, Chem. Listy 47 (1953) 1285. K. Fucik, S. Koristek, Chem. Listy 46 (1952) 190. Tripos Inc., Sybyl 6.8., 2003. M. J. Frisch et al., GAUSSIAN 03, Revision C.01, 2004. A.D. Becke, J. Chem. Phys. 98 (1993) 5648. V. Barone, M. Cossi, J. Phys. Chem. A 102 (1998) 1995. A.K. Rappe, C.J. Casewit, K.S. Colwell, J. Am. Chem. Soc. 114 (1992) 10024. R. Labbe-Bois, C. Laruelle, J.J. Godfroid, J. Med. Chem. 18 (1975) 85. M.L. Verdonk, J.C. Cole, M.J. Hartshorn, C.W. Murray, R.D. Taylor, Proteins 52 (2003) 609. E. Johansson, G.N. Parkinson, W.A. Denny, S. Neidle, J. Med. Chem. 46 (2003) 4009. K. Ito et al., J. Biol. Chem. 283 (2008) 13889.