The Role of Efflux Pumps and Environmental pH in Bacterial Multidrug Resistance.

BACKGROUND/AIM: One of the most studied bacterial resistance mechanisms is the resistance related to multidrug efflux pumps. In our study the pump activity of the Escherichia coli K-12 AG100 strain expressing the AcrAB-TolC pump system was investigated at pH 7 and pH 5 in the presence of the efflux pump inhibitor (EPI) promethazine (PMZ). MATERIALS AND METHODS: The EPI activity was assessed by real-time fluorimetry. The influence of PMZ treatment on the relative expression of the pump genes acrA, acrB and their regulators marA, marB, marR, the stress genes soxS, rob, as well as the bacterial growth control genes ftsI, and sdiA were determined by RT-qPCR. RESULTS: The EPI activity of PMZ was more effective at neutral pH. The PMZ treatment induced a significant stress response in the bacterium at acidic pH by the up-regulation of genes. CONCLUSION: The genetic system that regulates the activity of the main efflux pump is pH-dependent.

Possible Biological and Clinical Applications of Phenothiazines.

Phenothiazines have been used in many areas of medicine, mainly in psychopharmacology. These compounds are able to effectively inhibit dopamine, histamine, serotonin, acetylcholine, and α-adrenergic receptors; thus, their effect and side-effect profiles are extremely diverse. Besides their antipsychotic activity, phenothiazines have a significant antimicrobial effect as well, since they can enhance the bactericidal function of macrophages and inhibit efflux pumps. They are also able to eliminate bacterial resistance plasmids and destroy bacteria by their membrane-destabilizing effect. Their antiviral, antiprotozoal, antifungal, and antiprion activities have also been described. Phenothiazines have also been proven to destroy cancer cells and sensitize them to chemotherapy. Anti-angiogenesis and anticancer stem cell activities have also been reported, and they might be applied as adjuvants in the treatment of infections and tumors in the future. Finally, phenothiazines can also be effective in the treatment of neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease.

New Roads Leading to Old Destinations: Efflux Pumps as Targets to Reverse Multidrug Resistance in Bacteria.

Multidrug resistance (MDR) has appeared in response to selective pressures resulting from the incorrect use of antibiotics and other antimicrobials. This inappropriate application and mismanagement of antibiotics have led to serious problems in the therapy of infectious diseases. Bacteria can develop resistance by various mechanisms and one of the most important factors resulting in MDR is efflux pump-mediated resistance. Because of the importance of the efflux-related multidrug resistance the development of new therapeutic approaches aiming to inhibit bacterial efflux pumps is a promising way to combat bacteria having over-expressed MDR efflux systems. The definition of an efflux pump inhibitor (EPI) includes the ability to render the bacterium increasingly more sensitive to a given antibiotic or even reverse the multidrug resistant phenotype. In the recent years numerous EPIs have been developed, although so far their clinical application has not yet been achieved due to their in vivo toxicity and side effects. In this review, we aim to give a short overview of efflux mediated resistance in bacteria, EPI compounds of plant and synthetic origin, and the possible methods to investigate and screen EPI compounds in bacterial systems.

Thioridazine: A Non-Antibiotic Drug Highly Effective, in Combination with First Line Anti-Tuberculosis Drugs, against Any Form of Antibiotic Resistance of Mycobacterium tuberculosis Due to Its Multi-Mechanisms of Action.

This review presents the evidence that supports the use of thioridazine (TZ) for the therapy of a pulmonary tuberculosis infection regardless of its antibiotic resistance status. The evidence consists of in vitro and ex vivo assays that demonstrate the activity of TZ against all encountered Mycobacterium tuberculosis (Mtb) regardless of its antibiotic resistance phenotype, as well as in vivo as a therapy for mice infected with multi-drug resistant strains of Mtb, or for human subjects infected with extensively drug resistant (XDR) Mtb. The mechanisms of action by which TZ brings about successful therapeutic outcomes are presented in detail.

Identification of Important Compounds Isolated from Natural Sources that Have Activity Against Multidrug-resistant Cancer Cell Lines: Effects on Proliferation, Apoptotic Mechanism and the Efflux Pump Responsible for Multi-resistance Phenotype.

The focus of this mini-review is to identify non-toxic compounds isolated from natural sources (plants) that exhibit specific activity against efflux pumps of specific multidrug-resistant (MDR) cancer cell lines, inhibit proliferation of the MDR cancer cell lines and inhibit the activity of overexpressed efflux pumps of the MDR cancer cell line.

The Anticancer Activity of the Old Neuroleptic Phenothiazine-type Drug Thioridazine.

Thioridazine (TZ), an antipsychotic drug, renders multidrug-resistant (MDR) cancer cells susceptible to cytotoxic agents to which they were initially resistant, has anti-prolilferative activity and apoptosis-inducing properties in various tumor cell lines and cancer stem cells. Whereas the anti-proliferative activity takes place at high concentrations that ensure the intercalation of the compound between nucleic bases (especially rich in G/C bases), much lower concentrations inhibit the export function of the ABCB1 (P-glycoprotein), which is responsible for the MDR phenotype of the cancer cell. The co-administration of TZ with doxorubicin inhibits efflux of doxorubicin and, hence, increases the intracellular concentration of anticancer drug. The (+) and (-) enantiomers of TZ have the same activities as TZ. The main focus of this review is to present extensive evidence provided by our work, confirmed by much later studies, as it supports adjuvant use of TZ with an anticancer drug for MDR cancer therapy.

Identification of selenocompounds with promising properties to reverse cancer multidrug resistance.

In previous studies, 56 novel selenoesters and one cyclic selenoanhydride with chemopreventive, antiproliferative and cytotoxic activity were described. Herein, the selenoanhydride and selected selenoesters were evaluated for their ability to reverse the cancer multidrug resistance (MDR) using the ABCB1 efflux pump inhibition assay in mouse MDR T-lymphoma cells. Results showed that the selenoanhydride (1) and the selenoesters with ketone terminal fragments (9-11) exerted (1.7-3.6)-fold stronger efflux pump inhibitory action than the reference verapamil. In addition, those four derivatives triggered apoptotic events in more than 80% of the examined MDR mouse cells.

Thioridazine Alters the Cell-Envelope Permeability of Mycobacterium tuberculosis.

The increasing occurrence of multidrug resistant tuberculosis exerts a major burden on treatment of this infectious disease. Thioridazine, previously used as a neuroleptic, is active against extensively drug resistant tuberculosis when added to other second- and third-line antibiotics. By quantitatively studying the proteome of thioridazine-treated Mycobacterium tuberculosis, we discovered the differential abundance of several proteins that are involved in the maintenance of the cell-envelope permeability barrier. By assessing the accumulation of fluorescent dyes in mycobacterial cells over time, we demonstrate that long-term drug exposure of M. tuberculosis indeed increased the cell-envelope permeability. The results of the current study demonstrate that thioridazine induced an increase in cell-envelope permeability and thereby the enhanced uptake of compounds. These results serve as a novel explanation to the previously reported synergistic effects between thioridazine and other antituberculosis drugs. This new insight in the working mechanism of this antituberculosis compound could open novel perspectives of future drug-administration regimens in combinational therapy.

Ion Channel Blockers as Antimicrobial Agents, Efflux Inhibitors, and Enhancers of Macrophage Killing Activity against Drug Resistant Mycobacterium tuberculosis.

Given the ability of M. tuberculosis to survive as an intracellular pathogen and its propensity to develop resistance to the existing antituberculosis drugs, its treatment requires new approaches. Here the antimycobacterial properties of verapamil, thioridazine, chlorpromazine, flupenthixol and haloperidol were investigated against a panel of drug resistant M. tuberculosis strains, both in vitro and on human-infected macrophages. These compounds are efflux inhibitors that share among them the characteristic of being ion channel blockers. In vitro, all compounds exhibited synergistic inhibitory activities when combined with isoniazid and rifampicin, and were able to inhibit active efflux, demonstrating their role as efflux inhibitors. Gene expression analysis showed that M. tuberculosis efflux genes were overexpressed in response to antibiotic exposure, in vitro and within macrophages, irrespective of their resistance pattern. These compounds displayed a rapid and high killing activity against M. tuberculosis, associated with a decrease in intracellular ATP levels demonstrating that the bactericidal action of the ion channel blockers against M. tuberculosis clinical strains is associated with their interference with energy metabolism. The compounds led to a decrease in the intracellular mycobacterial load by increasing phagosome acidification and activating lysosomal hydrolases. The results presented in this study enable us to propose the following mechanism of action for these compounds: a) in the bacteria, the compounds generate a cascade of events involving the inhibition of the respiratory chain complexes and energy production for efflux activity. Indirectly, this reduce the resistance level to antituberculosis drugs potentiating their activity; b) on the host cell, the treatment with the ion channel blockers increases phagosome acidification and induces the expression of phagosomal hydrolases, leading to bacterial growth restriction irrespective of their resistance pattern. This work highlights the potential value ion channel blockers as adjuvants of tuberculosis chemotherapy, in particular for the development of new therapeutic strategies, with strong potential for treatment shortening against drug susceptible and resistant forms of tuberculosis. Medicinal chemistry studies are now needed to improve the properties of these compounds, increasing their M. tuberculosis efflux-inhibition and killing-enhancement activity and reduce their toxicity for humans, therefore optimizing their potential for clinical usage.

Fluorimetric Methods for Analysis of Permeability, Drug Transport Kinetics, and Inhibition of the ABCB1 Membrane Transporter.

The cell membrane P-glycoprotein (P-gp; MDR1, ABCB1) is an energy-dependent efflux pump that belongs to the ATP-binding cassette (ABC) family of transporters, and has been associated with drug resistance in eukaryotic cells. Multidrug resistance (MDR) is related to an increased expression and function of the ABCB1 (P-gp) efflux pump that often causes chemotherapeutic failure in cancer. Modulators of this efflux pump, such as the calcium channel blocker verapamil (VP) and cyclosporine A (CypA), can reverse the MDR phenotype but in vivo studies have revealed disappointing results due to adverse side effects. Currently available methods are unable to visualize and assess in a real-time basis the effectiveness of ABCB1 inhibitors on the uptake and efflux of ABCB1 substrates. However, predicting and testing ABCB1 modulation activity using living cells during drug development are crucial. The use of ABCB1-transfected mouse T-lymphoma cell line to study the uptake/efflux of fluorescent probes like ethidium bromide (EB), rhodamine 123 (Rh-123), and carbocyanine dye DiOC2, in the presence and absence of potential inhibitors, is currently used in our laboratories to evaluate the ability of a drug to inhibit ABCB1-mediated drug accumulation and efflux. Here we describe and compare three in vitro methods, which evaluate the permeability, transport kinetics of fluorescent substrates, and inhibition of the ABCB1 efflux pump by drugs of chemical synthesis or extracted from natural sources, using model cancer cell lines overexpressing this transporter, namely (1) real-time fluorimetry that assesses the accumulation of ethidium bromide, (2) flow cytometry, and (3) fluorescent microscopy using rhodamine 123 and DiOC2.

Imidazolidine-4-one derivatives in the search for novel chemosensitizers of Staphylococcus aureus MRSA: synthesis, biological evaluation and molecular modeling studies.

A series of amine derivatives of 5-aromatic imidazolidine-4-ones (7-19), representing three subgroups: piperazine derivatives of 5-arylideneimidazolones (7-13), piperazine derivatives of 5-arylideneimidazolidine-2,4-dione (14-16) and primary amines of 5-naphthyl-5-methylimidazolidine-2,4-diones (17-19), was evaluated for their ability to improve antibiotics effectiveness in two strains of Gram-positive Staphylococcus aureus: ATCC 25923 (a reference strain) and MRSA (methicillin resistant S. aureus) HEMSA 5 (a resistant clinical isolate). The latter compounds (17-19) were obtained by 4-step synthesis using Bucherer-Bergs condensation, two-phase bromoalkylation and Gabriel reactions. The naphthalen derivative: (Z)-5-(naphthalen-2-ylmethylene)-2-(piperazin-1-yl)-3H-imidazol-4(5H)-one (10) was the most potent in combination with ß-lactam antibiotics and ciprofloxacin against the resistant strain. The high potency to increase efficacy of oxacillin was noted for (Z)-5-(anthracen-10-ylmethylene)-2-(piperazin-1-yl)-3H-imidazol-4(5H)one (12) too. In order to explain the mechanism of action of the compounds 10 and 12, docking studies with the use of crystal structures of a penicillin binding protein (PBP2a) and MecR1 were carried out. Their outcomes suggested that the most probable mechanism of action of the active compounds is the interaction with MecR1. Molecular dynamic experiments performed for the active compounds and compound 13 (structurally similar to 12) supported this hypothesis and provided possible explanation of activity dependencies of the tested compounds in terms of the restoration of antibiotic efficacy in S. aureus MRSA HEMSA 5.

Ecdysteroids sensitize MDR and non-MDR cancer cell lines to doxorubicin, paclitaxel, and vincristine but tend to protect them from cisplatin.

Ecdysteroids, analogs of the insect molting hormone, are known for their various mild, nonhormonal bioactivities in mammals. Previously, we reported that less-polar ecdysteroids can modulate the doxorubicin resistance of a multidrug resistant (MDR) mouse lymphoma cell line expressing the human ABCB1 transporter. Here, we describe the ability of 20-hydroxyecdysone (1) and its mono- (2) and diacetonide (3) derivatives to sensitize various MDR and non-MDR cancer cell lines towards doxorubicin, paclitaxel, vincristine, or cisplatin. Drug IC50 values with or without ecdysteroid were determined by MTT assay. Compound 3 significantly sensitized all cell lines to each chemotherapeutic except for cisplatin, whose activity was decreased. In order to overcome solubility and stability issues for the future in vivo administration of compound 3, liposomal formulations were developed. By means of their combination index values obtained via checkerboard microplate method, a formulation showed superior activity to that of compound 3 alone. Because ecdysteroids act also on non-ABCB1 expressing (sensitive) cell lines, our results demonstrate that they do not or not exclusively exert their adjuvant anticancer activity as ABCB1 inhibitors, but other mechanisms must be involved, and they opened the way towards their in vivo bioactivity testing against various cancer xenografts.

Reversal of ABCB1-related Multidrug Resistance of Colonic Adenocarcinoma Cells by Phenothiazines.

BACKGROUND: The most common mechanism that reduces the efficacy of anticancer agents is overexpression of ATP-binding cassette (ABC) drug transporters. Phenothiazines and structurally-related compounds can sensitize multidrug-resistant (MDR) cells to chemotherapeutics. MATERIALS AND METHODS: Phenothiazine derivatives were investigated regarding their anticancer and MDR-reversing effect on colonic adenocarcinoma cells. The anti-proliferative and cytotoxic effects of the derivatives were assessed by the thiazolyl blue tetrazolium bromide (MTT) method, the modulation of the ABCB1 activity was measured by rhodamine 123 accumulation assay using flow cytometry. RESULTS: All phenothiazines exhibited potent cytotoxic effect on the sensitive and MDR colon adenocarcinoma cell lines. The inhibition of the ABCB1 transporter was greater in the presence of the phenothiazine derivatives than for the known ABCB1 inhibitor verapamil. CONCLUSION: It can be concluded that these derivatives show synergism in the presence of doxorubicin and could have potential as ABCB1 inhibitors.

Characterization of mixtures of compounds produced in chlorpromazine aqueous solutions by ultraviolet laser irradiation: their applications in antimicrobial assays.

The study reports an investigation of the photoproducts obtained by exposure of chlorpromazine hydrochloride in ultrapure water (concentration 2 mg/mL) to a 266-nm laser beam obtained by fourth harmonic generation from a Nd:YAG laser (6-ns full time width at half maximum, 10-Hz pulse repetition rate). The photoproducts were analyzed by steady-state UV-Vis absorption, laser-induced fluorescence, Fourier transform infrared spectroscopy, and liquid chromatography-tandem time-of-flight mass spectroscopy. Two figures showing pathways that take place during irradiation for obtaining the final products are shown. The quantum yield of singlet oxygen generation by chlorpromazine (CPZ) was determined relative to standard Zn-phthalocyanine in dimethyl sulfoxide. To outline the role of fluorescence in photoproducts formation rates, fluorescence quantum yield of CPZ during exposure to 355-nm radiation (third harmonic of the fundamental beam of Nd:YAG laser) was investigated relative to standard Coumarin 1 in ethanol. The CPZ solutions exposed 60 and 240 min to 266-nm laser beam, respectively, were tested against Staphylococcus aureus ATCC 25923 strain. For 25 µL of CPZ samples irradiated 240 min, a higher diameter of inhibition has obtained against the tested strain than for the 60-min exposed ones.

Rapid, laser-induced conversion of 20-hydroxyecdysone - a follow-up study on the products obtained.

We have recently reported the set-up of an experimental system for the laser-induced photochemical modification of bioactive substances, where two ecdysteroids, 20-hydroxyecdysone (20E) and its diacetonide derivative served as probes. As a direct continuation of our previous work, three new compounds together with five other ecdysteroid derivatives, have been identified from the novel, laser-induced photo-transformation reaction of 20E. The structures and NMR signal assignment were established by comprehensive one- and two-dimensional NMR spectroscopy supported by mass spectroscopy. Possible ways for the formation of each species is also discussed. Similar to their parental compound, the products obtained are potentially bioactive and worthy for further investigations; due to the low yields, however, a different approach for their higher scale production is suggested.

Multidrug resistance reversing activity of newly developed phenothiazines on P-glycoprotein (ABCB1)-related resistance of mouse T-lymphoma cells.

BACKGROUND: Phenothiazines have anticancer properties and are able to reverse the multidrug resistance of neoplastic cells by inhibiting the ATP-binding cassette, sub-family B (MDR/TAP), member 1 protein (ABCB1 or P-glycoprotein) activity. MATERIALS AND METHODS: A series of new phenothiazine derivatives was investigated regarding their ABCB1-modulating effect on multidrug resistant mouse T-lymphoma cells by rhodamine 123 accumulation assay and real-time ethidium bromide accumulation assay. RESULTS: The phenothiazine derivatives exhibited a potent anticancer effect on the parental cell line and on its multidrug-resistant mouse T-lymphoma subline overexpressing the ABCB1 transporter. The inhibition of the ABCB1 transporter in the presence of the newly-developed phenothiazines was greater than that for the known ABCB1 inhibitors thioridazine and verapamil. CONCLUSION: Based on the chemical structures and biological activity, compounds with bivalent sulfur atom in the phenothiazine ring demonstrated marked ABCB1-modulating effect, however, other derivatives with halogen or amide substitutions were ineffective.

Mechanisms by which thioridazine in combination with antibiotics cures extensively drug-resistant infections of pulmonary tuberculosis.

Advances recently introduced into the Clinical Mycobacteriology Laboratory of the Institute of Hygiene and Tropical Medicine, such that a multi-drug resistant infection of pulmonary tuberculosis (MDR TB) can be identified within one day of receiving the sputum specimen, have greatly contributed to the reduction of the frequency of these infections. However, approximately 50% of reduced infections exhibit a phenotype that is consistent with that presented by an extensively drug-resistant (XDR) infection. More effective agents were required and hence attention was attributed to the possibility that the old neuroleptic phenothiazine thioridazine (TZ), previously shown to inhibit the growth of all encountered strains of Mycobacterium tuberculosis (Mtb) regardless of their antibiotic resistance profile, could be eventually used for therapy of problematic MDR/XDR TB infections. This mini-review discusses the mechanisms that render TZ an effective adjuvant to antibiotics to which the initial infective agent Mtb was resistant.

Efflux pumps of Gram-negative bacteria: what they do, how they do it, with what and how to deal with them.

This review discusses the relationship of the efflux pump (EP) system of Gram-negative bacteria to other antibiotic resistance mechanisms of the bacterium such as quorum sensing, biofilms, two component regulons, etc. The genetic responses of a Gram-negative to an antibiotic that render it immune to an antibiotic are also discussed. Lastly, the methods that have been developed for the identification of bacteria that over-express their EP system are presented in detail. Phenothiazines are well-known antipsychotic drugs with reported activity against bacterial EPs and other ancillary antibiotic mechanisms of the organism. Therefore these compounds will also be discussed.

Synthesis and structure-activity relationships of novel ecdysteroid dioxolanes as MDR modulators in cancer.

Ecdysteroids, molting hormones of insects, can exert several mild, non-hormonal bioactivities in mammals, including humans. In a previous study, we have found a significant effect of certain derivatives on the ABCB1 transporter mediated multi-drug resistance of a transfected murine leukemia cell line. In this paper, we present a structure-activity relationship study focused on the apolar dioxolane derivatives of 20-hydroxyecdysone. Semi-synthesis and bioactivity of a total of 32 ecdysteroids, including 20 new compounds, is presented, supplemented with their complete 1H- and 13C-NMR signal assignment.