Mechanisms Underlying Allosteric Molecular Switches of Metabotropic

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Mechanisms Underlying Allosteric Molecular Switches of Metabotropic Glutamate Receptor 5 Claudia Llinas del Torrent, Nil Casajuana-Martin, Leonardo Pardo, Gary Tresadern, and Laura Pérez-Benito J. Chem. Inf. Model., Just Accepted Manuscript • DOI: 10.1021/acs.jcim.8b00924 • Publication Date (Web): 27 Feb 2019 Downloaded from http://pubs.acs.org on February 28, 2019

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Journal of Chemical Information and Modeling

Mechanisms Underlying Allosteric Molecular Switches of Metabotropic Glutamate Receptor 5 Claudia Llinas del Torrenta , Nil Casajuana-Martina, Leonardo Pardoa , Gary Tresadernb, Laura Pérez-Benitoa,b,*

a

Laboratori de Medicina Computacional Unitat de Bioestadistica, Facultat de Medicina, Universitat Autonoma de Barcelona, 08193 Bellaterra, Spain. b Computational Chemistry, Janssen Research & Development, Janssen Pharmaceutica N. V., Turnhoutseweg 30, B-2340 Beerse, Belgium

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Abstract

The metabotropic glutamate 5 (mGlu5) receptor is a class C G protein-coupled receptor (GPCR) that is implicated in several CNS disorders making it a popular drug discovery target. Years of research have revealed allosteric mGlu5 ligands showing an unexpected complete switch in functional activity despite only small changes in their chemical structure, resulting in positive allosteric modulators (PAM) or negative allosteric modulators (NAM) for the same scaffold. Up to now the origins of this effect are not understood, causing difficulties in a drug discovery context. In this work, experimental data was gathered and analyzed alongside docking and Molecular Dynamics (MD) calculations for three sets of PAM and NAM pairs. The results consistently show the role of specific interactions formed between ligand substituents and amino acid sidechains that block or promote local movements associated with receptor activation. The work provides an explanation for how such small structural changes lead to remarkable differences in functional activity. Whilst this work can greatly help drug discovery programs avoid these switches, it also provides valuable insight into the mechanisms of

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class C GPCR allosteric activation. Furthermore, the approach shows the value of applying MD to understand functional activity in drug design programs, even for such close structural analogues.

Introduction

G protein-coupled receptors (GPCRs) are the largest class of membrane proteins in the human genome and are crucial for cell signaling.1 The metabotropic glutamate (mGlu) receptors are family C GPCRs that participate in the modulation of synaptic transmission and neuronal excitability throughout the central nervous system (CNS). They are known to form dimers and contain a characteristic bilobular extracellular agonist-binding domain. The crystal structures of the 7 transmembrane (TM) domain of mGlu1 and mGlu5 have shown that these receptors allow binding of allosteric ligands in an analogous position to the orthosteric pocket of the class A receptors.2 The discovery of allosteric ligands has enriched the ways in which the functions of GPCRs can be manipulated for potential


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therapeutic benefit.3 Allosteric modulators of mGlu receptors avoid amino-acid (glutamate-like) chemistry that can be difficult for brain penetration.4 The allosteric site is less conserved, helping the identification of selective ligands. Furthermore, allosteric modulators typically only modify receptor response in the presence of the endogenous ligand thus avoiding receptor desensitization or other unwanted-effects of agonist action. Based on their effects, allosteric modulators can be divided in three groups: positive allosteric modulators (PAMs) or negative allosteric modulators (NAMs) that either enhance or inhibit the response to glutamate respectively, and also silent allosteric modulators (SAMs) that occupy the allosteric pocket but do not modify activity yet can be of interest as competitive inhibitors of endogenous allosteric modulation.5 Interestingly for the mGlu5 receptor, a new phenomenon is emerging, whereby very subtle structural changes to the ligand result in a complete change in functional response: for instance, a PAM changing to a NAM, or vice versa.6 These so called “molecular switches” can modulate the modes of pharmacology or interfere in the subtype selectivity.


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The very recent cryo-electron microscopy structures of full length mGlu5 receptor have demonstrated the large-scale motions of dimer activation.7 Surprisingly, in the inactive state the two monomers are only in contact via their extracellular Venus fly trap (VFT) domains but they undergo massive conformational change bringing the 7TM domains 1520 Å closer together, and into contact in the active state. Given the relatively low resolution of the structures (4 Å) this exciting breakthrough does not yet offer insights into the local interactions in the 7TM. In this regard, the computational study of the 7TM of class C GPCRs benefits greatly from the mGlu1 and mGlu5 7TM crystal structures first solved in 2014.8, 9 Despite their similarity, the mGlu5 structure was co-crystallized with a NAM (mavoglurant) binding deeper compared to the NAM (FITM) bound to mGlu1, Figure 1A. Two more recent studies provided additional mGlu5 7TM X-ray crystallographic information revealing subtle differences in binding modes for different NAMs. In general, the amino acids in the binding site show very little difference between the crystal structures of mGlu5, Figure 1,10, 11 with the latest M-MPEP structure having a different conformation of only W785. The common heavy atoms, in the lower chamber and acetylene pockets, between mavoglurant and M-MPEP, only had a root mean square


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deviation (RMSD) of 0.8 Å, confirming the minor structural differences. Using these structural templates, existing mutagenesis data and structure activity relationships (SAR) could be understood. For instance, Harpsøe et al.12 merged literature mutagenesis data with docking to derive binding modes for multiple mGlu ligands. In 2018, Fu et al.13 performed a computational study of the pharmacophoric binding of several mGlu5 NAMs identifying important amino acids for binding in the allosteric site. These examples helped to confirm the binding modes of mGlu5 allosteric ligands. However, to date there is no explanation of the difference in functional activity or PAM and NAM switch effect.

Activation of class C GPCRs occurs in tens of ms.14 This cannot be simulated with current computational resources by all atom molecular dynamics (MD) simulations. Nevertheless, a lot is now known about the local changes and initiation of activation within the 7TM units of GPCRs.15,

16

Furthermore, monomeric mGlu receptors couple to G

proteins upon activation by a PAM alone,17 and PAM binding in only one allosteric site in an mGlu homodimer can achieve maximal potentiation,18 suggesting parallels with class


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A GPCRs. Synergies between class A and class B have been reported and recently19 we have also shown how µs length simulations can reveal consistent ligand-induced local effects for class C mGlu2 PAMs and NAMs.20-23 In that previous work, experimental binding data and mutagenesis was combined with MD to analyze binding modes and conformational effects. The allosteric ligands directly alter the conformational behavior of the “trigger” switch amino acids (F6433.36a.40c, N7355.47a.47c, and W7736.48a.50c) affecting the “transmission” switch (Y6473.40a.44c, L7385.50a.50c, and T7696.44a.46c) one turn below, similar to class A.24, 25 According to the mutagenesis, none of the amino acids involved in the “trigger switch” influenced glutamate receptor activity, only allosteric modulation. In contrast, as predicted by the modeling and analysis, the prospective mutation of Y6473.40a.44cA and T7696.44a.46cS prevented glutamate induced receptor activation, indicating that the “transmission switch” is crucial for activation and links the extracellular (glutamate binding site) and intracellular (G protein binding site) environments.

We are interested in the design of allosteric modulators of the mGlu5 receptor and especially understanding the origins of their functional activity.26,

27

Therefore, in this


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report we applied MD simulations to study three pairs of ligands that represent mGlu5 receptor molecular switches, Figure 1.6,

28, 29

These molecules are ideal to study for

several reasons. Firstly, they are highly similar to known crystal structure ligands therefore providing confident binding mode predictions. Secondly, the structural differences between them is minimal, allowing us to focus only on the implications of this small change. The molecules contain two aromatic centers separated by a linear spacer. The two rings occupy a lower and upper chamber in the mGlu5 allosteric pocket, Figure 1. The change in functional activity arising from such small structural differences might be considered a challenge for structure-based modeling, but analysis of our simulations reveals a consistent explanation. We have also analyzed the large body of available mutagenesis data to further support the importance of specific amino acids. Results show that stabilizing the “blocking” interaction of the ligands with S6583.39a.43c and Y6593.40a.44c as well as water network preservation are crucial for functional activity. Destabilization of these interactions induces movements associated with receptor activation.


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Figure 1. (A) View of the mGlu5 7TM allosteric binding site showing selected amino acids in light grey sticks and the upper and lower chambers in dark and light blue surfaces, respectively. Crystalized NAMs in the mGlu1 structures are shown: FITM in light pink, and M-MPEP in magenta. (B) The three sets of molecular switches studied in this work. Each set represents a pair of molecules, with a very similar structure, but which have opposite allosteric functional activity. (C) Superposition of M-MPEP (6FFI, magenta) and 5CGC (pale cyan) crystal structures revealing binding site RMSD difference of 0.21 Å. (D) Superposition of M-MPEP (6FFI, magenta) and mavoglurant (4OO9, yellow) crystal structures the binding site RMSD of 0.46 Å. The RMSD between equivalent atoms of the two ligands was just 0.80 Å.


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Methods

Computational models of mGlu5: In this work, two models (inactive and active-like states) of the 7TM domain of human mGlu5 receptor (Uniprot code P41594) were built using a combination of structural templates. The crystal structures of inactive mGlu5 receptor (Protein Data Bank (PDB) code 4OO98 and 5CGC10) were used for the inactive state. The structural differences between them are minor, all way below the typical RMSD changes observed throughout MD simulations. The difference in W785 conformation may be a concern for static docking methods but MD based approaches have been shown to sample this movement for other mGlu receptors.20-22 Due to the absence of ECL2 and the presence of the lysozyme bound to the end of TM3 in the crystal structures of the mGlu5 receptor, ECL2 and ICL2 were modelled using the crystal structure of mGlu1 receptor (PDB 4OR29). The lysozyme was removed and the length of TM3 was modelled as in the crystal structure of mGlu1. An active-like 7TM model of the mGlu5 receptor in complex with Gq was constructed. This was modeled from the crystal structure of the β2AR-Gs complex (PDB ID 3SN630). First, the conformation of the intracellular part of TM6 of mGlu5


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(N7696.32a.34c -M7786.41a.43c) was changed to be similar to the active conformation of β2AR (amino acids 6.31-6.41). For β2AR, TM6 opens at the position of W286 that is enabled by P288 above. The alignment of the bottom of TM6 up to M778 in mGlu5 assumes the double T780-T781 residues that are conserved in mGlu receptors can enable the alpha helical kink, a known feature of sequential TT.31 This short TM6 structural template opens the intracellular cavity required for the binding of the C-terminal 5 helix of the G-protein. Second, the Mus musculus Gq subunit (PDB ID 2RGN32) was modelled using the RAS domain of Gs-protein from the 2-adrenergic receptor (PDB ID 3SN6). The  subunits of Gq were taken from 2-adrenergic receptor-Gs complex. The integration of the G protein into the active model of mGlu5 was performed by superposition with the crystallized structure of β2AR. The complex was submitted to additional rounds of minimization to improve contacts at the receptor G protein interface followed by equilibration of the system. Jalview software33 was used to perform the alignment between target and template and MODELLER34 was used to build the structural models where necessary. Amino acids are numbered according to an updated Ballesteros-Weinstein format that accounts for structural differences between classes. The format is X.YYa/c where X refers


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to the 7TM helix, YY is the position of the amino acid on the helix relative to the most conserved residue assigned as 50, and a/c clarifies differences between class A and C.24, 25

Computational docking procedure: Ligands in set-1 and set-3 were superposed to the allosteric modulator (mavoglurant) co-crystalized in the original crystal structure of the mGlu5 receptor (PDB 4OO9) using the Pymol pair fitting tool. The binding poses were also compared with the subsequent mGlu5 crystal structure bound to M-MPEP (PDB code 6FFI11) and found to share high overlap. For the set-2 ligands, due to their bigger size, an induced fit docking was performed. The MOE software was used with the Triangle matcher algorithm.35 The binding poses were scored based on the London dG approach. Again, we used one of the available 7TM crystal structures to pre-align to our receptor models and help guide the docking pose selection. In this case the mGlu5 crystal structure (PDB code 5CGC) was used. All protein and ligand preparation for the docking was performed using the appropriate MOE tools and AMBER forcefield.


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System preparation: Regarding protein preparation for MD calculations, hydrogens were added, and ionization of amino acids and correct rotamer conformations were considered. N- and C-termini were capped. Special attention was paid to cysteine bridges to ensure they were bonded as necessary in the simulation input files. For the previously required preprocessing of the molecules, AMBER software was used.36 Charge parametrization of the ligands was carried out with Gaussian v0937 obtained using HF/631G*-derived RESP atomic charges.38

Molecular dynamics: Molecular dynamics simulations were performed using GROMACS v2016.4.39 The complexes of NAM (1, 3, 5) with the inactive conformation of the mGlu5 receptor and of PAM (2, 4, 6) with the active-like conformation of the mGlu5 receptor in complex with Gq were embedded in a pre-equilibrated box (9x9x9 or 10x10x19 nm respectively) containing a lipid bilayer (205 or 297 POPC molecules), with explicit solvent (~14000 or ~47000 waters) and 0.15 M concentration of Na+ and Cl- calculated based on the volume of the box (~140 or ~490 ions). Each system was energy minimized


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and subjected to a 6 step MD equilibration (10+5+2+2+2+2 ns). In the first step the whole system was fixed except hydrogen atoms; in the second step, the protein loops were released from restraints; and in the final four steps the restraints on the ligand and alpha carbon atoms of the protein were relaxed from 100, 50, 25 to 10 kJ.mol-1nm-2, respectively. For comparison purposes, the same approach was used to simulate the apo mGlu5 active state with G protein and the mGlu5 inactive state bound with a PAM. The AMBER99SD-ILDN40 force field was used for the protein and the parameters described by Berger et al. for lipids.41 Three replicas of 1μs of each receptor-ligand or apo complex were run.

Results Binding modes of NAMs and PAMs in the mGlu5 receptor Figure 2 shows detailed views of NAMs (in red) and PAMs (in green) of sets 1-3. Compounds 1 and 2 were docked into the mGlu5 receptor inactive and “active-like” states respectively, using mavoglurant as a reference compound (Figure 2A). The ligands were


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oriented in the binding site with the pyrimidine ring pointing towards the extracellular part of the receptor in the upper chamber and the methyl phenyl group accommodated in the deep sub-pocket of the allosteric cavity, lower chamber. The final poses showed a clear overlap of the phenylacetylene groups with the crystallized ligand mavoglurant. This docking was further confirmed with the recent release of the M-MPEP bound to mGlu5 receptor (PDB ID 6FFI11), Figure 2. The pyrimidine ring of the set-1 ligands was modelled in the lower chamber, interacting with W7856.48a.50c (as M-MPEP) and surrounded by P6553.36a.40c, S6543.35a.39c and A8107.36a.40c. The other side of the set-1 ligands is located in the upper chamber interacting with amino acids such as F7886.51a.53c M8027.38a.32c and V8067.42a.36c. The different position of the methyl group in 1 and 2, leads to different interactions. The meta substituted methyl of NAM 1 is placed between Y6593.40a.44c and S6583.39a.43c located in TM3 making a “blocking” interaction between these two amino acids, whereas in PAM 2 the para methyl points towards TM2 making an interaction with I6252.50a.46c. The docking of the second set of ligands, which includes molecules 3 and 4, was carried out as described in the method section. The upper ring of set-2 ligands interacts with


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amino acids of the upper chamber such as F7886.51a.53c and W7856.48a.50c. The methoxy group of NAM 3 attached to the lower ring binds in the lower chamber, interacting with Y6593.40a.44c and S6583.39a.43c. The analogous fluorine atom of PAM 4, attached to the lower ring, could adopt two conformations, either pointing to TM2 and interacting with I6252.50a.46c and G6242.49a.45c, or pointing towards TM3 interacting with Y6593.40a.44c and S6583.39a.43c as the methoxy in NAM 3, Figure 2B. Both compounds in set-3 have the same meta methyl substituted phenyl group. However, the pyrimidine ring was modified in the 2 position with an ethoxy group resulting in NAM 5 or with a 2-aminomethyl group resulting in PAM 6. NAM 5 is suggested to bind in a similar manner as its NAM analogue 1, placing the meta methyl substituent between Y6593.40a.44c and S6583.39a.43c (lower chamber) and with the ethoxy group of the pyrimidine in the upper chamber interacting with F7886.51a.53c and W7856.48a.50c. In contrast, compound 6 presented two different possible binding modes, with the NHMe group placed in the lower chamber oriented towards I6252.50a.46c and G6242.49a.45c or with the molecule entirely flipped and the NHMe group located in the upper chamber.


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Figure 2. Binding mode of mGlu5 allosteric modulators. Detailed view of the binding modes for the compounds presented. (A) NAM 1 (red) and PAM 2 (green), inset to the right shows the overlay of both with crystal structure ligands M-MPEP (magenta) and mavoglurant (yellow). (B) NAM 3 (red) and PAM 4 (green): PAM 4 can adopt two possible binding modes with the fluorine towards TM2 (dark green) or down towards TM3 (light green). (C) NAM 5 (red) and PAM 6 (green): PAM 6 can adopt two possible flipped binding modes, the NHMe in the lower (dark green) or upper (light green) chambers.

Addressing molecular switches of mGlu5 ligands by molecular dynamics simulations NAMs 1, 3 and 5 in complex with the inactive model of mGlu5 and PAMs 2, 4 and 6 in complex with the active-like state of mGlu5, were submitted for MD simulations (see


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methods). These unbiased MD simulations were used to study how the different position of the substituent groups affected the movement of key residues in the allosteric site of the mGlu5 receptor. Analysis of the simulations of the set-1 compounds NAM 1 and PAM 2 showed that the position of the methyl group in the lower chamber directly influences the conformation of key side chains. The methyl group in meta position in NAM 1 is located between S6583.39a.43c and Y6593.40a.44c, blocking any putative conformational change of their side chain. Thus, the side chains of S6583.39a.43c, Y6593.40a.44c, and T7816.44a.46c are highly stabilized in the inactive-like conformations and remain fixed during the simulation, Figure 3A. In contrast, the para methyl group in PAM 2 does not make this “blocking” interaction (Figure 3B) thus, the side chain of S6583.39a.43c is more flexible. Analysis of the trajectory reveals subsequent change in the dihedral angle of T7816.44a.46c (from gauche+ to

gauche-) and large movement in its spatial position, something not observed for the NAM 1. The positional change of the three amino acids seen in Figure 3 are clearly different in the PAM and NAM simulations and this observation was consistent in all three replicas of


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1μs unbiased MD simulation, see Figure S1. Inspection of other amino acids in the binding site did not reveal differences between PAM and NAMs, see Figure S1. The ligands were highly stable in the selected docking poses throughout the MD simulations as shown by their low RMSD in plots also provided in supporting information, Figure S2. For set-1 therefore, the difference between NAM and PAM is the stabilizing effect between residues S6583.39a.43c and Y6593.40a.44c and the different secondary effect on T7816.44a.46c. These amino acids were previously shown to be important for mGlu2 receptor allosteric activity21, but also mGlu5 receptor mutations S658C (functional and binding assays), Y659A (functional assay), Y659V (functional and binding assays), T781A (functional and binding assays) were shown to reduce or eliminate the activity of MPEP,28,

42, 43

Table S1 in supporting information. A conformational change of

T7816.44a.46c from gauche+ to gauche- is known to induce the decisive bend in the TM6 helix required for receptor activation.21 In addition, the S658C mutation abolishes the activity of PAMs in both functional and radioligand binding assays.44 The side chain of Ser can adopt either the gauche+, gauche- or trans conformation, whereas Cys is restricted to gauche+ or trans conformation because of the steric clash between the S


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atom and the backbone carbonyl in gauche-. The significant change in activity in the S658C mutation points to the gauche- conformation of Ser as the active conformation (not possible in Cys). Therefore, this experimental evidence adds weight to the importance of these amino acids in the binding and functional activity of allosteric ligands 1 and 2. Thus, a simple chemical change of methyl from meta to para is sufficient to make a large difference to the receptor side chain flexibility in this region and therefore in the functional activity of these two ligands.


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Figure 3. The movement of S6583.39a.43c Y6593.40a.44c and T7816.44a.46c in the presence of NAM 1 (A) or PAM 2 (B). The top two figures show the position of the oxygen sidechain atoms of S6583.39a.43c Y6593.40a.44c and T7816.44a.46c at even snapshots during the MD simulation of the receptor in the presence of NAM and PAM respectively. The bottom panels show the χ1 dihedral angle throughout the same simulation. One replica is displayed for better visualization although additional replicas showed consistent behavior, see Figure S1.


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The selected docking poses of set-2 ligands were also stable during the 1µs MD simulations (three replicas) as shown by the relatively low movement in RMSD plots, Figure S3. NAM 3 positions its meta methoxy substituent in the lower chamber at the same position as the methyl substituent of NAM 1. Thus, the methoxy group of NAM 3 stabilizes the side chains of Ser6583.39a.43c, Tyr6593.40a.44c and Thr7816.44a.46c in the “inactive”-like conformations (Figure 4A). It prevents substantial movement of either the position or dihedral angle of these amino acid resulting in a fixed gauche+ orientation of S6583.39a.43c during the simulations. PAM 4 could adopt two binding modes that differed slightly in the position of the fluorine substituent in the lower chamber. Analysis of the trajectories of PAM 4 binding with the fluorine oriented towards TM2, Figure 4B, shows increased movement of S6583.39a.43c and T7816.44a.46c similar to the movements seen in PAM 2, Figure 3B. Analysis of the rotamers of S6583.39a.43c and T7816.44a.46c of PAM 4 with the fluorine group oriented down towards TM3 (Figure 4C) showed a NAM behavior. Thus, it seems that the PAM effect of 4 is achieved via the binding mode with the fluorine pointing towards TM2.


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Indeed, the rotameric states of T7816.44a.46c show a clear difference in the presence of PAM and NAM. For this set of compounds, the role of S6583.39a.43c in functional activity is again supported by experimental mutation of S658C that abolished the functional effect of PAM 4.44 The PAM 4 mechanism of promoting activation via S6583.39a.43c and T7816.44a.46c side chain rotations is analogous to the direct TM3-TM6 activation hypothesis of the class A GPCRs.45 In summary for set-2, NAM 3 stabilizes the position of S6583.39a.43c and Y6593.40a.44c. For PAM 4 the S6583.39a.43c moves, leading to Y6593.40a.44c adopting various conformations, also affecting the conformation of the functionally important T7816.44a.46c.all at the transmission switch level, one turn above the transmission switch amino acids.


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Figure 4. The movement of S6543.39a.43c S6583.39a.43c Y6593.40a.44c and T7816.44a.46c in the presence of NAM 3 (A) or PAM 4 (B and C). Rotamers, PAM 4 is shown in two possible binding modes, with the fluorine group pointing towards TM2 (panel B) or pointing between S6583.39a.43c and Y6593.40a.44c (panel C). The top panels show the position of the oxygen sidechain atoms of S6583.39a.43c Y6593.40a.44c and T7816.44a.46c at even snapshots during the MD simulation of the receptor in the presence of NAM and PAM respectively. The bottom panels show the χ1 dihedral angle throughout the same simulation as shown above. One replica is displayed for better visualization, additional replicas showed consistent behavior, see Figure S4.

The docking pose of NAM 5 and PAM 6 bound to mGlu5 receptor were stable throughout the simulations as shown by the RMSD plots, see Figure S5. The results for NAM 5 were


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very similar to those obtained for NAMs 1 and 3. In this sense, NAMs 1, 3 and 5 all contain a phenyl meta substituent in the lower chamber that is positioned in a small cavity between S6583.39a.43c and Y6593.40a.44c, (Figures 3A, 4A, 5A) thus stabilizing their sidechains as well as T7816.44a.46c in the inactive like conformations. PAM 6 was evaluated in two possible binding poses (see above). In the pose in which the meta methylphenyl occupies the lower chamber (like NAM 5) no significant change in the conformation of Ser6583.39a.43c, Tyr6593.40a.44c, and Thr7816.44a.46c can be observed (Figure 5B), therefore not explaining its PAM activity. However, in the alternative binding mode in which the para NHMe is located in the lower chamber significant changes are observed. Again, PAM 6 bound in this conformation promoted changes in the side chain conformation of T7816.44a.46c from gauche+ to gauche-. Moreover, the methyl substituent of the upper phenyl ring positions itself tightly between F7886.51a.53c and W7856.48a.50c altering their conformational behavior and leading to a knock-on effect on T7816.44a.46c below, see Figure S6. This is seen from the comparative analysis of dihedral angles in Figure 5. The simulations did not reveal any movement of these sidechains in the presence of NAM whereas the effects were consistent in three replicas with the PAM.


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Experimental mutagenesis performed in both radioligand binding and functional assays showed that F788A, W785A and T781A have all affected activity of MPEP although there is no data for exact compounds 5 and 6.28, 42, 46

Figure 5. The movement of S6583.39a.43c Y6593.40a.44c and T7816.44a.46c in the presence of NAM 5 (A) or PAM 6 (B and C). Red dots represent the 𝛘𝟏 angle of the side chain of the residue when NAM 5 is bound to mGlu5, light green corresponds to the 𝛘𝟏 angle of the residue when PAM 6 is bound to the receptor with the methylphenyl group oriented deeper in the pocket and dark green corresponds to the 𝛘𝟏 angle when PAM 6 is bound with the pyrimidine ring deeper in the cavity. In each case only one replica is displayed for better visualization, replicas showed consistent behavior, see Figure S6.


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These simulations have shown a difference in conformational behavior of side chains in the transmission switch when performed with NAMs or PAMs. To check the possibility that this was due to the alternative starting, active or inactive states, or the role of the G protein, additional simulations were performed. Three replicate simulations of the apo mGlu5 active state bound to the G protein shows no rotameric movement from gauche+ of T7816.44a.46c, Figure S7. Likewise, repeat simulations of the mGlu5 inactive state with a PAM also showed no movement of the same amino acid. Thus, the effects seen in previous simulations in the presence of PAMs are not a function of only the ligand, nor only a consequence of the active-state model, but the result of the combination of PAM and active-state receptor.

The water-mediated interaction between Y6593.40a.44c and T7816.44a.46c The role of water molecules has been shown to be important for activity of GPCR 7TM binding ligands.47, 48 In the case of mGlu5, several X-ray structures contain crystallized waters.8, 10, 11 One of these water molecules occurs directly at the level of the transmission switch, bridging the sidechains of Y6593.40a.44c and T7816.44a.46c. This has led to a


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hypothesis that this water molecule has a functional role. Figure 6 shows the percentage of presence of this water-mediated interaction between Y6593.40a.44c and T7816.44a.46c in the three MD replicas of mGlu5 bound to NAMs 1, 3 and 5 and PAMs 2, 4 and 6. A water bridge is defined when a water molecule simultaneously forms two hydrogen bonds (distance < 3 Å and angle > 120º) with the OH group of Y6593.40a.44c and the OH group of T7816.44a.46c, as calculated with the python module MDAnalysis.49 Clearly, NAMs (mean: 88.2%, standard error: 8.2%) maintain the water bridge in the transmission switch more frequent than PAMs (mean: 28.8%, standard error: 8.1%) (Mann Whitney test, two tailed: U=5, n1=n2=9, p=0.001). In our previous study of the functional effect of mGlu2 receptor allosteric modulators we used a homology model and therefore there was no experimental evidence of exact water positions for mGlu2.21 Nevertheless, the computational approach included the crystallographic waters from the mGlu5 X-ray structure into the mGlu2 simulations. The fact that NAM Ro-4491533 (93% for a single simulation) also maintains the water bridge more frequent than the PAM JNJ-46281222 (60% for a single simulation) offers additional support for the hypothesis that NAMs


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maintain the stable inactive-like conformation via this water molecule that is part of the transmission switch interaction network.

Figure 6. Analysis of the presence of a water bridge between Y6593.40a.44c and T7816.44a.46c. (A) Heat-map showing the presence of a water bridge between the OH group of Y6593.40a.44c and the OH group of T7816.44a.46c in the three MD replicas of mGlu5 bound to NAMs 1, 3 and 5 (n1=9) and PAMs 2, 4 and 6 (n2=9). Error bars represent standard errors (n1=n2=9). (B) Representation of NAM inactive-like configuration and PAM active-like. Spheres represent position of T7816.44a.46c sidechain oxygen during MD simulation.

Discussion and conclusions


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For class A GPCRs huge breakthroughs in crystallography revealed that specific amino acids are involved in a common mechanism of receptor activation. Amino acids located at positions 3.40a, 5.50a and 6.44a were described to be involved in the necessary rearrangements for receptor activation. This cluster of amino acids is known as the “transmission switch”.15,

21

However, receptor activation has also been described to

happen via only TM3 and TM6 movements, in brief the ligand induces TM3 rotation and then TM6 opening.45 Previous work for the mGlu2 receptor has shown how a similar mechanism can be important for allosteric modulators of this class C receptor.21 Amino acids also in TM3, TM5 and TM6: F6433.36a.40c, N7355.47a.47c and W7736.48a.50c form part of a “trigger switch”. They are in direct contact with the allosteric modulator and their movements couple to the “transmission switch” amino acids (Y6473.40a.44c, L7385.50a.50c and T7696.44a.46c) one level below and induce alternative conformational behavior. In the mGlu5 case the allosteric ligands access one turn deeper and interact directly with the transmission switch.


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The ligand-mGlu5 receptor MD simulations show a consistent difference in behavior of the amino acids located at the level of the transmission switch involving S6583.39a.43c Y6593.40a.44c and T7816.44a.46c in the presence of NAM or PAM. In general, NAMs present a methyl group (the meta methylphenyl of 1 and 5 or the meta methoxyphenyl of 3) that is positioned in a small cavity between S6583.39a.43c and Y6593.40a.44c. This interaction locks the side chains of S6583.39a.43c, Y6593.40a.44c and T7816.44a.46c in an “inactive”-like conformation, preventing the movement of amino acids that can be relevant to initiate TM6 activation. This interaction is seen in the experimental X-ray structures of mGlu5 NAMs, Figure 7 panel A. Also crucial to the stable “inactive”-like conformation is the crystallographic water that is significantly more present in NAM simulations stabilizing the transmission switch further. In contrast, PAMs do not block or prevent the movement of S6583.39a.43c and Y6593.40a.44c, nor in turn T7816.44a.46c, allowing these side chains to adopt the proposed “active”-like gauche- conformation. In this gauche- conformation the side chains of Ser and Thr are capable to hydrogen bond the backbone carbonyl in the previous turn of the helix, which is known to induce flexibility in the helix that could initiate the opening


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of TM6.31 These results are consistent for all the replica simulations of the ligands studied and in accordance with previous studies of allosteric mechanism of mGlu2 receptor.

Figure 7. The blocking interaction of NAMs. (A) The meta substituent on the aromatic ring in the lower chamber fills a small cavity between S6583.39a.43c and Y6593.40a.44c. This prevents the movement of their sidechains and they remain in their stable inactive like conformations. (B) SAR supports a small substituent in the cavity between S6583.39a.43c and Y6593.40a.44c.


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As mentioned in the results, the hypothesis is supported by mutagenesis. Briefly, S658C for instance was an important mutant for the NAM functional and binding activity of MPEP (analog of NAM 1), this mutation also abolished the PAM effect of PAM 4, TableS1 in supporting information. Furthermore, the mutation of Y659V, affected the NAM functional and binding activity for MPEP. Experimental SAR also corroborates the conclusions. The direct analogue of NAM 1, where the Me is replaced by a Chloro is also a potent NAM (34 nM compared to 7 nM for 1). However, the activity drops as this group increases in size, such as trifluoromethyl (450 nM).50 Searching ChEMBL and SureChEMBL51 for published or patented pyrimidoacetylphenyls returned over 200 molecules active (IC50 < 10 µM) versus the mGlu5 receptor. Interestingly, none of these have a substituent with two or more heavy atoms at the meta position of the phenyl ring (except the CF3 example above). This is despite many examples (>1400) being reported in other patents or publications suggesting these were synthesizable but inactive analogues as mGlu5 allosteric ligands. Furthermore, we also extracted 123 phenylacetylene containing NAMs with high reported activity, IC50

Mechanisms Underlying Allosteric Molecular Switches of Metabotropic

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