Revisiting the Molecular Interactions between the Tumor Protein TCTP and the Drugs Sertraline/Thioridazine.

TCTP protein is a pharmacological target in cancer and TCTP inhibitors such as sertraline have been evaluated in clinical trials. The direct interaction of TCTP with the drugs sertraline and thioridazine has been reported in vitro by SPR experiments to be in the ∼30-50 µM Kd range (Amson et al. Nature Med 2012), supporting a TCTP-dependent mode of action of the drugs on tumor cells. However, the molecular details of the interaction remain elusive although they are crucial to improve the efforts of on-going medicinal chemistry. In addition, TCTP can be phosphorylated by the Plk-1 kinase, which is indicative of poor prognosis in several cancers. The impact of phosphorylation on TCTP structure/dynamics and binding with therapeutical ligands remains unexplored. Here, we combined NMR, TSA, SPR, BLI and ITC techniques to probe the molecular interactions between TCTP with the drugs sertraline and thioridazine. We reveal that drug binding is much weaker than reported with an apparent ∼mM Kd and leads to protein destabilization that obscured the analysis of the published SPR data. We further demonstrate by NMR and SAXS that TCTP S46 phosphorylation does not promote tighter interaction between TCTP and sertraline. Accordingly, we question the supported model in which sertraline and thioridazine directly interact with isolated TCTP in tumor cells and discuss alternative modes of action for the drugs in light of current literature.

Interrogating the Inhibition Mechanisms of Human Aldehyde Oxidase by X-ray Crystallography and NMR Spectroscopy: The Raloxifene Case.

Human aldehyde oxidase (hAOX1) is mainly present in the liver and has an emerging role in drug metabolism, since it accepts a wide range of molecules as substrates and inhibitors. Herein, we employed an integrative approach by combining NMR, X-ray crystallography, and enzyme inhibition kinetics to understand the inhibition modes of three hAOX1 inhibitors-thioridazine, benzamidine, and raloxifene. These integrative data indicate that thioridazine is a noncompetitive inhibitor, while benzamidine presents a mixed type of inhibition. Additionally, we describe the first crystal structure of hAOX1 in complex with raloxifene. Raloxifene binds tightly at the entrance of the substrate tunnel, stabilizing the flexible entrance gates and elucidating an unusual substrate-dependent mechanism of inhibition with potential impact on drug-drug interactions. This study can be considered as a proof-of-concept for an efficient experimental screening of prospective substrates and inhibitors of hAOX1 relevant in drug discovery.

New insights about the monomer and homodimer structures of the human AOX1.

Human aldehyde oxidase (hAOX1) is a molybdenum dependent enzyme that plays an important role in the metabolism of various compounds either endogenous or xenobiotics. Due to its promiscuity, hAOX1 plays a major role in the pharmacokinetics of many drugs and therefore has gathered a lot of attention from the scientific community and, particularly, from the pharmaceutical industry. In this work, homology modelling, molecular docking and molecular dynamics simulations were used to study the structure of the monomer and dimer of human AOX. The results with the monomer of hAOX1 allowed to shed some light on the role played by thioridazine and two malonate ions that are co-crystalized in the recent X-ray structure of hAOX1. The results show that these molecules endorse several conformational rearrangements in the binding pocket of the enzyme and these changes have an impact in the active site topology as well as in the stability of the substrate (phthalazine). The results show that the presence of both molecules open two gates located at the entrance of the binding pocket, from which results the flooding of the active site. They also endorse several modifications in the shape of the binding pocket (namely the position of Lys893) that, together with the presence of the solvent molecules, favour the release of the substrate to the solvent. Further insights were also obtained with the assembled homodimer of hAOX1. The allosteric inhibitor (THI) binds closely to the region where the dimerization of both monomers occur. These findings suggest that THI can interfere with protein dimerization.

An optical sensor with specific binding sites for the detection of thioridazine hydrochloride based on ZnO-QDs coated with molecularly imprinted polymer.

Here, an optical sensor with specific binding sites for sensitive and selective detection of thioridazine hydrochloride (THZ) was prepared. The optosensor was developed based on ZnO quantum dots (QDs) coated with molecularly imprinted polymers (MIPs). Initially, ZnO quantum dots (QDs) were synthesized by precipitation from Zn(CH3COO)2 and NaOH then, reverse microemulsion method was applied for fixing the MIPs layer on the surface of QDs. It was perceived that the fluorescence intensity of the QDs-MIPs quenched with increasing THZ concentration. Several parameters affect the optical sensor response were studied and optimized. Under the optimal conditions, THZ could be determined with a linear dynamic range of 4-120 nmol L-1 and with a low detection limit of 0.43 nmol L-1. The relative standard deviations for 25 and 60 nmol L-1 of THZ were obtained as 4.9% and 3.1%, respectively (three times measurement). High selectivity, simplicity, and cost-efficient for THZ measurement are the most important advantages of the fluorimetric sensor.

Cocktail Strategy Based on Spatio-Temporally Controlled Nano Device Improves Therapy of Breast Cancer.

Metastatic breast cancer may be resistant to chemo-immunotherapy due to the existence of cancer stem cells (CSC). Also, the control of particle size and drug release of a drug carrier for multidrug combination is a key issue influencing the therapy effect. Here, a cocktail strategy is reported, in which chemotherapy against both bulk tumor cells and CSC and immune checkpoint blockade therapy are intergraded into one drug delivery system. The chemotherapeutic agent paclitaxel (PTX), the anti-CSC agent thioridazine (THZ), and the PD-1/PD-L1 inhibitor HY19991 (HY) are all incorporated into an enzyme/pH dual-sensitive nanoparticle with a micelle-liposome double-layer structure. The particle size shrinks when the nanoparticle transfers from circulation to tumor tissues, favoring both pharmacokinetics and cellular uptake, meanwhile achieving sequential drug release where needed. This nano device, named PM@THL, increases the intratumoral drug concentrations in mice and exhibits significant anticancer efficacy, with tumor inhibiting rate of 93.45% and lung metastasis suppression rate of 97.64%. It also reduces the proportion of CSC and enhances the T cells infiltration in tumor tissues, and thus prolongs the survival of mice. The cocktail therapy based on the spatio-temporally controlled nano device will be a promising strategy for treating breast cancer.

Fluoxetine and thioridazine inhibit efflux and attenuate crystalline biofilm formation by Proteus mirabilis.

Proteus mirabilis forms extensive crystalline biofilms on indwelling urethral catheters that block urine flow and lead to serious clinical complications. The Bcr/CflA efflux system has previously been identified as important for development of P. mirabilis crystalline biofilms, highlighting the potential for efflux pump inhibitors (EPIs) to control catheter blockage. Here we evaluate the potential for drugs already used in human medicine (fluoxetine and thioridazine) to act as EPIs in P. mirabilis, and control crystalline biofilm formation. Both fluoxetine and thioridazine inhibited efflux in P. mirabilis, and molecular modelling predicted both drugs interact strongly with the biofilm-associated Bcr/CflA efflux system. Both EPIs were also found to significantly reduce the rate of P. mirabilis crystalline biofilm formation on catheters, and increase the time taken for catheters to block. Swimming and swarming motilies in P. mirabilis were also significantly reduced by both EPIs. The impact of these drugs on catheter biofilm formation by other uropathogens (Escherichia coli, Pseudomonas aeruginosa) was also explored, and thioridazine was shown to also inhibit biofilm formation in these species. Therefore, repurposing of existing drugs with EPI activity could be a promising approach to control catheter blockage, or biofilm formation on other medical devices.

Sulpiride, Amisulpride, Thioridazine, and Olanzapine: Interaction with Model Membranes. Thermodynamic and Structural Aspects.

Neuroleptic drugs are widely applied in effective treatment of schizophrenia and related disorders. The lipophilic character of neuroleptics means that they tend to accumulate in the lipid membranes, impacting their functioning and processing. In this paper, the effect of four drugs, namely, thioridazine, olanzapine, sulpiride, and amisulpride, on neutral and negatively charged lipid bilayers was examined. The interaction of neuroleptics with lipids and the subsequent changes in the membrane physical properties was assessed using several complementary biophysical approaches (isothermal titration calorimetry, electron paramagnetic resonance spectroscopy, dynamic light scattering, and ζ potential measurements). We have determined the thermodynamic parameters, that is, the enthalpy of interaction and the binding constant, to describe the interactions of the investigated drugs with model membranes. Unlike thioridazine and olanzapine, which bind to both neutral and negatively charged membranes, amisulpride interacts with only the negatively charged one, while sulpiride does not bind to any of them. The mechanism of olanzapine and thioridazine insertion into the bilayer membrane cannot be described merely by a simple molecule partition between two different phases (the aqueous and the lipid phase). We have estimated the number of protons transferred in the course of drug binding to determine which of its forms, ionized or neutral, binds more strongly to the membrane. Finally, electron paramagnetic resonance results indicated that the drugs are localized near the water-membrane interface of the bilayer and presence of a negative charge promotes their burying deeper into the membrane.

Synthesis and SAR evaluation of novel thioridazine derivatives active against drug-resistant tuberculosis.

The neuroleptic drug thioridazine has been recently repositioned as possible anti-tubercular drug. Thioridazine showed anti-tubercular activity against drug resistant mycobacteria but it is endowed with adverse side effects. A small library of thioridazine derivatives has been designed through the replacement of the piperidine and phenothiazine moieties, with the aim to improve the anti-tubercular activity and to reduce the cytotoxic effects. Among the resulting compounds, the indole derivative 12e showed an antimycobacterial activity significantly better than thioridazine and a cytotoxicity 15-fold lower.

Co-release of dicloxacillin and thioridazine from catheter material containing an interpenetrating polymer network for inhibiting device-associated Staphylococcus aureus infection.

Approximately half of all nosocomial bloodstream infections are caused by bacterial colonization of vascular catheters. Attempts have been made to improve devices using anti-adhesive or antimicrobial coatings; however, it is often difficult to bind coatings stably to catheter materials, and the low amounts of drug in thin-film coatings limit effective long-term release. Interpenetrating polymer networks (IPNs) are polymer hybrid materials with unique drug release properties. While IPNs have been extensively investigated for use in tablet- or capsule-based drug delivery systems, the potential for use of IPNs in drug release medical devices remains largely unexplored. Here, we investigated the use of silicone-hydrogel IPNs as a catheter material to provide slow anti-bacterial drug-release functionality. IPN catheters were produced by the sequential method, using supercritical CO2 as a solvent to polymerize and crosslink poly(2-hydroxyethyl methacrylate) (PHEMA) in silicone elastomer. The design was tested against Staphylococcus aureus colonization after loading with dicloxacillin (DCX) alone or in combination with thioridazine (TDZ), the latter of which is known to synergistically potentiate the antibacterial effect of DCX against both methicillin-sensitive and methicillin-resistant S. aureus. The hydrophilic PHEMA component allowed for drug loading in the catheters by passive diffusion and provided controlled release properties. The drug-loaded IPN material inhibited bacterial growth on agar plates for up to two weeks and in blood cultures for up to five days, and it withstood 24h of seeding with resilient biofilm aggregates. The combined loading of DCX+TDZ enhanced the antibacterial efficiency in static in vitro experiments, although release analyses revealed that this effect was due to an enhanced loading capacity of DCX when co-loaded with TDZ. Lastly, the IPN catheters were tested in a novel porcine model of central venous catheter-related infection, in which drug-loaded IPN catheters were found to significantly decrease the frequency of infection.

Synthesis and Application of an Aldazine-Based Fluorescence Chemosensor for the Sequential Detection of Cu²âº and Biological Thiols in Aqueous Solution and Living Cells.

A fluorescence chemosensor, 2-hydroxy-1-naphthaldehyde azine (HNA) was designed and synthesized for sequential detection of Cu(2+) and biothiols. It was found that HNA can specifically bind to Cu(2+) with 1:1 stoichiometry, accompanied with a dramatic fluorescence quenching and a remarkable bathochromic-shift of the absorbance peak in HEPES buffer. The generated HNA-Cu(2+) ensemble displayed a "turn-on" fluorescent response specific for biothiols (Hcy, Cys and GSH) based on the displacement approach, giving a remarkable recovery of fluorescence and UV-Vis spectra. The detection limits of HNA-Cu(2+) to Hcy, Cys and GSH were estimated to be 1.5 µM, 1.0 µM and 0.8 µM, respectively, suggesting that HNA-Cu(2+) is sensitive enough for the determination of thiols in biological systems. The biocompatibility of HNA towards A549 human lung carcinoma cell, was evaluated by an MTT assay. The capability of HNA-Cu(2+) to detect biothiols in live A549 cells was then demonstrated by a microscopy fluorescence imaging assay.

Thioridazine in PLGA nanoparticles reduces toxicity and improves rifampicin therapy against mycobacterial infection in zebrafish.

Encapsulating antibiotics such as rifampicin in biodegradable nanoparticles provides several advantages compared to free drug administration, including reduced dosing due to localized targeting and sustained release. Consequently, these characteristics reduce systemic drug toxicity. However, new nanoformulations need to be tested in complex biological systems to fully characterize their potential for improved drug therapy. Tuberculosis, caused by infection with the bacterium Mycobacterium tuberculosis, requires lengthy and expensive treatment, and incomplete therapy contributes to an increasing incidence of drug resistance. Recent evidence suggests that standard therapy may be improved by combining antibiotics with bacterial efflux pump inhibitors, such as thioridazine. However, this drug is difficult to use clinically due to its toxicity. Here, we encapsulated thioridazine in poly(lactic-co-glycolic) acid nanoparticles and tested them alone and in combination with rifampicin nanoparticles, or free rifampicin in macrophages and in a zebrafish model of tuberculosis. Whereas free thioridazine was highly toxic in both cells and zebrafish embryos, after encapsulation in nanoparticles no toxicity was detected. When combined with rifampicin nanoparticles, the nanoparticles loaded with thioridazine gave a modest increase in killing of both Mycobacterium bovis BCG and M. tuberculosis in macrophages. In the zebrafish, the thioridazine nanoparticles showed a significant therapeutic effect in combination with rifampicin by enhancing embryo survival and reducing mycobacterial infection. Our results show that the zebrafish embryo is a highly sensitive indicator of drug toxicity and that thioridazine nanoparticle therapy can improve the antibacterial effect of rifampicin in vivo.

Rational Design and Synthesis of Thioridazine Analogues as Enhancers of the Antituberculosis Therapy.

Tuberculosis, caused by Mycobacterium tuberculosis, is still one of the leading infectious diseases globally. Therefore, novel approaches are needed to face this disease. Efflux pumps are known to contribute to the emergence of M. tuberculosis drug resistance. Thioridazine has shown good anti-TB properties both in vitro and in vivo, likely due to its capacity to inhibit efflux mechanisms. Here we report the design and synthesis of a number of putative efflux inhibitors inspired by the structure of thioridazine. Compounds were evaluated for their in vitro and ex vivo activity against M. tuberculosis H37Rv. Compared to the parent molecule, some of the compounds synthesized showed higher efflux inhibitory capacity, less cytotoxicity, and a remarkable synergistic effect with anti-TB drugs both in vitro and in human macrophages, demonstrating their potential to be used as coadjuvants for the treatment of tuberculosis.

A reversible fluorescence chemosensor for sequentially quantitative monitoring copper and sulfide in living cells.

We report a novel, selective and sensitive strategy for the sequentially "ON-OFF-ON" fluorescent detection of Cu(2+) and S(2-) based on a fluorescein derivative, FL. The specific binding of FL towards Cu(2+) in aqueous and biological media led to the intensive green fluorescence quenching and a notable increase of the absorbance maximum at 480 nm. In the presence of S(2-), the intensity and overall pattern of the fluorescence emission and UV-vis spectra of FL-Cu(2+) ensemble were recovered since the abolishment of paramagnetic Cu(2+). This displacement approach exhibited highly specificity, and sensitivity with detection limits of 3 nM for Cu(2+) and 150 nM for S(2-). The fluorescence "ON-OFF-ON" circle can be repeated to a minimum of 5 times by the alternative addition of Cu(2+) and S(2-), implying that FL is a renewable dual-functional chemosensor. The biocompatibility of FL toward breast carcinoma cells, MDA-MB-231 was confirmed by MTT assay. The reversible "ON-OFF-ON" fluorescent response of FL to Cu(2+) and S(2-) in living system was further confirmed by confocal fluorescence imaging of living cells. The quantification of Cu(2+) and S(2-) in single intact cell was realized by the flow cytometry analysis.

Differential effects of thioridazine enantiomers on action potential duration in rabbit papillary muscle.

The antipsychotic drug thioridazine has potential for treatment of multidrug-resistant microbes including tuberculosis but also causes cardiotoxic QT interval prolongation. Both thioridazine enantiomers have potent antimicrobial effects, but the neuroleptic effect primarily resides with (+)-thioridazine. In this study we for the first time investigate the cardiotoxicity of the isolated thioridazine enantiomers and show their effects on ventricular repolarization. The effects of (+)-thioridazine, (-)-thioridazine, and racemate on the rabbit ventricular action potential duration (APD) were investigated in a randomized controlled blinded experiment. Action potentials were measured in papillary muscles isolated from 21 female rabbits, and the drug effect on 90% APD in comparison with control (ΔΔ-APD90) was evaluated. Increasing concentrations of (+)-thioridazine and the racemate caused significant dose-dependent ΔΔ-APD90 prolongation, while (-)-thioridazine did not. At 0.5 and 2Hz pacing, (+)-thioridazine caused 19.5% and 20.1% ΔΔ-APD90 prolongation, the racemate caused 8.0% and 12.9%, and (-)-thioridazine caused 1.5% and 1.1%. The effect of (-)-thioridazine on APD90 was significantly less than that of the other drugs at both pacing rates (P<0.01 in all cases), and there was no significant difference between (-)-thioridazine and control. The results of this study indicate that the APD prolonging effect of thioridazine is primarily due to the (+)-thioridazine enantiomer. If these results are valid in humans, (-)-thioridazine may be a safer drug for treatment of multidrug-resistant tuberculosis and other microbes.

Reinforcing the membrane-mediated mechanism of action of the anti-tuberculosis candidate drug thioridazine with molecular simulations.

Thioridazine is a well-known dopamine-antagonist drug with a wide range of pharmacological properties ranging from neuroleptic to antimicrobial and even anticancer activity. Thioridazine is a critical component of a promising multi-drug therapy against M. tuberculosis. Amongst the various proposed mechanisms of action, the cell membrane-mediated one is peculiarly tempting due to the distinctive feature of phenothiazine drug family to accumulate in selected body tissues. In this study, we employ long-scale molecular dynamics simulations to investigate the interactions of three different concentrations of thioridazine with zwitterionic and negatively charged model lipid membranes. Thioridazine partitions into the interfacial region of membranes and modifies their structural and dynamic properties, however dissimilarly so at the highest membrane-occurring concentration, that appears to be obtainable only for the negatively charged bilayer. We show that the origin of such changes is the drug induced decrease of the interfacial tension, which ultimately leads to the significant membrane expansion. Our findings support the hypothesis that the phenothiazines therapeutic activity may arise from the drug-membrane interactions, and reinforce the wider, emerging view of action of many small, bioactive compounds.

In silico study on the inhibitory interaction of drugs with wild-type CYP2D6.1 and the natural variant CYP2D6.17.

The natural variant of the cytochrome P450 enzyme CYP2D6.1, CYP2D6.17, is most common in African populations, has three amino acid substitutions (T107I, R296C, and S486T) compared to the wild-type, and is known to have a different ligand preference from CYP2D6.1. It is becoming increasingly important to understand differences in the metabolism of medicines in different ethnic groups in order to assess the relevance of clinical data from different countries. This study investigated differences in the inhibition profiles of drugs for CYP2D6 with respect to gene polymorphisms. Firstly, we used computer docking with six drugs to several CYP2D6.1 structures, sampled from the trajectory of MD simulations, and calculated MM-GB/SA scores representing binding free energies. We then used regression analysis to predict the potency with which drugs inhibited CYP2D6.1 based on MM-GB/SA scores. The pKi-values obtained were in good agreement with experimental values measured for the six drugs (r(2) = 0.81). We carried out the same analysis for CYP2D6.17 and the pKi-values calculated were also in good agreement with experimental values (r(2) = 0.92). Finally, we were able to successfully explain the different abilities of CYP2D6.1 and CYP2D6.17 to metabolize drugs in different ethnic groups with reference to their 3D-structures.

UV-light effects on cytochrome c modulated by the aggregation state of phenothiazines.

The present study shows the factors that modulate the photodamage promoted by phenothiazines. Cytochrome c was irradiated with UV light for 120 min, over a pH range from 4.0 to 8.0, in the absence and in the presence of different concentrations of thioridazine (TR) and fluphenazine (FP). In the absence of phenothiazines, the maximal rate of a Soret band blue shift (nm/min) from 409 to 406 nm was obtained at pH 4.0 (0.028 nm/min). The presence of phenothiazines at the concentration range 10-25 µmol/L amplified and accelerated a cytochrome c blue shift (409 to 405 nm, at a rate = 0.041 nm/min). Above 25 µmol/L, crescent concentrations of phenothiazines contributed to cytochrome c protection with (maximal at 2500 µmol/L). Scanning electronic microscopy revealed the formation of nanostructures. The pH also influenced the effect of low phenothiazine concentrations on cytochrome c. Thus, the predominance of phenothiazine-promoted cytochrome c damage or protection depends on a balance of the following factors: the yield of photo-generated drug cation radicals, which is favored by acidic pH; the stability of the cation radicals, which is favored by the drug aggregation; and the cytochrome c structure, modulated by the pH.

The structures of two aldazines: [1,1'-(1E,1'E)-hydrazine-1,2-diylidenebis(methan-1-yl-1-ylidene)dinaphthalen-2-ol] (Lumogen) and 2,2'-(1E,1'E)-hydrazine-1,2-diylidenebis(methan-1-yl-1-ylidene)diphenol (salicylaldazine) in the solid state and in solution.

A combination of NMR spectroscopy and theoretical methods Density functional theory including dispersion corrections (DFT-D) was used to study the structures of Lumogen and salicylaldazine. In the solid state, Lumogen exists as the dihydroxy tautomer 1a (an azine, C=N-N=C) as was already known from an X-ray determination. In a deuterated dimethyl sulfoxide solution, another tautomer is observed besides 1a; its structure corresponds to the hydroxy-oxo tautomer 1b (a hydrazone, C=N-NH-Csp(2)). In what concerns salicylaldazine, we have observed only the dihydroxy tautomer 2a.

A comparative analysis of in vitro and in vivo efficacies of the enantiomers of thioridazine and its racemate.

A long list of chemotherapeutical drugs used in the treatment of the peripheral and the central nervous systems possess anti-microbial activity. Some of these neurotropic compounds are chiral, with the one stereo isomeric form exaggerating reduced neurotropism. This is the case for the levorotatory form of thioridazine. The phenothiazine thioridazine is an interesting compound, characterized by exhibiting a significant growth inhibiting activity on a wide array of micro-organisms. Thioridazine is characterized by another challenging feature, because the compound is concentrated in certain human tissue cells. The present study describes a comparative study of the two enantiomers as well as the racemic form of thioridazine. The study exploits the stereochemical aspect and the in vitro and in vivo potential of these compounds, with a focus on the effects on gram negative organism Salmonella enterica serover Typhimurium. In summary, the results of this study yielded a significant antibacterial activity of all forms of thioridazine, indicating the levorotatory (-)-form to be superior in terms of both its in vitro and in vivo efficacies.