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Binding of sulfoxaflor to Aplysia californica-AChBP: computational insights from multiscale approaches Zakaria Alamiddine, Balaji Selvam, Jerome Graton, Adèle D. Laurent, Elodie Landagaray, Jacques Lebreton, Monique Mathé-Allainmat, Steeve H. Thany, and Jean-Yves Le Questel J. Chem. Inf. Model., Just Accepted Manuscript • DOI: 10.1021/acs.jcim.9b00272 • Publication Date (Web): 30 Jul 2019 Downloaded from pubs.acs.org on July 31, 2019

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Journal of Chemical Information and Modeling

Binding of Sulfoxaflor to Aplysia californicaAChBP: Computational Insights from Multiscale Approaches Zakaria Alamiddine,a Balaji Selvam,b Jérôme Graton,*a Adèle D. Laurent, a Elodie Landagaray,c Jacques Lebreton,a Monique Mathé-Allainmat,a Steeve Thany,d and JeanYves Le Questel.* a

[a] Université de Nantes, CEISAM UMR CNRS 6230, Faculté des Sciences et des Techniques, Université de Nantes, 2 rue de la Houssinière, BP 92208, Nantes F-44322 France.

[b] University of Illinois at Urbana-Champaign, 600 S Mathews Ave Roger Adams Laboratory, Urbana, IL 61801, USA.

[c] Université d'Orléans, Institut de Chimie Organique Analytique, UMR CNRS 7311, rue de Chartres, BP 6759, 45067 Orléans Cedex 2 France.

ACS Paragon Plus Environment

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[d] Université d’Orléans, Laboratoire Biologie des Ligneux et des Grandes Cultures, USC INRA 1328. Rue de Chartres, BP 6759. 45067 Orléans Cedex 2, France.

ABSTRACT. Structural features and binding properties of sulfoxaflor (SFX) with AcAChBP, the surrogate of the insect nAChR ligand binding domain (LBD), are reported herein using various complementary molecular modeling approaches (QM, molecular docking, molecular dynamics and QM/QM’). The different SFX stereoisomers show distinct behaviors in terms of binding and interactions with Ac-AChBP. Molecular docking and Molecular Dynamics (MD) simulations highlight the specific intermolecular contacts involved in the binding of the different SFX isomers and the relative contribution of the SFX functional groups. QM/QM’ calculations provide further insights and a significant refinement of the geometric and energetic contributions of the various residues leading to a preference for the SS and RR stereoisomers. Notable differences in terms of binding


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interactions are pointed out for the four stereoisomers. The results point out the induced fit of the Ac-AChBP binding site according to the SFX stereoisomer. In this process, the water molecules mediated contacts play a key role, their energetic contribution being among the most important for the various stereoisomers. In all cases, the interaction with Trp147 is the major binding component, through CH… and … interactions. This study provides a rationale for the binding of SFX to insect nAChR, in particular with respect to the new class of sulfoximine based insect nAChR competitive modulators, and points out the requirements of various levels of theory for an accurate description of ligand-receptor interactions.

1. Introduction The resistance of important insect pests to a broad range of control agents, in particular to neonicotinoids which emerged as the leader class on the global insecticide sales (more


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than 25% in 2014), has brought chemists to develop new active compounds.1 Thus, based on the sulfoximine moiety,2 Dow AgroSciences has designed Sulfoxaflor (IsoclastTM, SFX, Figure 1), the first representative of sulfoximines on the market for commercial pest insect control. Two commercial pesticide products, CloserTM and TransformTM, that features Sulfoxaflor, have been marketed in 2013. These compounds represent a new subgroup (4C) within the IRAC classification, corresponding to insect nicotinic AcetylCholine Receptors (nAChR) competitive modulators.3 Indeed, the sulfoximine chemotype is new among insecticides and, interestingly, has been shown to interact with nAChRs in a different manner than other competitive modulators.4-6 Another major difference with neonicotinoids is the fact that SFX is characterized by two chiral centers. Therefore, four different stereoisomers can be defined (see Figure 1), clearly providing a further degree of differentiation and complexity with respect to its interaction with insect nAChRs.


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9

CH3 D1

4

3

5 7

F 3C

6

N

2

* 8

D2 10 CH3 12 S* D3

O

11

1

N 13

C 14

N 15

CH3

CH3

RR

RS

CH3

CH3

S O F 3C

S N

N

C

O N

F 3C

SR

CH3 O

N

CH3 S

S N

C

CH3

CH3

SS

F 3C

N

N

N

C

O N

F 3C

N

N

C

N

Figure 1. Chemical structure, numbering of SFX and its four stereoisomers. For better clarity, the three dihedrals angles considered (D1, D2 and D3) are also indicated.

Several studies have confirmed the high potency of SFX and its broad spectrum since it has been proven efficient on a wide range of sap-feeding insect pests including those resistant to currently available insecticides, in particular neonicotinoids.4, 7 Furthermore, excepted one instance of limited cross-resistance to SFX reported in a strain of M.

persicae possessing a mutation in a nAChR subunit, several investigations of metabolic and target site-based mechanisms of resistance showed little or no cross-resistance to SFX.7 It has been suggested that the new sulfoximine chemotype of SFX might be one of the factor limiting its susceptibility to known metabolic-based resistance mechanisms.8-


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In this context, the interest of this new chemotype has recently led to SAR

investigations.11

From a structural point of view, there is a lack of co-crystallized complexes of SFX with nAChRs or its models, such as Acetylcholine Binding Protein (AChBP), the recognized surrogate of the nAChR ligand binding domain (LBD). Therefore, to the best of our knowledge, the investigations reported so far are limited to molecular modeling studies.12, 13

Thus, using homology modeling, docking and QTAIM calculations on simplified models,

the binding of SFX to the active site of the Myzus persicae nAChR has recently been investigated.13 This study included SFX together with representative of the other chemical subclasses of group 4 (4A: neonicotinoids: imidacloprid, thiacloprid; 4B: nicotine; 4D: butenolides: flupyradifurone). It has highlighted significant differences in the binding interactions of the five compounds to the selected target, in agreement with the chemical subgroups defined in the IRAC classification. Furthermore, Sparks and coworkers have investigated through molecular modeling the binding of the four stereoisomers of SFX to wild type Mysus persicae nAChR homology models and the R81T mutant.12 In particular,


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the rank order of experimental binding affinity of neonicotinoids and SFX was correctly predicted by the molecular modeling calculations and all four stereoisomers were found to contribute to the activity of SFX.

NAChRs are pentameric ligand-gated ion channels (LGICs), the five subunits being symmetrically or pseudosymmetrically arranged around a central ion-conducting pore, forming homo- or heteropentamers of related subunits.14 15The functional organization of nAChRs, as well as their diversity in terms of subunit composition and stochiometries, is much better known in vertebrates than in insects.16 The agonist binding site of nAChRs is localized at interfacial regions between subunits and consists of several discontinuous loops (A–F), specific subunit combinations conferring differences in sensitivity to ACh and in pharmacological profiles.14 Today, a crystallographic structure of the human 42 nAChR is available17 but no corresponding information is available for insect nAChRs. Over the years, the determination of the X-ray crystallographic structures of the bacterial transmembrane proteins GLIC (Gloeobacter violaceus pentameric ligand-gated ion channel homologue) and ELIC (a bacterial homologue from Erwinia chrysan-themi),


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which are distant homologues of the nAChRs, have revealed many structural features of these membrane proteins, notably the architecture of the pore, including its gate and its selectivity filter.18 19 However, these structures do not allow a comprehensive description of the ligand–nAChRs interactions. In this context, the discovery and crystallization of ligand-free and ligand-bound structures of acetylcholine binding protein (AChBP) have allowed to gain deep insights into the details of the binding site and its relation to function.20

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In the field of insecticides, AChBP extracted from Aplysia californica (Ac-

AChBP) has been used as a plausible structural surrogate of insects nAChRs because it has been shown to be pharmacology reminiscent for the insect nAChR subtypes, that is, to present high neonicotinoid sensitivity.22 These data have provided the structural basis for the design of homology models for extracellular domains of specific nAChRs subunits combinations and the investigation of the binding of neonicotinoids on the corresponding receptor–ligand binding interfaces.23

With respect to the agonist binding site, several techniques, among which affinity labeling has a prominent place, were used24 to determine the amino acid residues involved in the


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binding (shown in Figure S1 in the supporting information). The binding site has afterwards been defined as a series of loops : A, B, C from the principal component and D, E, F from the complementary component (Figure S1 in the supporting information). In AChBP, the homologous residues mark out a compact pocket located at the interface between two subunits. aromatic amino acids:

20 21

24

The conserved residues of the AChBP binding site are the

Y94 (loop A), W147 and Y155 (loop B), Y188 and Y195 (loop

C), and the bridged cysteins from C-loop (C190 and C191) (see Figure S2). Expectedly, these residues establish contacts with nicotinic/neonicotinoid ligands in co-crystals of AChBP, for example with thiacloprid in complex with Ac-AChBP23 (3C84 pdb entry, see Figure S2 in the supporting information).

In the present study, we use a wide range of molecular modeling methodologies, from molecular docking to hybrid QM/QM’ calculations including molecular dynamic (MD) simulations, to complement the earlier works devoted to the elucidation of SFX binding and provide a detailed description of the structural features, dynamic and binding properties of SFX within Ac-AChBP. At first, Density Functional Theory (DFT) calculations


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are performed on relevant stereoisomers in the isolated state and in solution (continuum models describing dichloromethane and water) to realize conformational analyses. In a second step, the minima found through the DFT conformational analysis for each stereoisomer have been docked in the binding site of Ac-AChBP. The best poses found for the four stereoisomers have then been submitted to MD simulations on a 40 ns period. Finally, starting from the MD results, QM/QM’ calculations were performed to refine the geometrical parameters and energetics. All results were compared to THI, which presents several chemical and structural analogies with SFX and for which a X-ray structure with

Ac-AChBP, is available.23 On the whole, the results presented herein provide a detailed description and understanding of the structural and binding properties of SFX, representative of the new sulfoximine chemotype.

2. Method and computational details DFT calculations. All DFT calculations were performed using the GAUSSIAN09 program.25 We have selected the M06-2X26, 27 functional associated to the 6-311G(d) basis set, to perform our conformational investigations. Indeed, M06-2X has been proven to surpass other


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functionals for energy prediction and non-bonded interactions in the gas phase and in biological systems.28 29 30 A full geometry optimization of the four SFX stereoisomers has first been carried out and two stereoisomers (SS and SR) were considered for a systematic conformational analysis. In addition to gas phase optimizations and in order to probe the effect of the surroundings, the located minima have also been fully optimized under the influence of solvent effects (dichloromethane and water), accounted for with the Solvation Model Density (SMD).31 Further methodological details are given in the SI.

Molecular docking. The molecular docking was performed using the Glide v6.3 program of the Schrodinger suite 2014-132 using a flexible docking protocol with the SP (Standard Precision) mode. It is worth noticing that for this step, the various stereoisomers have been considered, using the low energy conformers identified through the DFT calculations in the different solvent models investigated (total of twenty three conformations). The assignment of the protonation state of the SFX molecule has been made using the LigPrep v3.0 module of the Schrodinger suite 2014-1. The protonation state of the amino acid residues of Ac-AChBP used for the docking (3C84 pdb entry) was assessed using the Protein Preparation wizard module of the Schrodinger suite 2014-1. In addition to the flexibility of the ligand, the amino acid residues included in a sphere of 6 Å radius of the binding pocket have been relaxed during the docking process. Finally, a minimization procedure was carried out to reach the best poses. It is worth reminding that AChBP are organized as pentamers,


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the five resulting identical ligand binding sites being found between the cleft of two subunits. The ligands were therefore docked in the five subunits interfaces of the AChBP models. The glide energy is the summation of total electrostatic (Coulombic) and van der Waals terms for protein– ligand interactions in a given complex whereas the docking scores are estimated as the ligands binding energies with the protein. The latter are of course approximate values and have to be interpreted with caution, but both parameters are generally considered when comparing docking results. The selection of the most favorable poses has been realized from computation of RMSDs between the theoretical structures (obtained through docking) and a reference experimental crystal structure. Since no crystal structure is available in the case of SFX, the PDB crystal data of a nitrile neonicotinoid (thiacloprid (THI), 3C84 PDB entry),23 has been considered as the reference structure for the various structural comparisons.

MD simulations. All-atom MD calculations were performed using the DESMOND v3.833 program with the CHARMM27 force field for the protein and SFX.34,

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The protein–ligand complexes were

solvated using the System Builder panel in Maestro 9.735 with TIP3P water molecules in an orthorhombic periodic box of 100˚Å × 100˚Å × 100˚Å dimension. Sodium and chloride ions were added to neutralize the system, the final model having approximately 90 000 atoms. In the first step, the MD system was energy minimized using a conjugate gradient algorithm for 2000 iterations up to a convergence value of 1.0 kcal mol-1 Å-1. In the second phase, the MD system was slowly heated to 10 K in isochore conditions (NVT) over 12 ps and later heated to 300 K in isothermal-isobaric ensemble (NPT) over 12 ps. The protein– ligand complex system was equilibrated for 5 ns and then the production runs were carried out up


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to 40 ns. The Particle-Mesh Ewald36 method was used for the calculation of the electrostatic interactions and the SHAKE algorithm37 was used to constrain the bonds. The temperature was kept constant at 300 K by a Berendsen thermostat. Relevant constraints were applied for “key” hydrogen bonds, (especially between the nitrile group of SFX and the water molecule), similarly to the procedure we used recently for THI.38 For the sake of comparison and to check the quality of the final models, the average SFX–AChBP MD structures have been compared to the corresponding relevant agonist-Ac-AChBP crystallographic (thiacloprid, 3C84) structures through the calculation of RMSDs taking into account, for the protein, the backbone atoms and for the ligands the heavy atoms. The pairwise interaction energies were calculated by selecting SFX and the respective binding site residues. The default DESMOND values were used as cutoff distances for the various interactions: hydrogen bond ≤ 2.5˚Å, aromatic ≤ 4.5˚Å and hydrophobic ≤ 3.6˚Å.

QM/QM’ calculations. Definition of the model system

In the absence of a crystallographic structure of a SFX-Ac-AChBP complex, the starting models for the QM/QM’ calculations have been selected from the lower potential energy conformations obtained from the production phase of the MD simulations for the four SFX stereoisomers, on the basis of their high flexible character. To take into account the SFX surroundings in Ac-AChBP, we have selected all the residues contained inside a sphere


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of 4 Å radius of any SFX atom. This selection includes 13 amino acids namely, Tyr55, Tyr93, Val108, Met116, Phe117, Ile118, Trp147, Val148, Tyr188, Ser189, Cys190, Cys191, and Tyr195 and three water molecules. Although outside the 4 Å sphere, we have also included the Gln57 residue, which has previously been shown to play a significant role in the binding of nAChR competitive modulators.39 The resulting model, composed on the whole of fourteen residues, is illustrated in Figure 2. The N- and Cterminal ends of cut residues were respectively capped with acetyl (H3CCO−) and Nmethyl amino (−NHCH3) groups as commonly done in quantum mechanics studies of receptor active sites.40,

41

The geometries of the capped groups were taken from the

backbone geometries of the removed residues. Finally, the defined model corresponds to the “real system” (372 atoms) investigated within the ONIOM framework.

Tyr195 Tyr188 Trp147

Cys190-191

Tyr93 Ile118 Gln57


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(a)

(b)

Figure 2. SFX-Ac-AChBP model system considered during the ONIOM calculations, (a) ONIOM-1, (b) ONIOM-2, on the example of the SS stereoisomer. The residues in the high layer are displayed in ball and sticks, the other ones in sticks. For more clarity the labels of some relevant amino acid residues are indicated in Figure 2a). The hydrogen atoms are not represented.

ONIOM calculations

High levels of theory such as wave function-based methods like CCSD(T),42-43 lead to a very accurate description of non-covalent interactions. Unfortunately, such simulations remain too computationally demanding for the number of atoms of the system studied here. DFT functionals, able to handle dispersion and stacking interactions, and to simulate precisely H-bonds, have been proven to achieve good accuracy and efficiency in large scale systems. The meta hybrid functional M06-2X, designed by Zhao and Truhlar,26 has been successfully used in the description of noncovalent interactions in biological systems.28 However, even if DFT methodologies require less computational resources than wave function-based approaches, the full DFT description of models such as the one shown in Figure 2 stays out of reach. Hybrid methodologies, able to combine different levels of theory in the same calculation, are especially appropriate to these situations. In the present contribution, two different QM methods have been combined in a hybrid QM/QM’ approach (ONIOM)44 as implemented in Gaussian 09. The M06-2X functional (QM) has been selected for the high layer, because of its excellent efficiency for the description of non-covalent interactions (see above), and the semi-empirical PM6 method45 (QM’) for the low layer, since it is recognized as very effective to simulate protein systems.46 The modeling of the SFX interactions with Ac-AChBP was studied with two ONIOM schemes. In the first, labelled ONIOM-1 hereafter, SFX and the three water molecules composed the high layer (at the M06-2X level of theory), while


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the rest of the system was inserted into the low layer (at the PM6 semi-empirical level of theory). In the second scheme (ONIOM-2 in the following), the amino acids presenting a significant pairwise energetic contribution (lower than -15 kJ mol-1) in the interaction with SFX (after calculations within the ONIOM-1 scheme) have subsequently been incorporated in the high layer in order to further refine the geometry and energy of the system. These amino acids are the following: Tyr93, Trp147, Val148, Tyr188, Ser189, Cys190-191, Tyr195 for the principal component of the Ac-AChBP binding site and Gln57, Ile118 for the complementary component. Starting from the lower potential energy conformations obtained from the production phase of the MD simulations for the four SFX stereoisomers, both ligand and lateral chains were fully optimized while the backbone atoms were frozen in space to retain the characteristic structure of the binding site.41 To ensure the minimum nature (no imaginary frequencies) of the stationary points, vibrational frequency calculations were used. The energy of the whole system is then computed as: E(ONIOM) = Ereal(PM6) + Emodel(M06-2X/6-311G(d)) - Emodel(PM6)

(1)

In equation (1), the subscript “real” means the whole system containing all atoms (high and low layers), whereas the subscript “model” notifies the system with a restricted number of atoms (high layer). The geometries of each SFX-amino acid pair in interaction were then extracted to compute the corresponding pairwise interaction energies E(i, j) according to the two-body approach (2):40 E(i, j) = E(i, j) - E(i) - E(j) +EBSSE

(2)

where E(i, j) is the energy of the SFX-residue pair, E(i) and E(j) are the energies of the isolated individual molecules, and EBSSE is the correction for the basis set superposition error (BSSE) computed through the counterpoise method (CP).47


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3. Results and discussion 3.1 Conformational features of SFX from DFT calculations To the best of our knowledge, no experimental information on the three dimensional structure of SFX is available. As specified above, the presence of the two chiral centers induces the existence of four stereoisomers: RR, RS, SS and SR. We have limited the conformational analysis to two of them, SS and SR, since the corresponding potential energy surface (PES) is symmetric to the ones of the RR and RS related enantiomers. Before carrying out the conformational analysis of D1 (C2C3-C8-S10) and D2 (C3-C8-S10-N13) dihedrals for SS and SR enantiomers (see Figure 1), we explored the position of the nitrile group with respect to the S10N13 bond. Two minima with D3 (C8-S10-N13-C14, see Figure 1) at 60 and 180° were found. For the first, the nitrile group is parallel to the pyridine ring, while for the second, the nitrile is oriented perpendicularly to the aromatic ring. We have then carried out a systematic conformational analysis around the D1 and D2 dihedrals for each of these minima. The results obtained (Tables and Figures in the SI) highlight the flexibility of SFX and the significant influence of the solvent surroundings on the conformational features. A total of twenty three low energy conformers is coming out from the conformational analysis that will be employed for the molecular docking.

3.2 Molecular Docking of SFX to Ac-AChBP


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Before presenting the molecular docking results, let us reminding the main features of SFX to emphasize its common characteristics and main differences with neonicotinoid insecticides. First, SFX, like some members of the neonicotinoid class (imidacloprid, acetamiprid, nitenpyram, thiacloprid), is composed of a pyridine heterocycle and carries a nitrile group, the new chemical scaffold introduced in SFX corresponding to a sulfoximine moiety.7 Secondly, it is worth noticing that SFX carries a methyl group on the methylene bridge linking the two fragments, at the origin of the first chiral center, and a trifluoromethyl group in place of a chlorine atom, on the pyridine ring. In the molecular docking investigations, we have studied the binding of the four stereoisomers of SFX to the Ac-AChBP model, using THI as the reference structure, which similarly carries a pyridine heterocycle and a nitrile group. For this investigation, the twenty three low energy conformers were docked to Ac-AChBP, and the binding interactions of the most favorable poses for each stereoisomer are illustrated in Figure 3. The different SFX stereoisomers exhibit a distinct behavior in terms of interactions with the Ac-AChBP binding site residues, and selected geometrical parameters are gathered in Table 1. The differences are essentially due to the orientations and interactions of the groups of atoms carried by


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the chiral centers as shown by the corresponding scores and energies in Table 2. In all isomers, the nitrogen of the pyridine ring (N1) is consistently involved in water mediated (W485) HB interactions with Ile106 (CO) and Ile118 (NH) (Figure 3). Another common feature revealed by these results is the importance of water mediated HB interactions (W463, W446) in the vicinity of the nitrile group nitrogen (N15) of SFX as already observed for THI embedded in Ac-AChBP.23, 38 These two water molecules are also involved in a network of HB interactions with the side chain hydroxyl and main chain NH groups of Ser189, the OH group of Tyr55 and the lateral chain NH of Gln57. In fact, the main differences between all stereoisomers involve, on the one hand, the orientation and contacts of the C9 and C12 methyl groups carried by the C8 and S10 chiral centers as emphasized recently by Sparks et al.12 in their modeling study and, on the other hand, the orientation of the SO moiety of the sulfoximine group. Indeed, in RS and SS stereoisomers, HB interactions between the SO group and the hydroxyl group of Tyr93 are observed (Figure 3). This is in contrast with the interactions computed in the binding site of Myzus persicae nAChR by molecular modeling12 and Quantum Theory of Atoms in Molecules (QTAIM) calculations on simplified models,13 in which no HB donor group of


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the surrounding amino acids appeared available. In fact, it is worth mentioning that, on the one hand, in their molecular modeling study, Sparks and coworkers12 mainly focused on the impact of the R81T mutation in a homology model of Myzus persicae (green peach aphid) built from AChBP on the binding of sulfoxaflor compared to neonicotinoids. No mention was made in their paper on the contribution of any tyrosine residue in the binding site, the main key components discussed being the Trp200 residue (homologous to Trp147), a water molecule in the vicinity of the pyridine nitrogen, the lateral chain of the Asn131 and, finally the lateral chain of Arg81 in the vicinity of the nitrile nitrogen. On the other hand, in their QTAIM investigation, Beck et al. simplify some of the key components of the insect nAChR by corresponding chemical groups, the contribution of aromatic amino acid side chains being simplified by a benzene ring.13 Any further comparison with the results obtained in these previous works is therefore impossible.

Table 1 and Figure 3 allow to apprehend the importance of the methyl groups (C9 and C12, see Figure 1 for the numbering). In fact, according to the stereoisomers, these groups are involved in contacts with aromatic rings of amino acid residues of the binding


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pocket. The methyl group in position 9 appears in contact with the indole ring of Trp147 (both five and six-membered rings) in RR and RS isomers. In the same vein, the C12 methyl group can interact with the Tyr188 aromatic ring in RR and SS isomers. From the selected parameters, each stereoisomer can be accommodated in the Ac-AChBP binding site. Nevertheless, the RS stereoisomer appears as the most favored, both in terms of energetic (Glide Emodel and score) and structural (RMSD) parameters (Table 2).

Table 1. Selected interatomic distances (in Å), of the different SFX stereoisomers (EA subunit interface) in Ac-AChBP.


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SFX/water molecule N1 W485 W485 N15 N15 W463 W463 W463 N15 N15 W446 W446 W446 O11 C12 C9 O11 C12 C12 C9 C9 O11 O11 C12 a

Residue W485 Ile118(N) Trp147(Nd1) Ser189(O) W463 Ser189(Oγ) Gln57(Nd1) Gln57(NH) W446 Cys190(N) Ser189(Oγ) Ser189(N) Tyr55(Oη) Tyr55(Ce) Tyr55(Ce) Tyr188Ce) Tyr188(Ce) Trp147(Ce5) Trp147(Ce6) Trp147(Ce5) Trp147(Ce6) Tyr93 Tyr55(Oη) Tyr188(Ce)

d(XH...residue) Exp 2.94 2.92 3.84 3.32 2.62 3.20 4.92 2.94 3.81 3.42 3.41 2.96 2.80 3.86 3.93 3.78 3.78 4.38 3.57

RRa 2.93 (1.94) 2.96(1.97) 3.86(3.27) 4.45(4.11) 3.39(3.25) 4.53(4.74) 5.11 2.91(1.97) 3.30(3.00) 3.41(3.10) 2.95(2.05) 2.98(1.97) 2.77(1.78) 4.25 4.32 5.05 5.46 4.39 3.65 3.76 6.69(7.58)

RSa 2.88(1.89) 2.94(1.96) 3.90(3.27) 2.88(3.53) 3.37(2.93) 2.85(1.85) 4.88 2.71(1.75) 5.26(4.54) 3.43(3.15) 3.99(3.42) 4.66(3.73) 2.77(1.9) 4.66 3.83 4.38 3.80 4.45 4.15 5.34 5.53 2.75(1.81) 3.92

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SRa 3.00(2.02) 3.00(2.01) 3.73(3.14) 4.54(4.08) 3.29(3.12) 4.98(4.98) 5.04 2.85(1.93) 3.30(2.88) 3.49(2.84) 2.81(1.88) 3.03(2.03) 2.76(1.78) 4.75 3.88 4.62 3.44 4.39 4.04 4.00 4.11 6.72(7.61)

3.89

SSa 2.88(1.88) 2.96(1.96) 3.96(3.36) 4.98(5.24) 3.30(3.22) 4.52(4.28) 5.12 2.92(1.99) 3.41(3.27) 3.36(3.24) 3.47(3.68) 2.98(1.96) 2.76(1.77) 4.09 4.28 4.16 6.31 5.24 4.11 5.19 5.24 2.75(1.81) q(2.75(1.81 4.97 )3.82

values in brackets correspond to the distances computed with respect to the hydrogen atom of the HB

donor.

Table 2. Glide Emodel and Docking score (DS) (kJ mol−1), of the different SFX stereoisomers (EA subunit interface) in Ac-AChBP. SFX/water Emodel DS RMSDa molecule RMSDb

a RMSD

RR

RS

-287.9 -37.2 -39.3 0.01 0.05 0.66 307.1 0.59

SRa (Cα) -289.1 -36.8 0.09 0.84

SSb (ligand) -284.9 -38.1 0.07 0.72

computed considering the C atoms of the binding site residues. b RMSD computed taking into

account the ligand atoms (10 pairs)


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(a)

(b)

(c)

(d)

(e)

(f)


23


(g)

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(h)

Figure 3. Predicted binding mode of SFX in the binding site of Ac-AChBP from molecular docking. For better clarity, the figure is shown according to two orientations of the ligand: one with the heterocyclic fragment (a, c, e and g) the other with the push-pull fragment (b, d, f and h) in the plane of the figure, respectively. The RR, RS, SR and SS have the purple, red, yellow and orange color respectively.

3.3 MD simulations of SFX - Ac-AChBP complexes

Significant differences in terms of the nature of the interactions and of their geometric features have been obtained through the docking of SFX stereoisomers to the model we have considered, Ac-AChBP. However, the differences in scores and energetics obtained for the various isomers are not significant and we have therefore completed this study by MD simulations,48 which provide a more realistic picture of the binding process, using a dynamic instead of a static picture of the system. Thus, the best docking pose of each


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isomer with Ac-AChBP has been used as a starting point for MD simulations over a 40 ns period, as we have done in previous studies on THI.49

Global motion

At first, we have considered the structural profile (through the Cα atoms RMSD) along the simulation time for the four SFX stereoisomers. Significantly different fluctuations are observed for the four stereoisomers, as illustrated on Figure 4. Indeed, RS and SS stereoisomers show, along the last 25 ns of the simulation time, the weakest structural fluctuations (less than 2 Å) and appear therefore, with a profile very close to that of THI, significantly more stabilized in the binding pocket than the two other ones. However, an increase of the RMSD values occurs at 35 ns (RMSD change greater than 2 Å) in the case of the SS stereoisomer. The SR stereoisomer also shows important fluctuations (higher than 2 Å) but along the whole simulation time. The last stereoisomer RR, embedded in Ac-AChBP model, has a similar profile than the SR one in terms of RMSD values. Nevertheless, during the last 10 nsthe fluctuations tend to decrease.


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Figure 4. Evolution of the C atoms RMSD values during the MD simulations for each SFX stereoisomer.

Binding site interactions

We have first analyzed the interaction energy profile over the simulation time for the four stereoisomers in complex with Ac-AChBP. Figure 5 shows that, in coherence with the structural fluctuation profiles discussed above, RS and SS stereoisomers are characterized by the strongest interaction energies along the simulation time. The profiles calculated for RR and SR are in line with the Cα RMSD fluctuations since they show significant changes of interaction energies (ranging from -180 to -70 kJ mol−1 and from 180 to -120 kJ mol−1 for RR and SR, respectively) all along the simulation time, in particular during the last 25 ns.


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Figure 5. Interaction energy plot along the simulation time of the various stereoisomers of SFX in complex with Ac-AChBP. The profile obtained for THI is also reported for the sake of comparison.

The 2D diagram depicting the frequency of interactions of the various stereisomers is represented in Figure 6. This figure first shows that, as for neonicotinoids, the pyridinic ring of SFX is involved in a water mediated interaction remarkably stable along the period of simulation, as observed during the molecular docking. This water mediated interaction reaches a value close to the maximum of interaction frequency, for the RS, SS and SR stereoisomers. Moreover, Figure 6 indicates that Trp147 appears as another major component of the binding site, with persistent in time  interactions (90 and 70%, respectively for SS and SR stereoisomers). Interestingly, Figure 6 also points out two significant differences between the various stereoisomers. The first is related to the


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interaction of the oxygen atom of the SFX sulfoximine group in the RS steroisomer with the phenolic hydroxyl of Tyr93, this interaction lasting 90% of the simulation time. The second highlights a particular behavior of the RR stereoisomer, which, according to the MD simulations, do not exhibit any long-lived interaction.

a)

b)

c)

d)

Figure 6. Diagram of the interactions of SFX bound with Ac-AChBP along the simulation time for the a) RS, b) SS, c) SR and d) RR stereoisomers.


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Binding energies The MD analysis was completed by the calculation of the contribution of the individual aminoacid residues in the interaction energy on the basis of the MD simulations results. The corresponding results are represented in Figure 7.

Figure 7. Average interaction energies of binding sites residues over the simulation time for the four stereoisomers of SFX. The various obained for thiacloprid are given in a sake of comparison.

Based on the percentage of interactions observed for the various stereoisomers along the simulation time, the SS stereoisomer appears stabilized in the binding pocket through, on the one hand, water mediated interactions, and, on the other hand, Trp147. The highly specific behavior of the RS stereoisomer is also clear from Figure 7, since the S=O…HO


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(Tyr93) H-bond comes out as the most relevant contribution of the interaction energy. The interactions involved in the binding of the RR and SR stereoisomers are similar to the ones observed in the case of neonicotinoids, with major roles of aromatic residues, in particular of Trp147, but also of Tyr188 and Tyr195. These results show that the specific orientations of the various SFX isomers lead to different

interactions.

Indeed,

for

Imidacloprid

(IMI)

and

THI

neonicotinoids,

crystallographic structures have shown that if Tyr93 is in close proximity with the ligands, it does not seem to be involved in any interaction.49 In contrast, its contribution in the binding of the SFX RS stereoisomer appears obvious, through a S=O…HO (Tyr93) HB. In line with the trends observed for other nitrile nAChR competitive modulators, water mediated interactions involving two water molecules at the vicinity of the SFX nitrile group are apparent during the simulations.23, 38 However, owing to their important fluctuations over the simulation time, they do not appear as important contributors for SFX binding.

3.4 QM/QM’ calculations of SFX - Ac-AChBP complexes


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For a more refined description of the structural and energetic parameters of the various SFX…Ac-AChBP complexes, QM/QM’ calculations were realized, starting from the most stabilized MD structure. Table 3 presents the relative total interaction energies obtained for the four stereoisomers at the best level level of theory, corresponding to ther ONIOM2 scheme.

Table 3. QM/QM’ (ONIOM2 level) relative total interaction energies (E, kJ mol−1) of the different SFX stereoisomers with the model system of the Ac-AChBP binding site. These values are calculated considering the most stabilized stereoisomer (SS) as the reference. SFX stereoisomers E

SS

RR

RS

SR

0.0

+37.7

+81.6

+92.4

The results show notable differences compared to the molecular docking and the MD simulations. Indeed, on the basis of the Glide energy and the RMSDs, the RS isomer was the preferred one (of about 20 kJ mol-1) after the docking whereas SS and RS stereoisomers led to the most negative interaction energies within the Ac-AChBP binding site through the MD simulations (from 50 to 80 kJ mol-1 compared to SR and RR ). We think that these discrepancies might find their origin in the limits of the description of the SFX…Ac-AChBP interactions through classical methodologies. Indeed, the SFX…Ac-


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AChBP complexes show several known challenging features (in terms of depiction accuracy) for classical (force field based) methodologies. More precisely, these features first concern the description of the interaction between the sulfoximine group of SFX and the phenolic OH of Tyr 55 (S=O…HO). Indeed, this interaction leads to strong interaction energies, especially responsible of the preference for the RS stereoisomer from the MD simulations. The intramolecular surroundings of the SO bond in a sulfoximine group is actually specific and the contribution of this interaction in the binding might be overestimated with classical methods. The total interaction energies computed at the QM/QM’ are in favor of this hypothesis since the two most stabilized isomers SS and RR don’t show this S=O…HO interaction. Without any experimental data allowing the characterization of the interaction, QM methods appear as an interesting alternative for a comprehensive investigation of the binding features of a sulfoximine compound like SFX. The other specific features of the SFX…Ac-AChBP that might be difficult to handle accurately through classical methods are the interactions involving  systems (of the ligand and/or the amino acid residues through …, XH… interactions) and water mediated interactions involving several water molecules.


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Since the QM/QM’ results lead unambiguously to the most accurate structural and energetic description and also for the sake of clarity, we only present and discuss in the manuscript the results obtained for the most stabilized SS and RR isomers. Of course, in order to have a complete description of the behavior of the various isomers, the results obtained for SR and RS are presented in the SI.

Influence of the protein surroundings on the SFX conformational features

Table 4 reports the three dihedral angle values of the two selected stereoisomers in the protein surroundings, and of the closest optimized structure in water (DFT calculations, see above). The same information is reported in the supporting information for the two other stereoisomers (Table S4 for SR and RS). Table 4 shows that the conformational features of the SS stereoisomer are very close in the two environments through the various calculations for the D1 and D2 dihedral angles (see Figure 1 for notation), with variations ranging between 10 and 20°. Discrepancies, ranging from 15 to 45°, become apparent comparing the results obtained for D3.


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Table 4. Representative dihedral angles (in °) of the SFX structures, predicted in a water solvated state (continuum SDM model), and within the AChBP environment through the MD simulations and ONIOM calculations.

MD ONIOM2-1 ONIOM2-2 a

Environment Water Ac-AChBP Ac-AChBP Ac-AChBP

D1 98 106 108 107

SFX SS a D2 -60 -58 -62 -65

D3 -172 141 173 164

D1 90 105 89 107

SFX RR b D2 64 71 39 39

D3 168 -177 -175 -179

conformer II’c in water. b conformer Vc in water.

The examination of the corresponding values for the RR stereoisomer points out a similar behavior, with variations for the D1 and D2 angles ranging from 1 to 25°. The same trend is predicted for the D3 angle, which remains remarkably constant, from the MD simulations to the ONIOM calculations, with differences at the maximum of about 15°. It is worth noting that this remains true for the other stereoisomers (see Table S4 in the SI), except for the SR one, which shows for the D3 dihedral angle a significant rotation, of about 70° according to the level of theory. In this case, the rearrangements occuring in the binding site concern mainly the water molecules around the nitrile nitrogen, Tyr55 and Ser189.


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Interactions of SFX with Ac-AChBP

The atomic interactions between the two selected stereoisomers of SFX and the components of the AChBP binding site are listed in Table 5, whereas the orientation of the two SS and RR isomers in the receptor surroundings is illustrated in Figure 8 (detailed views are given in the SI for the four stereoisomers).


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Table 5. Selected interatomic distances (in Å) of the ONIOM2-2 SFX-Ac-AChBP complex models for the RR and SS stereoisomers. The values in brackets correspond to the distance with respect to the hydrogen atom of the HB donor. SFX atom (or water molecule) N1 W485 W485 W485 W485 C8 C9 C9 C9 O11 O11 O11 C12 C12 C12 C12 C12 N15 N15 N15 N15 W463 W463 W446 W446 W446 W463

Residue (or water molecule) W485 Ile118(O) Ile118(N) Trp147(N1) Trp147(O) Trp147(Ce6) Tyr188(Ce) Tyr195(Ce) Tyr93(O(OH)) Tyr93(O) Tyr55 (C1) Tyr188 (C) Trp147(Ce5) Trp147(Ce6) Tyr188(Ce) Tyr93(O(OH)) W446 W446 W463 Ser189(NH) Gln57(N2H) Ser189(O) Gln57(N"H"21) Ser189(O) C8 Ser189(N) Ser189(N)

3C84

SS-ONIOM2-2

RR-ONIOM2-2

2.93 3.68 2.92 3.84

2.84(1.90) 2.75(1.80) 3.09(2.17) 2.87(1.88) 5.68 3.46(2.39) 5.45 3.60(2.62) 9.34 9.01 7.50 3.24(2.37) CH…O11 5.96 4.46 3.84 6.66 3.05(1.98) CH…O 3.88 2.88(1.97) 3.64 3.73(2.78) 2.75(1.90) OwH…O 5.28 5.30 5.62 4.71 2.84(1.93)

2.92(1.97) 3.17 3.55(2.75) 2.99(2.15) 5.02 4.71 5.51 7.59 3.15(2.33) C9H…O 4.77(5.50) 3.16(2.22) C1H…O11 5.91 7.48 6.52 3.49(2.47) 3.10(2.62) C12H…O 3.14(2.12) CH…O 3.31 2.74(1.79) 3.44(2.75) 5.95(6.91) 2.80 8.32 9.48 9.37 3.85 3.20(2.26) NH…O

2.62

3.2 2.94 3.41 5.29


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(b)

Tyr188

(a

Tyr188

)

Tyr93 Tyr93

Tyr55

Trp147 Ile118 Tyr55

(c)

(d)

Tyr188

Tyr55

Ser189

Gln57

Ser18 9

Gln57

Figure 8. Comparison of the binding interactions, calculated within the ONIOM2-2 procedure, of the RR (in purple) and SS (in orange) SFX stereoisomers in the Ac-AChBP binding site in two orientations: (a) and (b) with the pyridine ring (c) and (d) with the nitrile end of the second fragment in the plane of the figure. The carbon atom residues of the principal and complementary components of the Ac-AChBP binding site are colored in blue and green, respectively. For more clarity, only polar hydrogens are represented. The labels of relevant residues, involved in the


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binding site interactions, are specified. Detailed views for RS and SR stereoisomers are given in the SI.

With respect to the pyridine fragment, Table 5 and Figure 8 show that significant differences are noticeable for the two isomers. Hence, if the water mediated HB with the SFX pyridinic nitrogen is kept in both cases, with similar surroundings, modifications in terms of coordination of this water molecule are observed. Indeed, in the case of the SS stereoisomer, it uses its full H-bond potential, with a total of four interactions. As an Hbond acceptor, we can observe a first interaction with the main chain NH of Ile118 (dH…Ow = 2.17 Å), and a second with the NH of the indole ring of Trp147 (d(N1)H…Ow = 1.88 Å). As an H-bond donor, it is bonded to the pyridinic ring of SFX (d(Ow)H…N1 = 1.90 Å), and to the carbonyl main chain of Ile118 (d(Ow)H…O = 1.80 Å). In the case of the RR isomer, one H-bond interaction is lost. The water molecule behaves as an acceptor, with much longer distances than for the SS stereoisomer (dH…Ow = 2.75 Å with Ile118, and d(N1)H…Ow = 2.15 Å with the NH of the indole ring of Trp147) and as a donor with only the pyridinic Nsp2 nitrogen of SFX (d(Ow)H…N1 = 1.97 Å).


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The second fragment of the molecule is also subjected to significant variations in terms of interactions in the two stereoisomers. Figure 8 shows indeed notable rearrangements (rotations) of some residues (Tyr55, Tyr93, Tyr188) in order to optimize the various interactions involving this SFX moiety. The examination of the geometric parameters reported in Table 5 points out that most of the interactions are significantly shorter for the

SS stereoisomer compared to RR. Thus, it is worth noticing that for the SS stereoisomer, a CH… interaction between the C8 methylene and the centroid of the six membered ring of the Trp147 indole is visible (dH...Ce = 2.39 Å). A similar contact is apparent between the CH of the C9 methyl group and the centroid of Tyr195 (dH...Ce = 2.62 Å). Lastly, the CH of the C12 methyl group interacts with one of the water molecule in the vicinity (d(C12)H...Ow = 1.98 Å). For the RR isomer, the change of orientation of the various SFX interaction sites leads to different stabilizing contacts. In fact, this isomer appears less stabilized by the AChBP environment through aromatic residues, since only one CH… interaction is apparent between the CH of the C9 methyl group and the Tyr188 centroid (dH...Ce = 2.47 Å). As in the SS isomer, a CH…O contact is obtained between the CH of the C12 methyl group and a water molecule, albeit significantly longer (d(C12)H...Ow = 2.12


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Å). The surroundings of the sulfoximine oxygen are particularly interesting to explore. Thus, in the RR isomer, a CH…O H-bond is found between a phenolic Csp2H group of Tyr55 and the sulfoximine oxygen (d(C1)H...O11 = 2.22 Å) whereas for the SS isomer a similar H-bond is found in which the CH of Tyr188 plays the role of H-bond donor (d(C)H...O11 = 2.37 Å). In fact, the examination of the sulfoximine environment for the RS and SR isomers (see SI) shows that the oxygen atom of the sulfoximine group appears to use its H-bond potential in all isomers: the short H-bond between the phenolic hydroxyl of Tyr93 and the oxygen O11 (d(O)H...O11 = 1.72 Å) already observed through the previous levels of theory is conserved for the RS isomer whereas for the SR one, a weak CH…O11 interaction is predicted, the H-bond donor involved being the C2H group of Tyr55 (d(C2)H...O11 = 2.70 Å). As already mentioned, we will not further discuss the behavior of these two stereoisomers (RS and SR) since their corresponding interaction energies are much less favorable (81 and 92 kJ mol-1 above the preferred SS one, respectively, vide

supra). A common feature observed for the two most stabilized isomers is the role played by the water molecules in the vicinity of the nitrile group. Indeed, a remarkable HB network


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involving two water molecules is structured around the nitrile nitrogen, as observed with the nitrile containing insecticides (THI and ACE).38 However, the functional groups, and aminoacid residues engaged in these interactions, are different according to the isomer considered. Thus, a direct H-bond with the N15 nitrile nitrogen is predicted by the calculations in the two cases, but for the SS, the NH of the amide lateral chain of Gln57 is the H-bond donor whereas for RR the main chain NH of Ser189 plays this role. With respective values of 2.78 and 2.75 Å, these H-bonds appear weak. In contrast, the interactions established with the W463 water molecule with dOwH…N15 respective values of 1.97 and 1.79 Å for SS and RR isomers, are much shorter. In fact this water molecule is implied in a polarised network with the second water molecule (W446) and polar groups of aminoacids residues (e.g. OH of Ser189 and Tyr 55, Figure 8).

SFX-Ac-AChBP residues interaction energies

To get a deeper understanding of the binding features of the SS and RR SFX stereoisomers, pairwise interaction energies have been computed for each ligand-


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aminoacid (or water) pair. Owing to the particular behavior of the RS stereoisomer, which was the second preferred from the molecular docking and MD simulations, we have also included it in this discussion to give clues to interpret this result. These values, computed at the M06-2X/6-311++G(d) level of theory, are illustrated in Figure 9 (the corresponding Table is given in the SI (Table S5)).

Figure 9. Pairwise SFX…amino acid residue interaction energies computed through single point M06-2X/6-311++G(d) calculations for RR (orange), SS (blue) and RS (red) SFX stereoisomers.

Table S5 in the SI allows for a comparison of the QM/QM’ scheme on the SFX amino acids interaction energies. It can first be seen that the trends are the same at the ONIOM1


42

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level, that is to say the same order of stability for the stereoisomers is obtained, as well as in terms of ranking of the various residues contributing to the interaction. In fact, the ONIOM2 description leads to more stabilized interaction energies, the inclusion of the most relevant residues in the inner sphere leading to an optimization of the geometry and therefore in general to more favourable energetics.

In line with the trends suggested by the previous structural analysis, the interaction energies are for the most part stabilized preferently for the SS stereoisomer (-311 compared to -273 kJ mol-1 for RR), leading on the whole to a preference of about 38 kJ mol-1, the binding of the two main stereoisomers being stabilized by different residues. Remarkably, however, the main stabilization is due in both cases to the Trp147 residue, from about -65 and -61 kJ mol-1, respectively, for SS and RR isomers, in agreement with the major role played by this aminoacid in the function of nAChRs.14 Another interesting common point is that, as previously pointed out for THI38 and the above structural parameters, the water molecules appear as important contributors in the anchoring of the two fragments (pyridine ring and nitrile moiety) of SFX, with energetic contribution ranging


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from -16.7 to -30.7 kJ mol-1 for both isomers. In that respect, it is worth noting that the two water molecules in the vicinity of the nitrile nitrogen appear more tightly bound (average values of -30.0 and -24.0 kJ mol-1 for SS and RR) than the one hydrogen-bonded to the pyridine ring (values of -16.5 and -22.8 kJ mol-1 for SS and RR). This increased stability is rationalized through the characteristic network of H-bonds stabilizing the nitrile group pointed out by the structural analysis, in which the two water molecules are kept through several H-bonds involving different amino acid residues (Ser189 for RR and Ser189, Tyr55 and Tyr188 for SS) or fragments of the SFX ligand. The results are then markedly different according to the stereoisomer considered. Thus, the respective orders of energetic stabilization of the various binding site residues are respectively the following, for the SS and RR isomers (the common aminoacids have been underlined):

SS: Trp147 < W463 < W446 < Cys190 < Tyr195 < Ile118 < Tyr188 < Gln57 < W485

RR: Trp147 < Tyr188 < W446 < W463 < W485 < Ser189 < Val148 < Tyr93

Indeed, according to the stereoisomer and the corresponding orientation of the sulfoximine fragment, the SFX ligand is stabilized by various residues, among which


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“unusual” ones in the binding of neonicotinoids and related ligands are involved. Thus, for the SS isomer, the contribution of Gln57, through a direct H-bond interaction of the NH2 amide side chain with the nitrile nitrogen is noteworthy. In the same vein, for the RR isomer, the involvement of Tyr93 is unexpected. In fact, the significant stabilization brought by this residue (-16.4 kJ mol-1) is explained by CH…O hydrogen-bonds between the C9 and C12 SFX polarized CH and the phenolic OH. For both stereoisomers, the role played by aromatic residues is notable, since it represents respectively 50 and 37% of the interaction energy for RR and SS stereoisomers. It is worth noting that among the whole interaction energies, specific interactions are mainly brought by water molecules (contribution of 26 and 24% for RR and SS isomers) since only one direct H-bond involving a main chain NH (of Ser189) to the nitrile nitrogen is predicted for the RR isomer (accounting for 8% of the total interaction energy) whereas for the SS one, the NH of the Gln57 amide side chain plays this role (contributing to 7%). Lastly, it is interesting to note that the oxygen atom of the sulfoximine group of SFX appears to play its role as a H-bond acceptor since it is involved in such interactions in all stereoisomers, with, in three of them (RR, SS, SR) CH groups of aromatic residues behaving as H-bond donors, whereas in


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the last one (RS), the phenolic OH group of Tyr93 plays this role (d(O)H…O11 = 1.72 Å), this interaction amounting to 9% of the total interaction energy (see SI). From this point of view, the chemical modulation at the basis of the sulfoximine new class of nAChRs competitive modulators is therefore found to bring additional interactions with these receptors. To our knowledge, this is the first time that this property is highlighted at the atomic level despite the existence in the recent literature of structure activity relationships6, 11 or molecular modeling investigations12, 13 devoted to SFX. The case of the RS stereoisomer is worth to consider since it was among the “preferred” stereoisomers from the molecular docking and MD simlations results. Figure 9 allows rationalizing its specific behavior, compared to the SS and Tyrand RR stereoisomers. Thus, the contribution of the binding site residues are completely different, the pivotal Trp147 accounting for about 13% of the interaction energy (instead of 21-22% in the case of SS and RR). The same is true for Tyr195, accounting for 17% for the RR stereoisomer, whereas its contribution for the RS one is of 8%. In fact Figure 9 shows that in the case of the RS isomer, the main contributors of the interaction energy are the water molecules in direct contact with hydrogen bond acceptor sites of SFX, namely the nitrile nitrogen


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(W463, amounting to 18%) and the pyridinic nitrogen (W485: 16%). Despite appearing as the main contributors for this stereoisomer, it is of interest noting that their stabilizing contribution appears significantly weaker (not below -42 kJ/mol whereas for SS and RR the involvement of Trp147 amounts for -65 and -60 kJ/mol, respectively). Therefore, it appears that the specific behavior of the RS stereoisomer is due to (i) the weaker contribution of key aromatic amino acids residues (Trp147, Tyr188 and Tyr195) (ii) the fact that the main contributors to the interaction energy are indirect (water bridged) interactions.

4. Conclusion The structural and binding properties of SFX, the representative of sulfoximines, a new class of insect nAChR competitive modulators, have been investigated through a wide range of molecular modeling methods. The important flexibility of this insecticide was first established, from a comprehensive conformational analysis of SFX through M06-2X/6311G(d) calculations in the isolated state, SMD dichloromethane and water solvent


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models. Depending on the environment, from six to eleven conformers separated by surmountable energy barriers (ranging from 15 to 40 kJ mol-1) were indeed predicted. Docking studies of the four SFX stereoisomers in the binding site of Ac-AChBP show remarkable conserved features for the pyridinic fragment whereas significant differences are envisaged for the sulfoximine moiety. Thus, for RS and SR isomers, the oxygen atom of the sulfoximine group is engaged in strong H-bonds with the phenolic hydroxyl of Tyr93 whereas it is not involved in any interaction in the case of the RR and SS isomers. Among the four stereoisomers, the RS and SS ones appear as the most stabilized in the AcAChBP binding site from the docking. MD simulations allow confirming these trends, less structural fluctuations and increased interaction energies being computed for RS and SS isomers. For the first, this preference is in particular due to a persistent SO…HO (Tyr 93) H-bond along the simulation time, whereas for the second, the predilection is rationalized through interactions with the indole moiety of Trp 147. Importantly, the MD simulations delineate the key role played by H-bonds mediated through water molecules, both with the nitrogen atom of the pyridine ring and of the nitrile group. The refinement of these data through QM/QM’ calculations confirms some of the previous trends but provide a


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further structural and energetic refinement leading to a clear preference for the SS stereoisomer, followed by the RR (38 kJ mol-1 above) and the RS and SR ones (located respectively 82 and 92 kJ mol-1 beyond). The various methods used throughout the work have their own role and allow to tackle the behavior of this important new insecticide in the absence of structural data. At first, molecular docking allowed determining the best orientations of the various insecticide stereoisomers in the binding pocket. Then, MD simulations probed the stability of the corresponding complexes as a function of time and highlight the key ligand…receptor interactions for each stereoisomer. Finally, even if the previous methods have known deficiencies, the QM/QM’ methodology used in the final step, with two consecutive schemes, allowed for a refinement, a “correction” of the previous results, leading to accurate energetic and geometric parameters. The present work provides complementary pictures and a comprehensive description of the binding properties of SFX, the leading ligand of the new sulfoximine class of insecticides. SFX is found to share common features with the binding of neonicotoinoids, significant differences being pointed out, in particular with respect to the role of the


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sulfoximine moiety in the binding process. It is our opinion that the detailed pictures of SFX binding provided in this work will contribute to a better understanding of its binding to insect nAChRs. From a methodological point of view, our work points out the requirement of QM/QM’ methodologies for a proper description of weak interactions in such ligand-receptor complexes.

Supporting Information. Figures (S1 and S2), respectively, representing schematically the binding site residues of the 7 nAChR and of Ac-AChBP (from the 3C84 pdb entry) to identify the conserved residues. Methodological details for the DFT (M06-2X/6-311G(d) level) calculations. Tables and Figures for the various conformers of the SS and RS SFX stereoisomers in the various environments investigated (isolated state, dicholoromethane and water SMD models) with DFT (Figures S3 and S4). Detailed views of the QM/QM’ binding interactions for the RS and SR stereoisomers (Figure S5), together with the corresponding geometric and energetic parameters and coordinates. This material is available free of charge via the Internet at http://pubs.acs.org.


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AUTHOR INFORMATION

Corresponding Author Prof. Jean-Yves Le Questel, CEISAM UMR CNRS 6230, Faculté des Sciences et des Techniques, Université de Nantes, 2 rue de la Houssinière BP 92208, Nantes F-44322 France

E-mail: [email protected]

ORCID Jean-Yves Le Questel: 0000-0001-5307-2137 Jérome Graton: 0000-0002-1114-200X

Notes: The authors declare no competing financial interest.

ACKNOWLEDGMENT J.Y.L.Q. acknowledges the Région des Pays de la Loire for financial support in the framework of the ECRIN ‘‘Paris Scientifiques’’ Grant. This work was granted access to


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the HPC resources of [CCRT/CINES/IDRIS] under the allocation A0020805117 made by GENCI (Grand Equipement National de Calcul Intensif). The authors gratefully acknowledge the CCIPL (Centre de Calcul Intensif des Pays de la Loire) for grants of computer time.

ABBREVIATIONS nAChR, nicotinic acetylcholine receptor; AChBP: acetylcholine binding protein; SFX: sulfoxaflor; DFT, density functional theory

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Binding of Sulfoxaflor to Aplysia californica-AChBP ... - ACS Publications

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