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

Inhibitors of the serotonin transporter (SERT) and norepinephrine transporter (NET) are widely used in the treatment of major depressive disorder. Although SERT/NET selectivity is a key determinant for the therapeutic properties of these drugs, the molecular determinants defining SERT/NET selectivity are poorly understood. In this study, the structural basis for selectivity of the SERT selective inhibitor citalopram and the structurally closely related NET selective inhibitor talopram is delineated. A systematic structure-activity relationship study allowed identification of the substituents that control activity and selectivity toward SERT and NET and revealed a common pattern showing that SERT and NET have opposite preference for the stereochemical configuration of these inhibitors. Mutational analysis of nonconserved SERT/NET residues within the central substrate binding site was performed to determine the molecular basis for inhibitor selectivity. Changing only five residues in NET to the complementary residues in SERT transferred a SERT-like affinity profile for R- and S-citalopram into NET, showing that the selectivity of these compounds is determined by amino acid differences in the central binding site of the transporters. In contrast, the activity of R- and S-talopram was largely unaffected by any mutations within the central substrate binding site of SERT and NET and in the outer vestibule of NET, suggesting that citalopram and talopram bind to distinct sites on SERT and NET. Together, these findings provide important insight into the molecular basis for SERT/NET selectivity of antidepressants, which can be used to guide rational development of unique transporter inhibitors with fine-tuned transporter selectivity.     

Permeation and gating residues in serotonin transporter

The third transmembrane domain (TM3) of serotonin transporter (SERT) contains two isoleucine residues previously proposed to be involved in binding and transport of serotonin. When Ile-172 was replaced with cysteine, SERT became sensitive to inactivation by externally added [2-(trimethylammonium)ethyl]methanethio-sulfonate (MTSET). The disulfide product of this inactivation was not sensitive to reduction by externally added sulfhydryl compounds, but apparently reacted with intracellular reducing agents to spontaneously regenerate active SERT. The apparent accessibility of this residue to both external and cytoplasmic reagents is consistent with its localization near a serotonin binding site that is alternately exposed to both internal and external media. In another SERT mutant, I179C, transport also was inactivated by MTSET but substrate binding was resistant. External substrate bound to the inactivated I179C and enhanced its reactivation by free thiols. In norepinephrine transporter (NET), cysteine replacement of Ile-155 (corresponding to SERT Ile-179) also rendered the transporter sensitive to MTSET inactivation. In NET I155C, cocaine enhanced this inactivation, and the substrate, dopamine, apparently protected against inactivation. The characteristics of this protection suggest that dopamine was transported, converting NET to a form in which Ile-155 was occluded. The results support the proposal that TM3 of SERT and NET constitute part of the substrate permeation pathway, and that Ile-172 in SERT resides close to the substrate binding site. They also suggest that Ile-179 in SERT (and Ile-155 in NET) is in a conformationally sensitive part of TM3, which may act as part of an external gate.     

Abolished cocaine reward in mice with a cocaine-insensitive dopamine transporter

There are three known high-affinity targets for cocaine: the dopamine transporter (DAT), the serotonin transporter (SERT), and the norepinephrine transporter (NET). Decades of studies support the dopamine (DA) hypothesis that the blockade of DAT and the subsequent increase in extracellular DA primarily mediate cocaine reward and reinforcement. Contrary to expectations, DAT knockout (DAT-KO) mice and SERT or NET knockout mice still self-administer cocaine and/or display conditioned place preference (CPP) to cocaine, which led to the reevaluation of the DA hypothesis and the proposal of redundant reward pathways. To study the role of DAT in cocaine reward, we have generated a knockin mouse line carrying a functional DAT that is insensitive to cocaine. In these mice, cocaine suppressed locomotor activity, did not elevate extracellular DA in the nucleus accumbens, and did not produce reward as measured by CPP. This result suggests that blockade of DAT is necessary for cocaine reward in mice with a functional DAT. This mouse model is unique in that it is specifically designed to differentiate the role of DAT from the roles of NET and SERT in cocaine-induced biochemical and behavioral effects.     

Targeted rescue of a destabilized mutant of p53 by an in silico screened drug

The tumor suppressor p53 is mutationally inactivated in ≈50% of human cancers. Approximately one-third of the mutations lower the melting temperature of the protein, leading to its rapid denaturation. Small molecules that bind to those mutants and stabilize them could be effective anticancer drugs. The mutation Y220C, which occurs in ≈75,000 new cancer cases per annum, creates a surface cavity that destabilizes the protein by 4 kcal/mol, at a site that is not functional. We have designed a series of binding molecules from an in silico analysis of the crystal structure using virtual screening and rational drug design. One of them, a carbazole derivative (PhiKan083), binds to the cavity with a dissociation constant of ≈150 μM. It raises the melting temperature of the mutant and slows down its rate of denaturation. We have solved the crystal structure of the protein–PhiKan083 complex at 1.5-Å resolution. The structure implicates key interactions between the protein and ligand and conformational changes that occur on binding, which will provide a basis for lead optimization. The Y220C mutant is an excellent “druggable” target for developing and testing novel anticancer drugs based on protein stabilization. We point out some general principles in relationships between binding constants, raising of melting temperatures, and increase of protein half-lives by stabilizing ligands.     

Predicted 3D structure for the human beta 2 adrenergic receptor and its binding site for agonists and antagonists

We report the 3D structure of human beta2 adrenergic receptor (AR) predicted by using the MembStruk first principles method. To validate this structure, we use the HierDock first principles method to predict the ligand-binding sites for epinephrine and norepinephrine and for eight other ligands, including agonists and antagonists to beta 2 AR and ligands not observed to bind to beta 2 AR. The binding sites agree well with available mutagenesis data, and the calculated relative binding energies correlate reasonably with measured binding affinities. In addition, we find characteristic differences in the predicted binding sites of known agonists and antagonists that allow us to infer the likely activity of other ligands. The predicted ligand-binding properties validate the methods used to predict the 3D structure and function. This validation is a successful step toward applying these procedures to predict the 3D structures and function of the other eight subtypes of ARs, which should enable the development of subtype-specific antagonists and agonists with reduced side effects.     

Norepinephrine transporter inhibition prevents tilt-induced pre-syncope.

OBJECTIVES: We tested the hypothesis that pharmacological norepinephrine reuptake transporter (NET) inhibition delays the onset of head-up tilt-induced presyncope in healthy subjects. BACKGROUND: Treatment of neurally mediated syncope is unsatisfactory. In a previous study in a small number of healthy subjects, pharmacologic NET inhibition delayed the onset of head-up tilt-induced pre-syncope. METHODS: We combined data sets from 3 substudies comprising 51 healthy subjects without a history of syncope. In a double-blind, randomized, cross-over fashion, subjects underwent 2 head-up tilt tests, once with placebo and once with a NET inhibitor (sibutramine or reboxetine). Tilt testing was prematurely ended when pre-syncopal symptoms such as dizziness, nausea, or visual disturbances occurred together with a decrease in blood pressure and/or heart rate. RESULTS: The mean tolerated tilt test duration was 29 +/- 2 min with placebo and 35 +/- 1 min with NET inhibition (p = 0.001). The odds ratio for premature abortion of head-up tilt testing was 0.22 (95% confidence interval 0.09 to 0.55, p < 0.001) in favor of NET inhibition. Norepinephrine reuptake transporter inhibition elicited a pressor response and increased upright heart rate. CONCLUSIONS: In healthy subjects, NET inhibition prevents tilt-induced neurally mediated (pre)syncope. Therefore, NET inhibition may be a worthwhile target of drug intervention for larger trials in highly symptomatic patients with neurally mediated syncope.     

Ligand- and mutation-induced conformational selection in the CCR5 chemokine G protein-coupled receptor

We predicted the structural basis for pleiotropic signaling of the C-C chemokine type 5 (CCR5) G protein-coupled receptor (GPCR) by predicting the binding of several ligands to the lower-energy conformations of the CCR5 receptor and 11 mutants. For each case, we predicted the ∼20 most stable conformations for the receptor along with the binding sites for four anti-HIV ligands. We found that none of the ligands bind to the lowest-energy apo-receptor conformation. The three ligands with a similar pharmacophore (Maraviroc, PF-232798, and Aplaviroc) bind to a specific higher-energy receptor conformation whereas TAK-779 (with a different pharmacophore) binds to a different high-energy conformation. This result is in agreement with the very different binding-site profiles for these ligands obtained by us and others. The predicted Maraviroc binding site agrees with the recent structure of CCR5 receptor cocrystallized with Maraviroc. We performed 11 site-directed mutagenesis experiments to validate the predicted binding sites. Here, we independently predicted the lowest 10 mutant protein conformations for each of the 11 mutants and then docked the ligands to these lowest conformations. We found the predicted binding energies to be in excellent agreement with our mutagenesis experiments. These results show that, for GPCRs, each ligand can stabilize a different protein conformation, complicating the use of cocrystallized structures for ligand screening. Moreover, these results show that a single-point mutation in a GPCR can dramatically alter the available low-energy conformations, which in turn alters the binding site, potentially altering downstream signaling events. These studies validate the conformational selection paradigm for the pleiotropic function and structural plasticity of GPCRs.Since the finding that individuals lacking the C-C chemokine receptor type 5 (CCR5) gene are resistant to HIV, the CCR5 receptor has been established as a coreceptor for macrophage-tropic viruses, including HIV, to enter host cells (1). Several drugs aimed at disrupting the HIV–CCR5 coupling have been developed. To understand the structural mechanism of ligand binding to G protein-coupled receptors (GPCRs) and how these CCR5 targeting drugs work, we predicted the low-energy structures of the apo CCR5 receptor, which in turn were used to predict the binding sites for various ligands. The focus was on ligands already known to inhibit CCR5: Maraviroc (MVC) (2), PF-232798 (PF) (3), Aplaviroc (APL) (4), and TAK-779 (TAK) (5) because there are abundant experimental data on their binding to numerous CCR5 mutants, to test the predicted structures. The ligand structures are shown in Scheme S1. For three ligands (MVC, PF, and APL), we predict a similar binding site pharmacophore centered on a protonated nitrogen, whereas the other ligand (TAK) containing a quaternary nitrogen leads to a very different binding site pharmacophore. To validate our predictions, we identified 11 singly mutated CCR5 receptor forms selected to test the predicted binding region for the ligands. The predicted structures of ligand-bound CCR5 complexes presented here have already been used in two recent studies to provide a structural basis for (i) biophysical measurements (6) mapping the binding mode of MVC to CCR5 using genetically encoded photo–cross-linkers and (ii) viral entry experiments (7) probing the basis for G protein-coupled and -uncoupled CCR5 receptors by MVC-resistant and -sensitive HIV-1 viruses.While this work was being finalized for submission, the CCR5 receptor was crystallized with MVC (8), allowing a direct structural validation of the predicted structures as discussed in Prediction of Ligand–CCR5 Structures and Comparison with the Crystal Structure.     

去甲肾上腺素转运蛋白在心力衰竭中的作用

去甲肾上腺素转运蛋白是交感神经信号传递途径中的重要环节,主要功能是将神经元释放的去甲肾上腺素重新摄取回突触前膜中,通过这种方式调控神经递质信号强度。心脏交感神经去甲肾腺素转运蛋白功能异常对心力衰竭恶化、进展发挥重要作用。心力衰竭时去甲肾上腺素转运蛋白功能异常可能与交感神经去神经、神经元表面表达降低、内化、转录后异常、氧化负荷增加、细胞因子对心脏交感神经的损伤作用有关。     

Structures of ABCB10, a human ATP-binding cassette transporter in apo- and nucleotide-bound states

ABCB10 is one of the three ATP-binding cassette (ABC) transporters found in the inner membrane of mitochondria. In mammals ABCB10 is essential for erythropoiesis, and for protection of mitochondria against oxidative stress. ABCB10 is therefore a potential therapeutic target for diseases in which increased mitochondrial reactive oxygen species production and oxidative stress play a major role. The crystal structure of apo-ABCB10 shows a classic exporter fold ABC transporter structure, in an open-inwards conformation, ready to bind the substrate or nucleotide from the inner mitochondrial matrix or membrane. Unexpectedly, however, ABCB10 adopts an open-inwards conformation when complexed with nonhydrolysable ATP analogs, in contrast to other transporter structures which adopt an open-outwards conformation in complex with ATP. The three complexes of ABCB10/ATP analogs reported here showed varying degrees of opening of the transport substrate binding site, indicating that in this conformation there is some flexibility between the two halves of the protein. These structures suggest that the observed plasticity, together with a portal between two helices in the transmembrane region of ABCB10, assist transport substrate entry into the substrate binding cavity. These structures indicate that ABC transporters may exist in an open-inwards conformation when nucleotide is bound. We discuss ways in which this observation can be aligned with the current views on mechanisms of ABC transporters.     

Norepinephrine transporter function and autonomic control of metabolism

Genetic variability, numerous medications, and some illicit drugs influence norepinephrine transporter (NET) function; however, the metabolic consequences of NET inhibition are poorly understood. We performed a randomized, double-blind, cross-over trial in 15 healthy subjects who ingested 8 mg of the selective NET inhibitor reboxetine or placebo. Energy expenditure and substrate oxidation rates were determined by indirect calorimetry before and during iv infusion of 0.25, 0.5, 1, and 2 micro g isoproterenol/min. Adipose tissue metabolism was studied by microdialysis before and during local isoproterenol perfusion. At rest, energy expenditure and substrate oxidation rates did not differ between reboxetine and placebo treatment. At 1 micro g/min isoproterenol, energy expenditure was significantly increased in men (+15%) and women (+20%) with both reboxetine and placebo treatment. However, carbohydrate oxidation rate was significantly higher with reboxetine compared with placebo. Baseline and isoproterenol-stimulated adipose tissue blood flow was about 2-fold higher with reboxetine vs. placebo. Furthermore, glucose supply and metabolism was significantly increased and lipid mobilization much more stimulated in adipose tissue under reboxetine when compared with placebo at all isoproterenol concentrations used. We conclude that acute NET inhibition increases adipose tissue glucose uptake and metabolism. While lipid mobilization is increased, overall lipid oxidation is decreased during beta-adrenergic stimulation. This effect cannot be explained by increased systemic or adipose tissue norepinephrine concentrations. Instead, NET inhibition may sensitize adipose tissue to beta-adrenergic stimulation.     

Spironolactone preserves cardiac norepinephrine reuptake in salt-sensitive Dahl rats

An impairment of cardiac norepinephrine (NE) reuptake via the neuronal NE transporter (NET) enhances the effects of increased cardiac NE release in heart failure patients. Increasing evidence suggests that aldosterone and endothelins promote sympathetic overstimulation of failing hearts. Salt-sensitive Dahl rats (DS) fed a high-salt diet developed arterial hypertension and diastolic heart failure as well as elevated plasma levels of endothelin-1 and NE. Cardiac NE reuptake and NET-binding sites, as assessed by clearance of bolus-injected [(3)H]NE in isolated perfused rat hearts and [(3)H]mazindol binding, were reduced. Treatment of DS with the mineralocorticoid receptor antagonist spironolactone preserved the plasma levels of endothelin-1 and NE, cardiac NE reuptake, and myocardial NET density. Moreover, the ventricular function and survival of spironolactone-treated DS were significantly improved compared with untreated DS. The alpha(1)-inhibitor prazosin decreased blood pressure in DS similar to spironolactone treatment, but did not normalize the plasma levels of endothelin-1 and NE, NE reuptake, or ventricular function. In a heart failure-independent model, Wistar rats that were infused with aldosterone and fed a high-salt diet developed impaired cardiac NE reuptake. Treatment of these rats with the endothelin A receptor antagonist darusentan attenuated the impairment of NE reuptake. In conclusion, spironolactone preserves NET-dependent cardiac NE reuptake in salt-dependent heart failure. Evidence is provided that aldosterone inhibits NET function through an interaction with the endothelin system. Selective antagonism of the mineralocorticoid and/or the endothelin A receptor might represent therapeutic principles to prevent cardiac sympathetic overactivity in salt-dependent heart failure.     

Trapping of palindromic ligands within native transthyretin prevents amyloid formation

Transthyretin (TTR) amyloidosis is a fatal disease for which new therapeutic approaches are urgently needed. We have designed two palindromic ligands, 2,2'-(4,4'-(heptane-1,7-diylbis(oxy))bis(3,5-dichloro-4,1-phenylene)) bis(azanediyl)dibenzoic acid (mds84) and 2,2'-(4,4'-(undecane-1,11-diylbis(oxy))bis(3,5-dichloro-4,1-phenylene)) bis(azanediyl)dibenzoic acid (4ajm15), that are rapidly bound by native wild-type TTR in whole serum and even more avidly by amyloidogenic TTR variants. One to one stoichiometry, demonstrable in solution and by MS, was confirmed by X-ray crystallographic analysis showing simultaneous occupation of both T4 binding sites in each tetrameric TTR molecule by the pair of ligand head groups. Ligand binding by native TTR was irreversible under physiological conditions, and it stabilized the tetrameric assembly and inhibited amyloidogenic aggregation more potently than other known ligands. These superstabilizers are orally bioavailable and exhibit low inhibitory activity against cyclooxygenase (COX). They offer a promising platform for development of drugs to treat and prevent TTR amyloidosis.     

Induced allostery in the directed evolution of an enantioselective Baeyer–Villiger monooxygenase

The molecular basis of allosteric effects, known to be caused by an effector docking to an enzyme at a site distal from the binding pocket, has been studied recently by applying directed evolution. Here, we utilize laboratory evolution in a different way, namely to induce allostery by introducing appropriate distal mutations that cause domain movements with concomitant reshaping of the binding pocket in the absence of an effector. To test this concept, the thermostable Baeyer–Villiger monooxygenase, phenylacetone monooxygenase (PAMO), was chosen as the enzyme to be employed in asymmetric Baeyer–Villiger reactions of substrates that are not accepted by the wild type. By using the known X-ray structure of PAMO, a decision was made regarding an appropriate site at which saturation mutagenesis is most likely to generate mutants capable of inducing allostery without any effector compound being present. After screening only 400 transformants, a double mutant was discovered that catalyzes the asymmetric oxidative kinetic resolution of a set of structurally different 2-substituted cyclohexanone derivatives as well as the desymmetrization of three different 4-substituted cyclohexanones, all with high enantioselectivity. Molecular dynamics (MD) simulations and covariance maps unveiled the origin of increased substrate scope as being due to allostery. Large domain movements occur that expose and reshape the binding pocket. This type of focused library production, aimed at inducing significant allosteric effects, is a viable alternative to traditional approaches to “designed” directed evolution that address the binding site directly.     

Structure of influenza hemagglutinin in complex with an inhibitor of membrane fusion

The influenza surface glycoprotein hemagglutinin (HA) is a potential target for antiviral drugs because of its key roles in the initial stages of infection: receptor binding and the fusion of virus and cell membranes. The structure of HA in complex with a known inhibitor of membrane fusion and virus infectivity, tert-butyl hydroquinone (TBHQ), shows that the inhibitor binds in a hydrophobic pocket formed at an interface between HA monomers. Occupation of this site by TBHQ stabilizes the neutral pH structure through intersubunit and intrasubunit interactions that presumably inhibit the conformational rearrangements required for membrane fusion. The nature of the binding site suggests routes for the chemical modification of TBHQ that could lead to the development of more potent inhibitors of membrane fusion and potential anti-influenza drugs.     

Structure-based ligand discovery for the protein-protein interface of chemokine receptor CXCR4

G-protein-coupled receptors (GPCRs) are key signaling molecules and are intensely studied. Whereas GPCRs recognizing small-molecules have been successfully targeted for drug discovery, protein-recognizing GPCRs, such as the chemokine receptors, claim few drugs or even useful small molecule reagents. This reflects both the difficulties that attend protein-protein interface inhibitor discovery, and the lack of structures for these targets. Imminent structure determination of chemokine receptor CXCR4 motivated docking screens for new ligands against a homology model and subsequently the crystal structure. More than 3 million molecules were docked against the model and then against the crystal structure; 24 and 23 high-scoring compounds from the respective screens were tested experimentally. Docking against the model yielded only one antagonist, which resembled known ligands and lacked specificity, whereas the crystal structure docking yielded four that were dissimilar to previously known scaffolds and apparently specific. Intriguingly, several were potent and relatively small, with IC(50) values as low as 306 nM, ligand efficiencies as high as 0.36, and with efficacy in cellular chemotaxis. The potency and efficiency of these molecules has few precedents among protein-protein interface inhibitors, and supports structure-based efforts to discover leads for chemokine GPCRs.     

Reduced Gene Expression for Dopamine Biosynthesis and Transport in Midbrain Neurons of Adult Male Rats Exposed Prenatally to Ethanol

BACKGROUND: Prenatal ethanol exposure affects brain dopaminergic neuronal systems, and many of these alterations are permanent. METHODS: The primary objective of this study was to determine the effects of prenatal ethanol exposure on adult mRNA expression for two key regulatory proteins in the mesolimbic and nigrostriatal dopaminergic cell groups which mediate behavioral responses to alcohol and other drugs of abuse: tyrosine hydroxylase (TH) and dopamine transporter (DAT) in the substantia nigra pars compacta (SNpc) and ventral tegmental area (VTA). To also address the effects on noradrenergic regulation, we quantitated mRNA expression for TH and norepinephrine transporter (NET) in the noradrenergic loci of the locus coeruleus (LC). RESULTS: Daily dietary ethanol consumption by female Sprague-Dawley rats for 3 weeks before, and continuing throughout, pregnancy decreased both DAT (approximately 68%,p < 0.002) and TH (approximately 45%,p < 0.002) mRNA expression in the VTA of adult male offspring. This prenatal exposure also suppressed DAT mRNA expression in the SNpc (approximately 81 %;p < 0.03), although TH mRNA expression in this region was not significantly altered. Prenatal ethanol exposure did not alter significantly either TH or NET mRNA expression in the LC of adult male offspring, which suggests that this brain catecholaminergic response may be limited to DA neurons. CONCLUSION: These results demonstrated that prenatal maternal ethanol consumption suppresses mRNA expression for important regulatory proteins in the mesolimbic and nigrostriatal dopaminergic systems of adult male rat offspring. These persistent prenatal ethanol-induced changes in mRNA expression may thus contribute to the persistent effects of fetal ethanol exposure on the diverse behavioral and/or metabolic responses mediated by the mesolimbic and nigrostriatal dopaminergic systems in the adult.