X-ray structures and mechanism of the human serotonin transporter.

The serotonin transporter (SERT) terminates serotonergic signalling through the sodium- and chloride-dependent reuptake of neurotransmitter into presynaptic neurons. SERT is a target for antidepressant and psychostimulant drugs, which block reuptake and prolong neurotransmitter signalling. Here we report X-ray crystallographic structures of human SERT at 3.15 Å resolution bound to the antidepressants (S)-citalopram or paroxetine. Antidepressants lock SERT in an outward-open conformation by lodging in the central binding site, located between transmembrane helices 1, 3, 6, 8 and 10, directly blocking serotonin binding. We further identify the location of an allosteric site in the complex as residing at the periphery of the extracellular vestibule, interposed between extracellular loops 4 and 6 and transmembrane helices 1, 6, 10 and 11. Occupancy of the allosteric site sterically hinders ligand unbinding from the central site, providing an explanation for the action of (S)-citalopram as an allosteric ligand. These structures define the mechanism of antidepressant action in SERT, and provide blueprints for future drug design.

Assessment of Paroxetine Molecular Interactions with Selected Monoamine and γ-Aminobutyric Acid Transporters.

Thus far, many hypotheses have been proposed explaining the cause of depression. Among the most popular of these are: monoamine, neurogenesis, neurobiology, inflammation and stress hypotheses. Many studies have proven that neurogenesis in the brains of adult mammals occurs throughout life. The generation of new neurons persists throughout adulthood in the mammalian brain due to the proliferation and differentiation of adult neural stem cells. For this reason, the search for drugs acting in this mechanism seems to be a priority for modern pharmacotherapy. Paroxetine is one of the most commonly used antidepressants. However, the exact mechanism of its action is not fully understood. The fact that the therapeutic effect after the administration of paroxetine occurs after a few weeks, even if the levels of monoamine are rapidly increased (within a few minutes), allows us to assume a neurogenic mechanism of action. Due to the confirmed dependence of depression on serotonin, norepinephrine, dopamine and γ-aminobutyric acid levels, studies have been undertaken into paroxetine interactions with these primary neurotransmitters using in silico and in vitro methods. We confirmed that paroxetine interacts most strongly with monoamine transporters and shows some interaction with γ-aminobutyric acid transporters. However, studies of the potency inhibitors and binding affinity values indicate that the neurogenic mechanism of paroxetine's action may be determined mainly by its interactions with serotonin transporters.

A New Paroxetine-Based GRK2 Inhibitor Reduces Internalization of the µ-Opioid Receptor.

G protein-coupled receptor (GPCR) kinases (GRKs) play a key role in terminating signals initiated by agonist-bound GPCRs. However, chronic stimulation of GPCRs, such as that which occurs during heart failure, leads to the overexpression of GRKs and maladaptive downregulation of GPCRs on the cell surface. We previously reported the discovery of potent and selective families of GRK inhibitors based on either the paroxetine or GSK180736A scaffold. A new inhibitor, CCG258747, which is based on paroxetine, demonstrates increased potency against the GRK2 subfamily and favorable pharmacokinetic parameters in mice. CCG258747 and the closely related compound CCG258208 also showed high selectivity for the GRK2 subfamily in a kinome panel of 104 kinases. We developed a cell-based assay to screen the ability of CCG258747 and 10 other inhibitors with different GRK subfamily selectivities and with either the paroxetine or GSK180736A scaffold to block internalization of the µ-opioid receptor (MOR). CCG258747 showed the best efficacy in blocking MOR internalization among the compounds tested. Furthermore, we show that compounds based on paroxetine had much better cell permeability than those based on GSK180736A, which explains why GSK180736A-based inhibitors, although being potent in vitro, do not always show efficacy in cell-based assays. This study validates the paroxetine scaffold as the most effective for GRK inhibition in living cells, confirming that GRK2 predominantly drives internalization of MOR in the cell lines we tested and underscores the utility of high-resolution cell-based assays for assessment of compound efficacy. SIGNIFICANCE STATEMENT: G protein-coupled receptor kinases (GRKs) are attractive targets for developing therapeutics for heart failure. We have synthesized a new GRK2 subfamily-selective inhibitor, CCG258747, which has nanomolar potency against GRK2 and excellent selectivity over other kinases. A live-cell receptor internalization assay was used to test the ability of GRK2 inhibitors to impart efficacy on a GRK-dependent process in cells. Our data indicate that CCG258747 blocked the internalization of the µ-opioid receptor most efficaciously because it has the ability to cross cell membranes.

The quasi-irreversible inactivation of cytochrome P450 enzymes by paroxetine: a computational approach.

The mechanism-based inactivation (MBI) of P450 by paroxetine was investigated by computational analysis. The drug-enzyme interactions were figured out through studying energy profiles of three competing mechanisms. The potency of paroxetine as P450's inhibitor was estimated based on the availability of two active sites for the MBI in the paroxetine structure. The inactivation by the amino site of paroxetine mainly proceeds via the hydrogen atom transfer pathway because of the lower energy demand of its rate determining step. In addition, the low-spin state is the predominant route in the MBI at the methylenedioxo active site as a result of being rebound barrier-free mechanism. Our comparative investigation showed that inactivation at the secondary amine is thermodynamically more favorable because of the lower energy barrier of the dehydration mechanism of the hydroxylated paroxetine complex than its methylenedioxo counterpart. The results of docking analysis coincided with the outputs of DFT calculations since the docking pose with the lowest binding affinity is that for conformation with polar interaction between the amino group of paroxetine and the oxo moiety of P450's active site. Assessment of the molecular dynamics simulations trajectories revealed the favorable interaction of paroxetine with P450.

Inhibitory and Stimulatory Effects of Selective Serotonin Reuptake Inhibitors on Cytochrome P450 2D6-mediated Dopamine Formation from p-Tyramine.

PURPOSE: The effects of selective serotonin reuptake inhibitors (SSRIs) such as fluoxetine and paroxetine on dopamine formation from p-tyramine, mediated by cytochrome P450 (CYP) 2D6.2 (Arg296Cys, Ser486Thr) and CYP2D6.10 (Pro34Ser, Ser486Thr), were compared with their effects on CYP2D6.1 (wild type)-mediated dopamine formation, to investigate the influence of a CYP2D6 polymorphism on neuroactive amine metabolism in the brain. METHODS: The Michaelis constants (Km) and maximal velocity (Vmax) values of dopamine formation mediated by CYP2D6.1, CYP2D6.2, and CYP2D6.10 (expressed in recombinant Escherichia coli), and inhibition constants (Ki) of the SSRIs toward dopamine formation catalyzed by the CYP2D6 variants were estimated. RESULTS: The Km values for CYP2D6.2 and CYP2D6.10 decreased at lower fluoxetine concentrations, while the Vmax values for all CYP2D6 variants increased, indicating that fluoxetine stimulated dopamine formation. Conversely, paroxetine competitively inhibited dopamine formation mediated by CYP2D6.1, CYP2D6.2, and CYP2D6.10 with Ki values of 0.47, 1.33, and 31.3 µM, respectively. CONCLUSIONS: These results suggest that the inhibition/stimulation of CYP2D6-mediated dopamine formation by these SSRIs would be affected by CYP2D6 polymorphisms in the brain.

N-Methylparoxetine Blocked Autophagic Flux and Induced Apoptosis by Activating ROS-MAPK Pathway in Non-Small Cell Lung Cancer Cells.

The main mechanistic function of most chemotherapeutic drugs is mediated by inducing mitochondria-dependent apoptosis. Tumor cells usually respond to upregulate autophagy to eliminate impaired mitochondria for survival. Hypothetically, inhibiting autophagy might promote mitochondria-dependent apoptosis, thus enhancing the efficacy of chemotherapeutic therapies. We previously identified N-methylparoxetine (NMP) as an inducer of mitochondrial fragmentation with subsequent apoptosis in non-small cell lung cancer (NSCLC) cells. We discovered that ROS was accumulated in NMP-treated NSCLC cells, followed by c-Jun N-terminal kinase (JNK) and p38 MAP kinase (p38) activation. This was reversed by the application of a reactive oxygen species (ROS) scavenger, N-acetylcysteine (NAC), leading to a reduction in apoptosis. Our data suggested that NMP induced apoptosis in NSCLC cells by activating mitogen-activated protein kinase (MAPK) pathway. We further speculated that the remarkable increase of ROS in NMP-treated NSCLC cells might result from an inhibition of autophagy. Our current data confirmed that NMP blocked autophagy flux at late stage wherein lysosomal acidification was inhibited. Taken together, this study demonstrated that NMP could exert dual apoptotic functions-mitochondria impairment and, concomitantly, autophagy inhibition. NMP-related excessive ROS accumulation induced apoptosis by activating the MAPK pathway in NSCLC cells.

Exploring the enantiorecognition mechanism of Cinchona alkaloid-based zwitterionic chiral stationary phases and the basic trans-paroxetine enantiomers.

The enantiomers of trans-paroxetine (the selectand) were separated on four chiral stationary phases incorporating either quinine [ZWIX(+), ZWIX(+A)] or quinidine [ZWIX(-), ZWIX(-A)] and (R,R)-aminocyclohexanesulfonic acid [in ZWIX(-), and ZWIX(+A)] or (S,S)-aminocyclohexanesulfonic acid [in ZWIX(+), and ZWIX(-A)] chiral selectors. The zwitterion nature of the phases is due to the presence of either (R,R)- or (S,S)-aminocyclohexanesulfonic acid in the selector structure bearing the quinuclidine moiety. ZWIX(+) and ZWIX(-) phases are available on the market with the commercial names CHIRALPAK ZWIX(+) and CHIRALPAK ZWIX(-), respectively. With the aim of rationalizing the enantiomer elution order with the above chiral stationary phases, a molecular dynamic protocol was applied and two energetic parameters were initially measured: selectand conformational energy and selectand interaction energy. In the search for other descriptors allowing a better fitting with the experimental evidences, in the present work we consider an energetic parameter, defined as the selector conformational energy, which resulted to be relevant in the explanation of the experimental elution order in most of the cases. Very importantly, the computational data produced by the present study strongly support the outstanding role of the conformational energy of the chiral selector as it interacts with the analytes.

Crystal Structure of G Protein-coupled Receptor Kinase 5 in Complex with a Rationally Designed Inhibitor.

G protein-coupled receptor kinases (GRKs) regulate cell signaling by initiating the desensitization of active G protein-coupled receptors. The two most widely expressed GRKs (GRK2 and GRK5) play a role in cardiovascular disease and thus represent important targets for the development of novel therapeutic drugs. In the course of a GRK2 structure-based drug design campaign, one inhibitor (CCG215022) exhibited nanomolar IC50 values against both GRK2 and GRK5 and good selectivity against other closely related kinases such as GRK1 and PKA. Treatment of murine cardiomyocytes with CCG215022 resulted in significantly increased contractility at 20-fold lower concentrations than paroxetine, an inhibitor with more modest selectivity for GRK2. A 2.4 Å crystal structure of the GRK5·CCG215022 complex was determined and revealed that the inhibitor binds in the active site similarly to its parent compound GSK180736A. As designed, its 2-pyridylmethyl amide side chain occupies the hydrophobic subsite of the active site where it forms three additional hydrogen bonds, including one with the catalytic lysine. The overall conformation of the GRK5 kinase domain is similar to that of a previously determined structure of GRK6 in what is proposed to be its active state, but the C-terminal region of the enzyme adopts a distinct conformation. The kinetic properties of site-directed mutants in this region are consistent with the hypothesis that this novel C-terminal structure is representative of the membrane-bound conformation of the enzyme.

Enantioselective Synthesis of Chiral Piperidines via the Stepwise Dearomatization/Borylation of Pyridines.

We have developed a novel approach for the synthesis of enantioenriched 3-boryl-tetrahydropyridines via the Cu(I)-catalyzed regio-, diastereo-, and enantioselective protoborylation of 1,2-dihydropyridines, which were obtained by the partial reduction of the pyridine derivatives. This dearomatization/enantioselective borylation stepwise strategy provides facile access to chiral piperidines together with the stereospecific transformation of a stereogenic C-B bond from readily available starting materials. Furthermore, the utility of this method is demonstrated for the concise synthesis of the antidepressant drug (-)-paroxetine. A theoretical study of the reaction mechanism is also described.

Catalytic Michael/Ring-Closure Reaction of α,ß-Unsaturated Pyrazoleamides with Amidomalonates: Asymmetric Synthesis of (-)-Paroxetine.

A highly enantioselective tandem Michael/ring-closure reaction of α,ß-unsaturated pyrazoleamides and amidomalonates has been accomplished in the presence of a chiral N,N'-dioxide-Yb(OTf)3 complex (Tf: trifluoromethanesulfonyl) to give various substituted chiral glutarimides with high yields and diastereo- and enantioselectivities. Moreover, this methodology could be used for gram-scale manipulation and was successfully applied to the synthesis of (-)-paroxetine. Further nonlinear and HRMS studies revealed that the real catalytically active species was a monomeric L-PMe2 -Yb3+ complex. A plausible transition state was proposed to explain the origin of the asymmetric induction.

A dearth of data: the problem of phosphorus in prescription medications.

A high dietary intake of phosphorus is considered by most to be a significant health threat for dialysis patients. Efforts to include the phosphorus content of foods on the nutrition label in the US have, to date, been fruitless. Another source of phosphorus, largely unrecognized, is prescription medications. These may contain phosphorus as indicated on their package label; the amount is not quantified. We examined the labels of the branded forms of 200 of the most widely prescribed medications in Dialysis Clinic centers in the United States and found that 23 (11.5%) contained phosphorus. A sampling of different doses and manufacturers (generic and branded) of these drugs was analyzed for phosphorus content and found levels as high as 111.5 mg/dose (40 mg paroxetine). Notable were the phosphorus content of a generic 10 mg lisinopril (32.6 mg) and a generic 10 mg amlodipine (40.1 mg). The significant potential for iatrogenic injury accruing from the use of these drugs warrants efforts at remediation. Specific information on the phosphorus content of medications used by dialysis population needs to be made available to the dialysis community.

Deuterated Drugs.

Many drugs are carbon-based, and carbon-hydrogen bonding is particularly relevant for understanding important properties of drug molecules. Deuteration refers to the selective replacement of protium hydrogen isotope atoms in small-molecule drugs with deuterium hydrogen isotope atoms. Deuteration of a drug is most likely to affect pharmacokinetic properties, such as metabolism, rather than its pharmacodynamic effects. For this reason, the metabolism of certain drugs may be favorably influenced when deuterium is substituted for protium, resulting in improved safety, tolerability, or efficacy. Examples of deuterated drugs that have been evaluated in clinical studies include paroxetine, tetrabenazine, and dextromethorphan.

Structural and functional analysis of g protein-coupled receptor kinase inhibition by paroxetine and a rationally designed analog.

Recently we identified the serotonin reuptake inhibitor paroxetine as an inhibitor of G protein-coupled receptor kinase 2 (GRK2) that improves cardiac performance in live animals. Paroxetine exhibits up to 50-fold selectivity for GRK2 versus other GRKs. A better understanding of the molecular basis of this selectivity is important for the development of even more selective and potent small molecule therapeutics and chemical genetic probes. We first sought to understand the molecular mechanisms underlying paroxetine selectivity among GRKs. We directly measured the K(D) for paroxetine and assessed its mechanism of inhibition for each of the GRK subfamilies and then determined the atomic structure of its complex with GRK1, the most weakly inhibited GRK tested. Our results suggest that the selectivity of paroxetine for GRK2 largely reflects its lower affinity for adenine nucleotides. Thus, stabilization of off-pathway conformational states unique to GRK2 will likely be key for the development of even more selective inhibitors. Next, we designed a benzolactam derivative of paroxetine that has optimized interactions with the hinge of the GRK2 kinase domain. The crystal structure of this compound in complex with GRK2 confirmed the predicted interactions. Although the benzolactam derivative did not significantly alter potency of inhibition among GRKs, it exhibited 20-fold lower inhibition of serotonin reuptake. However, there was an associated increase in the potency for inhibition of other AGC kinases, suggesting that the unconventional hydrogen bond formed by the benzodioxole ring of paroxetine is better accommodated by GRKs.

New validated HPLC methodology for the determination of (-)-trans-paroxetine and its enantiomer in pharmaceutical formulations with use of ovomucoid chiral stationary phase.

A new chromatographic method for the enantioseparation and the determination of (-)-trans-paroxetine and (+)-trans-paroxetine has been developed with the aid of amylose ovomucoid-based chiral stationary phase. The method is faster and five times more sensitive than procedures recommended previously: limit of detection and limit of quantification are 5 and 16 ng/mL, respectively [modified (Ferretti et al. in J Chromatogr B 710:157-164, 1998): 20 and 60 ng/mL]. It was carefully validated and applied for the determination of (-)-trans-paroxetine and (+)-trans-paroxetine in Parogen (Mc Dermott Laboratories Ltd.) and Xetanor (Actavis) coated tablets.

Development and pharmacological evaluation of liposomes and nanocapsules containing paroxetine hydrochloride.

Depression is one of the most common psychiatric disorders. Nanotechnology has emerged to optimize the pharmacological response. Therefore, the aim of this work was to develop and characterize liposomes and nanocapsules containing paroxetine hydrochloride and evaluate their antidepressant-like effect using the open field and tail suspension tests in mice. Liposomes and nanocapsules were prepared using the reverse-phase evaporation and nanoprecipitation methods, respectively. The particle size of the formulation ranged from 121.81 to 310.73 nm, the polydispersity index from 0.096 to 0.303, the zeta potential from -11.94 to -34.50 mV, the pH from 5.31 to 7.38, the drug content from 80.82 to 94.36 %, and the association efficiency was 98 %. Paroxetine hydrochloride showed slower release when associated with liposomes (43.82 %) compared to nanocapsules (95.59 %) after 10 h. In Vero cells, in vitro toxicity showed a concentration-dependent effect for paroxetine hydrochloride nanostructures. Both nanostructures decreased the immobility time in the TST at 2.5 mg/kg without affecting the number of crossings in the open field test, suggesting the antidepressant-like effect of paroxetine. In addition, the nanocapsules decreased the number of groomings, reinforcing the anxiolytic effect of this drug. These results suggest that the nanostructures were effective in preserving the antidepressant-like effect of paroxetine hydrochloride even at low doses.

A structural snapshot of CYP2B4 in complex with paroxetine provides insights into ligand binding and clusters of conformational states.

An X-ray crystal structure of CYP2B4 in complex with the drug paroxetine [(3S,4R)-3-[(2H-1,3-benzodioxol-5-yloxy)methyl]-4-(4-fluorophenyl)piperidine] was solved at 2.14 Å resolution. The structure revealed a conformation intermediate to that of the recently solved complex with amlodipine and that of the more compact complex with 4-(4-chlorophenyl)imidazole in terms of the placement of the F-G cassette. Moreover, comparison of the new structure with 15 previously solved structures of CYP2B4 revealed some new insights into the determinants of active-site size and shape. The 2B4-paroxetine structure is nearly superimposable on a previously solved closed structure in a ligand-free state. Despite the overall conformational similarity among multiple closed structures, the active-site cavity volume of the paroxetine complex is enlarged. Further analysis of the accessible space and binding pocket near the heme reveals a new subchamber that resulted from the movement of secondary structural elements and rearrangements of active-site side chains. Overall, the results from the comparison of all 16 structures of CYP2B4 demonstrate a cluster of protein conformations that were observed in the presence or absence of various ligands.

The chemical fate of paroxetine metabolites. Dehydration of radicals derived from 4-(4-fluorophenyl)-3-(hydroxymethyl)piperidine.

Quantum chemical calculations have been used to model reactions which are important for understanding the chemical fate of paroxetine-derived radicals in the environment. In order to explain the experimental observation that the loss of water occurs along the (photo)degradation pathway, four different mechanisms of radical-induced dehydrations have been considered. The elimination of water from the N-centered radical cation, which results in the formation of an imine intermediate, has been calculated as the most feasible process. The predicted energy barrier (ΔG = 98.5 kJ mol(-1)) is within the barrier limits set by experimental measurements. All reaction intermediates and transition state structures have been calculated using the G3(MP2)-RAD composite procedure, and solvent effects have been determined using a mixed (cluster/continuum) solvation model. Several new products, which comply with the available experimental data, have been proposed. These structures could be relevant for the chemical fate of antidepressant paroxetine, but also for biologically and environmentally related substrates.

Optically pure γ-butyrolactones and epoxy esters via two stereocentered HKR of 3-substituted epoxy esters: a formal synthesis of (-)-paroxetine, Ro 67-8867 and (+)-eldanolide.

The HKR of racemic anti- or syn-3-substituted epoxy esters catalyzed by a Co(III)salen complex provides ready access to the corresponding enantioenriched 3,4-disubstituted γ-butyrolactones and 3-substituted epoxy esters. This strategy has been successfully employed in the formal synthesis of biologically active 3,4-disubstituted piperidine derivatives, (-)-paroxetine and Ro 67-8867 and a natural product, (+)-eldanolide.

Development and validation of a method for the analysis of paroxetine HCl by circular dichroism.

A simple, rapid, and sensitive method for the analysis of paroxetine, in tablets as well as the pure drug, by circular dichroism is described. The method was validated for repeatability, linearity, limit of detection, limit of quantification, and recovery according to the International Conference on Harmonization guidelines. Excellent results were obtained, within the globally accepted validation reference values, particularly taking into account the low concentration levels investigated. This is the first report of the quantitation of paroxetine, a chiral drug, without previous separation of the analyte. Additionally, the solid state CD spectrum of PXT was obtained.

Determination of paroxetine in serum treated with simple pretreatment by pre-column high-performance liquid chromatography using 4-(5,6-dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride as a fluorescent labeling reagent.

The therapeutic drug monitoring of paroxetine could be used to optimize the pharmacological treatment of depressed patients. A simple and sensitive high-performance liquid chromatography procedure was developed for the determination of paroxetine in serum. After simple pretreatment of serum (50 µL) with acetonitrile and o-phthalaldehyde, paroxetine was derivatized with 4-(5,6-dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride at 70°C for 20 min in borate buffer (0.1 mol/L, pH 8.0) to produce a fluorescent product. The derivative was separated on a reversed-phase column at 40°C for stepwise elution with (A) acetic acid (10 mmol/L) and (B) acetonitrile. The flow rate was 1.0 mL/min. The fluorescence intensity was monitored at excitation and emission wavelengths of 320 and 400 nm, respectively. The within-day and day-to-day relative standard deviations were 3.0-3.4 and 2.7-8.3%, respectively. The detection limit of paroxetine was 8.3 fmol at a signal-to-noise ratio of 3. As the proposed method that only requires a small quantity of serum (50 µL) is simple, sensitive and reproducible, it would be useful for clinical and biochemical research as well as drug monitoring.