Insights into the Structure and Pharmacology of the hTAAR1

Insights into the Structure and Pharmacology of the Human Trace Amine-Associated Receptor 1 (hTAAR1): Homology Modelling and Docking Studies

Chem Biol Drug Des 2013; 81: 509–516

Research Article

Insights into the Structure and Pharmacology of the Human Trace Amine-Associated Receptor 1 (hTAAR1): Homology Modelling and Docking Studies

Elena Cichero1, Stefano Espinoza2, Raul R. Gainetdinov2, Livio Brasili3 and Paola Fossa1,*

1Dipartimento di Scienze Farmaceutiche, Universita` degli Studi di Genova, Viale Benedetto XV n. 3, 16132, Genova 2Department of Neuroscience and Brain Technologies, Istituto Italiano di Tecnologia, Genova, Italy 3Dipartimento di Scienze Farmaceutiche, Universita` degli Studi di Modena e Reggio Emilia, Via Campi 183, 41100 Modena, Italy *Corresponding author: Paola Fossa, fossap@unige.it

Trace amine-associated receptor 1 (TAAR1) is a G pro- tein–coupled receptor that belongs to the family of TAAR receptors and responds to a class of com- pounds called trace amines, such as b-phenylethyl- amine (b-PEA) and 3-iodothyronamine (T

1

Trace amines (TAs), such as b-phenylethylamine (b-PEA), tyramine, 3-iodothyronamine (T

1

AM), octopamine, trypt- amine and synephrine, are found at low levels in multiple tissues in the periphery and brain of mammals, but their physiological functions remain enigmatic (1). A recent dis- covery of a family of rhodopsin-like G protein–coupled receptors (GPCRs), defined as Trace amine-associated receptors (TAARs), has provided an opportunity to explore the roles of TAs and their receptors in physiology and dis- ease (2,3). The human TAAR family consists of six genes and three pseudogenes and characterized by location on a single chromosome, high overall sequence homology to monoamine receptors and the presence of a TAAR-spe- cific peptide fingerprint motif with the seventh transmem- brane domain that is not found in all other known GPCRs. It is believed that the TAAR family most likely evolved from

AM). The receptor is known to have a very rich pharmacology

a common ancestor gene sharing closest similarity to the human gene encoding serotonin 5-HT

4 and could be also activated by other classes of com- pounds, including adrenergic and serotonergic ligands. It is expected that targeting TAAR1 could provide a novel pharmacological approach to correct monoamin- ergic dysfunctions found in several brain disorders, such as schizophrenia, depression, attention deficit hyperactivity disorder and Parkinson’s disease. Only recently, the first selective TAAR1 agonist RO5166017 has been identified. To explore the molecular mecha- nisms of protein–agonist interaction and speed up the identification of new chemical entities acting on this biomolecular target, we derived a homology model for the hTAAR1. The putative protein-binding site has been explored by comparing the hTAAR1 model with the b

2

receptor via a ser- ies of gene duplication events (4).

The most studied trace amine-associated receptor 1 (TAAR1) signals via the G

s

protein ⁄adenylyl cyclase system and could be activated not only by TAs but also by amphetamine derivatives, monoamine metabolites, iodo- thyronamines, ergolines as well as certain adrenergic and serotonergic drugs (3,5–11). Until recently, the lack of selective ligands has rendered a challenging task, the exploration of TAAR1 biological functions. Only in 2010– 2011, Hoener and co-workers have reported the identifica- tion of first selective TAAR1 ligands (12,13) with selective TAAR1 agonist RO5166017 being much more potent than -adrenoreceptor binding site, available by X-ray

the trace amine b-PEA (Fig. 1; 13). TAAR1 is expressed in crystallization studies, and with the homology mod- elled 5HT

1A

receptor. The obtained results, in tandem with docking studies performed with RO5166017, b- PEA and T

1

AM, provided an opportunity to reasonably identify the hTAAR1 key residues involved in ligand recognition and thus define important starting points to design new agonists.

several brain regions, including areas containing monoam- inergic nuclei and limbic regions. Accumulating evidence indicates that TAAR1 is involved in the modulation of dopaminergic and serotonergic systems, making this receptor as a promising novel target for drug discovery to manage monoaminergic disorders such as schizophrenia, depression, attention deficit hyperactivity disorders (ADHD)

Key words: GPCR, homology modelling, molecular docking,

and Parkinson’s disease (4,6,14–19).

TAAR1, trace amine-associated receptor

Up to now, experimental data highlighting the hTAAR1 key

Received 4 April 2012, revised 18 June 2012 and accepted for publication 30 July 2012

residues responsible for ligand recognition were not avail- able. The aim of this study was to perform an ’in silico’ investigation focused to explore which different amino acid

a 2012 John Wiley & Sons A/S. doi: 10.1111/cbdd.12018 509

Cichero et al.

Figure 1: Chemical structure of hTAAR1 agonists RO516607, b-PEA and T

1

residues can be involved in the binding of hTAAR1 agon- ists, so as to have a useful tool for the virtual identification of new chemical entities acting on this novel macromolec- ular target. In particular, we started our study from the fol- lowing preliminary considerations: (i) several serotonergic and adrenergic ligands also act as hTAAR1 agonists, (ii) X- ray data about the human b

2

-adrenoreceptor in complex with an agonist compound are available, (iii) site-directed mutagenesis data highlighting the 5HT

1A

key residues involved in ligand recognition are known.

Thus, by taking into account the correspondences between the derived homology model and the b

2

-adreno- receptor binding site (20), and also by performing compari- son between the hTAAR1 and the previously defined 5HT

1A

model (21,22), we identified the putative hTAAR1 binding site. Furthermore, docking studies with the known agonists RO5166017, b-PEA and T

1

AM allowed us to evaluate the reliability of the new model and also to high- light some interesting starting points for expediting the development of new selective TAAR1 agonists.

Methods and Materials

Ligand preparation RO5166017, b-PEA and T

1

AM were built, parameterized (Gasteiger-Hu ̈ckel method) and energy minimized within MOE using MMFF94 force field (the root mean square gra- dient has been set to 0.00001; 20).

Human TAAR1 homology modelling As most of the key residues characteristic of GPCRs are conserved in TAAR1 receptor, a hTAAR1 receptor homol- ogy model has been generated, starting from the X-ray structure of resolution = 510 Chem Biol Drug Des 2013; 81: 509–516

human 3.50 A

̊

b

2 -adrenoreceptor ), in complex (PDB with an code: 3PDS; agonist com- pound (20). The amino acid sequence of hTAAR1 (Q96RJ0) was retrieved from the SWISSPROT database (23), while the three-dimensional structure co-ordinates file of the GPCR template was obtained from the Protein Data Bank (24).

The amino acid sequences of hTAAR1 TM helices were aligned with the corresponding residues of 3PDS, on the

softwarea). basis of the Blosum62 matrix (

MOE

The con- necting loops were constructed by the loop search method implemented in MOE. The MOE output file included a series of ten hTAAR1 models that were inde- pendently built on the basis of a Boltzmann-weighted ran- domized procedure (25), combined with specialized logic for the handling of sequence insertions and deletions (26). Among the derived models, there were no significant main chain deviations. The model with the best packing quality function was selected for full energy minimization. The retained structure was minimized with MOE using the AMBER94 force field (27). The energy minimization was carried out by the 1000 steps of steepest descent fol- lowed by conjugate gradient minimization until the rms gradient of the potential energy was <0.1 kcal⁄mol⁄A ̊

.

The assessment of the final obtained model was per- formed using Ramachandran plots, generated within MOE and by the evaluation of an appropriate distribution of the hydrophobic and hydrophilic properties on the protein sur- face (Connolly surface, calculated with MOE).

hTAAR1 agonist-binding site and molecular docking studies Starting from the observation that several serotonergic and adrenergic ligands also act as TAAR1 agonists, the puta- tive receptor model binding site was identified on the basis of the corresponding regions of the 3PDS binding site, and also by highlighting the most interesting TAAR1 resi- dues which could be involved in the ligand recognition by a qualitative comparison with the 5HT

1A

model previously built by us (21,22). Successively, RO5166017, b-PEA and T

1

AM were docked into the hTAAR1 putative binding site, by means of the Surflex docking module implemented in S

YBYL

-X 1.0.b Then, for the three compounds, the best docking geometry (selected on the basis of the Surflex scoring functions) were refined by ligand–receptor complex energy minimization (CHARMM27), by means of the

MOE software.

To better refine the hTAAR1⁄RO5166017, the hTAAR1⁄ b-PEA and the TAAR1⁄T

1

AM complexes, a rotamer exploration of all side chains involved in the agonist bind- ing was carried out. The rotamer exploration methodology was implemented in the MOE suite.

AM.

]Structure and Pharmacology of the Human Trace Amine-Associated Receptor 1

Results and Discussion

Human TAAR1 homology modelling As shown in Fig. 2, the protein model was derived by the alignment of the hTAAR1 (Q96RJ0) FASTA sequence on the X-ray co-ordinates of human b

2

adrenoreceptor (3PDS), on the basis of the Blosum62 matrix (

MOE

soft- ware). The reliability of the alignment was verified by the high value of the pairwise percentage residue identity (PPRI = 30.5%).

Accordingly, a consistent number of hTAAR1 residues resulted to be present in the b

2

adrenoreceptor: (i) M30, L32, I33, L35, G40, N41, V44, I45 and I48 residues in TM1 (helix region: D21–I48), (ii) T58, N59, I62, S64, A66, D69, G73, V76 and P78 in TM2 (helix region: P57–L75), (iii) C96, T100, S101, D103, A109, S110, I111, L114, I117, D120, R121, Y122 (the DRY motif; 120–122 resi- dues) and A124 in TM3 (helix region: E93–Y123), (iv) the V142, I144, W148, V150, F156 and I159 residues in TM4 (helix region. I137–I159), (v) S198, F199, Y200, P201, I205, M206, V209, Y210, R212, A217, K218 and Q220 in TM5 (helix region: K188–K218), (vi) I255, M257, G258, F260, C263, W264, P266 (the CWXP motif; 263– 266 residues wherein hTAAR1 X: C265), F267, F268 and I269 in TM6 (helix region: E246–L277), (vii) L289, W291, G293, Y294, N296, S297, F299, N300, P301 and Y304 (the NPXZY sequence; 300–304 residues wherein hTAAR1 X: M302, hTAAR1 Z: V303) in TM7 (helix region: P283–L319).

The derived hTAAR1 model was superimposed to the co- ordinates of the human b

2

adrenoreceptor (Fig. 3), used as template for the homology modelling calculations, dis- playing a quite positive root mean square deviation value (RMSD = 1.073 A ̊

, calculated on the backbone carbon atom alignment).

Figure 2: Sequence alignment of the human TAAR1 receptor on the basis of the human b

2

Chem Biol Drug Des 2013; 81: 509–516 511

Structural analysis of the human TAAR1 model The final model backbone conformation was inspected by Ramachandran plot, showing the presence of two outliers in the general psi–phi plot (C178, and S242) and another in the glycine psi–phi plot report, G180 (see Fig. 4), which were reasonably far from the ligand binding site. In fact, C178 and G180 belong to the extracellular loop between the TM4 and TM5 regions, while the S242 residue was located in the loop connecting the TM5 and TM6 domains.

To explore the energetic profile of the whole selected hTAAR1 model, the contact energy values of the homol- ogy modelled protein were compared with those calcu- lated on the X-ray structure of the b

2

adrenoreceptor. The effective atomic contact energies (ACE) are calculated for heavy atoms of standard amino acids within a contact range of 6 A

̊

assigning energy terms (in kcal⁄mol) for each

A B

Figure 3: The superimposition of the final hTAAR1 model on the 3PDS co-ordinates is depicted as side view (A) and as top view (B). The conserved regions are highlighted in red.

adrenoreceptor (3PDS) co-ordinates.

contact pair, as described by Zhang et al. (28). These energies were summed up for each residue in the system. In general, a high negative value indicates that the residue is predominantly in contact with hydrophobic atoms and hence to be expected in a buried protein environment. Residues with positive energy terms indicated contacts with predominantly hydrophilic atoms and were expected in more solvent exposed areas of the protein.

As shown in Fig. 5, the energy trends observed for the two GPCRs resulted to be in good agreement.

In addition, quality estimates for the modelled protein side chain were also evaluated by the rotamer energy profile, displaying absence of outliers (Fig. 6). The rotamer strain energy was calculated on the basis of the backbone- dependent rotamer library published by Dunbrack and Co- hen (29). The rotamer statistics were collected for each u– w combination in 10° increments and smoothed with Bayesian methods. The backbone dependency of side- chain rotamers was restricted to the v1-angle; the remain- ing v angles are independent of the backbone u–w values. For a given backbone u–w combination, the resulting probabilities provide estimates of weakly or strongly popu- lated side-chain rotamers in the Protein Data Bank.

A qualitative assessment of the obtained model was also performed by evaluation of an appropriate distribution of

Cichero et al.

Figure 4: Ramachandran plots for the hTAAR1. Only three resi- dues (coloured in red) are in the disfavoured region. These resi- dues are located far from the putative binding site. Green: favoured region; yellow: allowed region.

the hydrophobic and hydrophilic properties on the hTAAR1 model surface. In fact, as shown in Fig. 7, the receptor transmembrane domain displayed hydrophobic surface properties (depicted in red), while the portions extended towards the extra-cellular (EC) or the intra-cellular (IC) envi- ronments were properly depicted in cyano (hydrophilic properties).

Taken together, these data generally validate the derived model.

hTAAR1 agonist-binding site and docking studies The putative hTAAR1 binding site was identified by super- imposition of the derived protein on the 3PDS-binding site and also on the 5HT

1A

model already built by us. The three aligned GPCR regions, surrounding the 3PDS com- plex agonist (5 A

̊

distance), resulted to be highly similar to each other. In fact, the pairwise percentage residue iden- tity (PPRI) evaluated between the hTAAR1 pocket residues and those of the 3PDS and 5HT

1A

binding sites proved to be of 40.7% and 38.5%, respectively. In particular, the hTAAR1 residues, W89, D113, C182, W264, F267, F268, N286, W291 and Y294, according to our calculations, were present in the three GPCRs (Table 1). Furthermore, the hTAAR1 T100 and S198 residues corresponded to the b

2

-adrenoreceptor T110 and S207 residues, while the hTAAR1 F199 corresponded to the 5HT

1A

F204.

512 Chem Biol Drug Des 2013; 81: 509–516

]Structure and Pharmacology of the Human Trace Amine-Associated Receptor 1

A

B

Figure 5: Contact energy profiles of hTAAR1 (A) and of the human b

2

adrenoreceptor (3PDS) (B). The positions of the amino acid residues are shown on the x-axis, while the contact energies are shown on the y-axis.

Figure 6: The derived hTAAR1 rotamer profile is depicted. The positions of the amino acid residues are shown on the x-axis, while the rotamer energy is shown on the y-axis.

Taking into account these experimental data, the agonist co-crystallized in the 3PDS complex displayed H-bond contacts with residues D113 and N312 and was also engaged in H-bonds with S203 and S207 (Fig. 8). Notably,

Chem Biol Drug Des 2013; 81: 509–516 513

the key residues D113 and N312 are maintained in all the three GPCRs here investigated, while S207 corresponded to the hTAAR1 residue S198 (Table 1). Moreover, site- directed mutagenesis data concerning the human seroto- ninergic receptor highlighted that all the 5HT

1A

ligands dis- played a salt bridge with D116 side chain, confirming the importance of this residue as an anchoring point for the ligand activity; probably this interaction is kept also for hTAAR1 ligands. In addition, molecular docking studies already published (21,22,30) underlined the relevance of the 5HT

1A

N386 residue for ligand recognition and allowed us to reasonably hypothesize that even the corresponding hTAAR1 N286 residue could be involved in the agonist interaction.

On the basis of our docking calculations, RO5166017, b- PEA and T

1

AM share the following interactions: (i) one H- bond between the two compound amino groups and the D103 side chain, (ii) p–p stacking between the phenyl ring (for T

1

AM, the ethylamino-phenyl one) and W264, F267 and F268 (Figs9 and 10). Furthermore, compound RO5166017 displays an additional H-bond with T100 by means of the oxazole ring, while T

1

AM is also engaged in H-bonds with N286 and D287 (by the two oxygen atoms).

Taking only into account the RO5166017 agonist, the ethyl group is projected towards the hTAAR1 F267, T271, N286, D287 and I290 residues. Among these, F267 and

Figure 7: Distribution of the hydrophobic and hydrophilic proper- ties on the hTAAR1 model surface is depicted. The receptor transmembrane domain displays hydrophobic surface properties (depicted in red), while the portions extended towards the extra- cellular (EC) or the intra-cellular (IC) environments are properly depicted in blue (hydrophilic properties).

Cichero et al.

Table 1: The residues (located 5 A

̊

far from the 3PDS ligand) belonging to the aligned 3PDS, 5HT

1A

and hTAAR1 receptors are listed

b

2

-adrenoreceptor

5HT

1A

putative

hTAAR1 putative binding site

binding site

binding site

G90 A93 S80 C93 Y96 R83 I94 Q97 S84 – – C88 W99 W102 W89 W109 H99 T110 I113 T100 D113 D116 D103 V114 V117 I104 V117 C120 S107 – – S108 – – K174 – – G181 C191 C187 C182 D192 T188 S183

Figure 8: MOE LigPlot of the agonist compound into the 3PDS F193 I189 V184

binding site. T195 K191 F186 Y199 – G191 A200 T196 V192 S203 S199 T194 S204 T200 F195 S207 A203 S198 – F204 F199 W286 W358 W264 F289 F361 F267 F290 F362 F268 N293 A385 T271 Y308 L381 V272 I309 G382 – N312 N386 N286 – – D287 – – I290 W313 W387 W291 Y316 Y390 Y294

Residues conserved among all the three GPCRs are reported in bold, residues that are conserved between the hTAAR1 receptor and one GPCR template are in italic.

Figure 9: RO516607 and b-PEA selected docking poses into the hTAAR1 putative binding site are depicted. The agonists are reported in stick; RO516607 and b-PEA C atom are coloured in N286 are conserved with respect to the b

2

-adrenoreceptor and the 5HT

1A

one, while T271 to the b

2

-adrenergic polar

pink and in white, respectively. Residues located 5 A

̊

from the ag- onists are shown and labelled. residue N293 and to the 5HT

1A

hydrophobic residue A385 (see Table 1). Interestingly, hTAAR1 D287 and I290 are in

tion of hydrophobic or aromatic group at this position correspondence of two gap regions, in comparison with

could be beneficial for the hTAAR1 agonist selectivity. the two GPCR used as template. Thus, the hTAAR1 pocket surrounding the RO5166017 ethyl substituent

Finally, the RO5166017 4,5-dihydro-1,3-oxazole ring results to be expended respect to the b

2

- and 5HT

1A

occupies a region including the hTAAR1 H99, T100, D103 receptors. Accordingly, the ethyl chain could be properly

and C182. Among these, D103 and C182 are conserved substituted with a bulkier group, also bearing a hydrophilic

with respect to the b

2

one, function, to enhance H-bond contacts with T271 and

while T100 is only conserved between the hTAAR1 and N286.

the adrenergic receptor (see Table 1). Notably, H99 corre- sponds to the b

2 The RO5166017 phenyl ring is oriented towards F195 and the two conserved residues W264 and F268. Notably, F195 corresponds to the b

2

-adrenergic and 5HT

1A

polar residues S204 and T200 (see Table 1). Thus, the introduc-

514 Chem Biol Drug Des 2013; 81: 509–516

-adrenoreceptor and the 5HT

1A

-adrenergic W109 residue. Accordingly, the introduction of proper substituents at the oxazole posi- tion 2 could enhance the interaction with the hTAAR1 basic residue, probably also achieving hTAAR1 agonist selectivity.

]Structure and Pharmacology of the Human Trace Amine-Associated Receptor 1

Figure 10: RO516607 and T

1

AM selected docking poses into the hTAAR1 putative binding site are depicted. The agonists are reported in stick; pink and in white, RO516607 and T

1 AM C respectively. Residues located atom are 5 A

̊

coloured in from the ag- onists are shown and labelled.

Conclusions and Future Directions

To gain a better understanding of the structural mecha- nisms of agonist-hTAAR1 receptor interactions, we present here a theoretical model of the human trace amine-associ- ated receptor 1 (hTAAR1) developed by homology model- ling. The protein putative binding site has been explored by comparing the derived hTAAR1 model with the b

2

- adrenoreceptor binding site and also with the homology modelled 5HT

1A

receptor. The obtained results allowed us to identify two residues, D103 and N286, as potential anchor-points for the ligand recognition process. Accord- ingly, docking studies performed on compounds RO5166017, b-PEA and T

1

AM proved that both the two agonists interact with D103. This study could represent an interesting starting point to design new selective hTAAR1 agonists.

Acknowledgments

This work was financially supported by the University of Genova. E.C. was financially supported by a post-doc fel- lowship, Area Chimica, University of Genova.

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516 Chem Biol Drug Des 2013; 81: 509–516