A Novel Bromine-Containing Paroxetine Analogue Provides Mechanistic Clues for Binding Ambiguity at the Central Primary Binding Site of the Serotonin Transporter.

The serotonin transporter (SERT) is the primary target for the selective serotonin reuptake inhibitors (SSRIs). However, the structural basis for the extraordinarily high binding affinity of the widely prescribed SSRI, paroxetine, to human SERT (hSERT) has not yet been fully elucidated. Our previous findings unveiled a plausible ambiguity in paroxetine's binding orientations that may constitute an integral component of this SSRI's high affinity for hSERT. Herein, we investigate factors contributing to paroxetine's high affinity by modifying both the ligand and the protein. We generated a series of bromine (Br)-containing derivatives and found that the one in which the 4-F of paroxetine had been replaced with the chemically similar but more electron-rich Br atom (13) had the highest affinity. By comparatively characterizing the binding of paroxetine and 13 to both wild type (WT) and a construct harboring a paroxetine-sensitive mutation in the binding cavity, we identified a mechanistic determinant responsible for the pose ambiguity of paroxetine, which can guide future drug design.

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.

Structure-Based Design of Highly Selective and Potent G Protein-Coupled Receptor Kinase 2 Inhibitors Based on Paroxetine.

In heart failure, the ß-adrenergic receptors (ßARs) become desensitized and uncoupled from heterotrimeric G proteins. This process is initiated by G protein-coupled receptor kinases (GRKs), some of which are upregulated in the failing heart, making them desirable therapeutic targets. The selective serotonin reuptake inhibitor, paroxetine, was previously identified as a GRK2 inhibitor. Utilizing a structure-based drug design approach, we modified paroxetine to generate a small compound library. Included in this series is a highly potent and selective GRK2 inhibitor, 14as, with an IC50 of 30 nM against GRK2 and greater than 230-fold selectivity over other GRKs and kinases. Furthermore, 14as showed a 100-fold improvement in cardiomyocyte contractility assays over paroxetine and a plasma concentration higher than its IC50 for over 7 h. Three of these inhibitors, including 14as, were additionally crystallized in complex with GRK2 to give insights into the structural determinants of potency and selectivity of these inhibitors.

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.

An HPLC method for the indirect quantification of a quinone adduct of the drug paroxetine.

An HPLC method has been developed which enables the quantification of low levels of a catechol derivative and a quinone adduct of paroxetine in the presence of excess drug substance. Due to its inherent instability, the paroxetine quinone adduct is not available as a pure compound so that an indirect method was developed for its quantification. This procedure is based on the assumption that one molecule of the catechol (or more precisely the corresponding 1,2-benzoquinone) reacts with one molecule of paroxetine to produce one molecule of paroxetine quinone adduct. In the presence of paroxetine excess, pseudo-first-order kinetics was used to study the formation of the unstable product. A detector response factor for the paroxetine quinone adduct was calculated as a function of the response factor for the paroxetine catechol derivative, after considering a mass balance of the reaction. Using the methodology outlined, quantitative analysis was carried out of the paroxetine catechol derivative and the paroxetine quinone adduct in batches of paroxetine drug substance.

Anhydrides as acylating agents in the enzymatic resolution of an intermediate of (-)-Paroxetine.

A new chemoenzymatic method for the preparation of an intermediate of (-)-Paroxetine is reported. Cyclic anhydrides are used as acylating agents in the lipase-catalyzed esterification of trans-4-(4'-fluorophenyl)-3-hydroxymethyl-N-phenyloxycarbonylpiperidine in organic solvents. The best enantioselectivities are obtained with two different lipases from Candida antarctica. These two lipases show opposite stereochemical preference in these processes, so that both enantiomers can be obtained in their optically pure forms. The (3S,4R) isomer is an intermediate for the synthesis of (-)-Paroxetine.

[Adverse effects after switching to a different generic form of paroxetine: paroxetine mesylate instead of paroxetine HCl hemihydrate]. / Bijwerkingen na overstappen op een ander generiek preparaat van paroxetine: paroxetine-mesilaat in plaats van paroxetine-HCl-hemihydraat.

Two patients, a 29-year-old man and a 32-year-old woman who were being treated with paroxetine HCl hemihydrate for depression and panic disorder with agoraphobia, respectively, unexpectedly developed complaints after switching to paroxetine mesylate. The symptoms were generalised itching with relapse of the depressive disorder in the man, and nausea with diarrhoea in the woman. The symptoms disappeared after the original treatment with paroxetine HCl hemihydrate was resumed. In the first patient, a rechallenge with the mesylate form of paroxetine led to the same complaints which subsided again after using the first product. Those treating such patients, and the patients themselves, should be informed if the delivery form of paroxetine is changed.

Synthesis and ligand binding of tropane ring analogues of paroxetine.

(3S,4R)-4-(4-Fluorophenyl)-3-[[3,4-(methylenedioxy)phenoxy]methyl] piperidine [(3S,9R)-3, paroxetine] is a selective serotonin reuptake inhibitor (SSRI) used as an antidepressant in humans. In previous studies, we reported that certain (1R)-3 beta-(substituted phenyl)nortropane-2 beta-carboxylic acid methyl esters (2a) exhibited high affinity and reasonable selectivity for the serotonin transporter (5-HTT). The major structural differences between 2a and (3S,4R)-3 are that 2a possesses a different absolute stereochemistry and has an ethylene bridge not present in 3. In addition, 2a possesses a carbomethoxy substituent adjacent to the aryl ring, whereas (3S,4R)-3 contains a [3,4-(methylenedioxy)phenoxy]methyl group. In this study, we present the synthesis and biological evaluations of six of the possible eight isomers of 3-(4-fluorophenyl)-2-[[3,4-(methylenedioxy)phenoxy]methyl]nortropane+ ++ (4). The data for inhibition of [3H]paroxetine binding show that (1R)-2 beta, 3 alpha-4c, which has the same stereochemistry as paroxetine, has the highest affinity at the 5-HTT. Strikingly, the most potent compounds for inhibition of [3H]WIN-35,428 binding were not the (1R)-2 beta, 3 beta-isomers but rather (1R)-2 beta, 3 alpha-4c and (1S)-2 beta, 3 alpha-4f. Conformational analyses show that these isomers exist in a flattened boat conformation with pseudoequatorial substituents. Thus, the binding data show that this conformation is recognized by the DAT-associated binding site and also suggest that this conformation of paroxetine is recognized by the 5-HTT-associated binding site.

Regional distribution of specific high affinity binding sites for 3H-imipramine and 3H-paroxetine in human brain.

The binding of 3H-paroxetine and 3H-imipramine has been compared in 17 different regions of 12 human control brains. Our findings reveal that the regional distribution is similar for both radioligands and their bindings tend to be parallel in the brain. The highest binding site density was found in basal ganglia (hypothalamus Bmax 780 +/- 102 fmol/mg protein for 3H-imipramine binding and Bmax 515 approximately 83 for 3H-paroxetine binding). The lowest values were found in cortical areas (cingulate cortex 191 +/- 18.5 fmol/mg for 3H-imipramine binding and 88 +/- 7.5 fmol/mg for 3H-paroxetine binding). The Kd values for 3H-paroxetine binding to neuronal membranes were similar in all brain regions (mean +/- s.d. Kd 0.07 +/- 0.007 nM) and also for 3H-imipramine binding (mean +/- s.d. Kd 1.05 +/- 0.12 nM). As these values are the same as in platelet membrane, the results obtained indicate that both binding sites are identical in neuronal and in platelet membranes. These findings suggest that both ligands are good markers of the 5HT transporter. However, the higher affinity of 3H-paroxetine confirms that this compound is a better radioligand for the 5HT uptake site.

Synthesis and characterization of an aryl-azidoparoxetine. A novel photo-affinity probe for serotonin-transporter.

Paroxetine is an effective antidepressant drug and potent serotonin (5-HT) uptake inhibitor. It selectively labels 5-HT transporter on platelets and neurons. We report here the synthesis of an aryl-azido derivative of paroxetine, which is a novel photoactive and irreversible ligand for the [3H]paroxetine binding site on the platelet 5-HT transporter. The compound inhibited [3H]paroxetine binding (IC50, 55 nM) and 5-HT uptake (IC50, 12 nM) at equilibrium conditions and inactivated 10-20% of [3H]paroxetine binding sites upon irradiation at 320 nm. SDS-PAGE of platelet protein extract labelled with the radioactive analogue of the synthesized probe revealed the presence of four radioactive bands of which the 71-kDa one was the most prominent.

Binding potency of paroxetine analogues for the 5-hydroxytryptamine uptake complex.

The in-vitro inhibition constants (Ki) of 14 structural analogues of the potent 5-hydroxytryptamine (5-HT)-uptake inhibitor paroxetine were determined to assess the structure-affinity relationship of these derivatives. A goal of these studies was to determine those positions on paroxetine which could be derivatized without significantly decreasing the affinity of the drug for the binding site, so that radiolabels such as [18F]fluoroalkyl groups might be appended for future in-vivo imaging studies of the 5-HT uptake system. Using the methyl moiety as a steric probe for these studies, it was found that the rank order of potency of various methyl-substituted paroxetine analogues for inhibiting the binding of [3H]paroxetine to the 5-HT re-uptake site was: 4'-approximately equal to 3'-approximately equal to 2''- > 2'-approximately equal to 1- > 5''- > 6''-methyl. The in-vitro equipotent molar ratios (EPMR, Ki(analogue)/Ki(paroxetine)) of the analogues were determined, and the EPMRs of the 4'-, 3'-, and 2''-methyl derivatives were 1.9, 2.2 and 2.2, respectively. The 4'- and 2''-fluoromethyl and -fluoroethyl analogues were synthesized, and the EPMRs of the 4'- and 2''-fluoromethyl derivatives were determined to be 2.0 and 3.5, and those of the 4'- and 2''-fluoroethyl analogues were 5.2 and 6.2, respectively. The 2''-fluoromethyl analogue was unstable in aqueous solutions, and it is not a promising ligand for in-vivo studies.(ABSTRACT TRUNCATED AT 250 WORDS)

Radiosynthesis and evaluation of N-(3-[18F]fluoropropyl)paroxetine as a radiotracer for in vivo labeling of serotonin uptake sites by PET.

To visualize serotonin uptake sites by positron emission tomography (PET), N-(3-[18F]fluoropropyl)-paroxetine ([18F]FPP), a derivative of the selective serotonin uptake blocker paroxetine, was synthesized from 3-[18F]fluoropropyltosylate and paroxetine via a one-pot procedure. The rate of formation of [18F]FPP was a function of the ratio of the initial amount of paroxetine to that of 1,3-propanediol bistosylate with which [18F]fluoropropyltosylate was synthesized. When the reaction mixture contained an excess amount of paroxetine over that of the propyl-bistosylate, the radiosynthesis followed by HPLC purification, which took approx. 90 min, gave [18F]FPP in a radiochemical yield of approx. 8%, and in high radiochemical and chemical purity. The specific activity was 2640 +/- 360 mCi/mumol. The brain biodistribution of [18F]FPP showed no distinguishable localization in regions with high density of serotonin uptake sites such as hypothalamus or olfactory tubercles. In vitro binding assays revealed that N-fluoropropylation of paroxetine reduced the affinity for the serotonin uptake site by three orders of magnitude.