N-phenylalkyl-substituted tropane analogs of boat conformation with high selectivity for the dopamine versus serotonin transporter

A series of N-phenylalkyl-substituted tropane analogs of boat conformation was synthesized, and these tropanes were evaluated for their ability to inhibit high affinity uptake of dopamine (DA) and serotonin (5-HT) into striatal nerve endings (synaptosomes). Some of these compounds exhibit high affinity for the DA transporter with a 5-HT/DA transporter selectivity ratio of >50.     

Synthesis of tropane and nortropane analogues with phenyl substitutions as serotonin transporter ligands.

The effects of structural modifications of 2 beta-carbomethoxy-3 beta-phenyl tropane analogues were evaluated on in vitro affinity to the dopamine (DAT) and serotonin (5-HTT) transporters in rat brain tissue. The introduction of a large alkyl group at the 4'-position of the phenyl ring, affording 2 beta-carbomethoxy-3 beta-(4'-alkylphenyl) tropane, diminished the affinity for the DAT whereas moderate 5-HTT affinity was obtained. The introduction of an iodine at the 3'-position of the 4'-alkylphenyl, affording 2 beta-carbomethoxy-3 beta-(3'-iodo-4'-alkylphenyl) tropane, and N-demethylation, affording 2 beta-carbomethoxy-3 beta-(3'-iodo-4'-alkylphenyl) nortropane, improved affinity and specificity for the 5-HTT. It could be assumed from these results that the combination of these three modifications of tropane structure yielded highly selective compounds for the 5-HTT. Of the new compounds synthesized, the most selective cocaine derivative, 2 beta-carbomethoxy-3 beta-(3'-iodo-4'-isopropylphenyl) nortropane (8d) labeled with iodine-123 or carbon-11, could be a potential ligand for exploration of the 5-HT transporter by SPET or PET.     

Comparative structure-activity relationships of benztropine analogues at the dopamine transporter and histamine H(1) receptors

Benztropine (BZT) and its analogues inhibit dopamine uptake and bind with moderate to high affinity to the dopamine transporter (DAT). However, many of these compounds, in contrast to other monoamine uptake inhibitors, lack cocaine-like behavioral effects and fail to potentiate the effects of cocaine. The BZT analogues also exhibit varied binding affinities for muscarinic M(1) and histamine H(1) receptors. In this study, a comparative analysis was conducted of pharmacophoric features with respect to the activities of BZT analogues at the DAT and at the histamine H(1) receptor. The BZT analogues showed a wide range of histamine H(1) receptor (K(i)=16-37,600 nM) and DAT (K(i)=8.5-6370 nM) binding affinities. A stereoselective histamine H(1)-antagonist pharmacophore, using a five-point superimposition of classical antagonists on the template, cyproheptadine, was developed. A series of superimpositions and comparisons were performed with various analogues of BZT. In general, smaller substituents were well tolerated on the aromatic rings of the diphenyl methoxy group for both the DAT and H(1) receptor, however, for the H(1) receptor, substitution at only one of the aromatic rings was preferred. The substituents at the 2- and N-positions of the tropane ring were preferred for DAT, however, these groups seem to overlap receptor essential regions in the histamine H(1) receptor. Molecular models at the DAT and the histamine H(1) receptor provide further insight into the structural requirements for binding affinity and selectivity that can be implemented in future drug design.     

Synthesis of 8-thiabicyclo[3.2.1]oct-2-enes and their binding affinity for the dopamine and serotonin transporters

The reinforcing and stimulant properties of cocaine have been primarily associated with its propensity to bind to monoamine transport systems, in particular the dopamine transporter. Inhibition of the dopamine transporter then leads to an increase of synaptic dopamine with substantial pharmacological consequences. The search for medications for cocaine abuse has had a particular focus on tropane analogs of cocaine, and the interchange of nitrogen for oxygen in this class has led to potent and selective inhibitors of monoamine transport. Herein we report that 8-thiatrop-2-enes are highly potent and quite selective inhibitors of the dopamine transporter. The 3,4-dichlorophenyl-8-thiabicyclo[3.2.1]oct-2-ene (4f) is particularly potent (IC₅₀ = 4.5 nM) and selective (800-fold) with respect to inhibition of the serotonin transporter.     

Synthesis and evaluation of a series of tropane analogues as novel vesicular monoamine transporter-2 ligands

A series of tropane derivatives has been synthesized as lobelane analogues and evaluated for their binding affinity at the vesicular monoamine transporter-2 (VMAT2), and at alpha4beta2* and alpha7* nicotinic acetylcholine receptors. The trop-2-ene analogues 4a and 4b exhibited good affinity and high selectivity for VMAT2.     

Synthesis of dopamine transporter selective 3-diarylmethoxymethyl-8-arylalkyl-8-azabicyclo[3.2.1]octane derivatives

A series of diarylmethoxymethyltropane-GBR hybrid analogues with all three possible stereochemical orientations at C3 were synthesized and evaluated at dopamine and serotonin transporters. The 3alpha derivatives were found to be the most potent compounds with the 3alpha-di(4-fluorophenyl)methoxymethyl-8-(3-phenylpropyl)-8-azabicyclo[3.2.1]octane 15b (Ki = 5 nM) being the most potent compound of the series. The corresponding 3-di(4-fluorophenyl)-methoxymethyl-8-(3-phenylpropyl)-8-azabicyclo[3.2.1]oct-2-ene 12b (Ki = 12 nM) was slightly less potent than the 3alpha-analogue, while the 3beta-di(4-fluorophenyl)methoxymethyl-8-(3-phenylpropyl)-8-azabicyclo[3.2.1]octane 23b (Ki = 78 nM) exhibited only modest affinity for the dopamine transporter. Only the 3alpha-analogue 15b (SERT/DAT = 48) exhibited higher SERT/DAT selectivity than GBR 12909. These results indicate that the dopamine transporter can tolerate some variability in proximity of the benzhydryl ether to the basic nitrogen atom of the tropane without loss in potency. In addition, the structure-activity data for these tropane-GBR 12909 hybrid analogues support previous findings that the stereochemical and conformational effects imparted by unsaturation at C3 are important for dopamine transporter selectivity over the serotonin transporter.     

Synuclein modulation of monoamine transporters

Although well-studied in the context of neurodegenerative disease, a clear biological function for the synuclein proteins remains elusive. Emerging data indicate a role for synucleins in monoamine neurotransmitter homeostasis. A key regulatory component of monoamine neurotransmission is re-uptake of neurotransmitter by the dopamine transporter, norepinephrine transporter, and serotonin transporter, which are common drug targets in the treatment of depression and other mood disorders. Through interactions with these transporters, the neuronal cytoskeleton, and pre-synaptic scaffolding proteins, α-synuclein, β-synuclein, and γ-synuclein modulate trafficking, expression and function of monoamine transporters at the cell surface, thus playing a central role in regulating monoamine re-uptake.     

The conserved glutamate (Glu136) in transmembrane domain 2 of the serotonin transporter is required for the conformational switch in the transport cycle

The alternate access model provides the theoretical framework for understanding how transporters translocate hydrophilic substrates across the lipid bilayer. The model postulates at least two conformations of a transporter, an outward and an inward facing conformation, which seal the translocation pathway to the interior and exterior of the cell, respectively. It is not clear how the conformational switch is triggered in neurotransmitter/sodium symporters, but Na+ is likely to play an essential role. Here, we focused on Glu136 of the serotonin transporter (SERT); this residue is conserved in transmembrane domain 2 of neurotransmitter/sodium symporters and related proteins. Three substitutions were introduced, resulting in SERT-E136D, SERT-E136Q, and SERT-E136A, which were all correctly inserted into the plasma membrane. SERT-E136Q and SERT-E136A failed to support substrate influx into cells, whereas SERT-E136D did so at a reduced rate. Binding experiments with the inhibitor 2beta-[3H]carbomethoxy-3beta-(4-iodophenyl)tropane (beta-[3H]CIT) supported the conjecture that the mutant transporters preferentially adopted the inward facing conformation: beta-[3H]CIT interacted with SERT in a manner consistent with binding to the outward facing state. Accordingly, the Na+-induced acceleration of beta-[3H]CIT association was most pronounced in wild-type SERT, followed by SERT-E136D > SERT-E136Q > SERT-E136A. Similarly, SERT-E136Q supported substrate efflux in a manner indistinguishable from wild-type SERT, whereas SERT-E136A was inactive. Thus, in the absence of Glu136, the conformational equilibrium of SERT is shifted progressively (SERT-E136D > SERT-E136Q > SERT-E136A) to the inward facing conformation.     

Synthesis and monoamine transporter affinity of new 2beta-carbomethoxy-3beta-[4-(substituted thiophenyl)]phenyltropanes: discovery of a selective SERT antagonist with picomolar potency

Preparation of cocaine analogues has been aimed largely at development of stable compounds with high affinity and selectivity for the dopamine transporter (DAT). We now report the synthesis and monoamine transporter affinity of 10 new 2beta-carbomethoxy-3beta-[4-(substituted thiophenyl)]phenyltropanes. Among these, compound 4b exhibited very high affinity for the serotonin transporter (SERT: K(i)=17 pM) and good selectivity over dopamine (DAT: 710-fold) and norepinephrine transporters (NET: 11,100-fold).     

Ligand Induced Conformational Changes of the Human Serotonin Transporter Revealed by Molecular Dynamics Simulations

The competitive inhibitor cocaine and the non-competitive inhibitor ibogaine induce different conformational states of the human serotonin transporter. It has been shown from accessibility experiments that cocaine mainly induces an outward-facing conformation, while the non-competitive inhibitor ibogaine, and its active metabolite noribogaine, have been proposed to induce an inward-facing conformation of the human serotonin transporter similar to what has been observed for the endogenous substrate, serotonin. The ligand induced conformational changes within the human serotonin transporter caused by these three different types of ligands, substrate, non-competitive and competitive inhibitors, are studied from multiple atomistic molecular dynamics simulations initiated from a homology model of the human serotonin transporter. The results reveal that diverse conformations of the human serotonin transporter are captured from the molecular dynamics simulations depending on the type of the ligand bound. The inward-facing conformation of the human serotonin transporter is reached with noribogaine bound, and this state resembles a previously identified inward-facing conformation of the human serotonin transporter obtained from molecular dynamics simulation with bound substrate, but also a recently published inward-facing conformation of a bacterial homolog, the leucine transporter from Aquifex Aoelicus. The differences observed in ligand induced behavior are found to originate from different interaction patterns between the ligands and the protein. Such atomic-level understanding of how an inhibitor can dictate the conformational response of a transporter by ligand binding may be of great importance for future drug design.     

Structural dynamics of the monoamine transporter homolog LeuT from accelerated conformational sampling and channel analysis

The bacterial leucine transporter LeuT retains significant secondary structure similarities to the human monoamine transporters (MAT) such as the dopamine and serotonin reuptake proteins. The primary method of computational study of the MATs has been through the use of the crystallized LeuT structure. Different conformations of LeuT can give insight into mechanistic details of the MAT family. A conformational sampling performed through accelerated molecular dynamics simulations testing different combinations of the leucine substrate and bound sodium ions revealed seven distinct conformational clusters. Further analysis has been performed to target salt‐bridge residues R30–D404, Y108–F253, and R5–D369 and transmembrane domains on both the seven isolated structures and the total trajectories. In addition, solvent accessibility of LeuT and its substrate binding pockets has been analyzed using a program for calculating channel radii. Occupation of the Na2 site stabilizes the outward conformation and should bind to the open outward conformation before the leucine and Na1 sodium while two possible pathways were found to be available for intracellular transport. Proteins 2014; 82:2289–2302. © 2014 Wiley Periodicals, Inc.     

3-(Arylamino)-3-phenylpropan-2-olamines as a new series of dual norepinephrine and serotonin reuptake inhibitors

A series of 3-(arylamino)-3-phenylpropan-2-olamines was prepared and screened for their ability to inhibit monoamine reuptake. A number of analogues displayed significant dual norepinephrine and serotonin reuptake inhibition. Compounds in this class exhibited minimal affinity for the dopamine transporter.     

3H]MFZ 2-12: a novel radioligand for the dopamine transporter.

In an effort to develop a tritiated dopamine transporter radioligand with higher affinity than the widely used [(3)H]WIN 35,428, we have synthesized [(3)H]2beta-carbomethoxy-3beta-(3',4'-dichlorophenyl)tropane ([(3)H]MFZ 2-12). Unlabeled MFZ 2-12 and the N-demethylated intermediate (MFZ 2-13) inhibited dopamine uptake by the human dopamine transporter with IC(50)'s of 1.1 and 1.4nM, respectively. The N-nor-intermediate (MFZ 2-13) was treated with CT(3)I resulting in [(3)H]MFZ 2-12; S.A.=80 Ci/mmol). [(3)H]MFZ 2-12 reversibly bound with a K(D) of 2.8nM to human dopamine transporter expressed heterologously in EM4 cells.     

Characterization of the bioactive conformation of the C-terminal tripeptide Gly-Leu-Met-NH2 of substance P using [3-prolinoleucine10]SP analogues.

Residue Leu10 of substance P (SP) is critical for NK-1 receptor recognition and agonist activity. In order to probe the bioactive conformation of this residue, cis- and trans-3-substituted prolinoleucines were introduced in position 10 of SP. The substituted SP analogues were tested for their affinity to human NK-1 receptor specific binding sites (NK-1M and NK-1m) and their potency to stimulate adenylate cyclase and phospholipase C in CHO cells transfected with the human NK-1 receptor. [trans-3-prolinoleucine10]SP retained affinity and potency similar to SP whereas [cis-3-prolinoleucine10]SP shows dramatic loss of affinity and potency. To analyze the structural implications of these biological results, the conformational preferences of the SP analogues were analyzed by NMR spectroscopy and minimum-energy conformers of Ac-cis-3-prolinoleucine-NHMe, Ac-trans-3-prolinoleucine-NHMe and model dipeptides were generated by molecular mechanics calculations. From NMR and modeling studies it can be proposed that residue Leu10 of SP adopts a gauche(+) conformation around the chi1 angle and a trans conformation around the chi2 angle in the bioactive conformation. Together with previously published results, our data indicate that the C-terminal SP tripeptide should preferentially adopt an extended conformation or a PPII helical structure when bound to the receptor.     

Cocaine inhibition of ligand binding at dopamine, norepinephrine and serotonin transporters: a structure-activity study

Structure-activity relationships for cocaine and analog binding at the dopamine, norepinephrine and serotonin transporters were determined. Cocaine inhibition of ligand binding to each of these sites has a stereospecific requirement for the levorotatory isomer. Binding potencies of cocaine derivatives involving N-substitution, C2 and C3 substituent modifications, however, revealed differences in structure-activity relationships for cocaine binding at the transporters. Removal of the N-methyl groups produced little change in binding potency at the dopamine transporter site but produced increases in binding potency at norepinephrine and serotonin transporter sites. Changes in structure at the C2 substituent produced changes in binding potency at the dopamine transporter which were generally similar in direction, but not necessarily in magnitude at the norepinephrine and serotonin transporters. Modifications to the C3 substituent, especially substitution of a hydroxyl moiety, produce changes in affinity at norepinephrine and serotonin transporters which are much larger than those observed at dopamine transporters. In general, our results indicate that unique structural requirements exist for each transporter site, but that cocaine binding at norepinephrine and dopamine transporters can be described by more similar structure-activity relationships than those found for the serotonin transporter. Requirements for cocaine binding to the dopamine transporter, which we have previously shown to be associated with the reinforcing effects of cocaine, include levorotatory stereospecificity, the benzene ring at C3, at least some portions of the tropane ring, and the presence of the C2 methyl ester group in the beta conformation.     

Cysteine-scanning mutagenesis of serotonin transporter intracellular loop 2 suggests an alpha-helical conformation

Like other proteins involved in neurotransmitter transport, serotonin transporter (SERT) activity is regulated by multiple intracellular signal transduction pathways. The second intracellular loop (IL2) of SERT contains consensus sequences for cGMP-dependent protein kinase and protein kinase C. A 24-residue region of SERT including IL2, from Ile-270 through Ser-293, was analyzed by cysteine-scanning mutagenesis and chemical modification. 2-(Aminoethyl)methanethiosulfonate hydrobromide (MTSEA) failed to inhibit serotonin transport or binding of the cocaine analog 2beta-carbomethoxy-3beta-(4-[125I]iodophenyl)tropane (beta-CIT) in intact cells expressing these mutants, but it inactivated beta-CIT binding in membrane preparations. From the pattern of sensitivity, IL2 appears to extend from Trp-271 through Ile-290, a significantly longer region than that initially predicted by hydropathy analysis. Six mutants reacted with MTSEA much faster than the others, and the pattern of the more reactive mutations suggested that IL2 is in an alpha-helical conformation. Some of the mutants had significantly elevated transport rates, suggesting a possible mechanism for the regulation of SERT activity.     

Functional coupling of serotonin and noradrenaline transporters

Re-uptake of the neurotransmitters serotonin and noradrenaline out of the synaptic cleft is mediated by selective transporter proteins, the serotonin transporter and the noradrenaline transporter respectively. Both are integral membrane proteins that are have a high degree of homology and represent members of a larger neurotransmitter transporter superfamily. Several studies have indicated that the serotonin transporter has an an oligomeric structure. To determine whether monoamine transporters can also function in oligomeric structures in situ, we constructed a concatenate consisting of one molecule of serotonin transporter covalently linked to one molecule of noradrenaline transporter. Heterologous expression of this hybrid construct allowed us to analyse the function, i.e. transport activity, and the structure, i.e. the molecular weight of the total construct and of its single components, at the same time. We showed that serotonin-noradrenaline transporter fusion proteins are fully active and exhibit the pharmacological profile of both their individual components. These findings support the hypothesis that monoamine transporters are expressed and may function as oligomeric proteins composed of non-interacting monomers.     

Dehydroquinate synthase: the use of substrate analogues to probe the early steps of the catalyzed reaction

The early steps of the proposed mechanistic pathway for dehydroquinate synthase have been probed with a series of substrate analogues. These analogues, 3-9, are structurally prohibited from undergoing the beta-elimination of inorganic phosphate that represents the committed step in the conversion of the substrate 3-deoxy-D-arabino-heptulosonate 7-phosphate (1) to dehydroquinate (2). In agreement with previous observations, the analogues that possess shortened side chains (3,5, and 6) bind more tightly to the enzyme than those (4 and 7-9) that are more nearly isosteric with the substrate. Two hitherto unrecognized factors that influence binding have been identified: (i) carbacylic analogues bind 25-100 times more tightly than the corresponding oxacyclic materials (indeed, the carbacyclic phosphonate 5 has a Ki value of 8 x 10(-10)M) and (ii) the side chain appears to be bound in a gauche conformation similar to the most stable conformation of the cis-vinylhomophosphonate 8. These trends in binding can be rationalized by considering the behavior of the analogues in the first two chemical steps of the mechanism: NAD+-mediated oxidation at C-5 and enolization at C-6 (the first part of the E1cB elimination of inorganic phosphate). Direct spectrophotometric determination of the equilibrium level of enzyme-bound NADH indicates that the carbacyclic analogues are more readily oxidized than the oxacyclic compounds, and this predictable difference in redox behavior is reflected in the observed differences in binding. The gauche conformation of the C-7 side chain appears to be required for proton abstraction from C-6, since only those analogues that can adopt this conformation undergo enzyme-catalyzed exchange of the C-6 proton with the solvent. This conformation positions one of the peripheral oxygens of the phosphate (or phosphonate) group close to the C-6 proton. Taken together with other data, these results suggest that the enzyme exploits this substrate base in the enolization, which occurs through an intramolecular proton transfer. The loss of Pi then completes the beta-elimination.     

Synthesis and monoamine transporter affinity of 3'-analogs of 2-beta-carbomethoxy-3-beta-(4'-iodophenyl)tropane (beta-CIT)

The 3'-iodo positional isomer of 2-beta-carbomethoxy-3-beta-(4'-iodophenyl)tropane (beta-CIT) and other 3'-substituted analogs were synthesized and evaluated for binding to monoamine transporters in rat forebrain and membranes of cell lines selectively expressing human transporter genes. All 3'-substituted compounds displayed affinity for both serotonin (SERT) and dopamine (DAT), but much less for norepinephrine transporters (NET), with selectivity for rat (r) or human (h) SERT over NET, but only 3'-iodo-substituted phenyltropanes showed selectivity for SERT versus DAT. The 3'-iodo, N-methyl analog of beta-CIT (7) displayed 29-fold selectivity and high affinity for hSERT (K(i) =9.6 nM) over hDAT (K(i) =279 nM), and its nor-congener (8) showed even higher hSERT potency (K(i) =1.2 nM) and selectivity over DAT (415-fold).