Thiorhodamines containing amide and thioamide functionality as inhibitors of the ATP-binding cassette drug transporter P-glycoprotein (ABCB1)

Twelve thiorhodamine derivatives have been examined for their ability to stimulate the ATPase activity of purified human P-glycoprotein (P-gp)-His(10), to promote uptake of calcein AM and vinblastine into multidrug-resistant, P-gp-overexpressing MDCKII-MDR1 cells, and for their rates of transport in monolayers of multidrug-resistant, P-gp-overexpressing MDCKII-MDR1 cells. The thiorhodamine derivatives have structural diversity from amide and thioamide functionality (N,N-diethyl and N-piperidyl) at the 5-position of a 2-thienyl substituent on the thiorhodamine core and from diversity at the 3-amino substituent with N,N-dimethylamino, fused azadecalin (julolidyl), and fused N-methylcyclohexylamine (half-julolidyl) substituents. The julolidyl and half-julolidyl derivatives were more effective inhibitors of P-gp than the dimethylamino analogues. Amide-containing derivatives were transported much more rapidly than thioamide-containing derivatives.     

The 5-aromatic hydantoin-3-acetate derivatives as inhibitors of the tumour multidrug resistance efflux pump P-glycoprotein (ABCB1): Synthesis,crystallographic and biological studies

A series of arylpiperazine derivatives of hydantoin-3-acetate, including previously obtained 5,5-diphenylhydantoin (1–7) and new-synthesized spirofluorene-hydantoin derivatives (8–12), were investigated in the search for new inhibitors of the tumour multidrug resistance (MDR) efflux pump P-glycoprotein (P-gp, ABCB1) overexpressed in mouse T-lymphoma cells. Synthesis of new compounds (8–12) was performed. Crystal structures of two compounds (8 and 11) were determined by X-ray diffraction method. The conformations of the investigated molecules (8 and 11) in the crystalline samples are different. The bent conformation seems to be more favourable for biological activity than the extended one. The efflux pump inhibitory properties of the compounds 1–12 were evaluated in the fluorescence uptake assay using rhodamine 123 dye in mouse T-lymphoma model in vitro. Their cytotoxic action was examined, too. All compounds with methyl acetate moiety displayed high potency to inhibit the MDR efflux pump. The most active compound, methyl 2-(1-(4-(4-(2,3-dichlorophenyl)piperazin-1-yl)butyl)-5,5-diphenylhydantoin-3-yl)acetate (5), tested at 1/10 of verapamil concentration displayed the 9-fold higher P-gp inhibitory action.     

Shedding light on drug transport: structure and function of the P-glycoprotein multidrug transporter (ABCB1).

P-glycoprotein (Pgp; ABCB1), a member of the ATP-binding cassette (ABC) superfamily, exports structurally diverse hydrophobic compounds from the cell, driven by ATP hydrolysis. Pgp expression has been linked to the efflux of chemotherapeutic drugs in human cancers, leading to multidrug resistance (MDR). The protein also plays an important physiological role in limiting drug uptake in the gut and entry into the brain. Substrates partition into the lipid bilayer before interacting with Pgp, which has been proposed to function as a hydrophobic vacuum cleaner. Low- and medium-resolution structural models of Pgp suggest that the 2 nucleotide-binding domains are closely associated to form a nucleotide sandwich dimer. Pgp is an outwardly directed flippase for fluorescent phospholipid and glycosphingolipid derivatives, which suggests that it may also translocate drug molecules from the inner to the outer membrane leaflet. The ATPase catalytic cycle of the protein is thought to proceed via an alternating site mechanism, although the details are not understood. The lipid bilayer plays an important role in Pgp function, and may regulate both the binding and transport of drugs. This review focuses on the structure and function of Pgp, and highlights the importance of fluorescence spectroscopic techniques in exploring the molecular details of this enigmatic transporter.     

Modulation of function of three ABC drug transporters, P-glycoprotein (ABCB1), mitoxantrone resistance protein (ABCG2) and multidrug resistance protein 1 (ABCC1) by tetrahydrocurcumin, a major metabolite of curcumin

Many studies have been performed with the aim of developing effective resistance modulators to overcome the multidrug resistance (MDR) of human cancers. Potent MDR modulators are being investigated in clinical trials. Many current studies are focused on dietary herbs due to the fact that these have been used for centuries without producing any harmful side effects. In this study, the effect of tetrahydrocurcumin (THC) on three ABC drug transporter proteins, P-glycoprotein (P-gp or ABCB1), mitoxantrone resistance protein (MXR or ABCG2) and multidrug resistance protein 1 (MRP1 or ABCC1) was investigated, to assess whether an ultimate metabolite form of curcuminoids (THC) is able to modulate MDR in cancer cells. Two different types of cell lines were used for P-gp study, human cervical carcinoma KB-3-1 (wild type) and KB-V-1 and human breast cancer MCF-7 (wild type) and MCF-7 MDR, whereas, pcDNA3.1 and pcDNA3.1-MRP1 transfected HEK 293 and MXR overexpressing MCF7AdrVp3000 or MCF7FL1000 and its parental MCF-7 were used for MRP1 and MXR study, respectively. We report here for the first time that THC is able to inhibit the function of P-gp, MXR and MRP1. The results of flow cytometry assay indicated that THC is able to inhibit the function of P-gp and thereby significantly increase the accumulation of rhodamine and calcein AM in KB-V-1 cells. The result was confirmed by the effect of THC on [³H]-vinblastine accumulation and efflux in MCF-7 and MCF-7MDR. THC significantly increased the accumulation and inhibited the efflux of [³H]-vinblastine in MCF-7 MDR in a concentration-dependent manner. This effect was not found in wild type MCF-7 cell line. The interaction of THC with the P-gp molecule was clearly indicated by ATPase assay and photoaffinity labeling of P-gp with transport substrate. THC stimulated P-gp ATPase activity and inhibited the incorporation of [¹²⁵I]-iodoarylazidoprazosin (IAAP) into P-gp in a concentration-dependent manner. The binding of [¹²⁵I]-IAAP to MXR was also inhibited by THC suggesting that THC interacted with drug binding site of the transporter. THC dose dependently inhibited the efflux of mitoxantrone and pheophorbide A from MXR expressing cells (MCF7AdrVp3000 and MCF7FL1000). Similarly with MRP1, the efflux of a fluorescent substrate calcein AM was inhibited effectively by THC thereby the accumulation of calcein was increased in MRP1-HEK 293 and not its parental pcDNA3.1-HEK 293 cells. The MDR reversing properties of THC on P-gp, MRP1, and MXR were determined by MTT assay. THC significantly increased the sensitivity of vinblastine, mitoxantrone and etoposide in drug resistance KB-V-1, MCF7AdrVp3000 and MRP1-HEK 293 cells, respectively. This effect was not found in respective drug sensitive parental cell lines. Taken together, this study clearly showed that THC inhibits the efflux function of P-gp, MXR and MRP1 and it is able to extend the MDR reversing activity of curcuminoids in vivo.     

Analogs of a 4-aminothieno[2,3-d]pyrimidine lead (QB13) as modulators of P-glycoprotein substrate specificity

P-glycoprotein (P-gp) is an important factor in the development of multidrug resistance (MDR) in cancer cells. In literature reports, a thieno[2,3-d]pyrimidine (QB13) was described as P-gp modulator and opposed effects on the cell accumulation of distinct P-gp substrates were postulated. On the basis of this lead structure, a series of 2-alkylthio-4-aminothieno[2,3-d]pyrimidines was prepared and tested in a daunorubicin accumulation assay. Modulation of substrate specificity was shown for selected compounds in cytotoxicity (MTT) assays.     

Inhibition of multidrug resistance-linked P-glycoprotein (ABCB1) function by 5'-fluorosulfonylbenzoyl 5'-adenosine: evidence for an ATP analogue that interacts with both drug-substrate-and nucleotide-binding sites

5'-Fluorosulfonylbenzonyl 5'-adenosine (FSBA) is an ATP analogue that covalently modifies several residues in the nucleotide-binding domains (NBDs) of several ATPases, kinases, and other proteins. P-glycoprotein (P-gp, ABCB1) is a member of the ATP-binding cassette (ABC) transporter superfamily that utilizes energy from ATP hydrolysis for the efflux of amphipathic anticancer agents from cancer cells. We investigated the interactions of FSBA with P-gp to study the catalytic cycle of ATP hydrolysis. Incubation of P-gp with FSBA inhibited ATP hydrolysis (IC(50 )= 0.21 mM) and the binding of 8-azido[α-(32)P]ATP (IC(50) = 0.68 mM). In addition, (14)C-FSBA cross-links to P-gp, suggesting that FSBA-mediated inhibition of ATP hydrolysis is irreversible due to covalent modification of P-gp. However, when the NBDs were occupied with a saturating concentration of ATP prior to treatment, FSBA stimulated ATP hydrolysis by P-gp. Furthermore, FSBA inhibited the photo-cross-linking of P-gp with [(125)I]iodoarylazidoprazosin (IAAP; IC(50) = 0.17 mM). As IAAP is a transport substrate for P-gp, this suggests that FSBA affects not only the NBDs but also the transport-substrate site in the transmembrane domains. Consistent with these results, FSBA blocked efflux of rhodamine 123 from P-gp-expressing cells. Additionally, mass spectrometric analysis identified FSBA cross-links to residues within or nearby the NBDs but not in the transmembrane domains, and docking of FSBA in a homology model of human P-gp NBDs supports the biochemical studies. Thus, FSBA is an ATP analogue that interacts with both the drug-binding and ATP-binding sites of P-gp, but fluorosulfonyl-mediated cross-linking is observed only at the NBDs.     

Ecophysiological basis of the Jack-and-Master strategy: Taraxacum officinale (dandelion) as an example of a successful invader

Aims Successful invasive plants are often assumed to display significant levels of phenotypic plasticity. Three possible strategies by which phenotypic plasticity may allow invasive plant species to thrive in changing environments have been suggested: (i) via plasticity in morphological or physiological traits, invasive plants are able to maintain a higher fitness than native plants in a range of environments, including stressful or low-resource habitats: a 'Jack-of-all-trades' strategy; (ii) phenotypic plasticity allows the invader to better exploit resources available in low stress or favorable habitats, showing higher fitness than native ones: a 'Master-of-some' strategy and (iii) a combination of these abilities, the 'Jack-and-Master' strategy.Methods We evaluated these strategies in the successful invader Taraxacum officinale in a controlled experiment mimicking natural environmental gradients. We set up three environmental gradients consisting of factorial arrays of two levels of temperature/light, temperature/water and light/water, respectively. We compared several ecophysiological traits, as well as the reaction norm in fitness-related traits, in both T. officinale and the closely related native Hypochaeris thrincioides subjected to these environmental scenarios.Important findings Overall, T. officinale showed significantly greater accumulation of biomass and higher survival than the native H. thrincioides, with this difference being more pronounced toward both ends of each gradient. T. officinale also showed significantly higher plasticity than its native counterpart in several ecophysiological traits. Therefore, T. officinale exhibits a Jack-and-Master strategy as it is able to maintain higher biomass and survival in unfavorable conditions, as well as to increase fitness when conditions are favorable. We suggest that this strategy is partly based on ecophysiological responses to the environment, and that it may contribute to explaining the successful invasion of T. officinale across different habitats.     

New screening strategy and analysis for identification of allosteric modulators for glucagon-like peptide-1 receptor using GLP-1 (9-36) amide

The glucagon-like peptide-1 receptor (GLP-1R) is an important physiologic regulator of insulin secretion and a major therapeutic target for diabetes mellitus. GLP-1 (7-36) amide (active form of GLP-1) is truncated to GLP-1 (9-36) amide, which has been described as a weak agonist of GLP-1R and the major form of GLP-1 in the circulation. New classes of positive allosteric modulators (PAMs) for GLP-1R may offer improved therapeutic profiles. To identify these new classes, we developed novel and robust primary and secondary high-throughput screening (HTS) systems in which PAMs were identified to enhance the GLP-1R signaling induced by GLP-1 (9-36) amide. Screening enabled identification of two compounds, HIT-465 and HIT-736, which possessed new patterns of modulation of GLP-1R. We investigated the ability of these compounds to modify GLP-1R signaling enhanced GLP-1 (9-36) amide- and/or GLP-1 (7-36) amide-mediated cyclic adenosine monophosphate (cAMP) accumulation. These compounds also had unique profiles with regard to allosteric modulation of multiple downstream signaling (PathHunter β-arrestin signaling, PathHunter internalization signaling, microscopy-based internalization assay). We found allosteric modulation patterns to be obviously different among HIT-465, HIT-736, and Novo Nordisk compound 2. This work may enable the design of new classes of drug candidates by targeting modulation of GLP-1 (7-36) amide and GLP-1 (9-36) amide.     

P-glycoprotein gene (MDR1) cDNA from human adrenal: normal P-glycoprotein carries Gly185 with an altered pattern of multidrug resistance

We isolated a full-length MDR1 cDNA from human adrenal where P-glycoprotein is expressed at high level. The deduced amino acid sequence shows two amino acid differences from the sequence of P-glycoprotein obtained from colchicine-selected multidrug resistant cultured cells. The amino acid substitution Gly----Val at codon 185 in P-glycoprotein from colchicine resistant cells occurred during selection of cells in colchicine. As previously reported, cells transfected with the MDR1 cDNA carrying Val185 acquire increased resistance to colchicine compared to other drugs. The other amino acid substitution Ser----Ala at codon 893 probably reflects genetic polymorphism. The MDR1 gene, the major member of the P-glycoprotein gene family expressed in human adrenal, is sufficient to confer multidrug-resistance on culture cells.     

Pharmacophore mapping of a series of pyrrolopyrimidines,indolopyrimidines and their congeners as multidrug-resistance-associated protein (MRP1) modulators

Pharmacophore mapping studies were undertaken for a series of molecules belonging to pyrrolopyrimidines, indolopyrimidines and their congeners as multidrug resistance-associated protein (MRP1) modulators. A five-point pharmacophore with two hydrogen bond acceptors (A), one lipophilic/hydrophobic group (H), one positive ionic feature (P) and one aromatic ring (R) as pharmacophoric features was developed. The pharmacophore hypothesis yielded a statistically significant 3D-QSAR model, with a correlation coefficient of r ² = 0.799 for training set molecules. The model generated showed excellent predictive power, with a correlation coefficient Q ² = 0.679 for an external test set of 20 molecules. The pharmacophore was further validated using four structurally diverse compounds with MRP1 modulatory activity. These compounds mapped well onto four of the five features of the pharmacophore. The pharmacophore proposed here was then utilised for the successful retrieval of active molecules with diverse chemotypes from database search. The geometry and features of pharmacophore are expected to be useful for the design of selective MRP1 inhibitors. Figure Alignment of multidrug resistance-associated protein (MRP1) inhibitors with the developed pharmacophore. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Recognition of sulfonylurea receptor (ABCC8/9) ligands by the multidrug resistance transporter P-glycoprotein (ABCB1): functional similarities based on common structural features between two multispecific ABC proteins

ATP-sensitive K(+) (K(ATP)) channels are the target of a number of pharmacological agents, blockers like hypoglycemic sulfonylureas and openers like the hypotensive cromakalim and diazoxide. These agents act on the channel regulatory subunit, the sulfonylurea receptor (SUR), which is an ABC protein with homologies to P-glycoprotein (P-gp). P-gp is a multidrug transporter expressed in tumor cells and in some healthy tissues. Because these two ABC proteins both exhibit multispecific recognition properties, we have tested whether SUR ligands could be substrates of P-gp. Interaction with P-gp was assayed by monitoring ATPase activity of P-gp-enriched vesicles. The blockers glibenclamide, tolbutamide, and meglitinide increased ATPase activity, with a rank order of potencies that correlated with their capacity to block K(ATP) channels. P-gp ATPase activity was also increased by the openers SR47063 (a cromakalim analog), P1075 (a pinacidil analog), and diazoxide. Thus, these molecules bind to P-gp (although with lower affinities than for SUR) and are possibly transported by P-gp. Competition experiments among these molecules as well as with typical P-gp substrates revealed a structural similarity between drug binding domains in the two proteins. To rationalize the observed data, we addressed the molecular features of these proteins and compared structural models, computerized by homology from the recently solved structures of murine P-gp and bacterial ABC transporters MsbA and Sav1866. Considering the various residues experimentally assigned to be involved in drug binding, we uncovered several hot spots, which organized spatially in two main binding domains, selective for SR47063 and for glibenclamide, in matching regions of both P-gp and SUR.     

Methods of brood protection as a basis for reclassification of the Spirorbinae (Serpulidae)

In a taxonomic review, nine previously defined subgenera are adopted and a tenth Marsipospira subgen. aov. is proposed for about four species, with S. striatus QuieVreux (1963) as the type. These all retain their embryos in a thoracic brood pouch, fixed to a modified dorsal tentacle, and also possess a translucent tube, often with transverse ridges, an opercular talon, with lateral lobes and/or lateral knobs, and vestiges of a fourth thoracic segment.
The remaining species, numbering more than 74, may be grouped in six subgenera with egg string incubation and three with opercular incubation. These three main methods of incubation are likely to have been derived independently, from ancestral serpulids spawning into their tubes.
The distribution of abdominal uncini helps to characterize subgenera. In Spirorbis, Spirorbella, Pileolaria, Leodora and Janua the abdominal tori are not very asymmetrical, those on the concave side being only slightly larger than the others. In Pileolaria the largest abdominal tori are usually found in the posterior half of the abdomen, but in other subgenera they usually lie in the anterior half of that region. Paralaeospira, Greets and Marsipospira have the abdominal tori very asymmetrically developed and usually absent from the convex side. This marked asymmetry is often associated with the tubes being translucent, smooth and presumably slippery on the inside. It may thus be related to the anchorage problem, during rapid withdrawal. The asymmetrically developed traces of a fourth thoracic segment, with which the abdominal asymmetry is often correlated, seem unlikely to be primitive survivals. They may rather be incidental expressions of genotypes evolved through selection pressure which was concerned primarily with anchorage problems in the abdominal region.     

Mutations in Intracellular Loops 1 and 3 Lead to Misfolding of Human P-glycoprotein (ABCB1) That Can Be Rescued by Cyclosporine A,Which Reduces Its Association with Chaperone Hsp70

P-glycoprotein (P-gp) is an ATP binding cassette transporter that effluxes a variety of structurally diverse compounds including anticancer drugs. Computational models of human P-gp in the apo- and nucleotide-bound conformation show that the adenine group of ATP forms hydrogen bonds with the conserved Asp-164 and Asp-805 in intracellular loops 1 and 3, respectively, which are located at the interface between the nucleotide binding domains and transmembrane domains. We investigated the role of Asp-164 and Asp-805 residues by substituting them with cysteine in a cysteine-less background. It was observed that the D164C/D805C mutant, when expressed in HeLa cells, led to misprocessing of P-gp, which thus failed to transport the drug substrates. The misfolded protein could be rescued to the cell surface by growing the cells at a lower temperature (27 °C) or by treatment with substrates (cyclosporine A, FK506), modulators (tariquidar), or small corrector molecules. We also show that short term (4–6 h) treatment with 15 μm cyclosporine A or FK506 rescues the pre-formed immature protein trapped in the endoplasmic reticulum in an immunophilin-independent pathway. The intracellularly trapped misprocessed protein associates more with chaperone Hsp70, and the treatment with cyclosporine A reduces the association of mutant P-gp, thus allowing it to be trafficked to the cell surface. The function of rescued cell surface mutant P-gp is similar to that of wild-type protein. These data demonstrate that the Asp-164 and Asp-805 residues are not important for ATP binding, as proposed earlier, but are critical for proper folding and maturation of a functional transporter.     

A mechanism for gamma-hydroxybutyrate (GHB) as a drug and a substance of abuse

Gamma-hydroxybutyrate (GHB) is mainly known because of its popularity as a drug of abuse among young individuals. However this substance increases slow-wave deep sleep and the secretion of growth hormone and besides its role in anaesthesia, it is used in several therapeutic indications including alcohol withdrawal, control of daytime sleep attacks and cataplexy in narcoleptic patients and is proposed for the treatment of fibromyalgia. GHB is also an endogenous substance present in several organs, including brain where it is synthesized from GABA in cells containing glutamic acid decarboxylase, the marker of GABAergic neurons. GHB is accumulated by the vesicular inhibitory aminoacid transporter (VIAAT) and released by depolarization via a Ca2+ dependent-mechanism. A family of GHB receptors exists in brain which possesses hyperpolarizing properties through Ca2+ and K+ channels. These receptors--one of them has been recently cloned from rat brain hippocampus--are thought to regulate GABAergic activities via a subtle balance between sensitized/desensitized states. Massive absorption of GHB desensitize GHB receptors and this modification, together with a direct stimulation of GABAB receptors by GHB, induce a perturbation in GABA, dopamine and opiate releases in several region of the brain. This adaptation phenomenon is probably responsible for the therapeutic and recreative effects of exogenous GHB.