Molecular determinants for selective recognition of in the human serotonin and norepinephrine transporters

Jacob Andersena, Nicolai Stuhr-Hansena, Linda Zachariassena, Søren Toubroa, Stinna M. R. Hansena, Jonas N. N. Eildala, Andrew D. Bondb, Klaus P. Bøgesøc, Benny Bang-Andersenc, Anders S. Kristensena,1, and Kristian Strømgaarda,1

aDepartment of Medicinal Chemistry, University of Copenhagen, DK-2100 Copenhagen, Denmark, bDepartment of Physics and Chemistry, University of Southern Denmark, DK-5230 Odense, Denmark; and cNeuroscience Drug Discovery Denmark, H. A/S, DK-2500 Valby, Denmark

Edited by Susan G. Amara, University of Pittsburgh School of Medicine, Pittsburgh, PA, and approved June 2, 2011 (received for review March 1, 2011) Inhibitors of the (SERT) and norepinephrine commonly prescribed for treatment of depression and anxiety transporter (NET) are widely used in the treatment of major de- disorders and are being used in an increasing number of unique pressive disorder. Although SERT/NET selectivity is a key determi- therapeutic applications (5). nant for the therapeutic properties of these drugs, the molecular Balancing the inhibitory activity at SERT and NET is a key determinants defining SERT/NET selectivity are poorly understood. determinant for the therapeutic properties of drugs targeting In this study, the structural basis for selectivity of the SERT selec- monoamine transporters (6). Importantly, delicate modifications tive inhibitor and the structurally closely related NET of the same chemical skeleton can tweak selectivity between the selective inhibitor talopram is delineated. A systematic structure- two transporters and have in several cases been used to develop activity relationship study allowed identification of the substitu- both SSRIs, SNRIs and NRIs, from the same chemical scaffold ents that control activity and selectivity toward SERT and NET and (5). Although the clinical significance of compounds with distinct revealed a common pattern showing that SERT and NET have op- SERT/NET selectivity profiles has been recognized for more posite preference for the stereochemical configuration of these than 30 years, the molecular basis for SERT/NET selectivity is inhibitors. Mutational analysis of nonconserved SERT/NET residues poorly understood, mainly due to limited insight into the local- within the central substrate binding site was performed to deter- ization and structure of the inhibitor binding pockets in SERT mine the molecular basis for inhibitor selectivity. Changing only and NET. High-resolution X-ray structures of a bacterial ho- fi ve residues in NET to the complementary residues in SERT trans- molog to SLC6 transporters (LeuT) have been reported (7–11), fi fi R S ferred a SERT-like af nity pro le for - and -citalopram into NET, providing insight into the tertiary structure of this class of pro- showing that the selectivity of these compounds is determined by teins. The structures revealed a topology of 12 transmembrane amino acid differences in the central binding site of the transport- R S (TM) spanning regions connected by short intra- and extracel- ers. In contrast, the activity of - and -talopram was largely un- lular loops with a high-affinity substrate binding site (denoted the affected by any mutations within the central substrate binding site S1 site) centrally located in the core of the transporter protein of SERT and NET and in the outer vestibule of NET, suggesting that (12). LeuT has proved to be an excellent structural template for citalopram and talopram bind to distinct sites on SERT and NET. construction of homology models of SERT and NET, facilitating Together, these findings provide important insight into the molec- identification of the location and molecular structure of binding ular basis for SERT/NET selectivity of antidepressants, which can pockets for substrates, ions, and inhibitors (13–18). be used to guide rational development of unique transporter In this study, we delineate the structural basis for SERT/NET inhibitors with fine-tuned transporter selectivity. selectivity of the SSRI citalopram and the structurally closely related NRI talopram. Through systematic structure-activity re- monoamine | neurotransmitter | SLC6 transporter lationship studies, we identify specific substituents as key deter- minants for inhibitory activity and selectivity toward SERT and mbalances in neurotransmission involving the monoamines NET. Furthermore, we find that switching nonconserved SERT/ Iserotonin (5-hydroxytryptamine; 5-HT) and norepinephrine NET residues within the S1 site fully transfers citalopram sen- (NE) are implicated in depression and anxiety disorders (1). In fi sitivity to NET and renders SERT insensitive to citalopram, the brain, speci c monoamine transporters, the 5-HT transporter thereby demonstrating that the selectivity of citalopram is de- (SERT) and the NE transporter (NET), curtail the lifetime of termined solely by the nature of the central substrate binding extracellular monoamines by performing active uptake (or re- fi

pocket in SERT and NET. In contrast, we nd that the NRI PHARMACOLOGY uptake) from the extracellular space into neurons. properties of talopram are remarkably unaffected both by per- for the treatment of depression and anxiety disorders act by in- turbations of the S1 pockets in SERT and NET, as well as in the creasing the extracellular concentration of 5-HT and/or NE by outer vestibule in NET, which has been proposed to harbor an inhibiting SERT and/or NET mediated transmitter reuptake (2). inhibitor binding site (denoted the S2 site) (10, 11), suggesting SERT and NET belong to the solute carrier 6 (SLC6) trans- porter family, and they are integral membrane proteins that use cotransport of sodium as an energy source to convey neuro- Author contributions: J.A., B.B.-A., A.S.K., and K.S. designed research; J.A., N.S.-H., L.Z., transmitters from the extracellular space to the cytoplasm (3). S.T., S.M.R.H., and J.N.N.E. performed research; J.A., A.D.B., K.P.B., B.B.-A., A.S.K., and K.S. The first generation of drugs targeting SERT and NET were the analyzed data; and J.A., A.S.K., and K.S. wrote the paper. antidepressants (TCAs), but their activity across a vari- The authors declare no conflict of interest. ety of other neurotransmitter receptor systems (4) associate their This article is a PNAS Direct Submission. use with severe side effects. Development of newer generations Data deposition: The atomic coordinates reported in this paper have been deposited in of drugs have focused on compounds the Cambridge Structural Database, Cambridge Crystallographic Data Centre, Cambridge with an improved selectivity toward SERT and/or NET, exem- CB2 1EZ, United Kingdom (CSD reference nos. 811835 and 811836). plified by the selective 5-HT reuptake inhibitors (SSRIs), the 1To whom correspondence may be addressed: E-mail: [email protected] or [email protected]. selective NE reuptake inhibitors (NRIs), and the dual inhibitors This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. of SERT and NET (SNRIs). These drugs are among the most 1073/pnas.1103060108/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1103060108 PNAS | July 19, 2011 | vol. 108 | no. 29 | 12137–12142 Downloaded by guest on September 24, 2021 that talopram is accommodated at a site distinct from the S1 and subtle perturbations of the same chemical scaffold, the selectivity S2 binding sites. Thus, we demonstrate that two structurally ratio between SERT and NET can be controlled. closely related compounds possibly can have distinct binding sites on the same transporter protein. SERT and NET Have Opposite Stereochemical Requirements. Cit- alopram and talopram are racemic mixtures, and it is well- Results established that the inhibitory potency of citalopram toward Structural Features of Inhibitors Underlying Activity and Selectivity. SERT resides in the S-enantiomer (16, 20, 21), as exemplified by Citalopram is among the most selective SERT inhibitors and the Lexapro. Accordingly, we found that the affinity of S-citalopram structurally related counterpart, talopram, is a potent and selec- was 35-fold higher for SERT compared with R-citalopram (4 nM tive inhibitor of NET (Fig. 1). The binding affinity of citalopram versus 136 nM), whereas both enantiomers displayed low affinity and talopram was determined by displacement of 125I-labeled binding to NET (3,025 nM versus 1,516 nM) (Fig. 2 and Table (-)-2β-carbomethoxy-3β-(4-iodophenyl) (β-CIT) binding S1). The stereochemical properties of talopram had not been to recombinantly expressed human SERT or NET and, as ex- examined. We therefore resolved talopram into the R- and S- pected, citalopram had high affinity and selectivity toward SERT enantiomers by preparative supercritical fluid chromatography over NET (4 nM versus 1,414 nM), whereas talopram had high (SFC), crystallized one of the enantiomers as a hydrotosylate affinity and selectivity toward NET over SERT (9 nM versus 719 salt, and used X-ray analysis to unequivocally identify this isomer nM) (Fig. 1 and Table S1). The two chiral compounds share a as the R-enantiomer (Fig. 2 and SI Experimental Procedures). phenyl-substituted phthalane skeleton and a propylamine moiety, Subsequent characterization of R- and S-talopram showed that and they are distinguished by four chemical substituents only the R-enantiomer has a remarkable 685-fold higher affinity for (Fig. 1). To delineate the role of these four diverging structural NET compared with S-talopram (3 nM versus 1,986 nM), whereas elements for activity at SERT and NET, we used a previously both enantiomers displayed low affinity binding to SERT prepared set of 16 compounds comprising all possible combina- (1,052 nM versus 2,752 nM) (Fig. 2 and Table S1). Interestingly, tions of the differing substituents (19) (Fig. 1) and determined SERT and NET display opposite enantioselectivity, because S- the inhibitory potency (Ki) of each compound at SERT and NET citalopram has a 775-fold preference for SERT over NET (4 nM in a functional uptake inhibition assay (Fig. 1, Fig. S1, Table S2, versus 3,025 nM) and R-talopram has almost 1,000-fold prefer- and SI Experimental Procedures). The systematic structure-activity ence for NET over SERT (3 nM versus 2,752 nM). In contrast, analysis allowed us to dissect the role of each of the four sub- both R-citalopram and S-talopram are low affinity SNRI com- stituents for inhibitory activity toward SERT and NET (Fig. S1). pounds with <12-fold selectivity between SERT and NET (Fig. 2 Furthermore, we found a striking relationship between the sub- and Table S1). Hence, the R/S-configuration of the inhibitors is stitution pattern of the 16 compounds and their SERT/NET se- a critical determinant for SERT/NET selectivity, which had not lectivity profile (Fig. 1C), leading to succinct information on the been appreciated. To explore the generality of this finding for this structural determinants for selectivity. Citalopram was the most compound series, we resolved the R- and S-enantiomers of potent and selective SERT inhibitor, whereas talopram was the compound 10 by preparative SFC, crystallized one of the enan- most potent and selective NET inhibitor of the 16 compounds tiomers as a hydrotosylate salt and identified this isomer as the S- (Fig. 1, Fig. S1, and Table S2). We observe that the cyano- and enantiomer by X-ray analysis (Fig. 2 and SI Experimental Pro- phthalane methyl groups are key determinants for inhibitory ac- cedures). Subsequent characterization showed that S-10 was tivity and selectivity toward SERT and NET, respectively. Nota- a high affinity SSRI, with 47-fold selectivity for SERT over NET bly, compound 10 is a SNRI with high affinity for both SERT and (28 nM versus 1,288 nM), whereas R-10 was a high affinity SNRI NET (32 nM versus 44 nM) (Table S1), which reaffirms that by compound, with sixfold selectivity for NET over SERT (29 nM

Fig. 1. (A) Chemical structures of citalopram, talopram, and the 14 compounds (2–15) comprising all possible combinations of the four substituents that distinguish citalopram from talopram. The differing substituents are colored blue (citalopram substituents) or green (talopram substituents), respectively. (B) Citalopram (Left), compound 10 (Center) and talopram (Right) were assessed for their ability to displace binding of [125I]β-CIT to SERT and NET. Data points

represent the mean ± SEM from triplicate determinations. (C) The inhibitory potency (Ki) was determined for the 16 inhibitors in a functional uptake in- hibition assay (Table S2), and the SERT/NET selectivity ratio were calculated as Ki(NET)/Ki(SERT) or -Ki(SERT)/Ki(NET) for SERT and NET preferring inhibitors, respectively. Inhibitors with >10-fold selectivity are highlighted in blue (SERT selective) or green (NET selective).

12138 | www.pnas.org/cgi/doi/10.1073/pnas.1103060108 Andersen et al. Downloaded by guest on September 24, 2021 Fig. 2. (A) The binding affinity (Ki) of pure S- and R-enantiomers (black and white squares, respectively) of citalopram, talopram, and compound 10 was determined at SERT and NET (Table S1). (B) Single crystal X-ray structures of hydrotosylate salts of S-10 (Left) and R-talopram (Right). C, gray; H, white; F, green; N, blue; O, red. Displacement ellipsoids are shown at 30% probability level for non-H atoms, and the counterion has been omitted for clarity.

versus 171 nM). These results suggest a pattern for this set of citalopram/talopram analogs, where SERT inhibition resides in the S-enantiomer and NET inhibition resides in the R-enantio- mer (Fig. 2). This difference in enantioselectivity between SERT and NET has recently also been observed for other citalopram Fig. 3. (A) Amino acid sequence alignment of nonconserved residues lo- analogs (21). cated within 6 Å of the central substrate binding site in human SERT and NET (24). (B) Homology model of NET (Left) and SERT (Right). The substrate is Residues in the Central Substrate Binding Site Control SERT/NET shown in yellow, sodium ions in magenta, and nonconserved residues are Inhibitor Selectivity. It has been demonstrated that S-citalopram highlighted in green (NET) or blue (SERT). (C) The inhibitory potency of has a competitive mode of inhibition in SERT (22, 23), which is talopram (black bars) and citalopram (gray bars) was determined at single point SERT and NET mutants (Table S4). Data represent the mean ± SEM consistent with overlapping binding sites for 5-HT and S- from at least three independent experiments each performed in triplicate. citalopram (13, 16). To elucidate the mode of inhibition by R- The stipulated line indicates the Ki value for the inhibitors at WT trans- talopram, we performed uptake-saturation experiments with porters. Asterisks denote significant different Ki value compared with WT SERT and NET in the absence or presence of inhibitors (SI transporters (Student’s t test; P < 0.05). Experimental Procedures), which revealed a competitive mode of inhibition for S-citalopram and R-talopram in both SERT and NET (Table S3). Hence, it is tempting to speculate that the SERT without important interactions to any of the 15 mutated determinants for inhibitor selectivity are located within or in residues. close proximity of the substrate binding site (S1). From a LeuT- Identification of Specific Residues That Control Citalopram Selectivity. based homology model of SERT (13), we identified 47 residues To test whether inhibitor selectivity is conferred by a single located within 6 Å of the S1 site, whereof 15 are nonconserved residue among the 15 nonconserved residues in the S1 pockets of between SERT and NET (Fig. 3 and Fig. S2). Initially, we mu- SERT and NET, we mutated each of the nonconserved residues tated all of the 15 residues in SERT and NET to the corre- to the corresponding residue in the other transporter (Fig. 3). sponding residues in the other transporter, leading to a 15-fold The 30 point mutants showed activity ranging from 10% to 97% SERT mutant (SERT-15) and a 15-fold NET mutant (NET-15) compared with WT transporters, and substrate KM were generally and envisioned that these mutations would generate a NET-like comparable to WT transporters (Table S4). The point mutants in S1 binding pocket in SERT and vice versa. SERT-15 and NET- SERT generally had a small effect on Ki for citalopram (<fivefold 15 were transfected into COS-7 cells, but did not display mea- changes), except for A173G and A441G, which induced a 14- and 125 β surable uptake. However, [ I] -CIT was able to bind SERT-15 a sevenfold increase in citalopram potency, respectively. None of PHARMACOLOGY in membrane preparations from transfected cells with KD com- the point mutants in SERT had a significant effect on the Ki of parable to wild-type (WT) SERT (3.9 nM versus 5.2 nM), talopram. Similarly, the point mutants in NET generally induced whereas no measurable binding was observed in similar prepa- <fivefold changes in the potency of citalopram and talopram, rations of NET-15. Citalopram and its enantiomers showed up to except for A77G and A426G, which induced a 16- and a 10-fold 753-fold loss of affinity (as observed for S-citalopram) at SERT- decrease in K for citalopram, respectively, and V148I and F150S, fi fi i 15 compared with WT SERT (Table S1). Speci cally, the af nity which increased the Ki of talopram by nine- and sixfold, re- for R-citalopram decreased toward the affinity observed in WT spectively (Fig. 3 and Table S4). NET (750 nM versus 1,516 nM), whereas the affinities for cit- Because single substitutions of nonconserved residues could alopram and S-citalopram were completely reversed to those not reverse the SERT/NET selectivity of citalopram and talo- observed in WT NET (1,305 nM versus 1,414 nM for citalopram; pram, we tested whether inhibitor selectivity could be changed by 2,938 nM versus 3,025 nM for S-citalopram). In contrast, SERT- combining the existing single mutants into 10 multiple SERT 15 induced

Andersen et al. PNAS | July 19, 2011 | vol. 108 | no. 29 | 12139 Downloaded by guest on September 24, 2021 Fig. 4. Topology diagram of SERT (blue) and NET (green) and graphical representation of multiple mutants. The identity, TM location, and numbering of the 15 nonconserved SERT/NET residues within the central substrate binding site are shown. SERT mutants are shown on a blue background, with mutations

indicated in green (Upper), and NET mutants are shown on a green background, with mutations indicated in blue (Lower). The inhibitory potency (Ki)for citalopram (gray bars) and talopram (black bars) were determined in a functional uptake inhibition assay (Table S4). The stipulated line indicates the Ki value for the inhibitors at WT transporters. Asterisks denote significantly different Ki value compared with WT transporters (Student’s t test; P < 0.05).

double SERT mutant S1 (Y95F-G100A). Further elaboration of duced

12140 | www.pnas.org/cgi/doi/10.1073/pnas.1103060108 Andersen et al. Downloaded by guest on September 24, 2021 close proximity of the S2 site (Fig. S4 and Table S5). Eighteen of the 32 mutants displayed functional activity that allowed determination of inhibitory potency, and we found that all tested mutations induced

Andersen et al. PNAS | July 19, 2011 | vol. 108 | no. 29 | 12141 Downloaded by guest on September 24, 2021 that SERT and NET have opposite preference for the stereo- development of unique inhibitors with tailor-made transporter chemical configuration of these inhibitiors was found (Fig. 2). selectivity and, possibly, an improved clinical efficacy. Similar patterns have been observed for sertraline and ven- lafaxine (38, 39), whereas SERT and NET inhibition resides in Experimental Procedures the same enantiomer for (40), thus demonstrating Molecular Biology, Transport Assays, and Radioligand Binding Experiments. that chirality is generally a key determinant for both inhibitory Generation of point-mutations in human SERT and NET was performed activity and selectivity toward monoamine transporters. In- by site-directed mutagenesis using the QuikChange mutagenesis kit (SI R S Experimental Procedures). Transport measurements were performed essen- terestingly, 3D superimposition of the - and -enantiomers of 125 β citalopram and analogs display a considerable structural overlap tially as described (13) (SI Experimental Procedures). Binding of [ I] -CIT to fl membrane preparations of COS-7 cells expressing SERT or NET mutants was (21, 37), which likely re ect that the observed enantioselectivity performed as described in SI Experimental Procedures. at SERT is primarily due to subtle differences in binding of the two aromatic ring systems as suggested recently (16). Resolution of R- and S-Enantiomers of Talopram and Compound 10. The R-and In summary, we have demonstrated how systematic and subtle S-enantiomers of talopram and compound 10 were resolved by preparative changes on the same chemical scaffold can tweak selectivity SFC and crystallized as hydrotosylate salts (SI Experimental Procedures). The between SERT and NET, possibly by controlling where the in- absolute configuration of the crystallized enantiomers was determined by hibitor binds on the transporter proteins. Taken together with single crystal X-ray analysis (SI Experimental Procedures). the identification of molecular determinants in SERT and NET that control selectivity of these compounds, new insight is ACKNOWLEDGMENTS. Peter Brøsen, Valentina Lauritzen, and Krestian Larsen are acknowledged for excellent technical assistance. Dr. Olivier obtained into the principal molecular features that govern Taboureau is acknowledged for providing us with the homology model of SERT/NET selectivity for structurally closely related inhibitors. human NET. We thank Dr. Jan Egebjerg for critical comments on the man- These findings provide an important framework to guide rational uscript and Dr. Claus J. Loland for sharing unpublished data.

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