A Multi-Species Phenotypic Screening Assay for Leishmaniasis Drug Discovery Shows That Active Compounds Display a High Degree of Species-Specificity.

High genetic and phenotypic variability between Leishmania species and strains within species make the development of broad-spectrum antileishmanial drugs challenging. Thus, screening panels consisting of several diverse Leishmania species can be useful in enabling compound prioritization based on their spectrum of activity. In this study, a robust and reproducible high content assay was developed, and 1280 small molecules were simultaneously screened against clinically relevant cutaneous and visceral species: L. amazonensis, L. braziliensis, and L. donovani. The assay is based on THP-1 macrophages infected with stationary phase promastigotes and posterior evaluation of both compound antileishmanial activity and host cell toxicity. The profile of compound activity was species-specific, and out of 51 active compounds, only 14 presented broad-spectrum activity against the three species, with activities ranging from 52% to 100%. Notably, the compounds CB1954, Clomipramine, Maprotiline, Protriptyline, and ML-9 presented pan-leishmanial activity, with efficacy greater than 70%. The results highlight the reduced number of compound classes with pan-leishmanial activity that might be available from diversity libraries, emphasizing the need to screen active compounds against a panel of species and strains. The assay reported here can be adapted to virtually any Leishmania species without the need for genetic modification of parasites, providing the basis for the discovery of broad spectrum anti-leishmanial agents.

Discovery of aryl sulfonamide-selective Nav1.7 inhibitors with a highly hydrophobic ethanoanthracene core.

Nav1.7 channels are mainly distributed in the peripheral nervous system. Blockade of Nav1.7 channels with small-molecule inhibitors in humans might provide pain relief without affecting the central nervous system. Based on the facts that many reported Nav1.7-selective inhibitors contain aryl sulfonamide fragments, as well as a tricyclic antidepressant, maprotiline, has been found to inhibit Nav1.7 channels, we designed and synthesized a series of compounds with ethanoanthracene and aryl sulfonamide moieties. Their inhibitory activity on sodium channels were detected with electrophysiological techniques. We found that compound 10o potently inhibited Nav1.7 channels stably expressed in HEK293 cells (IC50 = 0.64 ± 0.30 nmol/L) and displayed a high Nav1.7/Nav1.5 selectivity. In mouse small-sized dorsal root ganglion neurons, compound 10o (10, 100 nmol/L) dose-dependently decreased the sodium currents and dramatically suppressed depolarizing current-elicited neuronal discharge. Preliminary in vivo experiments showed that compound 10o possessed good analgesic activity: in a mouse visceral pain model, administration of compound 10o (30-100 mg/kg, i.p.) effectively and dose-dependently suppressed acetic acid-induced writhing.

Exploring the Inhibitory Mechanism of Approved Selective Norepinephrine Reuptake Inhibitors and Reboxetine Enantiomers by Molecular Dynamics Study.

Selective norepinephrine reuptake inhibitors (sNRIs) provide an effective class of approved antipsychotics, whose inhibitory mechanism could facilitate the discovery of privileged scaffolds with enhanced drug efficacy. However, the crystal structure of human norepinephrine transporter (hNET) has not been determined yet and the inhibitory mechanism of sNRIs remains elusive. In this work, multiple computational methods were integrated to explore the inhibitory mechanism of approved sNRIs (atomoxetine, maprotiline, reboxetine and viloxazine), and 3 lines of evidences were provided to verify the calculation results. Consequently, a binding mode defined by interactions between three chemical moieties in sNRIs and eleven residues in hNET was identified as shared by approved sNRIs. In the meantime, binding modes of reboxetine's enantiomers with hNET were compared. 6 key residues favoring the binding of (S, S)-reboxetine over that of (R, R)-reboxetine were discovered. This is the first study reporting that those 11 residues are the common determinants for the binding of approved sNRIs. The identified binding mode shed light on the inhibitory mechanism of approved sNRIs, which could help identify novel scaffolds with improved drug efficacy.

Synthesis of Chlorinated Tetracyclic Compounds and Testing for Their Potential Antidepressant Effect in Mice.

The synthesis of the tetracyclic compounds 1-(4,5-dichloro-9,10-dihydro-9,10-ethanoanthracen-11-yl)-N-methylmethanamine (5) and 1-(1,8-dichloro-9,10-dihydro-9,10-ethanoanthracen-11-yl)-N-methylmethanamine (6) as a homologue of the anxiolytic and antidepressant drugs benzoctamine and maprotiline were described. The key intermediate aldehydes (3) and (4) were successfully synthesized via a [4 + 2] cycloaddition between acrolein and 1,8-dichloroanthracene. The synthesized compounds were investigated for antidepressant activity using the forced swimming test. Compounds (5), (6) and (3) showed significant reduction in the mice immobility indicating significant antidepressant effects. These compounds significantly reduced the immobility times at a dose 80 mg/kg by 84.0%, 86.7% and 71.1% respectively.

Screen of FDA-approved drug library identifies maprotiline, an antibiofilm and antivirulence compound with QseC sensor-kinase dependent activity in Francisella novicida.

Development of new therapeutics against Select Agents such as Francisella is critical preparation in the event of bioterrorism. Testing FDA-approved drugs for this purpose may yield new activities unrelated to their intended purpose and may hasten the discovery of new therapeutics. A library of 420 FDA-approved drugs was screened for antibiofilm activity against a model organism for human tularemia, Francisella (F.) novicida, excluding drugs that significantly inhibited growth. The initial screen was based on the 2-component system (TCS) dependent biofilm effect, thus, the QseC dependence of maprotiline anti-biofilm action was demonstrated. By comparing their FDA-approved uses, chemical structures, and other properties of active drugs, toremifene and polycyclic antidepressants maprotiline and chlorpromazine were identified as being highly active against F. novicida biofilm formation. Further down-selection excluded toremifene for its membrane active activity and chlorpromazine for its high antimicrobial activity. The mode of action of maprotiline against F. novicida was sought. It was demonstrated that maprotiline was able to significantly down-regulate the expression of the virulence factor IglC, encoded on the Francisella Pathogenicity Island (FPI), suggesting that maprotiline is exerting an effect on bacterial virulence. Further studies showed that maprotiline significantly rescued F. novicida infected wax worm larvae. In vivo studies demonstrated that maprotiline treatment could prolong time to disease onset and survival in F. novicida infected mice. These results suggest that an FDA-approved drug such as maprotiline has the potential to combat Francisella infection as an antivirulence agent, and may have utility in combination with antibiotics.

Atypical tetracyclic antidepressant maprotiline is an antagonist at cardiac hERG potassium channels.

Maprotiline is an antidepressant compound with an atypical tetracyclic structure that is widely used in elderly patients due to its favourable side-effect profile. However, there have been reports of proarrhythmia associated with maprotiline and in vitro studies of its electrophysiological properties have been lacking. Therefore, we characterised the effects of maprotiline on cardiac hERG channels. hERG channels were expressed in HEK cells and in the Xenopus oocyte expression system. Currents were measured using a whole-cell patch clamp and a two-microelectrode voltage-clamp. Maprotiline inhibited hERG currents with an IC(50) of 8.2 micromol/l in HEK cells and 29.2 micromol/l in Xenopus oocytes. Onset of the effect was rather slow and took several minutes. No wash-out of effect was observed. Maprotiline blocked hERG channels in the open and inactivated states, but not in the closed states. In mutant hERG channels Y652A and F656A, the effect was markedly attenuated (hERG-F656A) or completely abolished (hERG-Y652A). Voltage dependence of hERG current activation and inactivation was not affected by maprotiline. hERG inactivation was accelerated at positive potentials. The effect of maprotiline on hERG currents was voltage-dependent with a marked reduction at a more positive potential. hERG blockade by maprotiline was not frequency-dependent. Maprotiline is an antagonist of cardiac hERG potassium channels that preferably accesses the putative pore binding site Y652/F656. Although the affinity of maprotiline to hERG channels is low, its use in patients with risk factors for acquired long QT syndrome should be monitored appropriately.

Synthesis and C-11 labeling of three potent norepinephrine transporter selective ligands ((R)-nisoxetine, lortalamine, and oxaprotiline) for comparative PET studies in baboons.

Three potent antidepressants, (R)-nisoxetine, lortalamine, and oxaprotiline, with high affinity and high selectivity for the norepinephrine transporter (NET) were synthesized and radiolabeled with C-11 via [11C]methylation. The reference compounds and their corresponding normethyl precursors were synthesized via multi-step synthetic approaches. The radiochemical syntheses were performed by simple alkylation of the corresponding normethyl precursors with no-carrier-added [11C]CH3I in DMF. After HPLC purification, (R)-[N-11CH3]nisoxetine, [11C]lortalamine, and [11C]oxaprotiline were obtained in 63-97% radiochemical yields, whereas (R)-[O-11CH3]nisoxetine was obtained in 23-29% radiochemical yields due to substantial formation of the undesired N-[11C]methylated byproduct (64-70%). These C-11 labeled tracers allowed us to carry out comparative studies of NET in baboons with positron emission tomography (PET) and evaluate their potential as PET tracers for imaging brain NET.

Cytochrome P450 enzymes contributing to demethylation of maprotiline in man.

From case reports of patients treated with the tetracyclic antidepressant drug maprotiline, it appears that this drug is subject to polymorphic metabolism. Thus, we studied formation of the major maprotiline metabolite desmethylmaprotiline to identify the human cytochrome P-450 enzymes (CYP) involved. In incubations with human liver microsomes from two different donors, the substrate maprotiline was used at five different concentrations (5 to 500 microM). For selective inhibition of CYPs, quinidine (0.5-50 microM; CYP2D6), furafylline (0.3-30 microM; CYP1A2), ketoconazole (0.2-20 microM; CYP3A4), mephenytoin (20-200 microM; CYP2C19), chlorzoxazone (1-100 microM; CYP2E1), sulphaphenazole (0.2-100 microM; CYP2C9) and coumarin (0.2-100 microM; CYP2A6) were used. Desmethylmaprotiline concentrations were measured by HPLC, and enzyme kinetic parameters were estimated using extended Michaelis-Menten equations with non-linear regression. Relevant inhibition of the desmethylmaprotiline formation rate was observed in incubations with quinidine, furafylline and ketoconazole only. Formation rates of desmethylmaprotiline were consistent with a two enzyme model with a high (K(M)=71 and 84 microM) and a low (K(M)=531 and 426 microM) affinity site for maprotiline in the two samples, respectively. The high affinity site was competitively inhibited by quinidine (K(i,nc) 0.13 and 0.61 microM), the low-affinity site was non-competitively inhibited by furafylline (K(i,nc) 0.11 and 1.3 microM). Thus it appears that CYP2D6 and CYPIA2 contribute to maprotiline demethylation. Based on the parameters obtained, for plasma concentrations of 1 microM 83% (mean) of desmethylmaprotiline formation in vivo is expected to be mediated by CYP2D6 while 17% only may be attributed to CYPIA2 activity.

Identification of selected psychopharmaceuticals and their metabolites in hair by LC/ESI-CID/MS and LC/MS/MS.

Hair samples of patients of psychiatry and hair samples of suicide cases were analysed by liquid-chromatography/ionspray-mass spectrometry (LC/MS) for antidepressants and neuroleptics. Electrospray ionisation (ESI) with in-source collision induced dissociation (ESI/CID) and tandem-mass spectrometry (MS/MS) were used for drug and metabolite identification. Mass spectra library searching was performed using an ESI/CID mass spectra library and a MS/MS spectra library. Furthermore, extracted ion chromatograms were used for the detection of N-desmethyl-metabolites, which were also identified by their fragment-ion spectra. Three examples using these methods are shown: The tricyclic antidepressant maprotiline, the selective serotonin receptor inhibitor (SSRI) citalopram and their desmethylmetabolites as well as the neuroleptic pipamperone were detected and identified in hair extracts. For extraction powdered hair was treated by ultrasonication in methanol and solid-phase extraction was used for sample clean-up prior to LC/MS or MS/MS analysis. These examples demonstrate the power of LC/MS and LC/MS/MS for the detection and identification of drugs in hair extracts using full-scan mode and ESI/CID with library searching or using highly selective LC/MS/MS-analysis with library searching or in multiple reaction monitoring mode.

Interactions between six psychotherapeutic drugs and plastic containers. Influence of plastic material and infusion solutions.

The interactions of chlorpromazine, clomipramine, maprotiline and viloxazine hydrochlorides, and of clorazepate dipotassium salt and diazepam with polyvinyl chloride (PVC) and Stedim 6 infusion bags were studied. Stedim 6, is anew multilayer film whose inner layer is made of polyethylene. The drugs were in 5% dextrose and 0.9% sodium chloride isotonic solutions and the influence of these was also considered. The remaining concentrations of each drug were determined at regular time intervals in a 24-h period, by a spectrofluorometric method for chlorpromazine hydrochloride and by ultraviolet spectrophotometric methods for the other drugs. No binding was observed for viloxazine and maprotiline hydrochlorides whatever the infusion solution and the plastic container. A slight retention in PVC bags, but not in Stedim 6 ones, was noted for clomipramine hydrochloride and clorazepate dipotassium salt. This was more marked in the sodium chloride solution than in the dextrose one. Diazepam and chlorpromazine hydrochloride were bound both in PVC and Stedim 6 bags, but more in the former and more again in the sodium chloride solution than in the dextrose one. The results were explained in terms of the degree of crystallinity of the plastic material and the degree of lipophilicity of the drugs. Practical consequences are discussed.

Comparative sorption of clomipramine, viloxazine, and maprotiline hydrochlorides in polyvinylchloride bags and glass vials.

The sorption of clomipramine, viloxazine, and maprotiline hydrochlorides was studied comparatively in polyvinylchloride (PVC) bags and glass flasks for up to 72 hours. When stored in glass flasks, no decrease in concentration was observed for any of the three drugs, either in dextrose or sodium chloride isotonic solution. When stored in PVC bags, a slight loss (about 7% within 72 hours), was noted for clomipramine hydrochloride in both isotonic solutions, but not for the other two drugs.