Structure, function, and inhibition of catalytically asymmetric ABC transporters: Lessons from the PDR subfamily.

ATP-binding cassette (ABC) superfamily comprises a large group of ubiquitous transmembrane proteins that play a crucial role in transporting a diverse spectrum of substrates across cellular membranes. They participate in a wide array of physiological and pathological processes including nutrient uptake, antigen presentation, toxin elimination, and drug resistance in cancer and microbial cells. ABC transporters couple ATP binding and hydrolysis to undergo conformational changes allowing substrate translocation. Within this superfamily, a set of ABC transporters has lost the capacity to hydrolyze ATP at one of their nucleotide-binding sites (NBS), called the non-catalytic NBS, whose importance became evident with extensive biochemistry carried out on yeast pleiotropic drug resistance (PDR) transporters. Recent single-particle cryogenic electron microscopy (cryo-EM) advances have further catapulted our understanding of the architecture of these pumps. We provide here a comprehensive overview of the structural and functional aspects of catalytically asymmetric ABC pumps with an emphasis on the PDR subfamily. Furthermore, given the increasing evidence of efflux-mediated antifungal resistance in clinical settings, we also discuss potential grounds to explore PDR transporters as therapeutic targets.

ABC-F proteins in mRNA translation and antibiotic resistance.

The ATP binding cassette protein superfamily comprises ATPase enzymes which are, for the most part, involved in transmembrane transport. Within this superfamily however, some protein families have other functions unrelated to transport. One example is the ABC-F family, which comprises an extremely diverse set of cytoplasmic proteins. All of the proteins in the ABC-F family characterized to date act on the ribosome and are translation factors. Their common function is ATP-dependent modulation of the stereochemistry of the peptidyl transferase center (PTC) in the ribosome coupled to changes in its global conformation and P-site tRNA binding geometry. In this review, we give an overview of the function, structure, and theories for the mechanisms-of-action of microbial proteins in the ABC-F family, including those involved in mediating resistance to ribosome-binding antibiotics.

Antioxidant, Anti-Inflammatory, and Multidrug Resistance Modulation Activity of Silychristin Derivatives.

Silychristin A is the second most abundant compound of silymarin. Silymarin complex was previously described as an antioxidant with multidrug resistance modulation activity. Here, the results of a classical biochemical antioxidant assay (ORAC) were compared with a cellular assay evaluating the antioxidant capacity of pure silychristin A and its derivatives (anhydrosilychristin, isosilychristin and 2,3-dehydrosilychristin A). All the tested compounds acted as antioxidants within the cells, but 2,3-dehydro- and anhydro derivatives were almost twice as potent as the other tested compounds. Similar results were obtained in LPS-stimulated macrophages, where 2,3-dehydro- and anhydrosilychristin inhibited NO production nearly twice as efficiently as silychristin A. The inhibition of P-glycoprotein (P-gp) was determined in vitro, and the respective sensitization of doxorubicin-resistant ovarian carcinoma overproducing P-gp was detected. Despite the fact that the inhibition of P-gp was demonstrated in a concentration-dependent manner for each tested compound, the sensitization of the resistant cell line was observed predominantly for silychristin A and 2,3-dehydrosilychristin A. However, anhydrosilychristin and isosilychristin affected the expression of both the P-gp (ABCB1) and ABCG2 genes. This is the first report showing that silychristin A and its 2,3-dehydro-derivative modulate multidrug resistance by the direct inhibition of P-gp, in contrast to anhydrosilychristin and isosilychristin modulating multidrug resistance by downregulating the expression of the dominant transmembrane efflux pumps.

Multidrug transporters of Candida species in clinical azole resistance.

Over-expression of the human P-glycoprotein (P-gp) in tumor cells is a classic example of an ABC protein serving as a hindrance to effective chemotherapy. The existence of proteins homologous to P-gp in organisms encompassing the entire living kingdom highlights extrusion of drugs as a general mechanism of multidrug resistance. Infections caused by opportunistic human fungal pathogens such as Candida species are very common and has intensified in recent years. The typical hosts, who possess suppressed immune systems due to conditions such as HIV and transplantation surgery etc., are prone to fungal infections. Prolonged chemotherapy induces fungal cells to eventually develop tolerance to most of the antifungals currently in clinical use. Amongst other prominent mechanisms of antifungal resistance such as manipulation of the drug target, rapid efflux achieved through overexpression of multidrug transporters has emerged as a major resistance mechanism for azoles. Herein, the azole-resistant clinical isolates of Candida species utilize a few select efflux pump proteins belonging to the ABC and MFS superfamilies, to deter the toxic accumulation of therapeutic azoles and thus, facilitating cell survival. In this article, we summarize and discuss the clinically relevant mechanisms of azole resistance in Candida albicans and non-albicans Candida (NAC) species, specifically highlighting the role of multidrug efflux proteins in the phenomenon.

The E-helix is a central core in a conserved helical bundle involved in nucleotide binding and transmembrane domain intercalation in the ABC transporter superfamily.

ABC transporter proteins are involved in active transport, both in prokaryotes and eukaryotes. Sequence analysis of nucleotide binding domains (NBDs) of ABC proteins from all taxa revealed a well-conserved new motif having the signature: xT/ShxE/DNhxF, located between Q-loop and ABC signature sequence. A recent structure of an ABC transporter, ABCG5/G8 highlighted the motif as an essential structural determinant of inter-domain crosstalk and termed it as E-helix. We carried out an extensive computational analysis to unravel important structural entities alongside E-helix which plausibly play role in the interlocking mechanism of NBD with TMD. We identified E-helix to be a central structural moiety which interacts with three helices and an intracellular loop that leads to the transmembrane domain. Considering its wide occurrence, we examined the importance of this motif in one representative multidrug ABC transporter of Candida albicans, Cdr1p. The motif residues were replaced by alanines both individually as well as in combinations. The GFP-tagged versions of mutant proteins were overexpressed in Saccharomyces cerevisiae. Overall, our mutational data suggested that this motif plays a role in the maintenance of proper structural fold and/or inter-domain contacts in Cdr1p. We, thus, unveil an essential structural motif in ABC superfamily transporters.

Targeted nanomedicine for cancer therapeutics: Towards precision medicine overcoming drug resistance.

Intrinsic anticancer drug resistance appearing prior to chemotherapy as well as acquired resistance due to drug treatment, remain the dominant impediments towards curative cancer therapy. Hence, novel targeted strategies to overcome cancer drug resistance constitute a key aim of cancer research. In this respect, targeted nanomedicine offers innovative therapeutic strategies to overcome the various limitations of conventional chemotherapy, enabling enhanced selectivity, early and more precise cancer diagnosis, individualized treatment as well as overcoming of drug resistance, including multidrug resistance (MDR). Delivery systems based on nanoparticles (NPs) include diverse platforms enabling a plethora of rationally designed therapeutic nanomedicines. Here we review NPs designed to enhance antitumor drug uptake and selective intracellular accumulation using strategies including passive and active targeting, stimuli-responsive drug activation or target-activated release, triggered solely in the cancer cell or in specific organelles, cutting edge theranostic multifunctional NPs delivering drug combinations for synergistic therapy, while facilitating diagnostics, and personalization of therapeutic regimens. In the current paper we review the recent findings of the past four years and discuss the advantages and limitations of the various novel NPs-based drug delivery systems. Special emphasis is put on in vivo study-based evidences supporting significant therapeutic impact in chemoresistant cancers. A future perspective is proposed for further research and development of complex targeted, multi-stage responsive nanomedical drug delivery systems for personalized cancer diagnosis and efficacious therapy.

Papel de la superfamilia ABC en la resistencia farmacológica / Role of the superfamily of ABC transports in pharmacological resistance / Papel da superfamília ABC na resistência a drogas / Rôle de la superfamille ABC dans la résistance pharmacologique

Resumen Objetivo: Actualizar los conocimientos acerca de los transportadores de la superfamilia ABC vinculados con la resistencia farmacológica. Materiales y métodos: Se realizó un estudio donde se aplicó el método cualitativo, mediante una revisión bibliográfica y documental sobre el tema en fuentes de datos digitales. Se usaron los descriptores DeCs-MeSH: transportadores ABC, superfamilia ABC, resistencia farmacológica, ATP binding- cassette. Se revisaron artículos publicados sobre el tema, a través de los buscadores habituales (Google, PubMed, Cochrane, Future Medicine, Scielo, entre otros), teniendo en cuenta la calidad y la actualidad de ellos. Resultados: Se destaca la importancia clínica de estos transportadores que se relacionan con la aterosclerosis, enfermedades hepáticas, enfermedad de Alzheimer, entre otras. Esto los convierte en dianas atractivas para el diseño de nuevos medicamentos; pero al mismo tiempo, pueden expulsarlos de la célula, haciéndola resistente como a los antitumorales, antimicrobianos y antivirales. Conclusiones: Los transportadores ABC tienen una función central en los efectos adversos que diferentes sustancias, entre ellas los fármacos, tienen sobre la célula. Además, el polimorfismo genético en esta superfamilia se asocia con alteraciones farmacocinéticas que pueden desencadenar resistencia farmacológica; el impacto de estas modificaciones es el centro de recientes investigaciones que las sitúan como posible blanco terapéutico.

Abstract Objective: To update the knowledge about the ABC transporters superfamily linked to drug resistance. Materials and methods: A qualitative study where the method was applied, using a bibliographical and documentary review on the subject of digital data sources was conducted. ABC transporters, ABC superfamily, and drug resistance, binding- ATP cassette: the DECS-MeSH descriptors were using published articles on the subject through the usual search engines (Google, PubMed, Cochrane, Future Medicine, Scielo, among others), counting on the quality and timeliness of them were review. Results: The clinical significance of these transporters is stress as they relate to atherosclerosis, liver disease, Alzheimer's disease, among other conditions. This makes them attractive targets for new drug design; but at the same time, they can expel the cell making it resistant as antitumor, antimicrobial and antiviral. Conclusions: ABC transporters have central role in the adverse effects of different substances, including drugs, have on the cell. In addition, the genetic polymorphism in this superfamily is associated with pharmacokinetic changes that can trigger drug resistance; the impact of these changes is the focus of recent research that place them as a possible therapeutic target.

Resumo Objectivo: Atualizar o conhecimento sobre os transportadores de superfamilia ABC ligada a resistência a drogas. Materiais e métodos: Foi realizado um estudo qualitativo, através de uma revisão da literatura e documental sobre o tema em fontes de dados digitais. Foram utilizados os descritores DeCs-MeSH: transportadores ABC, superfamilia ABC, resistência farmacológica, ATP binding- cassette. Foram pesquisados artigos sobre o tema, através dos motores de busca (Google, PubMed, Cochrane, Future Medicine, Scielo, entre outros), tendo em conta a qualidade e a sua atualidade. Resultados: Destacou-se a importãncia clínica destes transportadores que se relacionam com a aterosclerose, doen9as hepáticas, de Alzheimer, entre outras. Isso os torna alvos atraentes para o design de novos fármacos, mas ao mesmo tempo, os fármacos podem ser expulsos da célula, tornando-a resistente aos antitumorais, antimicrobianos e antivirais. Conclusões: Os transportadores ABC têm uma função central nos efeitos adversos que diferentes substãncias, tais como os fármacos, possuem sobre a célula. Além disso, o polimorfismo genético desta superfamilia está associado as alterações farmacocinéticas que podem desencadear a resistência aos medicamentos; o impacto dessas mudanças é o centro de pesquisas recentes que os coloca como um possível alvo terapéutico.

Résumé Objectif: Actualiser les connaissances sur les transporteurs de la superfamille ABC liés a la résistance pharmacologique. Matériaux et méthodes: Une étude qualitative a été réalisée au moyen d'une recherche bibliographique et documentaire dans différentes sources de données numériques avec les moteurs de recherche habituels (Google, PubMed, Cochrane, Medicine Future, Scielo, entre autres). Les descripteurs DeCs-MeSH utilisés ont été: transporteurs ABC, superfamille ABC, résistance pharmacologique, ATP binding- cassette. Un certain nombre d'articles relatifs au sujet ont été sélectionnés en fonction de leur qualité et de leur actualité. Résultats: Les résultats mettent en avant l'importance clinique de ces transporteurs du fait de leur relation avec l'athérosclérose, certaines maladies hépatiques, la maladie d'Alzheimer, et d'autres affections. Cela en fait des cibles attrayantes pour la conception de nouveaux médicaments; mais en meme temps, ils peuvent expulser des médicaments de la cellule et la rendre résistante aux antitumoraux, antimicrobiens et antiviraux, par exemple. Conclusions: Les transporteurs ABC jouent un role central dans les conséquences néfastes de différentes substances, y compris les médicaments, sur la cellule. En outre, le polymorphisme génétique dans cette superfamille est associé a des modifications pharmacocinétiques qui peuvent déclencher une résistance aux médicaments; l'impact de ces modifications est l'objet de recherches récentes qui les placent comme cible thérapeutique possible.

Inverse association between microRNA-124a and ABCC4 in HepG2 cells treated with antiretroviral drugs.

The ATP-binding cassette (ABC) super-family of drug transporters regulates efflux of xenobiotic compounds. The subfamily, multi-drug resistance proteins (MRPs) transports cyclic nucleotides and xenobiotics. Epigenetic modulation of drug transporters is scarcely described. The regulatory role of microRNA (miR)-124a on drug transporter gene ABCC4 was only recently reported. Our study investigated the differential regulation of miR-124a by nucleoside reverse transcriptase inhibitors (NRTIs): Zidovudine (AZT), Stavudine (d4T) and Tenofovir (TFV); at 24 h and 120 h treatments in HepG2 cells. ABCC4 mRNA (qPCR) and ABCC4 protein (western blot) were quantified. Cytotoxicity was evaluated by lactate dehydrogenase (LDH) levels. All NRTIs elevated miR-124a levels at 24 h, with a concomitant decline in ABCC4 mRNA levels (p<0.05). At 120 h, d4T and TFV elevated miR-124a and depleted ABCC4 mRNA levels (p<0.0001), while the inverse was observed with AZT (p<0.005). ABCC4 protein was increased by d4T and TFV at 24h. A significant reduction in protein levels was observed at 120 h in all three treatments (p<0.005). The disjoint in mRNA and protein levels is likely due to ABCC4 being a membrane bound protein. Following prolonged exposure, membrane integrity was compromised as evidenced by increased LDH leakage (p<0.005). We conclude antiretroviral drugs have varying effects on miR-124a and ABCC4.