Interrogating the Molecular Basis for Substrate Recognition in

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Interrogating the molecular basis for substrate recognition in serotonin and dopamine transporters with high-affinity substrate-based bivalent ligands Jacob Andersen, Lucy Kate Ladefoged, Trine N. Bjerre Kristensen, Lachlan Munro, Julie Grouleff, Nicolai Stuhr-Hansen, Anders S. Kristensen, Birgit Schiøtt, and Kristian Strømgaard ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.6b00164 • Publication Date (Web): 18 Jul 2016 Downloaded from http://pubs.acs.org on July 19, 2016

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ACS Chemical Neuroscience

Interrogating the molecular basis for substrate recognition in serotonin and dopamine transporters with high-affinity substrate-based bivalent ligands

Jacob Andersen,*,† Lucy Kate Ladefoged,‡ Trine N. Bjerre Kristensen,‡ Lachlan Munro,† Julie Grouleff,‡,§ Nicolai Stuhr-Hansen,†,# Anders S. Kristensen,† Birgit Schiøtt,‡ and Kristian Strømgaard*,†

†

Department of Drug Design and Pharmacology, University of Copenhagen, DK-2100

Copenhagen, Denmark. ‡

Interdisciplinary Nanoscience Center (iNANO), Department of Chemistry, Aarhus

University, DK-8000 Aarhus C, Denmark.

1 Environment ACS Paragon Plus


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ABSTRACT The transporters for the neurotransmitters serotonin and dopamine (SERT and DAT, respectively) are targets for drugs used in the treatment of mental disorders and widely used drugs of abuse. Studies of prokaryotic homologs have advanced our structural understanding of SERT and DAT, but it still remain enigmatic whether the human transporters contain one or two high-affinity substrate binding sites. We have designed and employed 24 bivalent ligands possessing a highly systematic combination of substrate moieties (serotonin and/or dopamine) and aliphatic or poly(ethylene glycol) spacers to reveal insight into substrate recognition in SERT and DAT. An optimized bivalent ligand comprising two serotonin moieties binds SERT with 3,800-fold increased affinity compared to serotonin, suggesting that the human transporters have two distinct substrate binding sites. We show that the bivalent ligands are inhibitors of SERT and an experimentally validated docking model suggests that the bivalent compounds bind with one substrate moiety in the central binding site (the S1 site) whereas the other substrate moiety is binding in a distinct binding site (the S2 site). A systematic study of non-conserved SERT/DAT residues surrounding the proposed binding region showed that non-conserved binding site residues do not contribute to selective recognition of substrates in SERT or DAT. This study provides novel insight into the molecular basis for substrate recognition in human transporters, and provides an improved foundation for development of new drugs targeting SERT and DAT.

KEYWORDS serotonin transporter, dopamine transporter, alternating access mechanism, neurotransmitter transport, molecular pharmacology, induced fit docking


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INTRODUCTION Neurotransmitter transporters play a central role in regulation of synaptic transmission by performing rapid reuptake of released neurotransmitters from the synapse into presynaptic neurons or neighbouring glial cells.1 The majority of neurotransmitter transporters belong to the solute carrier 6 family of transporters, which includes transporters for the biogenic monoamines, serotonin (5HT), dopamine (DA) and norepinephrine, as well as γ-aminobutyric acid and glycine.2 These transporters utilize the sodium gradient across the plasma membrane to facilitate the reuptake of neurotransmitters from the synapse against their concentration gradient.3 The closely related monoamine transporters (MATs) for 5HT, DA and norepinephrine (SERT, DAT and NET, respectively) are of particular interest because they are targeted by drugs used in the treatment of mental disorders and widely abused psychostimulants.4 Current structural understanding of human MATs is mainly based on X-ray crystal structures of bacterial transporters (LeuT and MhsT) and the Drosophila melanogaster DAT (dDAT).5-8 The transporter topology consists of 12 transmembrane domains (TMs) arranged in a barrellike bundle with a pseudosymmetric fold, and X-ray crystal structures of human SERT (hSERT) recently confirmed that this structural organization is evolutionarily conserved from bacterial to human transporters.9 Biochemical and computational studies have suggested that the high-affinity ligand binding site (denoted S1) is located in the central core of human MATs.10-23 Structures of LeuT and hSERT have furthermore suggested that inhibitors can also bind to a second binding site (denoted S2) in an extracellular facing vestibule,9, 24-26 which is in agreement with biochemical studies of hSERT showing that the S2 region harbours a low-affinity allosteric inhibitor binding site.27-28 Bivalent compounds are composed of two pharmacophoric units tethered through a linker, which may be capable of bridging independent recognition sites resulting in a



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thermodynamically favourable binding interaction compared to binding of two monovalent congeners. Interestingly, compounds containing two spacer-linked phenylpiperidines, which are structurally related to the antidepressant drug paroxetine, bind SERT with up to 1,000-fold improved affinity compared to the monovalent congeners,29-31 and bivalent ligands based on DAT substrates (e.g. DA and amphetamine) have increased affinity for human DAT (hDAT) compared to the parent substrates.32 The improved binding interaction of the bivalent ligands further support the idea that MATs contain two distinct ligand binding sites. However, bivalent compounds based on citalopram, imipramine, or the cocaine-like phenyltropane scaffold generally decreased activity towards MATs,33-38 showing that bivalent ligands must possess specific structural features to obtain high-affinity binding to MATs. The transport process of MATs is believed to follow the alternating access mechanism, implying that the transporters can alternate between at least two conformations in which binding sites for substrate and ions are accessible from either the extracellular or intracellular side of the membrane. Substrate binding within the central S1 site is key for driving the alternating access mechanism, but despite a series of extensive studies on bacterial transporters it still remains unclear if the S2 site also needs to be occupied with substrate to allosterically trigger substrate translocation.5-6, 39-48 Importantly, a major problem is whether studies of prokaryotic transporters can be directly correlated to its mammalian homologs, which emphasizes the urgent need for experimentation to resolve the molecular basis for substrate recognition in human MATs. To provide further insight into the molecular basis for substrate recognition in human MATs, we have employed a set of homo- and heterobivalent ligands possessing two substrate moieties (5HT and/or DA) linked by aliphatic or poly(ethylene glycol) (PEG) spacers. We demonstrate that bivalent 5HT ligands can bind hSERT with up to almost 4,000-fold higher affinity compared to 5HT, and that bivalent DA ligands can bind hDAT with up to ~80-fold


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higher affinity compared to DA. Our data strongly support the idea that human MATs contain more than a single domain for substrate binding, and combined with a systematic analysis of non-conserved hSERT/hDAT residues surrounding the predicted binding pockets, we report novel insight into the molecular basis for selective substrate recognition in human MATs.

RESULTS AND DISCUSSION Structure-activity relationship study of substrate-based bivalent ligands. We have systematically designed and synthesized a set of 24 bivalent substrate-based ligands in which two substrate moieties (5HT and/or DA) are tethered via PEG (1–12) or alkyl linkers (13–24) (Figure 1 and Figure S1).49 The binding affinity of the 24 compounds for wild-type (WT) hSERT and hDAT was determined in a [125I]RTI-55 competition binding assay. Generally, the homobivalent compounds containing 5HT or DA moieties generally exhibit substantially higher potency at hSERT or hDAT, respectively, than their monovalent counterparts. We confirm previous findings32, and show that bivalent DA-based molecules can yield up to ~80fold gain in binding affinity for hDAT compared to DA (as observed for 18). The affinity of the bivalent compounds for hDAT was largely independent of the linker length, whereas a distinct correlation was found between linker length and hSERT affinity, particularly for the 5HT-containing bivalent compounds. The presence of a PEG4 linker or the corresponding C10H20 alkyl linker resulted in the most potent compounds, and thus appear to be optimal spacer-length to favour bivalent binding in hSERT. Notably, compound 2 (5HT-PEG4-5HT) had ~3,800-fold higher affinity compared to 5HT for hSERT and bind the transporter with sub-nanomolar affinity (Ki = 0.64 nM), showing that hSERT is more sensitive towards bivalency compared to hDAT. This was also reflected in the selectivity profile of the 24 bivalent compounds, which generally had a more pronounced selectivity for hSERT compared to hDAT. Specifically, DA-PEG3-DA (9) was the most hDAT selective compound



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and had ~12-fold higher affinity for hDAT over hSERT, whereas 5HT-PEG5-5HT (3) had > 10,000-fold selectivity for hSERT over hDAT (Figure 1B). [INSERT FIGURE 1] [INSERT TABLE 1] The selectivity pattern was more distinct for the PEG-linked series of compounds (Figure 1B) compared to the corresponding alkyl-linked compounds (Figure 1C), indicating that the nature of the linker also has an effect on the pharmacological properties of the compounds. To examine the effect of the linker, we determined the binding affinity of 5HT and DA attached to a PEG linker (25–28, 33–36) or an alkyl linker (29–32, 37–40) for hSERT and hDAT (Figure 2 and Table 1). Addition of a linker to 5HT generally induced an increase in affinity for hSERT relative to 5HT (Figure 2B,D). The effect was most pronounced for the alkyl linkers, which induced up to 50-fold gain of affinity, whereas the PEG linkers induced up to 5-fold gain of affinity. Correspondingly, addition of an akyl linker to DA generally increased the binding affinity for hDAT (up to 14-fold) whereas addition of a PEG linker to DA induced up to 63-fold decreased binding affinity for hDAT (Figure 2C,E). Interestingly, the binding affinity of the alkyl-linked homobivalent compounds is comparable to the binding affinity of the substrate attached to the corresponding alkyl linkers (Figure 2D,E). This suggests that the improved binding affinity of the alkyl-linked bivalent compounds could be induced by the linker alone and not by bivalency as such, which could be due to unspecific hydrophobic interactions between the alkyl linker and the transporter protein. In contrast, the binding affinity of the PEG-linked bivalent compounds is in all cases higher compared to the binding affinity of the substrate attached to the corresponding PEG linker (Figure 2B,C), which suggests that the improved binding affinity of the PEG-linked bivalent compounds is induced by bivalency rather than the PEG linker itself.


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In summary, from the systematically designed mono- and bivalent substrate-based compounds we identified 5HT-PEG4-5HT (2) which bind hSERT with almost 4,000-fold higher affinity compared to 5HT, whereas addition of the linker alone (5HT-PEG3-Et; 26) induced a 5-fold gain of affinity for hSERT (Figure 2D). The corresponding homobivalent DA compound (DA-PEG4-DA; 10) had 10-fold higher affinity for hDAT compared to DA, and addition of the linker alone (DA-PEG3-Et; 34) led to a 3-fold decrease in the binding affinity (Figure 2E). [INSERT FIGURE 2] Substrate-based bivalent compounds are inhibitors of hSERT. When expressed heterologously in Xenopus oocytes, SERT is source of a constitutive inward membrane current due to spontaneous ion channel activity of the transporter.50-52 Administration of a substrate (e.g. 5HT) to a cell expressing SERT will increase membrane current, whereas application of an inhibitor will block the membrane current and thus induce an outward deflection of the current trace.51 We applied 10 µM of 5HT, one of the bivalent PEG4-linked compounds (2, 6 and 10) or the monovalent 5HT-PEG3-Et compound (26) to hSERT expressed in Xenopus oocytes under two-electrode voltage clamp conditions. Representative trace recordings are shown in Figure 3A, and demonstrate that application of 2, 6, 10 and 26 blocked the membrane current of hSERT, thus showing that these compounds are inhibitors, and not substrates, of hSERT. To investigate how these compounds affected the conformational equilibrium of hSERT, we determined if they affected accessibility of a cysteine in the extracellular permeation pathway. Specifically, we replaced the only reactive endogenous cysteine accessible from the extracellular side (Cys109) with alanine,53 and in this background we replaced Ser404 with cysteine (C109A-S404C). Ser404 is located in the extracellular loop 4 of hSERT, and the reactivity of S404C towards (2-aminoethyl)methanethiosulfonate (MTSEA) is decreased



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when SERT is stabilised in an inward-facing conformations by ibogaine or 5HT.54-55 In COS7 cells expressing C109A-S404C, we determined MTSEA-induced inhibition of [3H]5HT uptake in the absence (control) or in the presence of 5HT, 2, 6, 10 or 26 (Figure 3B, C). In agreement with previous observations, the presence of 5HT decreased reactivity of S404C. In contrast, the reactivity of S404C was increased in the presence of 2, 6, 10 or 26 compared to control (Figure 3B), which is characteristic of outward-facing conformations. Combined with the electrophysiological recordings, our results suggest that 2, 6, 10 and 26 inhibit uptake function by binding to hSERT and stabilise an outward-facing conformation of the transporter. [INSERT FIGURE 3] Induced-fit docking calculations of 5HT-PEG4-5HT into a homology model of hSERT. To gain insight into the high-affinity binding interaction between 5HT-PEG4-5HT and hSERT, we performed induced-fit docking (IFD) calculations of 5HT-PEG4-5HT into a dDAT-based homology model of hSERT in an outward-open conformation.20 Importantly, the hSERT model used for the IFDs is very similar to recently determined X-ray structure of hSERT9 (Figure S2). Specifically, the root-mean-square deviation between alpha carbons in TMs 1-12 is 1.38Å between the hSERT homology model and the X-ray crystal structure of hSERT. Noteworthy, hSERT has been suggested to form oligomeric complexes in the membrane,56 and the improved binding affinity of 5HT-PEG4-5HT could be induced by simultaneous binding of the two 5HT moieties in the S1 sites of two adjacent hSERT protomers. However, this seem unlikely since the PEG4 linker would not be long enough to bridge two central S1 sites in adjacent protomers. The bivalent compound could also be binding just with one of the 5HT moieties at a time in the S1 site of a single hSERT protomer, and soon after the binding site is vacated, the second substrate moiety (which is tethered in close proximity by the linker) could quickly get into the same binding site. This would


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increase the relative association rate, and increase the binding affinity of the bivalent compound. In this case, we would expect the compounds with the shortest linkers to have the highest affinity. Since the shortest linkers did not provide the most potent bivalent compounds (Figure 1), we assume that the bivalent compounds simultaneously occupy two distinct binding sites within a single hSERT protomer, similarly to what has previously been proposed for hDAT.32 Accordingly, from the IFDs of 5HT-PEG4-5HT in the hSERT model, we identified three clusters (Cluster 1 – 3) in which one of the 5HT moieties of 5HT-PEG4-5HT bind in the central binding site in a similar pose as the previously determined binding pose of 5HT (Figure 4).57-58 Specifically, the secondary ethylamino group of the 5HT moiety in the central site interacts with the side chain of Asp98, the indole nitrogen points towards Phe341, and the hydroxyl group on the indole ring is located in a hydrophilic pocket formed by Ser438 and Thr439. The PEG linker and the second 5HT moiety are oriented differently in the three binding clusters. In Cluster 1, there is a kink in the PEG linker, which places the second 5HT moiety close to the S1 site where it interacts with Tyr176 through hydrophobic π-π interactions. The PEG linker is more extended in Cluster 2 and 3, which places the second 5HT moiety in between Pro403 and Lys490 in S2, but with reversed orientations in the two clusters (Figure 4). To examine if computational docking studies could provide information about substrate specificity within the S1 and S2 sites in hSERT, we also performed IFDs of 5HT-PEG4-DA (6) and DA-PEG4-DA (10) into the hSERT model. We obtained three binding clusters of the homobivalent DA compound (10) and five binding clusters of 5HTPEG4-DA (Table S1), and overall these IFDs revealed that 6 and 10 can obtain similar binding poses as found for 5HT-PEG4-5HT. The predicted binding mode of DA-PEG4-DA in hSERT is comparable to the proposed binding mode of DA-(CH2)8-DA in hDAT.32 Both bivalent DA compounds span the S1-S2 region in the two transporters, but whereas the DA moieties in the S1 sites assume similar orientations in hSERT and hDAT, there seem to be



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minor differences between the DA moieties in the S2 sites. Specifically, whereas the second DA moiety was predicted to bind close to TMs 1 and 6 in hDAT, it seems to bind in close proximity of TMs 3 and 10 in hSERT. Among the five clusters for 5HT-PEG4-DA, two predicted that the 5HT moiety binds within the central site and the DA moiety in the allosteric site, and three predicted a reversed orientation of the ligand. Since all clusters had comparable docking scores (Table S1), these IFD calculations are not predictive of substrate specificity within the S1 and S2 sites of hSERT. [INSERT FIGURE 4] Mutational analysis of 5HT-PEG4-5HT binding in hSERT. To distinguish between the three proposed binding modes of 5HT-PEG4-5HT in hSERT, we performed a mutational analysis of residues surrounding the poses in Cluster 1 – 3 (Figure 5). We determined the potency of 5HT-PEG4-5HT at 52 functional point mutations across 24 different positions within the S1/S2 binding region of hSERT, and calculated the mutation-induced effect on 5HT-PEG4-5HT for each mutant (Figure 5A and Table S2). At five positions (Ala173, Asn177, Ile179, Thr439 and Lys490), point mutations induced >5-fold changes in the potency of 5HT-PEG4-5HT (ranging from 5-fold increase to 23-fold decrease in potency), suggesting that these residues are important for binding of the bivalent ligand (Figure 5A). These five residues are located within the S1 (Ala173, Asn177, Thr439) and the S2 (Ile179, Lys490) site, suggesting that the ligand occupies both binding pockets in hSERT. Notably, Lys490 is located on the top of TM10 in the S2 site and the amino group of the Lys490 side chain is located > 11Å away from 5HT-PEG4-5HT in Cluster 1 (Figure 4), indicating that this cluster do not represent a functionally relevant binding pose. In contrast, the five residues are located in vicinity of the predicted binding poses in Cluster 2 and 3 (Figure 4), suggesting that one of these binding clusters represent the bioactive binding conformation of 5HT-PEG4-5HT in hSERT.


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[INSERT FIGURE 5] Mutation of Ala173, Asn177 and Thr439 within the central S1 site have previously been shown to affect binding of 5HT,50, 57 and since mutation of these residues also affect 5HTPEG4-5HT, our mutational data supports that one of the 5HT moieties of the bivalent compound bind similarly as 5HT itself in S1. Interestingly, although mutation of Asn177 and Thr439 induced >10-fold decrease in potency of 5HT-PEG4-5HT, the docking models collectively predicted that these residues do not form direct interactions to the ligand, but instead engage in a putative H-bond interaction to each other (Figure 4 and Figure 5). Notably, for N177E and T439S, in which the putative H-bond can be preserved, 5HT-PEG45HT retained high-affinity binding, substantiating that the suggested H-bond interaction could be important for binding of the bivalent compound. To address whether the putative Asn177Thr439 interaction influence binding of 5HT-PEG4-5HT, we performed a mutant cycle analysis59 to probe for a functional coupling between Asn177 and Thr439. Mutant cycle analyses are commonly quantified by the coupling coefficient (Ω) which is calculated by: Ω = [Ki(mutant a) × Ki(mutant b)] / [Ki(WT) × Ki(mutant a-b)]. When Ω =1, there is no coupling between two residues, whereas if Ω ≠ 1 it suggests coupling between the two mutated residues. To probe for coupling between Asn177 and Thr439 we determined the potency of 5HT-PEG4-5HT at N177A and T439V and the corresponding double mutant (N177AT439V) and found Ω = 5.7 (Figure 5C). We subsequently performed another mutant cycle analysis by using N177C and T439C to test for coupling between Asn177 and Thr439. Here, we found a comparable coupling coefficient (Ω = 6.6; Table S2), thus providing experimental evidence that binding of 5HT-PEG4-5HT is dependent on a coupling between Asn177 and Thr439. Within the S2 binding site, the I179C mutation induced 5-fold gain of potency for 5HTPEG4-5HT (Figure 5). In Cluster 2, the side-chain of Ile179 is located ~ 4Å from the



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ethylamino group of the 5HT moiety in the allosteric site, and the increased potency could be induced by an interaction between I179C and the ethylamino group of 5HT-PEG4-5HT. In contrast, the side-chain of Ile179 is located > 7Å away from the corresponding ethylamino group in Cluster 3, making a favourable interaction to I179C less likely. In Cluster 2 and 3, the 5HT moiety in the S2 site is sandwiched in between Pro403 and Lys490 (Figure 4), and introducing mutations into these positions (P403S, K490T or K490D) induce an increased potency of 5HT-PEG4-5HT (Figure 5A). For P403S, the gain of potency could arise from an interaction between P403S and the indole nitrogen of 5HT-PEG4-5HT in Cluster 2. A similar favourable interaction is not compatible with the proposed binding mode in Cluster 3, which further substantiate that Cluster 2 represent the bioactive binding conformation. Since the 5HT moiety in S2 is binding in between Pro403 and Lys490 in Cluster 2, we predicted that binding of 5HT-PEG4-5HT is dependent on a functional coupling between Pro403 and Lys490. To examine this hypothesis, we performed a mutant cycle analysis with P403S and K490D, and obtained a coupling coefficient of Ω = 0.13, which confirmed a coupling between these two S2 residues (Figure 5). Taken together, our mutational analysis suggests that Cluster 2 represent the bioactive binding mode of 5HT-PEG4-5HT in hSERT. In the proposed binding mode, the two 5HT moieties are ~16 Å apart (measured as the distance between the two indole nitrogen atoms) with one 5HT moiety binding in the S1 site and the second 5HT moiety binding in the S2 site (Figure 5). Our data thus strongly support that human MATs contain two distinct substrate binding sites, but since the bivalent compounds and the monovalent congeners are inhibitors of hSERT and stabilize an outward-facing conformational state (Figure 3), these molecules cannot be used to predict if the S2 constitute a transient permeation site for substrates en route to the S1 site, or if the S2 site needs to be occupied with substrate to trigger the conformational change from outward- to inward-facing conformations. Interestingly, comparison of the proposed binding


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mode of 5HT-PEG4-5HT with X-ray crystal structures of hSERT9 and LeuT25 suggests that the 5HT moiety of 5HT-PEG4-5HT in S2 partially overlaps with imipramine in the S2 site of LeuT, but the 5HT moiety share no overlap with the allosteric inhibitor binding site in hSERT (Figure S2). Importantly, our mutational analysis substantiated that the 5HT moiety in S2 is sandwiched in between the side chains of Pro403 and Lys490 (Figure 5), and our data thus indicate that it may be possible to target distinct subpockets within the large extracellular vestibule of hSERT. Notably, the sub-nanomolar affinity found for 5HT-PEG4-5HT indicates that it may be possible to design ligands with unprecedented high-affinity for the allosteric site in hSERT.

Putative TM3-TM8 interaction in hSERT is not conserved in hDAT. The putative H-bond interaction between Asn177 (TM3) and Thr439 (TM8) could be critical for binding of 5HTPEG4-5HT by maintaining the structural integrity of the S1 site. Notably, the suggested intramolecular interaction in hSERT is not conserved in hDAT (Asn177/Thr439 in hSERT is equivalent to Asn157/Ala423 in hDAT) (Figure 6A), and the suggested interaction could therefore be an important determinant for the distinct selectivity of 5HT-PEG4-5HT. To examine if mutations of Asn177 and Thr439 affects the conformational equilibrium of hSERT, we determined if N177A and T439V altered the accessibility of S404C (Figure 6B). Specifically, we introduced N177A or T439V into C109A-S404C, and found that neither the N177A nor the T439V mutation affected the reactivity of S404C towards MTSEA, suggesting that these mutations do not grossly affect the conformational equilibrium of hSERT (Figure 6B). Notably, from these experiments we cannot rule out that the N177A or the T439V mutation disrupts the structural integrity locally within the S1 site and/or the dynamic properties for the transition to inward-facing conformations. [INSERT FIGURE 6]



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Next, we examined if we could transfer the putative H-bond interaction from hSERT to hDAT, and thereby introduce a high-affinity binding site for 5HT-PEG4-5HT in hDAT, by mutating Ala423 in hDAT to a threonine (the identity of the equivalent residue in hSERT). However, the A423T mutation in hDAT did not affect the potency of 5HT-PEG4-5HT, and the N157A mutation in hDAT also induced a smaller effect on 5HT-PEG4-5HT compared to the equivalent N177A mutation in hSERT (Figure 6, Table S2 and Table S3). To gain further insight into how these mutations affected function of hSERT and hDAT, we performed [3H]5HT (for hSERT mutants) or [3H]DA (for hDAT mutants) saturation assays (Figure 6). Whereas N177A and T439V in hSERT significantly increased the Km value for 5HT [Km values in µM (mean ± SEM): 13.7 ± 2.9 for N177A, 20.4 ± 4.4 for T439V and 2.2 ± 0.3 for hSERT WT], N157A and A423T in hDAT did not affect the Km value for DA [Km values in µM (mean ± SEM): 1.3 ± 0.2 for N153A, 3.8 ± 1.1 for A423T and 1.6 ± 0.2 for hDAT WT]. Collectively, this demonstrates that Asn177/Thr439 in hSERT and Asn157/Ala423 in hDAT have differential roles for ligand recognition and uptake kinetics, thus highlighting a previously unappreciated difference between hSERT and hDAT.

Non-conserved binding site residues do not control selective binding of substrates. We have previously systematically examined the role of non-conserved residues within the central S1 site of hSERT and human NET (hNET), and found that they are important determinants for selective recognition of MAT inhibitors.16, 60-61 However, the role of non-conserved binding site residues for substrate selectivity in MATs has not yet been reported, and this prompted us to systematically examine the role of non-conserved S1 residues in hSERT and hDAT for substrate recognition. We employed an approach as previously described,61 and identified 19 residues within 8Å of the central S1 site that are non-conserved between hSERT and hDAT (Figure 7 and Table S4). We generated two mutant constructs, in which all non-


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conserved S1 residues were simultaneously interchanged between hSERT and hDAT, and thereby in principle transplanting the S1 binding site from hSERT into hDAT and vice versa [the two constructs were denoted SERT-(DAT S1) and DAT-(SERT S1), respectively]. The constructs bound [125I]RTI-55 with high affinity (Table S4), which allowed us to determine the affinity of 5HT, DA and 5HT-PEG4-5HT for the two hSERT/hDAT binding site chimeras. Surprisingly, the affinity of the three compounds was unaffected by introducing 19 hSERT-to-hDAT mutations into the S1 site of hSERT and vice versa (Figure 7). Similarly, to examine the role of non-conserved S2 residues for substrate recognition, we simultaneously interchanged 13 non-conserved residues surrounding the S2 sites in hSERT and hDAT (for specific mutations, see Table S4), and the selectivity for 5HT, DA and 5HT-PEG4-5HT was not reversed by the two S2 mutants [SERT-(DAT S2) and DAT-(SERT S2), respectively] (Table S4). Hence, although the binding site chimeras generally lost the capacity to complete substrate translocation (Table S5), they are capable of binding ligands and our data demonstrate that selective binding of 5HT, DA or 5HT-PEG4-5HT are not determined by non-conserved residues surrounding their proposed binding sites. [INSERT FIGURE 7] In summary, we have systematically designed and characterized a set of 24 substrate-based bivalent ligands, and in combination with mutagenesis, electrophysiology and cysteine accessibility measurements, we have revealed novel insight into the molecular basis for substrate recognition in hSERT and hDAT. Our study strongly suggests that human MATs contain two distinct substrate binding sites, and a systematic mutational analysis revealed that non-conserved binding site residues in hSERT and hDAT do not contribute to selective binding of substrates. Our study reveals that it may be possible to design ligands that target distinct subpockets within the large extracellular vestibule of hSERT, which could constitute



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an unexploited opportunity for development of drugs targeting the S2 site of hSERT with high affinity and selectivity.

SUPPORTING INFORMATION Supporting Figures (Figure S1-S2), Supporting Tables (Table S1-S5) and Methods.

ABBREVIATIONS 5HT, serotonin; hDAT, human dopamine transporter; hSERT, human serotonin transporter; IFD, induced-fit docking; MTSEA, (2-aminoethyl)methanethiosulfonate; TM, transmembrane segment; WT, wild-type.

AUTHOR INFORMATION *

Jacob Andersen. E-mail: [email protected].

*

Kristian Strømgaard. E-mail: [email protected].

§

Present address: Ontario Institute for Cancer Research, MaRS Centre, Ontario M5G 0A3,

Toronto, Canada. #

Present address: Department of Chemistry, University of Copenhagen, DK-1871

Frederiksberg C, Denmark.

Author contributions: J.A., A.S.K. and K.S. conceptualized the study. J.A., L.K.L., T.N.B.K., L.M. and J.G. performed the experiments. N.S.-H. provided new reagents and analytical tools. A.S.K., B.S. and K.S. supervised the research. J.A. prepared the figures and wrote the manuscript. All authors provided input to the manuscript and approved the final version.


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ACKNOWLEDGEMENTS We are grateful for financial support from the Lundbeck Foundation, the Carlsberg Foundation and the Danish Research Council for Independent Research, Medical Sciences. Computations were made possible through allocations at the Center for Scientific Computing, Aarhus (CSC-Aa).



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Page 24 of 33

Table 1. Binding affinity of 5HT, DA and compounds 1 – 40 for hSERT and hDAT. The binding affinity (Ki) was determined in [125I]RTI-55 competition binding assays. Data are presented as mean ± SEM from n independent experiments each performed in duplicate. The selectivity ratio was calculated as Ki(hSERT WT)/Ki(hDAT WT). Compound

hSERT WT nM

hDAT WT n

nM

hSERT/hDAT n

selectivity

Serotonin (5HT)

2,426

±

456

16

143,412

±

27,423

6

59

Dopamine (DA)

587,210

±

80,744

6

4,877

±

539

5

0.008

5HT-PEG3-5HT (1)

20.6

±

17.1

4

1,305

±

266

4

63

5HT-PEG4-5HT (2)

0.64

±

0.1

9

1,676

±

128

7

2,619

5HT-PEG5-5HT (3)

1.6

±

0.5

8

16,778

±

1,614

7

10,486

5HT-PEG6-5HT (4)

4.3

±

4.6

4

15,908

±

2,652

4

3,700

5HT-PEG3-DA (5)

13.6

±

2.0

3

90.9

±

26.1

3

7

5HT-PEG4-DA (6)

7.9

±

1.4

4

408

±

128

3

52

5HT-PEG5-DA (7)

80.7

±

5.2

4

1,040

±

216

4

13

5HT-PEG6-DA (8)

17.0

±

3.5

3

1,005

±

389

3

59

DA-PEG3-DA (9)

14,879

±

4,426

3

1,254

±

567

3

0.1

DA-PEG4-DA (10)

1,056

±

131

5

454

±

73.0

3

0.4

DA-PEG5-DA (11)

1,330

±

125

4

612

±

78.0

4

0.5

DA-PEG6-DA (12)

3,689

±

517

4

2,432

±

646

4

0.7

5HT-C8H16-5HT (13)

189

±

139

3

12,787

±

4,222

3

68

5HT-C10H20-5HT (14)

1.1

±

0.2

4

641

±

92.6

4

593

5HT-C12H24-5HT (15)

59.0

±

21.4

3

1,580

±

469

3

27

5HT-C14H28-5HT (16)

80.9

±

25.9

4

890

±

210

4

11

5HT-C8H16-DA (17)

15.0

±

4.7

6

717

±

74.4

6

48

5HT-C10H20-DA (18)

0.43

±

0.2

3

60.2

±

27.8

3

140

5HT-C12H24-DA (19)

3.3

±

1.2

4

179

±

52.9

4

54

5HT-C14H28-DA (20)

17.6

±

8.9

5

243

±

38.1

4

14

DA-C8H16-DA (21)

109

±

35.0

6

721

±

132

7

7

DA-C10H20-DA (22)

191

±

127

5

643

±

178

5

3

DA-C12H24-DA (23)

50.1

±

7.9

4

395

±

83.8

4

8

DA-C14H28-DA (24)

203

±

60.0

4

209

±

43.0

3

1

5HT-PEG2-Et (25)

29,859

±

3,216

4

82,342

±

5,966

4

3

5HT-PEG3-Et (26)

455

±

55.1

3

72,813

±

25,095

3

160

5HT-PEG4-Me (27)

1,910

±

783

4

82,683

±

20,199

4

43

5HT-PEG5-Me (28)

907

±

159

3

127,392

±

54,426

3

140

5HT-C8H17 (29)

137

±

36.4

3

19,633

±

4,230

3

143

5HT-C10H21 (30)

47.3

±

7.7

3

1,519

±

377

3

32

5HT-C12H25 (31)

103

±

30.7

3

2,381

±

178

3

23

5HT-C14H29 (32)

163

±

59.7

4

4,795

±

1,228

4

29

DA-PEG2-Et (33)

1,213,757

±

253,980

4

304,984

±

33,957

4

0.3

DA-PEG3-Et (34)

114,153

±

16,359

3

15,593

±

835

3

0.1

DA-PEG4-Me (35)

28,192

±

3,801

3

49,828

±

7,449

3

2

DA-PEG5-Me (36)

1,350

±

322

3

77,949

±

16,975

3

58

DA-C8H17 (37)

10,085

±

4,822

4

2,287

±

684

4

0.2

DA-C10H21 (38)

2,178

±

228

3

360

±

58.0

3

0.2

DA-C12H25 (39)

2,103

±

251

3

699

±

179

3

0.3

DA-C14H29 (40)

709

±

275

3

2,079

±

361

3

3


Page 25 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 1. Structure activity relationship study of bivalent compounds. (A) Left: Structure of the bivalent compounds. 5HT moieties are shown in red and DA moieties are shown in blue. See also Figure S1. Right: Binding affinity (Ki) of the PEG-linked (1 – 12) and alkyl-linked (13 – 24) compounds for hDAT (blue triangles) and hSERT (red circles). The binding affinities were determined in [125I]RTI-55 competition binding assays. See Table 1 for Ki values. (B–C) hDAT/hSERT selectivity ratio [calculated as Ki(hDAT / Ki(hSERT)] for the PEG-linked (B) and the alkyl-linked (C) compounds. Dashed lines indicate equal affinity for hSERT and hDAT (selectivity ratio = 1).



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 2. Structure activity relationship study of monovalent compounds. (A) Structure of 5HT-based (25 – 32) and DA-based (33 – 40) monovalent compounds. See also Figure S1. (B–C) Binding affinity of PEG-linked 5HT and DA compounds for hSERT and hDAT, respectively. Dashed lines indicate binding affinity of 5HT for hSERT (Ki = 2,426 nM) and DA for hDAT (Ki = 4,877 nM), respectively. (D–E) Binding affinity of alkyl-linked 5HT and DA compounds for hSERT and hDAT, respectively. Dashed lines indicate binding affinity of 5HT for hSERT and DA for hDAT, respectively. The binding affinities for hSERT (B and D) and hDAT (C and E) were determined in [125I]RTI-55 competition binding assays. See Table 1 for Ki values.


Page 26 of 33

Page 27 of 33

A

5HT-PEG4-5HT 5HT-PEG4-DA (2) (6)

5HT

DA-PEG4-DA (10)

5HT-PEG3-Et (26)

5 nA 20 s 5HT (10µM)

[3H]5HT uptake (% of control)

B 100

5HT-PEG4-5HT (2; 10 µM)

Control 5HT 5HT-PEG4-5HT (2) 5HT-PEG4-DA (6) DA-PEG4-DA (10) 5HT-PEG3-Et (26)

75 50 25 0 1

10

100

[MTSEA], µM

C Rate constant (% of MTSEA alone)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


250

*

200 150 100 50

*

*

* *

Control 5HT 5HT-PEG4-5HT (2) 5HT-PEG4-DA (6) DA-PEG4-DA (10) 5HT-PEG3-Et (26)

0

Figure 3. Mechanism of action for bivalent compounds at hSERT. (A) Representative twoelectrode voltage clamp recordings from Xenopus oocytes expressing hSERT during application of 10 µM of 5HT, 2, 6, 10 or 26. The experiments were repeated at least three times and showed similar results. (B) COS-7 cells transfected with hSERT C109A-S404C were treated with the indicated concentrations of MTSEA either alone or in the presence of 5HT (10 µM), 2 (1 µM), 6 (1 µM), 10 (10 µM) or 26 (20 µM), and the residual [3H]5HT uptake activity was determined. Data points represent mean ± SEM from a representative experiment performed with triplicate determinations. (C) From the IC50 values of MTSEA, rate constants for the reaction of MTSEA with S404C was calculated and expressed as the rate constant of MTSEA alone (rate constant for MTSEA alone was 269 ± 37 M-1s-1). Bars 27 Environment ACS Paragon Plus


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

represent mean ± SEM from at least four independent experiments each performed in triplicate. Asterisks indicate that the compound induce a significant change in the reaction rate of MTSEA (one-way analysis of variance; p < 0.05).


Page 28 of 33

Page 29 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


A

B EL4 allosteric site (S2)

out

A486 3 K490 P403

EL4

8 D400

1

D98

6

10 I179 V489 N177

central site (S1)

A173

3 8 10 6 1

Y176 T439

F335

A169

Y95

in

C

Na1 Na2

F341

D

A486 3 K490 P403 EL4

8 D400

1

A486 3 K490 P403 EL4

8 D400

1

10

10

I179

I179 V489

V489 D98

6

N177 Y176 A173

T439

A169

Na2

Y95

6

Y176

Na1

Na1 Na2

T439

F335

F341

D98 N177

A173

A169

F335 Y95 F341

Figure 4. Docking models of 5HT-PEG4-5HT in hSERT. (A) Schematic representation of hSERT showing TM and loop regions surrounding the S1 and S2 binding sites. 5HT is shown as stick representation within the central binding site in the proposed bioactive binding conformation.57 (B-D) Global binding clusters obtained from IFD simulations of 5HT-PEG45HT into hSERT homology model. A representative binding pose of 5HT-PEG4-5HT from Cluster 1 (B), Cluster 2 (C) and Cluster 3 (D) is shown in yellow, and selected residues in proximity of the of the proposed binding modes are shown as sticks. See Table S1 for data from IFDs.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

A

B

E493 K490 V489 A486 T439 S438 P403 D400 V343 F341 F335 Y232 W182 I179 N177 Y176 Y175 A173 I172 F170 A169 W103 D98 Y95

OH

A E QF T D F EI V

C

HO

HN

O

N H

O

O

C

A I AE S K N

D 8

YW IW A AF A

P403 3

10 K490

10

D400

180° N177

D98

I

100

Na2 Na1

E F F I G YQF M L S D A E A WF

10

1

8

I179 V489

N177 T439

T439

Y95

F335 Y95

F341

13nM

279nM

hSERT WT

N177A

100

T439V

Fold decrease in Ki

F335 F341

3 13nM

hSERT WT

Ω = 5.7 10

1

D98

1

151nM

Fold increase in Ki

D400

P403

V489

C GA C S

NH

N H

5HT-PEG4-5HT (2)

AS T

Page 30 of 33

6.6nM

P403S

Ω = 0.13

N177AT439V 571nM

K490D 2.5nM

P403SK490D 10nM

Figure 5. Mutational analysis of binding site residues in hSERT. (A) Graphical summary of fold-change in potency of 5HT-PEG4-5HT (shown on x-axis) induced by point mutations in residues surrounding the proposed binding modes of the bivalent compound (shown on yaxis). Data represent mean fold-change from at least three independent experiments. The fold change was calculated as Ki(WT)/Ki(mutant) or Ki(mutant)/Ki(WT) for mutations increasing or decreasing 5HT-PEG4-5HT potency, respectively. Grey shaded region indicates < 3-fold change in potency. Grey shading of data points specifies that the mutation induce a significant change in 5HT-PEG4-5HT Ki (Student’s t-test; p < 0.05). See also Table S2. (B) Structure of 5HT-PEG4-5HT. (C-D) Top: Close-up views of a representative binding pose of 5HT-PEG45HT in hSERT (Cluster 2). Selected binding site residues are shown as stick representations, and the putative H-bond between Asn177 and Thr439 is indicated by stippled line. Bottom: Schematic of mutant cycle analyses of N177A/T439V (C) and P403S/K490D (D). Ki for 5HT-PEG4-5HT is shown in grey, and the coupling coefficient (Ω) is shown at the center of the schematics.


D

C

TM3

hSERT CIIAFYIASYYNTIMAWALYYLIS hDAT ILISLYVGFFYNVIIAWALHYLFS dDAT VLIAFYVDFYYNVIIAWSLRFFFA +

TM8 hSERT FLMLITLGLDSTFAGLEGVITAVL hDAT FIMLLTLGIDSAMGGMESVITGLI dDAT FMMLLTLGLDSSFGGSEAIITALS +

100 75 50 25 0

hSERT WT N177A T439V

E S404C S404C-N177A S404C-T439V

1 10 100 [MTSEA], µM

10 2 104 106 [5HT-PEG4-5HT], nM

F 100

[3H]DA uptake (in % of Vmax)

100 75 50 25 0

hDAT WT N157A A423T

100 75 50 25 0

100 102 10 4 [5HT-PEG4-5HT], nM

[3H]5HT uptake (in % of Vmax)

B

[3H]5HT uptake (% of control)

A

[3H5HT uptake (% of control)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


[3H]DA uptake (% of control)

Page 31 of 33

75 50 25 0 0 4 8 12 16 20 [5HT], µM

100 75 50 25 0 0 2 4 6 8 10 [DA], µM

Figure 6. Putative intramolecular interaction in hSERT is not conserved in hDAT. (A) Amino acid sequence alignment of hSERT, hDAT and dDAT of residues in TM3 (top) and TM8 (bottom). Positions corresponding to Asn177 (TM3) and Thr439 (TM8) in hSERT are indicated. (B) COS-7 cells transfected with hSERT S404C, S404C-N177A or S404C-T439V in the C109A background were treated with MTSEA and the residual [3H]5HT uptake activity was subsequently determined. Data points represent mean ± SEM from a representative experiment performed with triplicate determinations. (C-D) WT or mutant transporters was expressed in COS-7 cells, and potency of 5HT-PEG4-5HT was determined in [3H]5HT or [3H]DA uptake inhibition assays for hSERT and hDAT, respectively. Data points represent mean ± SEM from a representative experiment performed with triplicate determinations. See also Table S2 and Table S3. (E-F) WT or mutant transporters was expressed in COS-7 cells, and the uptake kinetics was determined in [3H]5HT or [3H]DA uptake saturation assays for hSERT and hDAT, respectively.



A

B out

Central site (S1)

TM3

3

Y175

G100 A173

S174 C473 F170

TM10

T439

L443 I172 T497 A169 C166 G445 V501

TM8 TM6

10

F334

8 A441 Y95

1 I333

I165

in

C Binding affinity (nM)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

104

ns

103

100

ns

105

102 101

106

104 ns

103

6

ns hSERT WT SERT-(DAT S1) ns

104

hDAT WT DAT-(SERT S1)

103

102

5HT-PEG4-5HT

106 105

ns

Page 32 of 33

102

5HT

DA

Figure 7. Role of non-conserved binding site residues for substrate selectivity. (A) Crosssectional illustration of the proposed binding mode of 5HT-PEG4-5HT in hSERT (Cluster 2). (B) Close up view of the S1 site in the 5HT-PEG4-5HT binding model. The 19 nonconserved hSERT/hDAT S1 residues are shown as sticks (hSERT numbering). The nonconserved residues were identified from the X-ray crystal structure of dDAT as described previously.61 (C) The 19 non-conserved hSERT/hDAT S1 residues were simultaneously mutated to the identity of the corresponding residues in the other transporter, and the binding affinity of 5HT-PEG4-5HT, 5HT and DA was determined in [125I]RTI-55 competition binding assays. Bars represent mean ± SEM from at least three independent experiments each performed in duplicate. ns = no significant difference (Student’s t-test; p > 0.05). See also Table S4.


Page 33 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


TOC GRAPHIC OH

S2 site

HN NH 2

Serotonin human SERT K i = 2,426 nM HO

OH

HN N H

O

O

O

NH N H

S1 site

Bivalent serotonin ligand human SERT K i = 0.64 nM


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