5-Oxo-hexahydroquinoline derivatives as modulators of P-gp, MRP1 and BCRP transporters to overcome multidrug resistance in cancer cells.

Multidrug resistance (MDR) in cancer cells is often associated with overexpression of ATP-binding cassette (ABC) transporters, including P-glycoprotein (P-gp/ABCB1), multidrug resistance-associated protein 1 (MRP1/ABCC1) and breast cancer resistance protein (BCRP/ABCG2). Modulators of these transporters might be helpful in overcoming MDR. Moreover, exploiting collateral sensitivity (CS) could be another approach for efficient treatment of cancer. Twelve novel 5-oxo-hexahydroquinoline derivatives bearing different aromatic substitutions at C4, while having 2-pyridyl alkyl carboxylate substituents at the C3 were synthesized and evaluated for MDR reversal activity by flow cytometric determination of rhodamine 123, calcein and mitoxantrone accumulations in P-gp, MRP1 and BCRP-overexpressing cell lines, respectively. Furthermore, to confirm the P-gp inhibitory activity, the effect of compounds on the reduction of doxorubicin's IC50 of drug-resistant human uterine sarcoma cell line, MES-SA/DX5, was evaluated. Compounds D6, D5 and D3 (bearing 3-chlorophenyl, 2,3-dichlorophenyl and 4-chlorophenyl substituents at C4 position of 5-oxo-hexahydroquinoline core) were the most potent P-gp, MRP1 and BCRP inhibitors, respectively, causing significant MDR reversal at concentrations of 1-10 µM. Additionally, D4 (containing 3-flourophenyl) was the most effective MRP1-dependent CS inducing agent. Overall, chlorine containing compounds D6, C4 and D3 were capable of significant inhibition of all 3 important efflux pumps in cancer cells. Moreover, D6 also induced CS triggered by reducing glutathione efflux. In conclusion, some of the 5-oxo-hexahydroquinoline derivatives are effective efflux pump inhibitors capable of simultaneously blocking 3 important ABC transporters involved in MDR, and represent promising agents to overcome MDR in cancer cells.

W1038 near D-loop of NBD2 is a focal point for inter-domain communication in multidrug transporter Cdr1 of Candida albicans.

Candida drug resistance 1 (Cdr1), a PDR subfamily ABC transporter mediates efflux of xenobiotics in Candida albicans. It is one of the prime factors contributing to multidrug resistance in the fungal pathogen. One hallmark of this transporter is its asymmetric nature, characterized by peculiar alterations in its nucleotide binding domains. As a consequence, there exists only one canonical ATP-binding site while the other is atypical. Here, we report suppressor analysis on the drug-susceptible transmembrane domain mutant V532D that identified the suppressor mutation W1038S, close to the D-loop of the non-catalytic ATP-binding site. Introduction of the W1038S mutation in the background of V532D mutant conferred resistance for most of the substrates to the latter. Such restoration is accompanied by a severe reduction of ATPase activity, of about 85%, while that of the V532D mutant is half-reduced. Conversely, alanine substitution of the highly conserved aspartate D1033A in that D-loop rendered cells selectively hyper-susceptible to miconazole without an impact on steady-state ATPase activity, suggesting altogether that ATP hydrolysis may not hold the key to restoration mechanism. Analysis of the ABCG5/ABCG8-based 3D-model of Cdr1p suggested that the W1038S substitution leads to the loss of hydrophobic interactions and H-bond with residues of the neighbor NBD1, in the non-catalytic ATP-binding site area. The compensatory effect within TMDs accounting for transport restoration in the V532D-W1038S variant may, therefore, be mainly due to an increase in NBDs mobility at the non-catalytic interface.

Methyl-cyclopentadienyl Ruthenium Compounds with 2,2'-Bipyridine Derivatives Display Strong Anticancer Activity and Multidrug Resistance Potential.

New ruthenium methyl-cyclopentadienyl compounds bearing bipyridine derivatives with the general formula [Ru(η5-MeCp)(PPh3)(4,4'-R-2,2'-bpy)]+ (Ru1, R = H; Ru2, R = CH3; and Ru3, R = CH2OH) have been synthesized and characterized by spectroscopic and analytical techniques. Ru1 crystallized in the monoclinic P21/ c, Ru2 in the triclinic P1̅, and Ru3 in the monoclinic P21/ n space group. In all molecular structures, the ruthenium center adopts a "piano stool" distribution. Density functional theory calculations were performed for all complexes, and the results support spectroscopic data. Ru1 and Ru3 were poor substrates of the main multidrug resistance human pumps, ABCB1, ABCG2, ABCC1, and ABCC2, while Ru2 displayed inhibitory properties of ABCC1 and ABCC2 pumps. Importantly, all compounds displayed a very high cytotoxic profile for ovarian cancer cells (sensitive and resistant) that was much more pronounced than that observed with cisplatin, making them very promising anticancer agents.

Monoterpene indole alkaloid azine derivatives as MDR reversal agents.

Aiming at generating a library of bioactive indole alkaloid derivatives as multidrug resistance (MDR) reversers, two epimeric indole alkaloids (1 and 2) were submitted to chemical transformations, giving rise to twenty-four derivatives (5-28), bearing new aromatic or aliphatic azine moieties. The structure of the compounds was established by 1D and 2D NMR (COSY, HMBC, HMQC and NOESY) experiments. Two different strategies were employed for assessing their anti-MDR potential, namely through the evaluation of their activity as inhibitors of typical MDR ABC transporters overexpressed by cell transfection, such as ABCB1 (P-gp), ABCC1 (MRP1), and ABCG2 (BCRP), or by evaluating their ability as collateral sensitivity (CS) agents in cells overexpressing MRP1. A considerable MDR reversing activity was observed for compounds bearing the aromatic azine moiety. The strongest and most selective P-gp inhibition was found for the epimeric azines 5 and 6, bearing a para-methylbenzylidene moiety. Instead, compounds 17 and 18 that possess a di-substituted benzylidene portion with methoxy and hydroxyl groups, selectively inhibited MRP1 drug-efflux. None of these compounds inhibited BCRP. Compounds 5, 6 and 18 were further investigated in drug combination experiments, which corroborated their anti-MDR potential. Moreover, it was observed that compound 12, with an aromatic azine moiety, and compounds 23-26, sharing a new aliphatic substituent, displayed a CS activity, selectively killing MRP1-overexpressing cells. Among these last compounds, it could be established that addition of 19, 23 and 25 to MRP1-overexpressing cells led to glutathione depletion triggering cell death through apoptosis.

Atomic modelling and systematic mutagenesis identify residues in multiple drug binding sites that are essential for drug resistance in the major Candida transporter Cdr1.

The ABC (ATP-Binding Cassette) transporter Cdr1 (Candida drug resistance 1) protein (Cdr1p) of Candida albicans, shows promiscuity towards the substrate it exports and plays a major role in antifungal resistance. It has two transmembrane domains (TMDs) comprising of six transmembrane helices (TMH) that envisage and confer the substrate specificity and two nucleotide binding domains (NBDs), interconnected by extracellular loops (ECLs) and intracellular loops (ICLs) Cdr1p. This study explores the diverse substrate specificity spectrum to get a deeper insight into the structural and functional features of Cdr1p. By screening with the variety of compounds towards an in-house TMH 252 mutant library of Cdr1p, we establish new substrates of Cdr1p. The localization of substrate-susceptible mutants in an ABCG5/G8 homology model highlights the common and specific binding pockets inside the membrane domain, where rhodamines and tetrazoliums mainly engage the N-moiety of Cdr1p, binding between TMH 2, 11 and surrounded by TMH 1, 5. Whereas, tin chlorides involve both N and C moieties located at the interface of TMH 2, 11, 1 and 5. Further, screening of the in house TMH mutant library of Cdr1p displays the TMH12 interaction with tetrazolium chloride, trimethyltin chloride and a Ca2+ ionophore, A23187. In silico localization reveals a binding site at the TMH 12, 9 and 10 interface, which is widely exposed to the lipid interface. Together, for the first time, our study shows the molecular localization of Cdr1p substrates-binding sites and demonstrates the participation of TMH12 in a peripheral drug binding site.

pHluorin enables insights into the transport mechanism of antiporter Mdr1: R215 is critical for drug/H+ antiport.

Multidrug resistance 1 (MDR1) is a member of the major facilitator superfamily that contributes to MDR of Candida albicans This antiporter belongs to the drug/H(+) antiporter 1 family, pairing the downhill gradient of protons to drug extrusion. Hence, drug efflux from cytosol to extracellular space and the parallel import of H(+) towards cytosol are inextricably linked processes. For monitoring the drug/H(+) antiporter activity of Mdr1p, we developed a new system, exploiting a GFP variant pHluorin, which changes its fluorescence properties with pH. This enabled us to measure the cytosolic pH correlated to drug efflux. Since protonation of charged residues is a key step in proton movement, we explored the role of all charged residues of the 12 transmembrane segments (TMSs) of Mdr1p in drug/H(+) transport by mutational analysis. This revealed that the conserved residue R(215), positioned close to the C-terminal end of TMS-4, is critical for drug/H(+) antiport, allowing protonation over a range of pH, in contrast with its H(215) or K(215) variants that failed to transport drugs at basic pH. Mutation of other residues of TMS-4 highlights the role of this TMS in drug transport, as confirmed by in silico modelling of Mdr1p and docking of drugs. The model points to the importance of R(215) in proton transport, suggesting that it may adopt two main conformations, one oriented towards the extracellular face and the other towards the centre of Mdr1p. Together, our results not only establish a new system for monitoring drug/H(+) transport, but also unveil a positively charged residue critical to Mdr1p function.

SERIAL ULTRASOUND TO ESTIMATE FETAL GROWTH CURVES IN SOUTHERN TAMANDUA (TAMANDUA TETRADACTYLA).

From 2012 to 2015, 16 pregnancies were monitored by ultrasonography in nine tamanduas ( Tamandua tetradactyla ) housed in three zoological facilities. Sonographic measurements were recorded to establish fetal growth curves using thoracic and skull landmarks described for giant anteaters ( Myrmecophaga tridactyla ). All pregnancies resulted in the uncomplicated delivery of healthy offspring, thus gestational development was considered normal. These data may be used as a reference for normal fetal development with potential for estimating parturition date in the absence of breeding data.

Purification and characterization of Cdr1, the drug-efflux pump conferring azole resistance in Candida species.

Candida albicans and C. glabrata express exporters of the ATP-binding cassette (ABC) superfamily and address them to their plasma membrane to expel azole antifungals, which cancels out their action and allows the yeast to become multidrug resistant (MDR). In a way to understand this mechanism of defense, we describe the purification and characterization of Cdr1, the membrane ABC exporter mainly responsible for such phenotype in both species. Cdr1 proteins were functionally expressed in the baker yeast, tagged at their C-terminal end with either a His-tag for the glabrata version, cgCdr1-His, or a green fluorescent protein (GFP) preceded by a proteolytic cleavage site for the albicans version, caCdr1-P-GFP. A membrane Cdr1-enriched fraction was then prepared to assay several detergents and stabilizers, probing their level of extraction and the ATPase activity of the proteins as a functional marker. Immobilized metal-affinity and size-exclusion chromatographies (IMAC, SEC) were then carried out to isolate homogenous samples. Overall, our data show that although topologically and phylogenetically close, both proteins display quite distinct behaviors during the extraction and purification steps, and qualify cgCdr1 as a good candidate to characterize this type of proteins for developing future inhibitors of their azole antifungal efflux activity.

Non-ionic cholesterol-based additives for the stabilization of membrane proteins.

We report herein the synthesis of two non-ionic amphiphiles with a cholesterol hydrophobic moiety that can be used as chemical additives for biochemical studies of membrane proteins. They were designed to show a high similarity with the planar steroid core of cholesterol and small-to-medium polar head groups attached at the C3 position of ring-A on the sterol skeleton. The two Chol-Tris and Chol-DG have a Tris-hydroxymethyl and a branched diglucose polar head group, respectively, which provide them sufficient water solubility when mixed with the "gold standard" detergent n-Dodecyl-ß-D-Maltoside (DDM). The colloidal properties of these mixed micelles were investigated by means of surface tension (SFT) measurements and dynamic light scattering (DLS) experiments and showed the formation of globular micelles of about 8 nm in diameter with a critical micellar concentration of 0.20 mM for DDM:Chol-DG and 0.22 mM for DDM:Chol-Tris. We showed that mixed micelles do not alter the extraction potency of a G-protein coupled receptor (GPCR): the human adenosine A2A receptor (A2AR). The thermostabilizing effect of the mixed micelles was confirmed on two GPCRs, A2AR and the growth hormone secretagogue receptor (GHSR). Finally, these two mixed micelles were found suitable for the purification of an active form of A2AR which remained able to bind two ligands of different class i.e. the specific agonist CGS-21680 and the specific inverse agonist ZM-241385. This suggests that Chol-Tris and Chol-DG may be used as a non-ionic alternative to the cholesteryl hemisuccinate (CHS) stabilizing agent.

Complete Chloroplast Genome of an Endophytic Ostreobium sp. (Ostreobiaceae) from the U.S. Virgin Islands.

We present the complete chloroplast genome sequence of an endophytic Ostreobium sp. isolated from a 19th-century coralline red algal specimen from St. Croix, U.S. Virgin Islands. The chloroplast genome is 84,848 bp in length, contains 114 genes, and has a high level of gene synteny to other Ostreobiaceae.

Spontaneous Suppressors against Debilitating Transmembrane Mutants of CaMdr1 Disclose Novel Interdomain Communication via Signature Motifs of the Major Facilitator Superfamily.

The Major Facilitator Superfamily (MFS) drug:H+ antiporter CaMdr1, from Candida albicans, is responsible for the efflux of structurally diverse antifungals. MFS members share a common fold of 12−14 transmembrane helices (TMHs) forming two N- and C-domains. Each domain is arranged in a pseudo-symmetric fold of two tandems of 3-TMHs that alternatively expose the drug-binding site towards the inside or the outside of the yeast to promote drug binding and release. MFS proteins show great diversity in primary structure and few conserved signature motifs, each thought to have a common function in the superfamily, although not yet clearly established. Here, we provide new information on these motifs by having screened a library of 64 drug transport-deficient mutants and their corresponding suppressors spontaneously addressing the deficiency. We found that five strains recovered the drug-resistance capacity by expressing CaMdr1 with a secondary mutation. The pairs of debilitating/rescuing residues are distributed either in the same TMH (T127ATMH1- > G140DTMH1) or 3-TMHs repeat (F216ATMH4- > G260ATMH5), at the hinge of 3-TMHs repeats tandems (R184ATMH3- > D235HTMH4, L480ATMH10- > A435TTMH9), and finally between the N- and C-domains (G230ATMH4- > P528HTMH12). Remarkably, most of these mutants belong to the different signature motifs, highlighting a mechanistic role and interplay thought to be conserved among MFS proteins. Results also point to the specific role of TMH11 in the interplay between the N- and C-domains in the inward- to outward-open conformational transition.

Production and Purification of a GFP-Tagged ABC Transporter CaCdr1p.

The production and purification are the first steps required in any functional or structural study of a protein of interest. In the case of membrane proteins, these tasks can be difficult due to low expression levels and the necessity to extract them from their membrane environment. This chapter describes a convenient method based on GFP tagged to the membrane protein to facilitates these steps. Production is carried out in the yeast S. cerevisiae and purification steps are carried out and monitored taking advantage of an anti-GFP nanobody. We show how GFP can be a very helpful tool for controlling the correct addressing of the protein and for probing and optimizing purification. These methods are described here for producing and purifying CaCdr1p, an ABC exporter conferring multiantifungal resistance to C. albicans. This purification method can be amenable to any other GFP-tagged protein.

ABCG: a new fold of ABC exporters and a whole new bag of riddles!

ATP-binding cassette (ABC) superfamily comprises membrane transporters that power the active transport of substrates across biological membranes. These proteins harness the energy of nucleotide binding and hydrolysis to fuel substrate translocation via an alternating-access mechanism. The primary structural blueprint is relatively conserved in all ABC transporters. A transport-competent ABC transporter is essentially made up of two nucleotide-binding domains (NBDs) and two transmembrane domains (TMDs). While the NBDs are conserved in their primary sequence and form at their interface two nucleotide-binding sites (NBSs) for ATP binding and hydrolysis, the TMDs are variable among different families and form the translocation channel. Transporters catalyzing the efflux of substrates from the cells are called exporters. In humans, they range from A to G subfamilies, with the B, C and G subfamilies being involved in chemoresistance. The recently elucidated structures of ABCG5/G8 followed by those of ABCG2 highlighted a novel structural fold that triggered extensive research. Notably, suppressor genetics in the orthologous yeast Pleiotropic Drug Resistance (PDR) subfamily proteins have pointed to a crosstalk between TMDs and NBDs modulating substrate export. Considering the structural information provided by their neighbors from the G subfamily, these studies provide mechanistic keys and posit a functional role for the non-hydrolytic NBS found in several ABC exporters. The present chapter provides an overview of structural and functional aspects of ABCG proteins with a special emphasis on the yeast PDR systems.

Uncompetitive nanomolar dimeric indenoindole inhibitors of the human breast cancer resistance pump ABCG2.

Multidrug resistance membrane pumps reduce the efficacy of chemotherapies by exporting a wide panel of structurally-divergent drugs. Here, to take advantage of the polyspecificity of the human Breast Cancer Resistance Protein (BCRP/ABCG2) and the dimeric nature of this pump, new dimeric indenoindole-based inhibitors from the monomeric α,ß-unsaturated ketone 4b and phenolic derivative 5a were designed. A library of 18 homo/hetero-dimers was synthesised. Homo-dimerization shifted the inhibition efficacy from sub-micromolar to nanomolar range, correlated with the presence of 5a, linked by a 2-6 methylene-long linker. Non-toxic, the best dimers displayed a therapeutic ratio as high as 70,000. It has been found that the high potency of the best compound 7b that displays a KI of 17 nM is due to an uncompetitive behavior toward mitoxantrone efflux and specific for that drug, compared to Hoechst 33342 efflux. Such property may be useful to target such anticancer drug efflux mediated by ABCG2. Finally, at a molecular level, an uncompetitive mechanism by which substrate promotes inhibitor binding implies that at least 2 ligands should bind simultaneously to the drug-binding pocket of ABCG2.

Chromones bearing amino acid residues: Easily accessible and potent inhibitors of the breast cancer resistance protein ABCG2.

The Breast Cancer Resistance Protein (BCRP/ABCG2) belongs to the G class of ABC (ATP-Binding Cassette) proteins, which is known as one of the main transporters involved in the multidrug resistance (MDR) phenotype that confer resistance to anticancer drugs. The aim of this study was to design, synthesize and develop new potent and selective inhibitors of BCRP that can be used to abolish MDR and potentialize clinically used anticancer agents. In previous reports, we showed the importance of chromone scaffold and hydrophobicity for the inhibition of ABC transporters. In the present study we report the design and development of chromones linked to one or two amino acids residues that are either hydrophobic or found in the structure of FTC, one of most potent (but highly toxic) inhibitors of BCRP. Herewith, we report the synthesis and evaluation of 13 compounds. The studied molecules were found to be not toxic and showed strong inhibition activity as well as high selectivity toward BCRP. The highest activity was obtained with the chromone bearing a valine residue (9c) which showed an inhibition activity against BCRP of 50 nM. The rationalization of the inhibition potential of the most active derivatives was performed through docking studies. Taken together, the ease of synthesis and the biological profile of these compounds render them as promising candidates for further development in the field of anticancer therapy.

Cdr1p highlights the role of the non-hydrolytic ATP-binding site in driving drug translocation in asymmetric ABC pumps.

ATP-binding cassette (ABC) transporters couple ATP binding and hydrolysis to the translocation of allocrites across membranes. Two shared nucleotide-binding sites (NBS) participate in this cycle. In asymmetric ABC pumps, only one of them hydrolyzes ATP, and the functional role of the other remains unclear. Using a drug-based selection strategy on the transport-deficient mutant L529A in the transmembrane domain of the Candida albicans pump Cdr1p; we identified a spontaneous secondary mutation restoring drug-translocation. The compensatory mutation Q1005H was mapped 60 Å away, precisely in the ABC signature sequence of the non-hydrolytic NBS. The same was observed in the homolog Cdr2p. Both the mutant and suppressor proteins remained ATPase active, but remarkably, the single Q1005H mutant displayed a two-fold reduced ATPase activity and a two-fold increased drug-resistance as compared to the wild-type protein, pointing at a direct control of the non-hydrolytic NBS in substrate-translocation through ATP binding in asymmetric ABC pumps.

Mortality of four captive-born crested screamer chicks (Chauna torquata).

Crested screamers, a unique, mainly terrestrial avian species native to South America, are known to have a markedly high chick mortality rate in captivity, ranging from 61% to 94%; however, there is very limited information on this species' natural history within the literature, and even less about common diseases that affect them. Four captive-born crested screamer chicks (Chauna torquata) at a U.S. zoological institution died acutely from different causes over the course of 2.5 months. Although a hands-off approach was initially taken, each chick became acutely weak on exhibit and medical intervention was deemed necessary, but proved unsuccessful in all cases. Necropsy results of the chicks revealed various causes of death, including acute Escherichia coli colitis, aspiration pneumonia complicated by concurrent gastrointestinal Candidiasis, severe dehydration and emaciation, and acute amoebic gastroenteritis. No direct associations were found between these deaths and diet or husbandry; however, the limited literature on this topic suspects inadequate husbandry and immunosuppression to be the greatest cause of chick mortality in this species. The cases presented here are consistent with this hypothesis, but further exemplify the limited knowledge of this species and the need to optimize their survivability and proliferation in captivity.

PDR-like ABC systems in pathogenic fungi.

ABC transporters of the Pleiotropic Drug Resistance (PDR) family are the main actors of antifungal resistance in pathogenic fungi. While their involvement in clinical resistant strains has been proven, their transport mechanism remains unclear. Notably, one hallmark of PDR transporters is their asymmetry, with one canonical nucleotide-binding site capable of ATP hydrolysis while the other site is not. Recent publications reviewed here show that the so-called "deviant" site is of crucial importance for drug transport and is a step towards alleviating the mystery around the existence of non-catalytic binding sites.

Unprecedented inhibition of P-gp activity by a novel ruthenium-cyclopentadienyl compound bearing a bipyridine-biotin ligand.

Two new ruthenium complexes, [Ru(η5-Cp)(PPh3)(2,2'-bipy-4,4'-R)]+ with R = -CH2OH (Ru1) or dibiotin ester (Ru2) were synthesized and fully characterized. Both compounds were tested against two types of breast cancer cells (MCF7 and MDA-MB-231), showing better cytotoxicity than cisplatin in the same experimental conditions. Since multidrug resistance (MDR) is one of the main problems in cancer chemotherapy, we have assessed the potential of these compounds to overcome resistance to treatments. Ru2 showed exceptional selectivity as P-gp inhibitor, while Ru1 is possibly a substrate. In vivo studies in zebrafish showed that Ru2 is well tolerated up to 1.17 mg/L, presenting a LC50 of 5.73 mg/L at 5 days post fertilization.

Loss aversion in schizophrenia.

BACKGROUND: Loss aversion in decision-making refers to a higher sensitivity to losses than to gains. Loss aversion is conceived as an affective interference in cognitive processes such as judgment and decision-making. Loss aversion in non-risky choices has not been studied in schizophrenia. METHOD: Forty-two individuals with schizophrenia and 42 non-patient control subjects, matched by gender and age, were randomized to two different scenarios (a buying scenario and a selling scenario). Subjects were asked to evaluate the price of a decorated mug. Schizophrenia subjects were re-tested four weeks later with the other scenario. RESULTS: Contrary to non-patient controls, schizophrenia subjects did not show loss aversion. In the schizophrenia group, absence of loss aversion was correlated with age, duration of illness, number of months in State hospitals, and poorer performance in the Wisconsin Card Sorting Test, but not with current psychopathology and two domains of emotional experience. CONCLUSIONS: Absence of loss aversion in schizophrenia represents a deficit in the processing of emotional information during decision-making. It can be interpreted as a lack of integration between the emotional and the cognitive systems, or to a more diffuse and de-differentiated impact of emotional information on decision-making. Future studies should bring more clarity to this question.