SUPPLEMENTARY MATERIAL

Molecular modelling studies on the interactions of 7-methoxytacrine-4-

pyridinealdoxime with VX-inhibited HssAChE. A near attach approach to

assess different spacer-lengths.

Jorge Alberto Valle da Silva, capalberto05@yahoo.com.br a,1, Eugenie Nepovimova,

eugenie.nepovimova@uhk.cz b, Teodorico Castro Ramalho, teo@dqi.ufla.br c, Kamil Kuca,

kamil.kuca@uhk.cz d, Tanos Celmar Costa França, tanos@ime.eb.br a, d.

aLaboratory of Molecular Modelling Applied to the Chemical and Biological Defense (LMCBD), Military Institute of Engineering, Rio de Janeiro/RJ, Brazil;

bBiomedical Research Centre, University Hospital Hradec Kralove, Czech Republic;

cLaboratory of Molecular Modelling, Chemistry Department, Federal University of Lavras, Lavras, MG, Brazil;

dCenter for Basic and Applied Research, Faculty of Informatics and Management, University of Hradec Kralove, Hradec Kralove, Czech Republic.

1 Present address: Chemical, Biological, Radiological and Nuclear Institute, Avenida das

Americas, 28705, Barra de Guaratiba Rio de Janeiro, RJ, Brazil.

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Figure S1. Protonation states of hybrids nC. Molecular structures of ligands predicted

through protonation analysis as the highest percentage of micro species. Result of the original

hybrid 5C [11] is highlighted in blue.

Figure S2. Re-docking results. Re-docking evaluation of HI-6 by overlapping of pose over

conformation within HssAChE/VX/HI-6 complex (modeled from MmAChE/GB/HI-6

crystallographic complex).

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Table S1. Docking results for hybrids from 1C to 10C. Results of the best pose selected for

each compound as NAC approach (Figure 2) [6,14-16].

Hybrid

s nC

The best pose selected regarding the highest ratio θOPO/dOP

dOP

(nm)θOPO

Einter

(kJ/mol)

EH−bond

(kJ/mol)

H-bond

interaction

residues a

Hydrophobic interactions

( π-π ) a, b

1C 0.57 154° - 520.11 - 30.00Tyr124,

Ser125

Tyr72, Trp86, Phe123,

Tyr124, Trp286,

Phe295, Phe297, Tyr337,

Phe338, Tyr341, His447

2C 0.50 151° - 517.15 - 31.38 Tyr124

Tyr72, Tyr124, Trp286,

His287, Phe295, Phe297,

Tyr337, Phe338, Tyr341

3C 0.67 148° - 671.84 - 34.79

Tyr124,

Ser125,

Ser298

Tyr72, Trp86, Tyr124,

Trp286, His287, Phe295,

Phe297, Phe299, Tyr337,

Phe338, Tyr341

4C 0.48 158° - 560.68 - 19.95 Tyr124

Tyr72, Tyr124, Trp286,

His287, Phe295, Phe297,

Tyr337, Phe338, Tyr341

5C 0.51 159° - 514.65 - 24.86 Tyr124

Tyr72, Tyr124, Trp286,

His287, Phe295, Phe297,

Tyr337, Phe338, Tyr341

6C 0.53 148° - 551.36 - 14.07VX-

Ser203

Tyr72, Tyr124, Trp286,

His287, Phe295, Phe297,

3

Hybrid

s nC

The best pose selected regarding the highest ratio θOPO/dOP

dOP

(nm)θOPO

Einter

(kJ/mol)

EH−bond

(kJ/mol)

H-bond

interaction

residues a

Hydrophobic interactions

( π-π ) a, b

Tyr337, Phe338, Tyr341

7C 0.40 149° - 594.30 - 5.17

Tyr72,

Tyr124,

His287

Tyr72, Tyr124, Trp286,

His287, Phe295, Phe297,

Tyr337, Phe338, Tyr341

8C 0.42 163° - 530.91 - 13.41

Tyr124,

VX-

Ser203

Tyr72, Phe123, Tyr124,

Trp286, Phe295,

Phe297, Phe299, Tyr337,

Phe338, Tyr341, His447

9C 0.38 174° - 473.14 - 13.89Tyr124,

Ser125

Tyr72, Trp86, Tyr124,

Trp286, His287, Phe295,

Phe297, Tyr337, Phe338,

Tyr341, His447

10C 0.59 158° - 615.71 - 15.49Tyr124,

Ser125

Tyr72, Trp86, Phe123,

Tyr124, Trp286,

Phe295, Phe297, Tyr337,

Phe338, Tyr341, His447

a Residues in bold highlight the interactions with PAS (Tyr72, Tyr124, Trp286 and Ser298) and CAS (VX-

Ser203 adduct and His447).

b Hydrophobic interactions amongst aromatic rings roughly in parallel.

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Figure S3. Dynamic behaviour of hybrids 4C and 5C. Plots of dOP throughout 50 ns of MD

simulation and the last frame of each ligand.

Figure S4. Temporal RMSD. Plots of RMSD throughout 50 ns of MD simulation.

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Figure S5. Behaviour of dOP. Plots of dOP per ligand throughout 50 ns of MD simulation.

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Figure S6. Last frames after 50 ns of MD simulation. Comparisons amongst last frames.

Ligands are shown in green, residues of CAS in yellow and others in blue. H-bonds and dOP

are shown in blue and brown dashed lines respectively.

Figure S7. H-bond interactions of hybrid 4C (PAS). (a) and (c) plots of H-bond

interactions with the atoms of Glu202, Tyr124 and Glu285 throughout 50 ns of MD

simulation. (b) and (d) variation of distances d of the H-bonds formed. “⟨ ⟩” represents the

mean of d values.

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Figure S8. H-bond interactions of hybrid 5C (PAS) with PAS. (a) plot of H-bond

interactions with the atoms of Tyr124 throughout 50 ns of MD simulation. (b) variation of

distances d of the H-bond formed. “⟨ ⟩” represents the mean of d values.

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Figure S9. H-bond interactions of hybrid 5C (PAS). (a) and (b) plots of H-bond

interactions with amongst the ligand, Asp74, Thr83, and Tyr341 throughout 50 ns of MD

simulation. (c) and (d) variation of distances d of the H-bonds formed. “⟨ ⟩” represents the

average d values.

Figure S10. H-bond interactions of hybrid 4C (PAS) deprotonated. (a) and (b) plots of H-

bond interactions with the atoms of Tyr72, Tyr124 and His447 throughout 50 ns of MD

simulation. (c) and (d) variation of distances d of the H-bonds formed. “⟨ ⟩” represents the

mean of d values.

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Figure S11. H-bond interactions of hybrid 4C (PAS) deprotonated within PAS. (a) plots

of H-bond interactions formed between Glu285 and Ser298 throughout 50 ns of MD

simulation of hybrid 4C (PAS) deprotonated. (b) variation of distances d of the H-bonds

formed. “⟨ ⟩” represents the average d values.

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Figure S12. H-bond interactions of hybrid 5C deprotonated. (a) and (c) plots of H-bond

interactions with Tyr124 and Glu292 throughout 50 ns of MD simulation. (b) and (d)

variation of distances d of the H-bonds formed. “⟨ ⟩” represents the average d values.

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Table S2. Relative binding free energies per key residues. The most significant

contributions ⟨ ∆ GResidue⟩ of key amino acid residues to ⟨ ∆ GBinding ⟩.

Contributions

per residue a, b, c

Hybrid 4C

(PAS)

Hybrid 5C

(PAS)

Hybrid 4C

(PAS)

deprotonated

Hybrid 5C

deprotonated

Asp74:

⟨ ∆ EMM ⟩ d -62.02 ± 8.48 -101.99 ± 5.99 -45.30 ± 7.70 -29.04 ± 5.91

⟨ ∆ GPolar ⟩ d 17.58 ± 5.17 64.59 ± 13.13 14.96 ± 8.25 6.67 ± 8.23

⟨ ∆ GNonpolar ⟩ d -0.19 ± 0.20 -0.64 ± 0.22 -0.40 ± 0.34 -0.06 ± 0.13

⟨ ∆ GResidue⟩ e -44.63 ± 5.29 -38.04 ±9.72 -30.74 ± 3.74 -22.44 ± 5.06

% of ⟨ ∆ GBinding ⟩ f 5.88% 5.29% 6.77% 5.16%

Glu84:

⟨ ∆ EMM ⟩ d - -52.45 ± 4.72 - -

⟨ ∆ GPolar ⟩ d - 13.31 ± 4.30 - -

⟨ ∆ GNonpolar ⟩ d - -0.02 ± 0.04 - -

⟨ ∆ GResidue⟩ e - -39.16 ± 1.73 - -

% of ⟨ ∆ GBinding ⟩ f - 5.44% - -

Tyr124 (PAS g):

⟨ ∆ EMM ⟩ d - - - -1.41 ± 1.00

⟨ ∆ GPolar ⟩ d - - - 1.35 ± 1.36

⟨ ∆ GNonpolar ⟩ d - - - -0.19 ±0.20

⟨ ∆ GResidue⟩ e - - - -0.25 ±0.59

% of ⟨ ∆ GBinding ⟩ f - - - 0.06%

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Contributions

per residue a, b, c

Hybrid 4C

(PAS)

Hybrid 5C

(PAS)

Hybrid 4C

(PAS)

deprotonated

Hybrid 5C

deprotonated

Glu202 (PAS g):

⟨ ∆ EMM ⟩ d -88.60 ± 10.14 -55.06 ± 1.71 -17.15 ± 1.41 -

⟨ ∆ GPolar ⟩ d 39.07 ± 7.52 13.89 ± 3.12 -4.70 ± 4.56 -

⟨ ∆ GNonpolar ⟩ d - 0.06 ± 0.08 - - -

⟨ ∆ GResidue⟩ e -49.59 ± 8.64 -41.18 ± 3.20 -12.46 ± 4.95 -

% of ⟨ ∆ GBinding ⟩ f 6.54% 5.72% 2.74% -

a Some key amino acid residues.

b Calculated in kJ/mol.

c Values in bold for key contributions discussed in the text.

d Calculated through MM-PBSA methodology [53].

e Calculated as shown in equation 5.

f Calculated as shown in equation 4.

g Interactions within PAS or CAS as indicated in Figure 2.

Table S2. Relative binding free energies per key residues. The most significant

contributions ⟨ ∆ GResidue⟩ of key amino acid residues to ⟨ ∆ GBinding ⟩ (Cont.).

Contributions

per residue a, b, c

Hybrid 4C

(PAS)

Hybrid 5C

(PAS)

Hybrid 4C

(PAS)

deprotonated

Hybrid 5C

deprotonated

Glu285 (PAS g):

⟨ ∆ EMM ⟩ d -112.94 ± 14.04 - -86.79 ± 15.31 -48.41 ± 12.27

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Contributions

per residue a, b, c

Hybrid 4C

(PAS)

Hybrid 5C

(PAS)

Hybrid 4C

(PAS)

deprotonated

Hybrid 5C

deprotonated

⟨ ∆ GPolar ⟩ d 68.98 ± 11.93 - 71.21 ± 11.50 26.32 ± 19.49

⟨ ∆ GNonpolar ⟩ d -0.29 ± 0.12 - -0.23 ± 0.10 -0.33 ± 0.30

⟨ ∆ GResidue⟩ e -44.26 ± 9.98 - -15.81 ± 9.70 -22.41 ± 9.72

% of ⟨ ∆ GBinding ⟩ f 5.83% - 3.48% 5.16%

Trp286 (PAS g):

⟨ ∆ EMM ⟩ d - - - -19.27 ± 3.84

⟨ ∆ GPolar ⟩ d - - - 6.96 ± 1.36

⟨ ∆ GNonpolar ⟩ d - - - -2.29 ±0.49

⟨ ∆ GResidue⟩ e - - - -14.59 ± 3.77

% of ⟨ ∆ GBinding ⟩ f - - - 3.36%

Glu292:

⟨ ∆ EMM ⟩ d -59.41 ± 7.43 -57..39 ± 5.88 -36.68 ± 3.43 -46.34 ± 18.60

⟨ ∆ GPolar ⟩ d 12.64 ± 7.81 10.88 ± 5.87 5.31 ± 3.97 16.93 ± 22.06

⟨ ∆ GNonpolar ⟩ d -0.02 ± 0.06 - 0.03 ± 0.08 - 0.01 ± 0.02 -0.66 ± 0.50

⟨ ∆ GResidue⟩ e -46.78 ± 4.42 -46.54 ± 2.72 -31.38 ± 1.94 -30.07 ± 5.07

% of ⟨ ∆ GBinding ⟩ f 6.17% 6.47% 6.91% 6.92%

a Some key amino acid residues.

b Calculated in kJ/mol.

c Values in bold for key contributions discussed in the text.

d Calculated through MM-PBSA methodology [53].

e Calculated as shown in equation 5.

f Calculated as shown in equation 4.

g Interactions within PAS and CAS as indicated in Figure 2.

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Figure S13. Relative binding free energies per key residues. Comparisons amongst the

most significant contributions ⟨ ∆ GResidue⟩ of key amino acid residues to ⟨ ∆ GBinding ⟩.

Table S3. Relative binding free energies through NAC approach. Relative binding

energies computed for the NAC frames selected.

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LIGAND Figure Frame a t(ns)∆ GNAC frame

(kJ/mol) b

∆ G1st frame

(kJ/mol) b

∆ ∆ GBinding

(kJ/mol) c

Hybrid 4C (PAS)

deprotonated7 (a) 162 3.24 - 443.367 - 442.177 - 1.190

Hybrid 5C

deprotonated7 (b) 140 2.80 - 481.755 - 381.415 - 100.340

a Selected with the highest ratio θOPO /dOP.b Calculated as equation 3 just for the respective frame.c Calculated as equation 6.

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