Allosteric modulation of human P-glycoprotein. Inhibition of transport by preventing substrate translocation and dissociation

The human multidrug transporter P-glycoprotein (Pgp, ABCB1) contributes to the poor bioavailability of many anticancer and antimicrobial agents as well as to drug resistance at the cellular level. For rational design of effective Pgp inhibitors, a clear understanding of its mechanism of action and functional regulation is essential. In this study, we demonstrate that inhibition of Pgp-mediated drug transport by cis-(Z)-flupentixol, a thioxanthene derivative, occurs through an allosteric mechanism. Unlike competitive inhibitors, such as cyclosporin A and verapamil, cis-(Z)-flupentixol does not interfere with substrate ([(125)I]iodoarylazidoprazosin) recognition by Pgp, instead it prevents substrate translocation and dissociation, resulting in a stable but reversible Pgp-substrate complex. cis-(Z)-Flupentixol-induced complex formation requires involvement of the Pgp substrate site, because agents that either physically compete (cyclosporin A) for or indirectly occlude (vanadate) the substrate-binding site prevent formation of the complex. Allosteric modulation by cis-(Z)-flupentixol involves a conformational change in Pgp detectable by monoclonal antibody UIC2 binding to a conformation-sensitive external epitope of Pgp. The conformational change observed is distinct from that induced by Pgp substrates or competitive inhibitors. A single amino acid substitution (F983A) in TM12 of Pgp that impairs inhibition by cis-(Z)-flupentixol of Pgp-mediated drug transport also affects stabilization of the Pgp-substrate complex as well as the characteristic conformational change. Taken together, our results describe the molecular mechanism by which the Pgp modulator cis-(Z)-flupentixol allosterically inhibits drug transport.     

WU-Blast2 server at the European Bioinformatics Institute

Since 1995, the WU-BLAST programs (http://blast.wustl.edu) have provided a fast, flexible and reliable method for similarity searching of biological sequence databases. The software is in use at many locales and web sites. The European Bioinformatics Institute's WU-Blast2 (http://www.ebi.ac.uk/blast2/) server has been providing free access to these search services since 1997 and today supports many features that both enhance the usability and expand on the scope of the software.     

Significance of low-dose and standard-dose ACTH tests compared to overnight metyrapone test in the diagnosis of adrenal insufficiency in childhood

OBJECTIVE: To discover the value of low-dose (LDAT) and standard-dose ACTH tests (SDAT) as compared with the metyrapone test in the diagnosis of secondary adrenal insufficiency. PATIENTS AND METHODS: LDAT (0.5 microg/m2), SDAT (250 microg/m2) and overnight metyrapone (30 mg/kg) tests were carried out in 29 patients with suspected adrenal insufficiency. LDAT and SDAT were also performed in 36 control subjects. RESULTS: 18 of 29 patients were grouped in the adrenal-sufficient (AS) group and 11 of 29 patients in the adrenal-deficient (AD) group according to the metyrapone test results. The control group had significantly higher cortisol responses than the AS and AD groups during LDAT. The control group had similar cortisol responses to the AS group but higher cortisol responses than the AD group during SDAT. The AS group was divided into 2 subgroups: AS patients with multiple pituitary hormone deficiencies (AS-multiple) and AS patients with idiopathic growth hormone deficiencies (AS-isolated). The AS-multiple group had statistically lower cortisol responses than the control group during LDAT. Receiver-operating characteristics analysis revealed that the cortisol cutoff value in LDAT was 19.8 microg/dl (100% sensitivity, 89% specificity) and 30.4 microg/dl in SDAT (82% sensitivity, 78% specificity). CONCLUSION: LDAT is capable of identifying patients with adrenal insufficiency more effectively than SDAT. The cortisol cutoff value in LDAT was calculated as 19.8 microg/dl with 100% sensitivity. AS patients with multiple pituitary hormone deficiencies had lower cortisol responses to LDAT than the control group implying that these patients might have a lower cortisol secretory capacity than healthy subjects.     

Various responses of male pituitary–gonadal axis to different intensities of long-term exercise: Role of expression of <Emphasis Type="Italic">KNDY</Emphasis>-related genes

The essential role of regular physical activity has been emphasized for maintaining a healthy life. However, unfortunately, during the last few decades, the lifestyle of people has led to a decrease in physical activity. Research studies have shown that exercise of different intensities is applied on reproductive performance indices, luteinizing hormone (LH) and testosterone (T), with different effects. Nevertheless, the molecular and cellular mechanisms underlying its function are not completely understood. Therefore, this study aimed to evaluate the role of kisspeptin, neurokinin-B and pro-dynorphin (KNDY) gene-expression changes located in the upstream of GnRH neurons in transferring the effects of different long-term exercise intensities on male reproductive axis. Twenty-one adult Wistar rats were randomly divided into control, 6-month regular moderate exercise (RME-6) and 6-month regular intensive exercise (RIE-6). In moderate and intensive exercise groups, rats were treated 5 days a week for 60 min, at 22 and 35 m/min, respectively. Finally, the hypothalamic arcuate nucleus was isolated and the relative gene expression of kisspeptin (Kiss1), neurokinin-B (Nkb), pro-dynorphin (Pdyn) and gonadotropin-releasing hormone (Gnrh) genes were measured by real-time polymerase chain reaction method. The results showed that RIE-6 treatment decreased Gnrh and increased Pdyn mRNA levels in the arcuate nucleus. Furthermore, although RME-6 treatment decreased Nkb and increased Pdyn mRNA levels, the Gnrh mRNA was not affected. Regarding the Gnrh mRNA levels and serum concentrations of reproductive indices (LH and T), moderate exercise did not impose harmful effects on the hypothalamic–pituitary–gonadal axis than intensive exercise. The different impacts of diverse long-term exercise intensities on the male pituitary–gonadal axis maybe relay by the various changes in hypothalamic Nkb and Pdyn gene expressions.     

Modulator-induced interference in functional cross talk between the substrate and the ATP sites of human P-glycoprotein

The human P-glycoprotein (Pgp, ABCB1) is an ATP-dependent efflux pump for structurally unrelated hydrophobic compounds, conferring simultaneous resistance to and restricting bioavailability of several anticancer and antimicrobial agents. Drug transport by Pgp requires a coordinated communication between its substrate binding/translocating pathway (substrate site) and the nucleotide binding domains (NBDs or ATP sites). In this study, we demonstrate that certain thioxanthene-based Pgp modulators, such as cis-(Z)-flupentixol and its closely related analogues, effectively disrupt molecular cross talk between the substrate, and the ATP, sites without affecting the basic functional aspects of the two domains, such as substrate recognition, binding, and hydrolysis of ATP and dissociation of ADP following ATP hydrolysis. The allosteric modulator cis-(Z)-flupentixol has no effect on [alpha-(32)P]-8-azido-ATP binding to Pgp under nonhydrolytic conditions or on the K(m) for ATP during ATP hydrolysis. Both hydrolysis of ATP and vanadate-induced [alpha-(32)P]-8-azido-ADP trapping (following [alpha-(32)P]-8-azido-ATP breakdown) by Pgp are stimulated by the modulator. However, the ability of Pgp substrates (such as prazosin) to stimulate ATP hydrolysis and facilitate vanadate-induced trapping of [alpha-(32)P]-8-azido-ADP is substantially affected in the presence of cis-(Z)-flupentixol. Substrate recognition by Pgp as determined by [(125)I]iodoarylazidoprazosin ([(125)I]IAAP) binding both in the presence and in the absence of ATP is facilitated by the modulator, whereas substrate dissociation in response to vanadate trapping is considerably affected in its presence. In the Pgp F983A mutant, which is impaired in modulation by cis-(Z)-flupentixol, the modulator has a minimal effect on substrate-stimulated ATP hydrolysis as well as on substrate dissociation coupled to vanadate trapping. Finally, cis-(Z)-flupentixol has no effect on dissociation of [alpha-(32)P]-8-azido-ADP (or ADP) from vanadate-trapped Pgp, which is essential for subsequent rounds of ATP hydrolysis. Taken together, our results demonstrate a distinct mechanism of Pgp modulation that involves allosteric disruption of molecular cross talk between the substrate, and the ATP, sites without any direct interference with their individual functions.     

Computer aided prediction and identification of potential epitopes in the receptor binding domain (RBD) of spike (S) glycoprotein of MERS-CoV

Middle East Respiratory Syndrome Coronavirus (MERS-CoV) belongs to the coronaviridae family. In spite of several outbreaks in the very recent years, no vaccine against this deadly virus is developed yet. In this study, the receptor binding domain (RBD) of Spike (S) glycoprotein of MERS-CoV was analyzed through Computational Immunology approach to identify the antigenic determinants (epitopes). In order to do so, the sequences of S glycoprotein that belong to different geographical regions were aligned to observe the conservancy of MERS-CoV RBD. The immune parameters of this region were determined using different in silico tools and Immune Epitope Database (IEDB). Molecular docking study was also employed to check the affinity of the potential epitope towards the binding cleft of the specific HLA allele. The N-terminus RBD (S367-S606) of S glycoprotein was found to be conserved among all the available strains of MERS-CoV. Based on the lower IC₅₀ value, a total of eight potential T-cell epitopes and 19 major histocompatibility complex (MHC) class-I alleles were identified for this conserved region. A 9-mer epitope CYSSLILDY displayed interactions with the maximum number of MHC class-I molecules and projected the highest peak in the B-cell antigenicity plot which concludes that it could be a better choice for designing an epitope based peptide vaccine against MERSCoV considering that it must undergo further in vitro and in vivo experiments. Moreover, in molecular docking study, this epitope was found to have a significant binding affinity of -8.5 kcal/mol towards the binding cleft of the HLA-C*12:03 molecule.     

Fimbria targeting superparamagnetic iron oxide nanoparticles enhance the antimicrobial and antibiofilm activity of ciprofloxacin against quinolone-resistant E. coli

High quinolone resistance of Escherichia coli limits the therapy options for urinary tract infection (UTI). In response to the urgent need for efficient treatment of multidrug-resistant infections, we designed a fimbriae targeting superparamagnetic iron oxide nanoparticle (SPION) delivering ciprofloxacin to ciprofloxacin-resistant E. coli. Bovine serum albumin (BSA) conjugated poly(acrylic acid) (PAA) coated SPIONs (BSA@PAA@SPION) were developed for encapsulation of ciprofloxacin and the nanoparticles were tagged with 4-aminophenyl-α-D-mannopyrannoside (mannoside, Man) to target E. coli fimbriae. Ciprofloxacin-loaded mannoside tagged nanoparticles (Cip-Man-BSA@PAA@SPION) provided high antibacterial activity (97.1 and 97.5%, respectively) with a dose of 32 μg/mL ciprofloxacin against two ciprofloxacin-resistant E. coli isolates. Furthermore, a strong biofilm inhibition (86.9% and 98.5%, respectively) was achieved in the isolates at a dose 16 and 8 times lower than the minimum biofilm eradication concentration (MBEC) of ciprofloxacin. Weaker growth inhibition was observed with untargeted nanoparticles, Cip-BSA@PAA@SPIONs, confirming that targeting E. coli fimbria with mannoside-tagged nanoparticles increases the ciprofloxacin efficiency to treat ciprofloxacin-resistant E. coli. Enhanced killing activity against ciprofloxacin-resistant E. coli planktonic cells and strong growth inhibition of their biofilms suggest that Cip-Man-BSA@PAA@SPION system might be an alternative and/or complementary therapeutic option for the treatment of quinolone-resistant E. coli infections.     

Immobilization of Ene Reductase in Polyvinyl Alcohol Hydrogel

The Protein Journal - In this study, ene reductase (ER) was entrapped in polyvinyl alcohol hydrogel, adsorbed on montmorillonite and immobilized covalently on glutaraldehyde activated...