Resistance reversing activity of plant-derived and novel synthetic compounds in bacterial models

Multidrug resistance is a major concern in the treatment of bacterial infections due to the reduced or missing response of microorganisms to the applied antimicrobial agents. One of the most important mechanisms of multidrug resistance is the increased expression of efflux pumps (EPs) that can extrude distinct classes of antibiotics from bacteria to the environment. In order to overcome this process, efflux pump inhibitors (EPIs) could be designed and applied alone or in combination with antibiotics as adjuvants. For this reason, the objective of this study was to investigate the antimicrobial and resistance modifying activity of natural compounds isolated from Cleistochlamys kirkii, fluorinated β-diketo phosphorus ylides and selenocompounds on different Gram-positive and Gram-negative strains. The effects of compounds on bacterial growth were determined by microdilution method. The EP inhibiting activity of compounds on the accumulation and/or efflux of the general EP substrate ethidium bromide was assessed by real-time fluorimetry on Escherichia coli, Salmonella Typhimurium and Staphylococcus aureus strains. The anti-biofilm effect of selenocompounds was investigated by crystal violet assay using S. Typhimurium 14028s strain. The combined effects of antibiotics (tetracycline and ciprofloxacin) and compounds were examined by minimum inhibitory concentration reduction assay or checkerboard microdilution method. The quorum sensing inhibition of natural compounds and phosphorus ylides was studied by agar diffusion method, furthermore the relative gene expression level of EP and/or quorum sensing genes were determined by reverse transcriptase quantitative polymerase chain reaction. Results showed that chamanetin (CK2) and dichamanetin (CK4) from C. kirkii showed synergism with antibiotics on methicillin sensitive S. aureus, all the natural compounds except isochamanetin (CK3) were able to inhibit the quorum sensing. The most potent representative of phosphorus ylides was 4,4,4-trifluoro-3-oxo-2-(triphenylphosphoranylidene)butanal (PY4) and its effect was more pronounced on the AcrAB-TolC efflux system expressing E. coli strain. According to the results the methylketone selenoester (EDA9) had remarkable antibacterial activity against Gram-positive strains. The most selenocompounds were able to inhibit the biofilm formation of S. Typhimurium. The selenoanhydride (EDA1) and the meta-substituted benzene selenodiester (EDA4) were active inhibitors of the AcrAB-TolC system in E. coli AG100. These results suggested that natural compounds could be effective adjuvants in the antibiotic treatment of infections. Phosphorus ylides might be valuable EPI compounds to reverse efflux related multidrug resistance in bacteria and selenocompounds can be effective antibacterials and EPIs

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.publishersversionpublishe

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

Reversal of Multidrug Resistance by Symmetrical Selenoesters in Colon Adenocarcinoma Cells

Recently, selenium containing derivatives have attracted more attention in medicinal chemistry. In the present work, the anticancer activity of symmetrical selenoesters was investigated by studying the reversal of efflux pump-related and apoptosis resistance in sensitive and resistant human colon adenocarcinoma cells expressing the ABCB1 protein. The combined effect of the compounds with doxorubicin was demonstrated with a checkerboard assay. The ABCB1 inhibitory and the apoptosis-inducing effects of the derivatives were measured with flow cytometry. Whole transcriptome sequencing was carried out on Illumina platform upon the treatment of resistant cells with the most potent derivatives. One ketone and three methyl ester selenoesters showed synergistic or weak synergistic interaction with doxorubicin, respectively. Ketone selenoesters were the most potent ABCB1 inhibitors and apoptosis inducers. Nitrile selenoesters could induce moderate early and late apoptotic processes that could be explained by their ABCB1 modulating properties. The transcriptome analysis revealed that symmetrical selenoesters may influence the redox state of the cells and interfere with metastasis formation. It can be assumed that these symmetrical selenocompounds possess toxic, DNA-damaging effects due to the presence of two selenium atoms in the molecule, which may be augmented by the presence of symmetrical groups

5-Arylidenerhodanines as P-gp modulators : an interesting effect of the carboxyl group on ABCB1 function in multidrug-resistant cancer cells

Multidrug resistance (MDR) is considered one of the major mechanisms responsible for the failure of numerous anticancer and antiviral chemotherapies. Various strategies to overcome the MDR phenomenon have been developed, and one of the most attractive research directions is focused on the inhibition of MDR transporters, membrane proteins that extrude cytotoxic drugs from living cells. Here, we report the results of our studies on a series newly synthesized of 5-arylidenerhodanines and their ability to inhibit the ABCB1 efflux pump in mouse T-lymphoma cancer cells. In the series, compounds possessing a triphenylamine moiety and the carboxyl group in their structure were of particular interest. These amphiphilic compounds showed over 17-fold stronger efflux pump inhibitory effects than verapamil. The cytotoxic and antiproliferative effects of target rhodanines on T-lymphoma cells were also investigated. A putative binding mode for 11, one of the most potent P-gp inhibitors tested here, was predicted by molecular docking studies and discussed with regard to the binding mode of verapamil

5-Arylidenerhodanines as P-gp Modulators: An Interesting Effect of the Carboxyl Group on ABCB1 Function in Multidrug-Resistant Cancer Cells

Multidrug resistance (MDR) is considered one of the major mechanisms responsible for the failure of numerous anticancer and antiviral chemotherapies. Various strategies to overcome the MDR phenomenon have been developed, and one of the most attractive research directions is focused on the inhibition of MDR transporters, membrane proteins that extrude cytotoxic drugs from living cells. Here, we report the results of our studies on a series newly synthesized of 5-arylidenerhodanines and their ability to inhibit the ABCB1 efflux pump in mouse T-lymphoma cancer cells. In the series, compounds possessing a triphenylamine moiety and the carboxyl group in their structure were of particular interest. These amphiphilic compounds showed over 17-fold stronger efflux pump inhibitory effects than verapamil. The cytotoxic and antiproliferative effects of target rhodanines on T-lymphoma cells were also investigated. A putative binding mode for 11, one of the most potent P-gp inhibitors tested here, was predicted by molecular docking studies and discussed with regard to the binding mode of verapamil

Search for ABCB1 modulators among 2-amine-5-arylideneimidazolones as a new perspective to overcome cancer multidrug resistance

Multidrug resistance (MDR) is a severe problem in the treatment of cancer with overexpression of glycoprotein P (Pgp, ABCB1) as a reason for chemotherapy failure. A series of 14 novel 5-arylideneimidazolone derivatives containing the morpholine moiety, with respect to two different topologies (groups A and B), were designed and obtained in a three- or four-step synthesis, involving the Dimroth rearrangement. The new compounds were tested for their inhibition of the ABCB1 efflux pump in both sensitive (parental (PAR)) and ABCB1-overexpressing (MDR) T-lymphoma cancer cells in a rhodamine 123 accumulation assay. Their cytotoxic and antiproliferative effects were investigated by a thiazolyl blue tetrazolium bromide (MTT) assay. For active compounds, an insight into the mechanisms of action using either the luminescent Pgp-Glo™ Assay in vitro or docking studies to human Pgp was performed. The safety profile in vitro was examined. Structure–activity relationship (SAR) analysis was discussed. The most active compounds, representing both 2-substituted- (11) and Dimroth-rearranged 3-substituted (18) imidazolone topologies, displayed 1.38–1.46 fold stronger efflux pump inhibiting effects than reference verapamil and were significantly safer than doxorubicin in cell-based toxicity assays in the HEK-293 cell line. Results of mechanistic studies indicate that active imidazolones are substrates with increasing Pgp ATPase activity, and their dye-efflux inhibition via competitive action on the Pgp verapamil binding site was predicted in silico

Pharmacophoric features for a very potent 5-spirofluorenehydantoin inhibitor of cancer efflux pump ABCB1, based on X-ray analysis

In order to extend knowledge about pharmacophoric features responsible for ABCB1 inhibitory properties of imidazolidin-2,4-dione derivatives, 1'-[4-(4-(o-methoxyphenyl)-piperazin-1-yl)butyl]-3'-methyl-spiro(fluoren-9,5'-imidazolidine)-2',4'-dione (3) and its salt (4) with rhodanine-3-acetic acid (RA) were prepared and investigated by X-ray diffraction method, as well as their efflux modulating effects in cancer cells (mouse T-lymphoma), cytotoxic and antiproliferative activities were evaluated in vitro. The molecular geometry, intermolecular interactions and crystal packing of base and acid forms of 3 were analyzed to see, if conformational changes influence the biological activities. The geometry of 2-methoxyphenylpiperazine and 5-spirofluorenehydantoin moieties was compared with other crystal structures containing these fragments. Our results indicated a very potent inhibitory action on ABCB1 pump, and significant cytotoxic and antiproliferative properties of 3 in T-lymphoma, even more potent in the case of MDR cells. Furthermore, the compound 3 converted into the salt 4 of inactive acid (RA) has maintained both, the efflux pump inhibitory and antiproliferative activities, showing strong synergism with doxorubicin. A comparison of geometry of 3 in both crystal structures (3 and 4) shows a significant difference in the arrangement of piperazine ring with respect to the aliphatic linker. This article is protected by copyright. All rights reserved

Indonesian Euphorbiaceae: Ethnobotanical Survey, In Vitro Antibacterial, Antitumour Screening and Phytochemical Analysis of Euphorbia atoto

Indonesia is among the countries with the most significant biodiversity globally. Jamu, the traditional medicine of Indonesia, predominantly uses herbal materials and is an integral component of the Indonesian healthcare system. The present study reviewed the ethnobotanical data of seven Indonesian Euphorbiaceae species, namely Euphorbia atoto, E. hypericifolia, Homalanthus giganteus, Macaranga tanarius, Mallotus mollissimus, M. rufidulus, and Shirakiopsis indica, based on the RISTOJA database and other literature sources. An antimicrobial screening of the plant extracts was performed in 15 microorganisms using the disk diffusion and broth microdilution methods, and the antiproliferative effects were examined in drug-sensitive Colo 205 and resistant Colo 320 cells by the MTT assay. The antimicrobial testing showed a high potency of M. tanarius, H. giganteus, M. rufidulus, S. indica, and E. atoto extracts (MIC = 12.5–500 µg/mL) against different bacteria. In the antitumour screening, remarkable activities (IC50 0.23–2.60 µg/mL) were demonstrated for the extracts of H. giganteus, M. rufidulus, S. indica, and E. atoto against Colo 205 cells. The n-hexane extract of E. atoto, with an IC50 value of 0.24 ± 0.06 µg/mL (Colo 205), was subjected to multistep chromatographic separation, and 24-methylene-cycloartan-3β-ol, jolkinolide E, tetra-tert-butyl-diphenyl ether, α-tocopherol, and β-sitosterol were isolated

Antiviral, antimicrobial and antibiofilm activity of selenoesters and selenoanhydrides

Selenoesters and the selenium isostere of phthalic anhydride are bioactive selenium compounds with a reported promising activity in cancer, both due to their cytotoxicity and capacity to reverse multidrug resistance. Herein we evaluate the antiviral, the biofilm inhibitory, the antibacterial and the antifungal activities of these compounds. The selenoanhydride and 7 out of the 10 selenoesters were especially potent antiviral agents in Vero cells infected with herpes simplex virus-2 (HSV-2). In addition, the tested selenium derivatives showed interesting antibiofilm activity against Staphylococcus aureus and Salmonella enterica serovar Typhimurium, as well as a moderate antifungal activity in resistant strains of Candida spp. They were inactive against anaerobes, which may indicate that the mechanism of action of these derivatives depends on the presence of oxygen. The capacity to inhibit the bacterial biofilm can be of particular interest in the treatment of nosocomial infections and in the coating of surfaces of prostheses. Finally, the potent antiviral activity observed converts these selenium derivatives into promising antiviral agents with potential medical applications.The study was supported by the project SZTE ÁOK-KKA 2018/270-62-2 of the University of Szeged, Faculty of Medicine. Gabriella Spengler was also supported by the János Bolyai Research Scholarship of the Hungarian Academy of Sciences. The authors of this paper received funding from the Márton Áron Research Programme financed by the Hungarian Ministry of Foreign Aairs and Trade. AK was supported by the New National Excellence Program (ÚNKP-18-3) of the Ministry of Human Capacities of Hungary and by the Campus mundi short-study program of the Tempus Public Foundation. EDA was supported by the Spanish “Consejo Superior de Investigaciones Científicas” (201780I027) (CSIC, Spanish National Research Council). CSM wishes to express gratitude to UNED-Pamplona, Fundación Bancaria “La Caixa”, and “Fundación Caja Navarra” for financial support for the project. JMAB and HEM are supported by a BBSRC David Phillips Fellowship to JMAB (BB/M02623X/1)