Effects of phosphorylation of P-glycoprotein on multidrug resistance

Cells expressing elevated levels of the membrane phosphoprotein P-glycoprotein exhibit a multidrug resistance phenotype. Studies involving protein kinase activators and inhibitors have implied that covalent modification of P-glycoprotein by phosphorylation may modulate its biological activity as a multidrug transporter. Most of these reagents, however, have additional mechanisms of action and may alter drug accumulation within multidrug resistant cells independent of, or in addition to their effects on the state of phosphorylation of P-glycoprotein. The protein kinase(s) responsible for P-glycoprotein phosphorylation has(ve) not been unambiguously identified, although several possible candidates have been suggested. Recent biochemical analyses demonstrate that the major sites of phosphorylation are clustered within the linker region that connects the two homologous halves of P-glycoprotein. Mutational analyses have been initiated to confirm this finding. Preliminary data obtained from phosphorylation- and dephosphorylation-defective mutants suggest that phosphorylation of P-glycoprotein is not essential to confer multidrug resistance.     

Triple-helix mediated excimer and exciplex formation

The synthesis and properties of triple-helical hybrids containing non-nucleosidic polyaromatic building blocks are described. Clamp-type oligonucleotides containing a non-nucleosidic pyrene linker form stable triple helices with a polypurine target strand containing a terminal pyrene or phenanthrene moiety. Stacking interactions between the unnatural building blocks enhance triplex stability and lead to strong excimer or exciplex formation, which is monitored by fluorescence spectroscopy.     

Investigation of the role of linker moieties in bifunctional tacrine hybrids

Alzheimer’s disease (AD) is a complex neurological disorder with multiple inter-connected factors playing roles in the onset and progression of the disease. One strategy currently being explored for the development of new therapeutics for AD involves linking tacrine, a known acetylcholinesterase (AChE) inhibitor, to another drug to create bifunctional hybrids. The role and influence on activity of the linker moiety in these hybrids remains ill-defined. In this study, three series of 6-chlorotacrine with linkers varying in terminal functional group and length were synthesized, evaluated for AChE inhibition, and compared to tacrine and 6-chlorotacrine–mefenamic acid hybrids. Out of the compounds with terminal amine, methyl, and hydroxyl moieties tested, several highly potent molecules (low nanomolar IC₅₀ values) comprised of linkers with terminal amines were identified. These 6-chlorotacrine with linkers were significantly more potent than tacrine alone and were often more potent than similar 6-chlorotacrine–mefenamic acid hybrids.     

Recent Progress in Understanding the Mechanism of P-Glycoprotein-mediated Drug Efflux

P-glycoprotein (P-gp) is an ATP-dependent drug pump that can transport a broad range of hydrophobic compounds out of the cell. The protein is clinically important because of its contribution to the phenomenon of multidrug resistance during AIDS/HIV and cancer chemotherapy. P-gp is a member of the ATP-binding cassette (ABC) family of proteins. It is a single polypeptide that contains two repeats joined by a linker region. Each repeat has a transmembrane domain consisting of six transmembrane segments followed by a hydrophilic domain containing the nucleotide-binding domain. In this mini-review, we discuss recent progress in determining the structure and mechanism of human P-glycoprotein.     

Design,synthesis, cholinesterase inhibition and molecular modelling study of novel tacrine hybrids with carbohydrate derivatives

A series of hybrids containing tacrine linked to carbohydrate-based moieties, such as d-xylose, d-ribose, and d-galactose derivatives, were synthesized by the nucleophilic substitution between 9-aminoalkylamino-1,2,3,4-tetrahydroacridines and the corresponding sugar-based tosylates. All compounds were found to be potent inhibitors of both acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) in the nanomolar IC₅₀ scale. Most of the d-xylose derivatives (6a-e) were selective for AChE and the compound 6e (IC₅₀?=?2.2?nM for AChE and 4.93?nM for BuChE) was the most active compound for both enzymes. The d-galactose derivative 8a was the most selective for AChE exhibiting an IC₅₀ ratio of 7.6 for AChE over BuChE. Only two compounds showed a preference for BuChE, namely 7a (d-ribose derivative) and 6b (d-xylose derivative). Molecular docking studies indicated that the inhibitors are capable of interacting with the entire binding cavity and the main contribution of the linker is to enable the most favorable positioning of the two moieties with CAS, PAS, and hydrophobic pocket to provide optimal interactions with the binding cavity. This finding is reinforced by the fact that there is no linear correlation between the linker size and the observed binding affinities. The majority of the new hybrids synthesized in this work do not violate the Lipinski's rule-of-five according to FAF-Drugs4, and do not demonstrated predicted hepatotoxicity according ProTox-II.     

On-line identification of P-glycoprotein substrates by monitoring of extracellular acidification and respiration rates in living cells

The influence of P-glycoprotein (ABCB1) in drug resistance as well as drug absorption and disposition is an important factor to be considered during the development of new drugs. Thus, the early identification and exclusion of compounds showing a high affinity towards P-glycoprotein can help to select drug candidates. The aim of our study was to implement a label-free assay for the identification of P-glycoprotein substrates in living cells. For this approach, a multiparametric, chip-based sensor system was used to determine extracellular acidification, cell respiration and adhesion upon stimulation with P-glycoprotein substrates. Using L-MDR1 cells, a human P-glycoprotein overexpressing cell line, the influence of P-glycoprotein activity was determined for seven different compounds, demonstrating the applicability of the system for P-glycoprotein substrate identification. Effects were concentration dependent, as shown for the P-glycoprotein substrate verapamil, and were associated with cellular acidification and respiration. P-glycoprotein ATPase activation by verapamil could be described by a Michaelis-Menten type kinetic profile showing saturation at high substrate concentrations. The Michaelis-Menten constants K(M) were determined to be 0.92μM (calculated based on extracellular acidification) and 4.9μM (calculated based on cellular respiration). Control experiments using 100nM of the P-glycoprotein inhibitor elacridar indicated that the observed effects were related to P-glycoprotein ATPase activity. In contrast, wild-type LLC-PK1 cells not expressing P-glycoprotein were not responsive towards stimulation with different P-glycoprotein substrates. Summarizing these findings, the used microsensor system is a generic system suitable for the identification of P-glycoprotein substrates. In contrast to biochemical P-glycoprotein assays, activation of the drug efflux pump can be monitored on-line in living cells to identify P-glycoprotein substrates and to study the molecular mechanisms of adenosintriphosphate-dependent active transport.     

Morphology of hair pigmentation in wild red foxes,silver foxes,and their hybrids

The effects of dominant allele Ar of locus Agouti on the morphology of hair pigmentation were described in foxes. The Ar allele was shown to determine the type of melanin and its content in hair with no effect on the morphology of pigment granules and their distribution throughout a hair. Using the method of electron spin resonance (ESR), the types of melanin (eumelanin and pheomelanin) and their content in the hair of red (ArArEE) and silver (aaEE) foxes and their hybrids (AraEE) were determined. In silver foxes, only one type of melanin (eumelanin) was found. In red foxes and their hybrids (which are phenotypically similar but darker than red foxes), both types of melanin (eu- and pheomelanin) were found. The highest melanin content was detected in the coat of silver foxes. In the hybrids, the total melanin content was lower than in silver foxes, but significantly higher than in red foxes. In red foxes, the contribution of pheomelanin to the total hair melanin content was twice as large as in the hybrids.     

Regulation of progenitor cell fusion by ABCB5 P-glycoprotein,a novel human ATP-binding cassette transporter

Cell fusion involving progenitor cells is a newly recognized phenomenon thought to contribute to tissue differentiation. The molecular mechanisms governing cell fusion are unknown. P-glycoprotein and related ATP-binding cassette transporters are expressed by progenitor cells, but their physiological role in these cell types has not been defined. Here, we have cloned ABCB5, a rhodamine efflux transporter and novel member of the human P-glycoprotein family, which marks CD133-expressing progenitor cells among human epidermal melanocytes and determines as a regulator of membrane potential the propensity of this subpopulation to undergo cell fusion. Our findings show that polyploid ABCB5+ cells are generated by cell fusion and that this process is specifically enhanced by ABCB5 P-glycoprotein blockade. Remarkably, multinucleated cell hybrids gave rise to mononucleated progeny, demonstrating that fusion contributes to culture growth and differentiation. Thus, our findings define a molecular mechanism for cell fusion involving progenitor cells and show that fusion and resultant growth and differentiation are not merely spontaneous events, but phenomena regulated by ABCB5 P-glycoprotein.     

Steroidal bivalent ligands for the estrogen receptor: Design,synthesis, characterization and binding affinities

Steroidal bivalent ligands for the estrogen receptor (ER) were designed using crystal structures of ERα dimers as a template. The syntheses of several 17α-ethynylestradiol-based bivalent ligands with varying linker compositions and lengths are described. The binding affinities of these bivalent ligands for ERα and ERβ were determined. In the two series of bivalent ligands that we synthesized, there is a clear correlation between linker length and binding affinity, both of which reach a maximum at the same tether length. Further studies are underway to explore aspects of bivalent ligand and control compound binding to the ERs and their effects on ER dimer formation; these results will be reported in a subsequent publication.     

Hybrids of bleak, Alburnus alburnus, and chub, Leuciscus cephalus in English rivers

The occurrence of hybrids between bleak, Alburnus alburnus , and chub, Leuciscus cephalus is reported from four discrete river systems in England. The hybrids are described and illustrated, and means of identification from the parent species are given. Bleak × chub hybrids appear to be moderately common and on superficial examination resemble the bleak parent species more than the chub.     

Immunogold localisation of P-glycoprotein in supported lipid bilayers by transmission electron microscopy and atomic force microscopy

In this study, purified P-glycoprotein molecules, a membrane drug pump responsible for the multidrug resistance phenomenon, were incorporated in model membranes deposited onto solid supports, according to the method described by Puu and Gustafson (1997). The insertion of proteins into planar supported model membranes is of interest, as the films are fundamental in biosensor applications and for the investigation of how proteins conform and aggregate in a lipid environment. In our investigation, two model membranes were prepared by transferring liposomes containing P-glycoprotein to different hydrophobic supports: (a) thin amorphous carbon films; (b) Langmuir–Blodgett lipid monolayers on mica. After the labelling of P-glycoprotein with two well-characterised monoclonal antibodies, MM4.17 and MRK-16, samples (a) were observed by transmission electron microscopy (TEM) and samples (b) by atomic force microscopy (AFM).The comparative analysis performed by TEM and AFM allowed us to demonstrate the successful insertion of P-glycoprotein in the model membranes and their stability under different environmental conditions (vacuum, air and water). P-glycoprotein appeared to maintain, after purification and insertion in lipid bilayers, a good part of its conformational features as shown by the P-glycoprotein segments bearing the specific monoclonal antibody epitopes.     

Variedly connected 1,8-naphthalimide-7-chloroquinoline conjugates: Synthesis,anti-mycobacterial and cytotoxic evaluation

Recent disclosures about anti-bacterial and anti-tubercular potential of naphthalimide and quinoline core respectively propelled us to synthesize a library of 1,8-naphthalimide-7-chloroquinoline hybrids. Different modes of linkage between two pharmacophoric units viz. simple alkyl chains and induction of amide bond were used and the substituents on the naphthalimide core were varied in order to determine Structure-Activity-Relationship (SAR). Our findings demonstrated that simple alkyl chain linked conjugates showed better activity profiles without any cytotoxicity, while the inclusion of amide bond enhanced the cytotoxic tendency. An interesting behaviour of conjugates in terms of activity and cytotoxicity was observed via switching over the nature of linker between two pharmacophores.     

The evolution of extracellular hemoglobins of annelids, vestimentiferans, and pogonophorans

The evolution of extracellular hemoglobins of annelids, vestimentiferans, and pogonophorans was investigated by applying cladistic and distance-based approaches to reconstruct the phylogenetic relationships of this group of respiratory pigments. We performed this study using the aligned sequences of globin and linker chains that are the constituents of these complex molecules. Three novel globin and two novel linker chains of Sabella spallanzanii described in an accompanying paper (Pallavicini, A., Negrisolo, E., Barbato, R., Dewilde, S., Ghiretti-Magaldi, A., Moens, L., and Lanfranchi, G. (2001) J. Biol. Chem. 276, 26384--26390) were also included. Our results allowed us to test previous hypotheses on the evolutionary pathways of these proteins and to formulate a new most parsimonious model of molecular evolution. According to this novel model, the genes coding for the polypeptides forming these composite molecules were already present in the common ancestor of annelids, vestimentiferans, and pogonophorans.     

Separation and Characterization of Two Pyridoxamine (Pyridoxine) 5′-Phosphate Oxidases from Baker’s Yeast

We have fused full length and the carboxyl-half of human MDR1 cDNA with the E. coli lacZ gene via a collagen linker and allowed their expression in yeast Saccharomyces cerevisiae. Using antibodies against β-galactosidase we partially purified the fusion proteins by immunoprecipitation and show here that the full length fusion protein has ATPase activity. By contrast, the fusion protein containing the carboxyl-half of P-glycoprotein did not show ATPase activity, indicating that both domains of P-glycoprotein are necessary. By treatment of the immunoprecipitated fusion protein with collagenase, P-glycoprotein was released from the β-galactosidase moiety. The results shown here open the possibility for a large scale purification of P-glycoprotein using this site specifically cleavable fusion protein.     

Development of a fast DNA-DNA hybridization method based on melting profiles in microplates

DNA-DNA hybridization is still the “gold standard” for the genotypic delineation of bacterial species. However, it is not widely used because traditional DNA-DNA hybridization techniques are rather time-consuming and not easy to perform in routine laboratories. In the present study, DNA of reference strains was digested with Sau3A, ligated with linker oligonucleotides S1/2 and in vitro amplified. The amplified DNA fragments were immobilized on MaxiSorb 96-well plates. DNA isolated from target strains was also digested with Sau3A, ligated with linker oligonuleotides P1/2 and in vitro amplified in the presence of digoxygenin modified dUTP. The labeled amplificate was hybridized to the immobilized reference DNA under isothermal conditions. Thermal denaturation curves of the DNA-DNA hybrids were obtained by using washing solutions of increasing stringency. Remaining hybrids were colorimetrically detected with anti-digoxygenin-horseradish peroxidase anti-bodies. The new method was validated with strains of the genus Pedioccocus for which DNA-DNA similarities have also been determined by the filter hybridization method. In addition, DNA-DNA hybridizations were performed with genotypically defined Enterobacter species.     

Proteolytic Cleavage of the Linker Region of the Human P-glycoprotein Modulates Its ATPase Function

P-glycoprotein (Pgp), an anticancer drug-translocating ATPase, is responsible for multidrug resistance in cancer. We have previously shown (Nuti, S. L., Mehdi, A., and Rao, U. S. (2000) Biochemistry 39, 3424-3432) that tryptic cleavage of Pgp results in the activation of basal and drug-stimulated ATPase functions of Pgp. To understand this phenomenon, we determined the sites cleaved by trypsin and further examined whether the modulation of Pgp function is trypsin-specific or the result of proteolysis in general. The effects of chymotrypsin and proteinase K on Pgp ATPase function were studied. The results show that proteolysis of Pgp irrespective of the protease employed resulted in the activation of basal ATPase activity. However, drug-stimulated ATPase activities were differentially modulated. Immunoblot analysis of proteolytic digests indicated that, irrespective of the protease employed, Pgp was predominantly cleaved in the middle of the molecule. N-terminal amino acid sequencing of Pgp tryptic and chymotryptic peptides indicated Arg(680) and Leu(682) as the sites of cleavage, respectively. These two cleavage sites are part of the predicted linker region that joins the two halves of Pgp. Together, these results suggest that the linker region in Pgp is primarily accessible to protease action and that cleavage of this region modulates Pgp ATPase function.     

New 1,3-dioxolane and 1,3-dioxane derivatives as effective modulators to overcome multidrug resistance

Multidrug resistance (MDR) to antitumor agents represents a major obstacle to a successful chemotherapy of cancer. Overexpression of P-glycoprotein (p-gp) seems to be the major factor responsible for MDR. A large number of chemically unrelated compounds are known to interact with p-gp resulting in a decreasing resistance. In our efforts related to structure-activity studies of new potential MDR reversal agents we synthesized a series of compounds that differ in the aromatic core structure, the linker, and the basic moiety. For our search of new aromatic core structures we synthesized novel 2,2-diphenyl-1,3-dioxolane, 2,2- diphenyl-1,3-dioxane, and 4,5-diphenyl-1,3-dioxolane derivatives. A range of lipophilic linker structures and protonable basic moieties were synthesized and investigated to optimize the structure of the potential MDR-modulators. The compounds were tested in vitro using human Caco-2 cells. Both the cytotoxicity of the synthons and their ability to resensitize the cells were determined with a MTT assay. The results show that at low concentration various substances reverse tumor cell MDR. Some of the new structures show better effects than established modulators like trifluoperazine.     

Morphology of Hair Pigmentation in Wild Red Foxes, Silver Foxes, and Their Hybrids

The effects of dominant allele A ^r of locus Agoution the morphology of hair pigmentation were described in foxes. The A ^r allele was shown to determine the type of melanin and its content in hair with no effect on the morphology of pigment granules and their distribution throughout a hair. Using the method of electron spin resonance (ESR), the types of melanin (eumelanin and pheomelanin) and their content in the hair of red (A ^r A ^r EE) and silver (aaEE) foxes and their hybrids (A ^r aEE) were determined. In silver foxes, only one type of melanin (eumelanin) was found. In red foxes and their hybrids (which are phenotypically similar but darker than red foxes), both types of melanin (eu- and pheomelanin) were found. The highest melanin content was detected in the coat of silver foxes. In the hybrids, the total melanin content was lower than in silver foxes, but significantly higher than in red foxes. In red foxes, the contribution of pheomelanin to the total hair melanin content was twice as large as in the hybrids.     

New type of linker useful for cloning experiments

A new class of linker oligodeoxynucleotide sequences is described which allows the original sequence of a foreign DNA to be restored after cloning. In the standard linker approach the terminal base pairs of the linker-sequences are irreversibly attached to the cloned DNA, thus altering the genetic information. The use of the new type of linker is demonstrated by the transformation of the unique Pvu II site in pBR322 into Bam HI site using the ISO ('in-site-out') linker d(GATCCGGATC). Possible further applications of these linker sequences are described.     

Acetophenone-based linker for solid-phase peptide synthesis.

A new and cost-effective linker for the generation of carboxylic acid end groups on Multipin supports (SynPhase crowns) has been developed. Synthesis of the linker was based on modification of grafted polystyrene (PS) crowns to generate a hydroxyethyl moiety which is acid labile in 10-20% trifluoroacetic acid (TFA) in dichloromethane (DCM). Solid-phase syntheses of model decapeptides using this linker are described.