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The virus SARS CoV-2, which causes the respiratory infection COVID-19, continues its spread across the world and to date has caused more than a million deaths. Although COVID-19 vaccine development appears to be progressing rapidly, scientists continue the search for different therapeutic options to treat this new illness. In this work, we synthesized five new 1-aryl-5-(3-azidopropyl)indol-4-ones and showed them to be potential inhibitors of the SARS CoV-2 main protease (3CLpro). The compounds were obtained in good overall yields and molecular docking indicated favorable binding with 3CLpro. In silico ADME/Tox profile of the new compounds were calculated using the SwissADME and pkCSM-pharmacokinetics web tools, and indicated adequate values of absorption, distribution and excretion, features related to bioavailability. Moreover, low values of toxicity were indicated for these compounds. And drug-likeness levels of the compounds were also predicted according to the Lipinski and Veber rules., (Copyright © 2020 Elsevier Inc. All rights reserved.)

In preclinical and phase I and II clinical studies, 2'-deoxy-2'-β-fluoro-4'-azidocytidine (FNC) displays a potent and long-lasting inhibition of HIV-1 infection. To investigate its mechanism of action, we compared it with the well-documented lamivudine (3TC). Pharmacokinetic studies revealed that the intracellular retention of FNC triphosphate in peripheral blood mononuclear cells was markedly longer than that of the 3TC triphosphate. FNC selectively enters and is retained in HIV target cells, where it exerts long-lasting prevention of HIV-1 infection. In addition to inhibition of HIV-1 reverse transcription, FNC also restores A3G expression in CD4 + T cells in FNC-treated HIV-1 patients. FNC binds to the Vif-E3 ubiquitin ligase complex, enabling A3G to avoid Vif-induced ubiquitination and degradation. These data reveal the mechanisms underlying the superior anti-HIV potency and long-lasting action of FNC. Our results also suggest a potential clinical application of FNC as a long-lasting pre-exposure prophylactic agent capable of preventing HIV infection.

The design of multiple stimuli-responsive, stable polymeric drug carriers is key for efficient drug release against solid tumors. Herein, core-crosslinked micelles were readily prepared from a pair of redox/pH-sensitive clickable copolymers. The two copolymers comprised the same poly(ethylene glycol) (PEG)-poly(ε-benzyloxycarbonyl-l-lysine) (PZLL) block but with either disulfide-linked azadibenzocyclooctyne (DBCO) or azide (AZ) group-tagged branched polyethylenimine (BPEI, 1.8 kDa). The data showed that an equivalent of the two copolymers could self-assemble into nanosized micelles with the crosslinked core via the DBCO-AZ click chemistry. The click-crosslinked micelles showed excellent size stability under multiple dilutions but destabilization in an acidic or reductive environment. Besides, they could load doxorubicin (DOX), an anticancer drug, and mediate slow drug release in a neutral environment but sufficient drug unloading under acidic plus reductive conditions. In vitro, DOX-loaded crosslinked micelles led to higher DOX accumulation in the cellular nucleus in comparison with non-crosslinked micelles from the PEG-PZLL-BPEI copolymer (PP), thus causing more marked cytotoxicity in SKOV-3 cells. In vivo, DOX-loaded crosslinked micelles caused significant growth inhibition of SKOV-3 tumors xenografted in BALB/c nude mice, and showed superior anticancer efficacy to non-crosslinked PP micelles. Chemotherapy with core-crosslinked micelles had no adverse side effects on the health (serum levels and body weight) of the mice. This study highlights the design of clickable block copolymers to easily construct core-crosslinked and multiple stimuli-responsive micelles for enhanced anticancer therapy.

Metabolic glycoengineering of unnatural monosaccharides provides a facile method to label cancer cells with chemical tags for glycan imaging and cancer targeting. Multiple types of monosaccharides have been utilized for metabolic cell labeling. However, the comparison of different types of monosaccharides in labeling efficiency and selectivity has not been reported. In this study, we compared N-azidoacetylgalactosamine (GalAz) and N-azidoacetylmannosamine (ManAz) for metabolic labeling of HepG2 hepatocellular carcinoma in vitro and in vivo. GalAz showed higher labeling efficiency at low concentrations, and outperformed ManAz in metabolic labeling of HepG2 tumors in vivo. GalAz mediated labeling of HepG2 tumors with azido groups significantly improved the tumor accumulation of dibenzocyclooctyne (DBCO)-Cy5 and DBCO-doxorubicin conjugate via efficient Click chemistry. This study, for the first time, uncovered the distinct labeling efficiency and selectivity of different unnatural monosaccharides in liver cancers.

Objective: Prenatal exposure to valproic acid (VPA) enhances the risk for later development of autism spectrum disorders (ASD). An altered gamma-aminobutyric acid (GABA) system may be a key factor in ASD. Here we investigated possible changes in the GABA system in rats exposed to a low dose of prenatal VPA., Method: We performed autoradiography with [3H]muscimol, (a GABAA receptor agonist), and [11C]Ro15-4513 (a partial agonist of the GABAA α1+5 receptor subtypes), in brain sections containing amygdala, thalamus and hippocampus of rats treated prenatally with 20 mg/kg VPA or saline from the 12th day of gestation. Result Prenatal VPA significantly increased [11C]Ro15-4513 binding in the left amygdala compared with controls (p<0.05). This difference was not observed in the hippocampus, thalamus or right amygdala. No differences were observed in [3H]muscimol binding., Conclusion: We observed an asymmetric increase in GABAA receptor binding. Disturbances in the GABAA receptor system have also been detected in human autism with [11C]Ro15-4513.

Introduction: Alteration of γ-aminobutyric acid "A" (GABA A ) receptor-mediated neurotransmission has been associated with various neurological and psychiatric disorders. [ 11 C]Ro15-4513 is a PET ligand with high affinity for α5-subunit-containing GABA A receptors, which are highly expressed in limbic regions of the human brain (Sur et al., 1998). We quantified the test-retest reproducibility of measures of [ 11 C]Ro15-4513 binding derived from six different quantification methods (12 variants)., Methods: Five healthy males (median age 40 years, range 38-49 years) had a 90-min PET scan on two occasions (median interval 12 days, range 11-30 days), after injection of a median dose of 441 MegaBequerels of [ 11 C]Ro15-4513. Metabolite-corrected arterial plasma input functions (parent plasma input functions, ppIFs) were generated for all scans. We quantified regional binding using six methods (12 variants), some of which were region-based (applied to the average time-activity curve within a region) and others were voxel-based: 1) Models requiring arterial ppIFs - regional reversible compartmental models with one and two tissue compartments (2kbv and 4kbv); 2) Regional and voxelwise Logan's graphical analyses (Logan et al., 1990), which required arterial ppIFs; 3) Model-free regional and voxelwise (exponential) spectral analyses (SA; (Cunningham and Jones, 1993)), which also required arterial ppIFs; 4) methods not requiring arterial ppIFs - voxelwise standardised uptake values (Kenney et al., 1941), and regional and voxelwise simplified reference tissue models (SRTM/SRTM2) using brainstem or alternatively cerebellum as pseudo-reference regions (Lammertsma and Hume, 1996; Gunn et al., 1997). To compare the variants, we sampled the mean values of the outcome parameters within six bilateral, non-reference grey matter regions-of-interest. Reliability was quantified in terms of median absolute percentage test-retest differences (MA-TDs; preferentially low) and between-subject coefficient of variation (BS-CV, preferentially high), both compounded by the intraclass correlation coefficient (ICC). These measures were compared between variants, with particular interest in the hippocampus., Results: Two of the six methods (5/12 variants) yielded reproducible data (i.e. MA-TD <10%): regional SRTMs and voxelwise SRTM2s, both using either the brainstem or the cerebellum; and voxelwise SA. However, the SRTMs using the brainstem yielded a lower median BS-CV (7% for regional, 7% voxelwise) than the other variants (8-11%), resulting in lower ICCs. The median ICCs across six regions were 0.89 (interquartile range 0.75-0.90) for voxelwise SA, 0.71 (0.64-0.84) for regional SRTM-cerebellum and 0.83 (0.70-0.86) for voxelwise SRTM-cerebellum. The ICCs for the hippocampus were 0.89 for voxelwise SA, 0.95 for regional SRTM-cerebellum and 0.93 for voxelwise SRTM-cerebellum., Conclusion: Quantification of [ 11 C]Ro15-4513 binding shows very good to excellent reproducibility with SRTM and with voxelwise SA which, however, requires an arterial ppIF. Quantification in the α5 subunit-rich hippocampus is particularly reliable. The very low expression of the α5 in the cerebellum (Fritschy and Mohler, 1995; Veronese et al., 2016) and the substantial α1 subunit density in this region may hamper the application of reference tissue methods., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Mitochondria are central to health and disease, hence there is considerable interest in developing mitochondria-targeted therapies that require the delivery of peptides or nucleic acid oligomers. However, progress has been impeded by the lack of a measure of mitochondrial import of these molecules. Here, we address this need by quantitatively detecting molecules within the mitochondrial matrix. We used a mitochondria- targeted cyclooctyne (MitoOct) that accumulates several- hundredfold in the matrix, driven by the membrane potential. There, MitoOct reacts through click chemistry with an azide on the target molecule to form a diagnostic product that can be quantified by mass spectrometry. Because the membrane potential-dependent MitoOct concentration in the matrix is essential for conjugation, we can now determine definitively whether a putative mitochondrion-targeted molecule reaches the matrix. This "ClickIn" approach will facilitate development of mitochondria-targeted therapies., (© 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

The importance of the GABA-benzodiazepine receptor complex and its subtypes are increasingly recognised in addiction. Using the α1/α5 benzodiazepine receptor PET radioligand [(11)C]Ro15 4513, we previously showed reduced binding in the nucleus accumbens and hippocampus in abstinent alcohol dependence. We proposed that reduced [(11)C]Ro15 4513 binding in the nucleus accumbens was a marker of addiction whilst the reduction in hippocampus and positive relationship with memory was a consequence of chronic alcohol abuse. To examine this further we assessed [(11)C]Ro15 4513 binding in another addiction, opiate dependence, and used spectral analysis to estimate contributions of α1 and α5 subtypes to [(11)C]Ro15 4513 binding in opiate and previously acquired alcohol-dependent groups. Opiate substitute maintained opiate-dependent men (n=12) underwent an [(11)C]Ro15 4513 PET scan and compared with matched healthy controls (n=13). We found a significant reduction in [(11)C]Ro15 4513 binding in the nucleus accumbens in the opiate-dependent compared with the healthy control group. There was no relationship between [(11)C]Ro15 4513 binding in the hippocampus with memory. We found that reduced [(11)C]Ro15 4513 binding was associated with reduced α5 but not α1 subtypes in the opiate-dependent group. This was also seen in an alcohol-dependent group where an association between memory performance and [(11)C]Ro15 4513 binding was primarily driven by α5 and not α1 subtype. We suggest that reduced α5 levels in the nucleus accumbens are associated with addiction since we have now shown this in dependence to two pharmacologically different substances, alcohol and opiates., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) click chemistry has inherent challenges for copper-labeled radiopharmaceuticals. An azide-modified phosphonate-based cross-bridged macrocyclic chelator was synthesized for click chemistry conjugation with azide-modified Y3-TATE (a somatostatin analogue) on resin, without the need for protecting the chelator. The (64)Cu-labeled bioconjugate shows favourable in vitro and in vivo behaviour.

Low affinity α1/α2 containing GABAA receptors are significantly less able to bind [(11) C]/[(3) H]Ro15-4513 following translocation to the endosomal environment. The alterations in [(11) C]Ro15-4513 binding observed in vivo following perturbations in endogenous GABA are likely driven by both alterations in receptor binding parameters following agonist induced internalisation and the GABA Shift., (© 2014 Wiley Periodicals, Inc.)

Unlabelled: Effective anticancer therapy induces tumor cell death through apoptosis. Noninvasive monitoring of apoptosis during therapy may provide predictive outcome information and help tailor treatment. A caspase-3-specific imaging radiotracer, (18)F-(S)-1-((1-(2-fluoroethyl)-1H-[1,2,3]-triazol-4-yl)methyl)-5-(2(2,4-difluorophenoxymethyl)-pyrrolidine-1-sulfonyl)isatin ((18)F-ICMT-11), has been developed for use in PET studies. We report the safety, biodistribution, and internal radiation dosimetry profiles of (18)F-ICMT-11 in 8 healthy human volunteers., Methods: (18)F-ICMT-11 was intravenously administered as a bolus injection (mean ± SD, 159 ± 2.75 MBq; range, 154-161 MBq) to 8 healthy volunteers (4 men, 4 women). Whole-body (vertex to mid thigh) PET/CT scans were acquired at 6 time points, up to 4 h after tracer injection. Serial whole blood, plasma, and urine samples were collected for radioactivity measurement and radiotracer stability. In vivo (18)F activities were determined from quantitative analysis of the images, and time-activity curves were generated. The total numbers of disintegrations in each organ normalized to injected activity (residence times) were calculated as the area under the curve of the time-activity curve, normalized to injected activities and standard values of organ volumes. Dosimetry calculations were then performed using OLINDA/EXM 1.1., Results: Injection of (18)F-ICMT-11 was well tolerated in all subjects, with no serious tracer-related adverse events reported. The mean effective dose averaged over both men and women was estimated to be 0.025 ± 0.004 mSv/MBq (men, 0.022 ± 0.004 mSv/MBq; women, 0.027 ± 0.004 mSv/MBq). The 5 organs receiving the highest absorbed dose (mGy/MBq), averaged over both men and women, were the gallbladder wall (0.59 ± 0.44), small intestine (0.12 ± 0.05), upper large intestinal wall (0.08 ± 0.07), urinary bladder wall (0.08 ± 0.02), and liver (0.07 ± 0.01). Elimination was both renal and via the hepatobiliary system., Conclusion: (18)F-ICMT-11 is a safe PET tracer with a dosimetry profile comparable to other common (18)F PET tracers. These data support the further development of (18)F-ICMT-11 for clinical imaging of apoptosis.

Niemann-Pick type C (NPC) disease is characterized by impaired cholesterol efflux from late endosomes and lysosomes and secondary accumulation of lipids. Although impaired trafficking of individual glycoproteins and glycolipids has been noted in NPC cells and other storage disorders, there is currently no effective way to monitor their localization and movement en masse. Using a chemical reporter strategy in combination with pharmacologic treatments, we demonstrate a disease-specific and previously unrecognized accumulation of a diverse set of glycoconjugates in NPC1-null and NPC2-deficient fibroblasts within endocytic compartments. These labeled vesicles do not colocalize with the cholesterol-laden compartments of NPC cells. Experiments using the endocytic uptake marker dextran show that the endosomal accumulation of sialylated molecules can be largely attributed to impaired recycling as opposed to altered fusion of vesicles. Treatment of either NPC1-null or NPC2-deficient cells with cyclodextrin was effective in reducing cholesterol storage as well as the endocytic accumulation of sialoglycoproteins, demonstrating a direct link between cholesterol storage and abnormal recycling. Our data further demonstrate that this accumulation is largely glycoproteins, given that inhibitors of O-glycan initiation or N-glycan processing led to a significant reduction in staining intensity. Taken together, our results provide a unique perspective on the trafficking defects in NPC cells, and highlight the utility of this methodology in analyzing cells with altered recycling and turnover of glycoproteins.

The capacity to evade apoptosis has been defined as one of the hallmarks of cancer and, thus, effective anti-cancer therapy often induces apoptosis. A biomarker for imaging apoptosis could assist in monitoring the efficacy of a wide range of current and future therapeutics. Despite the potential, there are limited clinical examples of the use of positron emission tomography for imaging of apoptosis. [(18)F]ICMT-11 is a novel reagent designed to non-invasively image caspase-3 activation and, hence, drug-induced apoptosis. Radiochemistry development of [(18)F]ICMT-11 has been undertaken to improve specific radioactivity, reduce content of stable impurities, reduce synthesis time and enable automation for manufacture of multi-patient dose. Due to the promising mechanistic and safety profile of [(18)F]ICMT-11, the radiotracer is transitioning to clinical development and has been selected as a candidate radiotracer by the QuIC-ConCePT consortium for further evaluation in preclinical models and humans. A successful outcome will allow use of the radiotracer as qualified method for evaluating the pharmaceutical industry's next generation therapeutics., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The current study investigated the possible inherent relationship between convulsions and sleep involving the GABA(A)/benzodiazepine site complex. The aim of this study was to determine if rats with high (HTR) and low (LTR) thresholds for clonic convulsions induced by DMCM, a benzodiazepine inverse agonist, differ in the following aspects: (1) sensitivity to the hypnotic effects of the GABA(A) positive allosteric modulators diazepam, pentobarbital and ethanol and (2) in the binding of [(3)H]-flunitrazepam, a benzodiazepine agonist, measured by autoradiography, and [(3)H]-Ro 15-4513, a benzodiazepine partial inverse agonist, to membranes from discrete brain regions. The LTR subgroup presented a shorter diazepam-induced sleeping time compared to that of the HTR subgroup. Biochemical assays revealed that the LTR subgroup did not differ in [(3)H]-flunitrazepam binding compared to the HTR subgroup. With respect to the binding of [(3)H]-Ro 15-4513, the LTR subgroup had higher binding in the brainstem and lower binding in the striatum compared to the HTR subgroup. These results suggest that differences in the benzodiazepine site on the GABA(A) receptor may underlie the susceptibility to DMCM-induced convulsions and sensitivity to the hypnotic effect of diazepam., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Preclinical evidence suggests the α5 subtype of the GABA-benzodiazepine receptor is involved in some of the actions of alcohol and in memory. The positron emission tomography (PET) tracer, [(11)C]Ro15 4513 shows relative selectivity in labelling the α5 subtype over the other GABA-benzodiazepine receptor subtypes in limbic regions of the brain. We used this tracer to investigate the distribution of α5 subtype availability in human alcohol dependence and its relationship to clinical variables. Abstinent (>6 weeks) alcohol-dependent men and healthy male controls underwent an [(11)C]Ro15 4513 PET scan. We report [(11)C]Ro15 4513 brain uptake for 8 alcohol-dependent men and 11 healthy controls. We found a significant reduction in [(11)C]Ro15 4513 binding in the nucleus accumbens, parahippocampal gyri, right hippocampus and amygdala in the alcohol-dependent compared with the healthy control group. Levels of [(11)C]Ro15 4513 binding in both hippocampi were significantly and positively associated with performance on a delayed verbal memory task in the alcohol-dependent but not the control group. We speculate that the reduced limbic [(11)C]Ro15 4513 binding seen here results from the effects of alcohol, though we cannot currently distinguish whether they are compensatory in nature or evidence of brain toxicity.

The copper-free click (CFC) reaction has been evaluated for its potential application to in vivo pre-targeting for PET imaging. A promising biodistribution profile is demonstrated when employing [(18)F]2-fluoroethylazide ([(18)F]1) and optimisation of the CFC reaction with a series of cyclooctynes shows that reactions proceed efficiently with tantalizing opportunities for application-specific tuning., (This journal is © The Royal Society of Chemistry 2012)

Radiopharmaceuticals for nuclear imaging are essentially targeting molecules, labeled with short-lived radionuclides (e.g., F-18 for PET). A significant drawback of radiopharmaceuticals development is the difficulty to access radiolabeled molecule libraries for initial in vitro evaluation, as radiolabeling has to be optimized for each individual molecule. The present paper discloses a method for preparing libraries of (18)F-labeled radiopharmaceuticals using both the fluorous-based (18)F-radiochemistry and the Huisgen 1,3-dipolar (click) conjugation reaction. As a proof of concept, this approach allowed us to obtain a series of readily accessible (18)F-radiolabeled nitroaromatic molecules, for exploring their structure-activity relationship and further in vitro evaluation of their hypoxic selectivity., (Crown Copyright © 2011. Published by Elsevier Ltd. All rights reserved.)

Multidrug resistance (MDR) is a major factor in the failure of chemotherapy in cancer patients. Resistance to chemotherapy has been correlated to the overexpression of ABC drug transporters including P-glycoprotein (P-gp) that actively efflux chemotherapeutic drugs from cancer cells. Our previous study showed that bitter melon (Momordica charantia) leaf extract (BMLE) was able to reverse the MDR phenotype by increasing the intracellular accumulation of chemotherapeutic drugs. In the present study, bioguided fractionation was used to identify the active component(s) of BMLE that is able to modulate the function of P-gp and the MDR phenotype in a human cervical carcinoma cell line (KB-V1). We found that kuguacin J, one of the active components in BMLE, increased sensitivity to vinblastine and paclitaxel in KB-V1 cells. A flow cytometry assay indicated that kuguacin J inhibits the transport function of P-gp and thereby significantly increases the accumulation of rhodamine 123 and calcein AM in the cells. These results were confirmed by [³H]-vinblastine transport assay. Kuguacin J significantly increases intracellular [³H]-vinblastine accumulation and decreased the [³H]-vinblastine efflux in the cells. Kuguacin J also inhibited the incorporation of [¹²⁵I]-iodoarylazidoprazosin into P-gp in a concentration-dependent manner, indicating that kuguacin J directly interacts with the drug-substrate-binding site on P-gp. These results indicate that kuguacin J modulates the function of P-gp by directly interacting at the drug-substrate-binding site, and it appears to be an effective inhibitor of P-gp activity in vitro and thus could be developed as an effective chemosensitizer to treat multidrug-resistant cancers., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The application of intact monoclonal antibodies (mAbs) as targeting agents in nuclear imaging and radioimmunotherapy is hampered by the slow pharmacokinetics of these molecules. Pretargeting with mAbs could be beneficial to reduce the radiation burden to the patient, while using the excellent targeting capacity of the mAbs. In this study, we evaluated the applicability of the Staudinger ligation as pretargeting strategy using an antibody-azide conjugate as tumor-targeting molecule in combination with a small phosphine-containing imaging/therapeutic probe. Up to 8 triazide molecules were attached to the antibody without seriously affecting its immunoreactivity, pharmacokinetics, and tumor uptake in tumor bearing nude mice. In addition, two (89)Zr- and (67/68)Ga-labeled desferrioxamine (DFO)-phosphines, a (177)Lu-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-phosphine and a (123)I-cubyl phosphine probe were synthesized and characterized for their pharmacokinetic behavior in nude mice. With respect to the phosphine probes, blood levels at 30 min after injection were <5% injected dose per gram tissue, indicating rapid blood clearance. In vitro Staudinger ligation of 3.33 μM antibody-azide conjugate with 1 equiv of radiolabeled phosphine, relative to the azide, in aqueous solution resulted in 20-25% efficiency after 2 h. The presence of 37% human serum resulted in a reduced ligation efficiency (reduction max. 30% at 2 h), while the phosphines were still >80% intact. No in vivo Staudinger ligation was observed in a mouse model after injection of 500 μg antibody-azide, followed by 68 μg DFO-phosphine at t = 2 h, and evaluation in blood at t = 7 h. To explain negative results in mice, Staudinger ligation was performed in vitro in mouse serum. Under these conditions, a side product with the phosphine was formed and ligation efficiency was severely reduced. It is concluded that in vivo application of the Staudinger ligation in a pretargeting approach in mice is not feasible, since this ligation reaction is not bioorthogonal and efficient enough. Slow reaction kinetics will also severely restrict the applicability of Staudinger ligation in humans.

Multidrug resistance protein 1 (MRP1) is an ATP-binding cassette transporter that effluxes drugs and organic anions across the plasma membrane. The 17 transmembrane helices of MRP1 are linked by extracellular and cytoplasmic loops (CLs), but their role in coupling the ATPase activity of MRP1 to the translocation of its substrates is poorly understood. Here we have examined the importance of CL5 by mutating eight conserved charged residues and the helix-disrupting Gly(511) in this region. Ala substitution of Lys(513), Lys(516), Glu(521), and Glu(535) markedly reduced MRP1 levels. Because three of these residues are predicted to lie at the interface of CL5 and the second nucleotide binding domain (NBD2), a critical role is indicated for this region in the plasma membrane expression of MRP1. Further support for this idea was obtained by mutating NBD2 amino acids His(1364) and Arg(1367) at the CL5 interface, which also resulted in reduced MRP1 levels. In contrast, mutation of Arg(501), Lys(503), Glu(507), Arg(532), and Gly(511) had no effect on MRP1 levels. Except for K503A, however, transport by these mutants was reduced by 50 to 75%, an effect largely attributable to reduced substrate binding and affinity. Studies with (32)P-labeled azido-ATP also indicated that whereas ATP binding by the G511I mutant was unchanged, vanadate-induced trapping of azido-ADP was reduced, indicating changes in the catalytic activity of MRP1. Together, these data demonstrate the multiple roles for CL5 in the membrane expression and function of MRP1.

The electronic absorption spectra of: 2-, 3-, and 4-azidopyridines have been investigated in a wide variety of polar and non-polar solvents. According to Onsager model, the studied spectra indicate that the orientation polarization of solvent dipoles affects the electronic spectrum much stronger than the induction polarization of solvent dipoles. The effect of solvent dipole moment predominates that of solvent refractive index in determining the values of band maxima of an electronic spectrum. The spectra of azidopyridines differ basically from these of pyridine or mono-substituted pyridine. Results at hand indicate that the azide group perturbs the pyridine ring in the case of 3-azidopyridine much more than it does in the case of 2-azidopyridine. This result agrees with the predictions of the resonance theory. Although the equilibrium <==> azide tetrazole is well known, yet the observed spectra prove that such an equilibrium does not exist at the studied conditions. The spectra of the studied azidopyridines are characterized by the existence of overlapping transitions. Gaussian analysis is used to obtain nice, resolved spectra. All the observed bands correspond to pi-->pi* transitions, n-->pi* may be overlapped with the stronger pi-->pi* ones.

Studies on animal models of epilepsy and cerebellar ataxia, e.g., stargazer mice (stg) have identified changes in the GABAergic properties of neurones associated with the affected brain loci. Whether these changes contribute to or constitute homeostatic adaptations to a state of altered neuronal excitability is as yet unknown. Using cultured cerebellar granule neurones from control [+/+; alpha-amino-3-hydroxyl-5-methyl-4-isoxazolepropionate receptor (AMPAR)-competent, Kainate receptor (KAR)-competent] and stg (AMPAR-incompetent, KAR-competent), we investigated whether non-NMDA receptor (NMDAR) activity regulates GABA(A) receptor (GABAR) expression. Neurones were maintained in 5 mmol/L KCl-containing basal media or depolarizing media containing either 25 mmol/L KCl or the non-NMDAR agonist kainic acid (KA) (100 micromol/L). KCl- and KA-mediated depolarization down-regulated GABAR alpha1, alpha6 and beta2, but up-regulated alpha4, beta3 and delta subunits in +/+ neurones. The KCl-evoked but not KA-evoked effects were reciprocated in stg neurones compatible with AMPAR-regulation of GABAR expression. Conversely, GABAR gamma2 expression was insensitive to KCl-mediated depolarization, but was down-regulated by KA-treatment in a 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX)-reversible manner in +/+ and stg neurones compatible with a KAR-mediated response. KA-mediated up-regulation of GABAR alpha4, beta3 and delta was inhibited by L-type voltage-gated calcium channel (L-VGCC) blockers and the Ca2+/calmodulin-dependent protein kinase inhibitor, 4-[(2S)-2-[(5-isoquinolinylsulfonyl)methylamino]-3-oxo-3-(4-phenyl-1-piperazinyl)propyl] phenyl isoquinoline sulfonic acid ester (KN-62). Up-regulation of GABAR alpha4 and beta3 was also prevented by calcineurin (CaN) inhibitors, FK506 and cyclosporin A. Down-regulation of GABAR alpha1, alpha6 and beta2 was independent of L-VGCC activity, but was prevented by inhibitors of CaN. Thus, we provide evidence that a KAR-mediated and at least three mutually exclusive AMPAR-mediated signalling mechanisms regulate neuronal GABAR expression.

In the present study, we investigated the co-localization pattern of the delta subunit with other subunits of GABA(A) receptors in the rat brain using immunoprecipitation and Western blotting techniques. Furthermore, we investigated whether low concentrations of ethanol affect the delta-subunit-containing GABA(A) receptor assemblies in the rat brain using radioligand binding to the rat brain membrane homogenates as well as to the immunoprecipitated receptor assemblies. Our results revealed that delta subunit is not co-localized with gamma(2) subunit but it is associated with the alpha(1), alpha(4) or alpha(6), beta(2) and/or beta(3) subunit(s) of GABA(A) receptors in the rat brain. Ethanol (1-50 mM) neither affected [(3)H]muscimol (3 nM) binding nor diazepam-insensitive [(3)H]Ro 15-4513 (2 nM) binding in the rat cerebellum and cerebral cortex membranes. However, a higher concentration of ethanol (500 mM) inhibited the binding of these radioligands to the GABA(A) receptors partially in the rat cerebellum and cerebral cortex. Similarly, ethanol (up to 50 mM) did not affect [(3)H]muscimol (15 nM) binding to the immunoprecipitated delta-subunit-containing GABA(A) receptor assemblies in the rat cerebellum and hippocampus but it inhibited the binding partially at a higher concentration (500 mM). These results suggest that the native delta-subunit-containing GABA(A) receptors do not play a major role in the pharmacology of clinically relevant low concentrations of ethanol.

Mice were forced to swim for 5 min in water at a temperature of 12 degrees C (cold water swim stress) or 32 degrees C (warm water swim stress), and stress-induced analgesia (SIA) was measured using the tail-flick test. The cold water swim stress induced non-opioid SIA as well as hypothermia, whereas the warm water swim stress caused opioid SIA. The in vivo binding of [(3)H]-Ro15-4513 was measured in the stressed mice and compared with that in control mice. The specific binding of [(3)H]-Ro15-4513 in the cerebral cortex, hippocampus, and cerebellum was significantly altered by forced swimming in cold water. Apparent association and dissociation rate of [(3)H]-Ro15-4513 binding were decreased, and the change in the dissociation rate was most pronounced in the hippocampus. In contrast, no significant alterations were observed in in vitro binding. The hypothermia induced by the cold water swim stress seems to be the main reason for alterations in the specific binding of [(3)H]-Ro15-4513. The kinetics of a saturable amount of [(3)H]-Ro15-4513 in the blood and brain were also measured. The relative ratio of the radioactivity concentration in the brain to that in the blood was significantly decreased by forced swimming in cold water, indicating that the cold water swim stress induced changes in the nonspecific binding of [(3)H]-Ro15-4513 in the brain. These results together with previous reports suggested that non-opioid SIA induced by the cold water swim stress might be related to alterations in the rates of general ligand-receptor interactions including GABA(A)/benzodiazepine system. Changes in the nonspecific binding might be also involved in non-opioid SIA., (Synapse 58:22-28, 2005. (c) 2005 Wiley-Liss, Inc.)

Following the recent disclosure of 3-methyl pyrrole-2,4-dicarboxylic acid 2-propyl ester 4-(1,2,2-trimethyl-propyl) ester, a potent and selective mGluR1 non-competitive antagonist, we report here a detailed exploration of the C-2 position of this scaffold with the preparation of differently substituted amides. Great improvement of the pharmacokinetic properties has been achieved through this exercise.

We performed in vitro and in vivo assays of the metabolism of [(11)C]Ro15-4513 over time in the plasma of mice, rats, monkeys and humans, using a radio-HPLC equipped with a sensitive positron detector, in order to compare the metabolic rates of the radiopharmaceutical agent among the different animal species and to establish a highly sensitive analytical method for the radiotracer agent. We also examined the metabolism of [(11)C]Ro15-4513 in the brain tissue of mice and rats. The analytical method used in this study permitted detection of even extremely low levels of radioactivity (approximately 5,000 dpm). In vitro experiments revealed that [(11)C]Ro15-4513 in the blood was metabolized to hydrolysate [(11)C]A. The species were classified in descending order of the metabolic rate of the radiotracer in vitro as follows; mice, rats, and monkeys/humans. In the in vitro experiment, the percentage of the unchanged drug in the plasma at 60 minutes postdose was 9% in mice, 70% in rats, 97% in monkeys, and 98% in humans. In vivo metabolite analysis in the blood showed the presence of two radioactive metabolites, consisting of one hydrolysate [(11)C]A and another unidentified substance. The species were classified in descending order of the metabolic rate of the radiotracer in vivo as follows; mice, rats/humans, and monkeys. The percentage of the unchanged drug in the plasma was 6% in mice, 21% in rats, 26% in humans, and 40% in monkeys. Furthermore, the in vitro and in vivo experiments conducted to analyze the metabolism of [(11)C]Ro15-4513 in the brain tissue of mice and rats revealed that the radiotracer was metabolized to some extent in the brain tissue of these animals. In the in vivo experiment, the percentage of the unchanged drug at 60 min postdose was 86% in the brain tissue of mice and 88% in the brain tissue of rats, while in the in vitro experiment, the corresponding percentage was 93% in mice, and 91% in rats.

The K1 capsule, an alpha(2,8)-linked polymer of sialic acid, is an important virulence determinant of invasive Escherichia coli. The 17-kb kps gene cluster of E. coli K1 encodes the information necessary for capsule expression at the cell surface. Two proteins, KpsM and KpsT, play a role in the transport of capsular polysaccharide across the cytoplasmic membrane, utilizing the energy from ATP hydrolysis. They belong to the ATP-binding cassette superfamily of transport proteins. In this study, we purified KpsT in its native form and show that the purified protein is able to bind ATP, undergo an ATP-dependent conformational change and hydrolyze ATP. Protease accessibility studies demonstrate the in vivo interaction between KpsM and KpsT.

Although [(11)C]Ro15-4513 and [(11)C]flumazenil both bind to the central benzodiazepine (BZ) receptors, the distributions of the two ligands are not identical in vivo. Moreover, the in vivo pharmacological properties of [(11)C]Ro15-4513 have not been thoroughly examined. In the present study, we examined the pharmacological profile of [(11)C]Ro15-4513 binding in the monkey brain using positron emission tomography (PET). [(11)C]Ro15-4513 showed relatively high accumulation in the anterior cingulate cortex, hippocampus, and insular cortex, with the lowest uptake being observed in the pons. Accumulation in the cerebral cortex was significantly diminished by the BZ antagonist flumazenil (0.1 mg/kg, i.v.), but not that in the pons. Using the pons as a reference region, the specific binding of [(11)C]Ro15-4513 in most of the cerebral cortex including the limbic regions clearly revealed two different affinity sites. On the other hand, specific binding in the occipital cortex and cerebellum showed only a low affinity site. Zolpidem with affinity for alpha1, alpha2, and alpha3 subunits of GABA(A)/BZ receptor fully inhibited [(11)C]Ro15-4513 binding in the occipital cortex and cerebellum, while only about 23% of the binding was blocked in the anterior cingulate cortex. Diazepam with affinity for alpha1, alpha2, alpha3, and alpha5 subunits inhibited the binding in all brain regions. Since Ro15-4513 has relatively high affinity for the alpha5 subunit in vitro, these in vivo bindings of [(11)C]Ro15-4513 can be interpreted as the relatively high accumulation in the fronto-temporal limbic regions representing binding to the GABA(A)/BZ receptor alpha5 subunit., (Copyright 2002 Wiley-Liss, Inc.)

The cystic fibrosis transmembrane conductance regulator (CFTR) contains two nucleotide-binding domains (NBDs) or ATP-binding cassettes (ABCs) that characterize a large family of membrane transporters. Although the three-dimensional structures of these domains from several ABC proteins have been determined, this is not the case for CFTR, and hence the domains are defined simply on the basis of sequence alignment. The functional C-terminal boundary of NBD1 of CFTR was located by analysis of chloride channel function [Chan, Csanady, Seto-Young, Nairn and Gadsby (2000) J. Gen. Physiol. 116, 163-180]. However, the boundary between the C-terminal end of NBD2 and sequences further downstream in the whole protein, that are important for its cellular localization and endocytotic turnover, has not been defined. We have now done this by assaying the influence of progressive C-terminal truncations on photolabelling of NBD2 by 8-azido-ATP, which reflects hydrolysis, as well as binding, at that domain, and on NBD2-dependent channel gating itself. The boundary defined in this way is between residues 1420 and 1424, which corresponds to the final beta-strand in aligned NBDs whose structures have been determined. Utilization of this information should facilitate the generation of monodisperse NBD2 polypeptides for structural analysis, which until now has not been possible. The established boundary includes within NBD2 a hydrophobic patch of four residues (1413-1416) previously shown to be essential for CFTR maturation and stability [Gentzsch and Riordan (2001) J. Biol. Chem. 276, 1291-1298]. This hydrophobic cluster is conserved in most ABC proteins, and on alignment with ones of known structure constitutes the penultimate beta-strand of the domain which is likely to participate in essential structure-stabilizing beta-sheet formation.

There is evidence of marked variation in the brain distribution of specific subtypes of the GABA-benzodiazepine receptor and that particular subtypes mediate different functions. The alpha5-containing subtype is highly expressed in the hippocampus, and selective alpha5 inverse agonists (which decrease tonic GABA inhibition) are being developed as potential memory-enhancing agents. Evidence for such receptor localization and specialization in humans in vivo is lacking because the widely used probes for imaging the GABA-benzodiazepine receptors, [11C]flumazenil and [123I]iomazenil, appear to reflect binding to the alpha1 subtype, based on its distribution and affinity of flumazenil for this subtype. The authors characterized for positron emission tomography (PET) a radioligand from Ro15 4513, the binding of which has a marked limbic distribution in the rat and human brain in vivo. Competition studies in vivo in the rat revealed that radiolabeled Ro15 4513 uptake was reduced to nonspecific levels only by drugs that have affinity for the alpha5 subtype (flunitrazepam, RY80, Ro15 4513, L655,708), but not by the alpha1 selective agonist, zolpidem. Quantification of [11C]Ro15 4513 PET was performed in humans using a metabolite-corrected plasma input function. [11C]Ro15 4513 uptake was relatively greater in limbic areas compared with [11C]flumazenil, but lower in the occipital cortex and cerebellum. The authors conclude that [11C]Ro15 4513 PET labels in vivo the GABA-benzodiazepine receptor containing the alpha5 subtype in limbic structures and can be used to further explore the functional role of this subtype in humans.

ATP-binding cassette (ABC) proteins transport a diverse collection of substrates. It is presumed that these proteins couple ATP hydrolysis to substrate transport, yet ATPase activity has been demonstrated for only a few. To provide direct evidence for such activity in Ste6p, the yeast ABC protein required for the export of a-factor mating pheromone, we established conditions for purification of Ste6p in biochemical quantities from both yeast and Sf9 insect cells. The basal ATPase activity of purified and reconstituted Ste6p (V(max) = 18 nmol/mg/min; K(m) for MgATP = 0.2 mm) compares favorably with several other ABC proteins and was inhibited by orthovanadate in a profile diagnostic of ABC transporters (apparent K(I) = 12 microm). Modest stimulation (approximately 40%) was observed upon the addition of a-factor either synthetic or in native form. We also used an 8-azido-[alpha-(32)P]ATP binding and vanadate-trapping assay to examine the behavior of wild-type Ste6p and two different double mutants (G392V/G1087V and G509D/G1193D) shown previously to be mating-deficient in vivo. Both mutants displayed a diminished ability to hydrolyze ATP, with the latter uncoupled from pheromone transport. We conclude that Ste6p catalyzes ATP hydrolysis coupled to a-factor transport, which in turn promotes mating.

The ATP-binding cassette (ABC) transporter TAP translocates peptides from the cytosol to awaiting MHC class I molecules in the endoplasmic reticulum. TAP is made up of the TAP1 and TAP2 polypeptides, which each possess a nucleotide binding domain (NBD). However, the role of ATP in peptide binding and translocation is poorly understood. We present biochemical and functional evidence that the NBDs of TAP1 and TAP2 are non-equivalent. Photolabeling experiments with 8-azido-ATP demonstrate a cooperative interaction between the two NBDs that can be stimulated by peptide. The substitution of key lysine residues in the Walker A motifs of TAP1 and TAP2 suggests that TAP1-mediated ATP hydrolysis is not essential for peptide translocation but that TAP2-mediated ATP hydrolysis is critical, not only for translocation, but for peptide binding.

P-glycoprotein (Pgp) is an ATP-dependent drug efflux pump whose overexpression confers multidrug resistance to cancer cells. Pgp exhibits a robust drug substrate-stimulable ATPase activity, and vanadate (Vi) blocks this activity effectively by trapping Pgp nucleotide in a non-covalent stable transition state conformation. In this study we compare Vi-induced [alpha-(32)P]8-azido-ADP trapping into Pgp in the presence of [alpha-(32)P]8-azido-ATP (with ATP hydrolysis) or [alpha-(32)P]8-azido-ADP (without ATP hydrolysis). Vi mimics P(i) to trap the nucleotide tenaciously in the Pgp.[alpha-(32)P]8-azido-ADP.Vi conformation in either condition. Thus, by using [alpha-(32)P]8-azido-ADP we show that the Vi-induced transition state of Pgp can be generated even in the absence of ATP hydrolysis. Furthermore, half-maximal trapping of nucleotide into Pgp in the presence of Vi occurs at similar concentrations of [alpha-(32)P]8-azido-ATP or [alpha-(32)P]8-azido-ADP. The trapped [alpha-(32)P]8-azido-ADP is almost equally distributed between the N- and the C-terminal ATP sites of Pgp in both conditions. Additionally, point mutations in the Walker B domain of either the N- (D555N) or C (D1200N)-terminal ATP sites that arrest ATP hydrolysis and Vi-induced trapping also show abrogation of [alpha-(32)P]8-azido-ADP trapping into Pgp in the absence of hydrolysis. These data suggest that both ATP sites are dependent on each other for function and that each site exhibits similar affinity for 8-azido-ATP (ATP) or 8-azido-ADP (ADP). Similarly, Pgp in the transition state conformation generated with either ADP or ATP exhibits drastically reduced affinity for the binding of analogues of drug substrate ([(125)I]iodoarylazidoprazosin) as well as nucleotide (2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate). Analyses of Arrhenius plots show that trapping of Pgp with [alpha-(32)P]8-azido-ADP (in the absence of hydrolysis) displays an approximately 2.5-fold higher energy of activation (152 kJ/mol) compared with that observed when the transition state intermediate is generated through hydrolysis of [alpha-(32)P]8-azido-ATP (62 kJ/mol). In aggregate, these results demonstrate that the Pgp.[alpha-(32)P]8-azido-ADP (or ADP).Vi transition state complexes generated either in the absence of or accompanying [alpha-(32)P]8-azido-ATP hydrolysis are functionally indistinguishable.

A rapid screening method was developed for the determination of the toxic volatile anions, cyanide and azide, in beverages. This method consisted of a microdiffusion extraction combined with spectrophotometry using the Konig cyanide reaction and ferric azide complex formation in conjugation with cerium azide oxido-reduction. The time required to achieve full recovery in the extraction of hydrogen cyanide and hydrazoic acid from samples was considerably shortened by increasing the diffusion temperature from 25 degrees C to 40 degrees C. The time required to achieve saturated color development in the Konig cyanide reaction was also shortened by increasing incubation temperature to 40 degrees C. The interference in both azide color reactions was examined for volatile compounds. Cyanide interfered only in the case of ferric azide complex formation. Sulfide, sulfate, nitrite, and acetic acid interfered in both the color reactions. The established method gave a detection limit of 6 microM for cyanide and 0.5mM for azide, and it required only 1 h to determine both anions. Cyanide and azide disappeared by evaporation from beverages during 25 degrees C storage under open conditions in a pH-dependent manner as a function of their respective pKa values of 9.2 and 4.6.

The transport of glucose across the mammalian blood-brain barrier is mediated by the GLUT1 glucose transporter, which is concentrated in the endothelial cells of the cerebral microvessels. Several studies supported an asymmetric distribution of GLUT1 protein between the luminal and abluminal membranes (1:4) with a significant proportion of intracellular transporters. In this study we investigated the activity and concentration of GLUT1 in isolated luminal and abluminal membrane fractions of bovine brain endothelial cells. Glucose transport activity and glucose transporter concentration, as determined by cytochalasin B binding, were 2-fold greater in the luminal than in the abluminal membranes. In contrast, Western blot analysis using a rabbit polyclonal antibody raised against the C-terminal 20 amino acids of GLUT1 indicated a 1:5 luminal:abluminal distribution. Western blot analysis with antibodies raised against either the intracellular loop of GLUT1 or the purified erythrocyte protein exhibited luminal:abluminal ratios of 1:1. A similar ratio was observed when the luminal and abluminal fractions were exposed to the 2-N-4[(3)H](1-azi-2,2,2,-trifluoroethyl)benzoxyl-1,3-bis-(d-mannos-4-yloxyl)-2-propylamine ([(3)H]ATB-BMPA) photoaffinity label. These observations suggest that either an additional glucose transporter isoform is present in the luminal membrane of the bovine blood-brain barrier or the C-terminal epitope of GLUT1 is "masked" in the luminal membrane but not in the abluminal membranes.

After phosphorylation by protein kinase A, gating of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is regulated by the interaction of ATP with its nucleotide binding domains (NBDs). Models of this gating regulation have proposed that ATP hydrolysis at NBD1 and NBD2 may drive channel opening and closing, respectively (reviewed in Nagel, G. (1999) Biochim. Biophys. Acta 1461, 263-274). However, as yet there has been little biochemical confirmation of the predictions of these models. We have employed photoaffinity labeling with 8-azido-ATP, which supports channel gating as effectively as ATP to evaluate interactions with each NBD in intact membrane-bound CFTR. Mutagenesis of Walker A lysine residues crucial for azido-ATP hydrolysis to generate the azido-ADP that is trapped by vanadate indicated a greater role of NBD1 than NBD2. Separation of the domains by limited trypsin digestion and enrichment by immunoprecipitation confirmed greater and more stable nucleotide trapping at NBD1. This asymmetry of the two domains in interactions with nucleotides was reflected most emphatically in the response to the nonhydrolyzable ATP analogue, 5'-adenylyl-beta,gamma-imidodiphosphate (AMP-PNP), which in the gating models was proposed to bind with high affinity to NBD2 causing inhibition of ATP hydrolysis there postulated to drive channel closing. Instead we found a strong competitive inhibition of nucleotide hydrolysis and trapping at NBD1 and a simultaneous enhancement at NBD2. This argues strongly that AMP-PNP does not inhibit ATP hydrolysis at NBD2 and thereby questions the relevance of hydrolysis at that domain to channel closing.

We previously reported that overexpression of GLUT4 in lean, nondiabetic C57BL/KsJ-lepr(db/+) (db/+) mice resulted in improved glucose tolerance associated with increased basal and insulin-stimulated glucose transport in isolated skeletal muscle. We used the diabetic (db/db) litter mates of these mice to examine the effects of GLUT4 overexpression on in vivo glucose utilization and on in vitro glucose transport and GLUT4 translocation in diabetic mice. We examined in vivo glucose disposal by oral glucose challenge and hyperinsulinemic-hyperglycemic clamps. We also evaluated the in vitro relationship between glucose transport activity and cell surface GLUT4 levels as assessed by photolabeling with the membrane-impermeant reagent 2-N-(4-(1-azi-2,2,2-trifluoroethyl)benzoyl)-1,3-bis(D-mannose-4-yloxy)-2-propylamine in extensor digitorum longus (EDL) muscles. All parameters were examined as functions of animal age and the level of GLUT4 overexpression. In young mice (age 10-12 weeks), both lower (two- to threefold) and higher (four- to fivefold) levels of GLUT4 overexpression were associated with improved glucose tolerance compared to age-matched nontransgenic (NTG) mice. However, glucose tolerance deteriorated with age in db/db mice, although less rapidly in transgenic mice expressing the higher level of GLUT4. Glucose infusion rates during hyperinsulinemic-hyperglycemic clamps were increased with GLUT4 overexpression, compared with NTG mice in both lower and higher levels of GLUT4 overexpression, even in the older mice. Surprisingly, isolated EDL muscles from diabetic db/db mice did not exhibit alterations in either basal or insulin-stimulated glucose transport activity or cell surface GLUT4 compared to nondiabetic db/+ mice. Furthermore, both GLUT4 overexpression levels and animal age are associated with increased basal and insulin-stimulated glucose transport activities and cell surface GLUT4. However, the observed increased glucose transport activity in older db/db mice was not accompanied by an equivalent increase in cell surface GLUT4 compared to younger animals. Thus, although in vivo glucose tolerance is improved with GLUT4 overexpression in young animals, it deteriorates with age; in contrast, insulin responsiveness as assessed by the clamp technique remains improved with GLUT4 overexpression, as does in vitro insulin action. In summary, despite an impairment in whole-body glucose tolerance, skeletal muscle of the old transgenic GLUT4 db/db mice is still insulin responsive in vitro and in vivo.

The yeast a-factor transporter Ste6 is a member of the ABC transporter family and is closely related to human MDR1. We constructed a set of 26 Ste6 mutants using a random mutagenesis approach. Cell fractionation experiments demonstrated that most of the mutants, with the notable exception of those with alterations in TM1, are transported to the plasma membrane, the presumptive site of action of Ste6. Trafficking, therefore, does not seem to be affected in most of the mutants. To identify regions in Ste6 that interact with the ABC transporter "signature motif" (LSGGQ) we screened for intragenic revertants of the LSGGQ mutant M68 (S507N). Suppressor mutations were identified in TM12 and upstream of TM6. Surprisingly, these mutations also suppressed the Walker A mutation G397D, which should be defective in ATP-binding and hydrolysis at NBD1. Photoaffinity labeling experiments with 8-azido-[alpha-32P]ATP showed that ATP binding at NBD2 is reduced by the suppressor mutation in TM12. The experiments further suggest that the two NBDs of Ste6 are not equivalent and affect each other's ability to bind and hydrolyze ATP.

Fatty acyl-CoA synthetase (FACS, fatty acid:CoA ligase, AMP-forming, EC ) catalyzes the esterification of fatty acids to CoA thioesters for further metabolism and is hypothesized to play a pivotal role in the coupled transport and activation of exogenous long-chain fatty acids in Escherichia coli. Previous work on the bacterial enzyme identified a highly conserved region (FACS signature motif) common to long- and medium-chain acyl-CoA synthetases, which appears to contribute to the fatty acid binding pocket. In an effort to further define the fatty acid-binding domain within this enzyme, we employed the affinity labeled long-chain fatty acid [(3)H]9-p-azidophenoxy nonanoic acid (APNA) to specifically modify the E. coli FACS. [(3)H]APNA labeling of the purified enzyme was saturable and specific for long-chain fatty acids as shown by the inhibition of modification with increasing concentrations of palmitate. The site of APNA modification was identified by digestion of [(3)H]APNA cross-linked FACS with trypsin and separation and purification of the resultant peptides using reverse phase high performance liquid chromatography. One specific (3)H-labeled peptide, T33, was identified and following purification subjected to NH(2)-terminal sequence analysis. This approach yielded the peptide sequence PDATDEIIK, which corresponded to residues 422 to 430 of FACS. This peptide is immediately adjacent to the region of the enzyme that contains the FACS signature motif (residues 431-455). This work represents the first direct identification of the carboxyl-containing substrate-binding domain within the adenylate-forming family of enzymes. The structural model for the E. coli FACS predicts this motif lies within a cleft separating two distinct domains of the enzyme and is adjacent to a region that contains the AMP/ATP signature motif, which together are likely to represent the catalytic core of the enzyme.

CD38 is a ubiquitous protein originally identified as a lymphocyte antigen and recently also found to be a multifunctional enzyme participating in the synthesis and metabolism of two Ca(2+) messengers, cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate. It is homologous to Aplysia ADP-ribosyl cyclase, where the crystal structure has been determined. Residues of CD38 corresponding to those at the active site of the Aplysia cyclase were mutagenized. Changing Glu-226, which corresponded to the catalytic residue of the cyclase, to Asp, Asn, Gln, Leu, or Gly eliminated essentially all enzymatic activities of CD38, indicating it is most likely the catalytic residue. Photoaffinity labeling showed that E226G, nevertheless, retained substantial NAD binding activity. The secondary structures of these inactive mutants as measured by circular dichroism were essentially unperturbed as compared with the wild type. Other nearby residues were also investigated. The mutants D147V and E146L showed 7- and 19-fold reduction in NADase activity, respectively. The cADPR hydrolase activity of the two mutants was similarly reduced. Asp-155, on the other hand, was crucial for the GDP-ribosyl cyclase activity since its substitution with either Glu, Asn, or Gln stimulated the activity 3-15-fold, whereas other activities remained essentially unchanged. In addition to these acidic residues, two tryptophans were also important, since all enzyme activities of W125F, W125Y, W189G and W189Y were substantially reduced. This is consistent with the two tryptophans serving a substrate positioning function. A good correlation was observed when the NADase activity of all the mutants was plotted against the cADPR hydrolase activity. Homology modeling revealed all these critical residues are clustered in a pocket near the center of the CD38 molecule. The results indicate a strong structural homology between the active sites of CD38 and the Aplysia cyclase.

Multidrug resistance protein 1 (MRP1) and P-glycoprotein, which are ATP-dependent multidrug efflux pumps and involved in multidrug resistance of tumor cells, are members of the ATP binding cassette proteins and contain two nucleotide-binding folds (NBFs). P-glycoprotein hydrolyzes ATP at both NBFs, and vanadate-induced nucleotide trapping occurs at both NBFs. We examined vanadate-induced nucleotide trapping in MRP1 stably expressed in KB cell membrane by using 8-azido-[alpha-(32)P]ATP. Vanadate-induced nucleotide trapping in MRP1 was found to be stimulated by reduced glutathione, glutathione disulfide, and etoposide and to be synergistically stimulated by the presence of etoposide and either glutathione. These results suggest that glutathione and etoposide interact with MRP1 at different sites and that those bindings cooperatively stimulate the nucleotide trapping. Mild trypsin digestion of MRP1 revealed that vanadate-induced nucleotide trapping mainly occurs at NBF2. Our results suggest that the two NBFs of MRP1 might be functionally nonequivalent.

Inhibitory neurotransmission in the brain is largely mediated by GABA(A) receptors. Potentiation of GABA receptor activation through an allosteric benzodiazepine (BZ) site produces the sedative, anxiolytic, muscle relaxant, anticonvulsant and cognition-impairing effects of clinically used BZs such as diazepam. We created genetically modified mice (alpha1 H101R) with a diazepam-insensitive alpha1 subtype and a selective BZ site ligand, L-838,417, to explore GABA(A) receptor subtypes mediating specific physiological effects. These two complimentary approaches revealed that the alpha1 subtype mediated the sedative, but not the anxiolytic effects of benzodiazepines. This finding suggests ways to improve anxiolytics and to develop drugs for other neurological disorders based on their specificity for GABA(A) receptor subtypes in distinct neuronal circuits.

Two different photoinsulins, the radioactive N(epsilonB29)-(4-azidosalicyloyl) insulin and the novel biotinylated N(epsilonB29)-(4-azidotetrafluorobenzoyl-biocytinyl) insulin, were synthesized in order to study the binding stoichiometry of insulin and the insulin receptor in a direct approach. Both derivatives were cross-linked simultaneously to the (alphabeta)(2) receptor. Insulin-receptor conjugates were formed that carry a radioactive label as well as a biotin label. The doubly labeled complexes were isolated by streptavidin-affinity chromatography. Analysis of both markers, the radioactive (125)I marker and the biotin marker, proved the existence of a covalent complex of one receptor molecule and two ligands. Thus, orthogonal photocross-linking is introduced as a method for the isolation and analysis of bivalent receptor complexes.

We have identified regions in poly(A) polymerases that interact with ATP. Conditions were established for efficient cross-linking of recombinant bovine and yeast poly(A) polymerases to 8-azido-ATP. Mn2+ strongly stimulated this reaction due to a 50-fold lower Ki for 8-azido-ATP in the presence of Mn2+. Mutations of the highly conserved Asp residues 113, 115, and 167, critical for metal binding in the catalytic domain of bovine poly(A) polymerase, led to a strong reduction of cross-linking efficiency, and Mn2+ no longer stimulated the reaction. Sites of 8-azido-ATP cross-linking were mapped in different poly(A) polymerases by CNBr-cleavage and analysis of tryptic peptides by mass spectroscopy. The main cross-link in Schizosaccharomyces pombe poly(A) polymerase could be assigned to the peptide DLELSDNNLLK (amino acids 167-177). Database searches with sequences surrounding the cross-link site detected significant homologies to other nucleotidyltransferase families, suggesting a conservation of the nucleotide-binding fold among these families of enzymes. Mutations in the region of the "helical turn motif" (a domain binding the triphosphate moiety of the nucleotide) and in the suspected nucleotide-binding helix of bovine poly(A) polymerase impaired ATP binding and catalysis. The results indicate that ATP is bound in part by the helical turn motif and in part by a region that may be a structural analog to the fingers domain found in many polymerases.

ADP-ribosyl cyclase synthesizes two Ca(2+) messengers by cyclizing NAD to produce cyclic ADP-ribose and exchanging nicotinic acid with the nicotinamide group of NADP to produce nicotinic acid adenine dinucleotide phosphate. Recombinant Aplysia cyclase was expressed in yeast and co-crystallized with a substrate, nicotinamide. x-ray crystallography showed that the nicotinamide was bound in a pocket formed in part by a conserved segment and was near the central cleft of the cyclase. Glu(98), Asn(107) and Trp(140) were within 3.5 A of the bound nicotinamide and appeared to coordinate it. Substituting Glu(98) with either Gln, Gly, Leu, or Asn reduced the cyclase activity by 16-222-fold, depending on the substitution. The mutant N107G exhibited only a 2-fold decrease in activity, while the activity of W140G was essentially eliminated. The base exchange activity of all mutants followed a similar pattern of reduction, suggesting that both reactions occur at the same active site. In addition to NAD, the wild-type cyclase also cyclizes nicotinamide guanine dinucleotide to cyclic GDP-ribose. All mutant enzymes had at least half of the GDP-ribosyl cyclase activity of the wild type, some even 2-3-fold higher, indicating that the three coordinating amino acids are responsible for positioning of the substrate but not absolutely critical for catalysis. To search for the catalytic residues, other amino acids in the binding pocket were mutagenized. E179G was totally devoid of GDP-ribosyl cyclase activity, and both its ADP-ribosyl cyclase and the base exchange activities were reduced by 10,000- and 18,000-fold, respectively. Substituting Glu(179) with either Asn, Leu, Asp, or Gln produced similar inactive enzymes, and so was the conversion of Trp(77) to Gly. However, both E179G and the double mutant E179G/W77G retained NAD-binding ability as shown by photoaffinity labeling with [(32)P]8-azido-NAD. These results indicate that both Glu(179) and Trp(77) are crucial for catalysis and that Glu(179) may indeed be the catalytic residue.

The imidazobenzodiazepines ethyl 8-iodo-5,6 dihydro-5-methyl-6-oxo-4H-imidazo[1,5a][1,4] benzodiazepine-3-carboxylate 1 and tert-butyl 8-iodo-5,6 dihydro-5-methyl-6-oxo-4H-imidazo [1,5a][1,4] benzodiazepine-3-carboxylate 2 were prepared to study the diazepam-insensitive (DI) benzodiazepine receptor (BZR) subtype. The [123I] analogues were prepared via iododestannylation reactions in radiochemical yields of 70-80% and a specific activity >2,500 Ci/mmol. The tert-butyl analogue [123I]-2 exhibited nanomolar affinity for BZRs in homogenate membranes of rat cerebellum with Kd values for the diazepam-sensitive (DS) and DI receptors of 3.18 +/- 0.58 and 13.55 +/- 2.72 nM, respectively. The Bmax for cerebellar DS and DI receptors were 1,276 +/- 195 and 518 +/- 26 fmol/mg protein, respectively.

The acridone carboxamide derivative GG918 (N-{4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)-ethyl]-pheny l}-9,10dihydro-5-methoxy-9-oxo-4-acridine carboxamide) is a potent inhibitor of MDR1 P-glycoprotein-mediated multidrug resistance. Direct measurements of ATP-dependent MDR1 P-glycoprotein-mediated transport in plasma membrane vesicles from human and rat hepatocyte canalicular membranes indicated 50% inhibition at GG918 concentrations between 8 nM and 80 nM using N-pentyl-[3H]quinidinium, ['4C]doxorubicin and [3H]daunorubicin as substrates. The inhibition constant K for GG918 was 35 nM in rat hepatocyte canalicular membrane vesicles with [3H]daunorubicin as the substrate. Photoaffinity labelling of canalicular and recombinant rat Mdr1b P-glycoprotein by [3H]azidopine was suppressed by 10 muM and 40 muM GG918. The high selectivity of GG918-induced inhibition was demonstrated in canalicular membrane vesicles and by analysis of the hepatobiliary elimination in rats using [3H]daunorubicin, [3H]taurocholate and [3H]cysteinyl leukotrienes as substrates for three distinct ATP-dependent export pumps. Almost complete inhibition of [3H]daunorubicin transport was observed at GG918 concentrations that did not affect the other hepatocyte canalicular export pumps. The high potency and selectivity of GG918 for the inhibition of human MDR1 and rat Mdr1b P-glycoprotein may serve to interfere with this type of multidrug resistance and provides a tool for studies on the function of these ATP-dependent transport proteins.

The ATP-sensitive potassium (KATP) channels in pancreatic beta cells are critical in the regulation of glucose-induced insulin secretion. Although electrophysiological studies provide clues to the complex control of KATP channels by ATP, MgADP, and pharmacological agents, the molecular mechanism of KATP-channel regulation remains unclear. The KATP channel is a heterooligomeric complex of SUR1 subunits of the ATP-binding-cassette superfamily with two nucleotide-binding folds (NBF1 and NBF2) and the pore-forming Kir6.2 subunits. Here, we report that MgATP and MgADP, but not the Mg salt of gamma-thio-ATP, stabilize the binding of prebound 8-azido-[alpha-32P]ATP to SUR1. Mutation in the Walker A and B motifs of NBF2 of SUR1 abolished this stabilizing effect of MgADP. These results suggest that SUR1 binds 8-azido-ATP strongly at NBF1 and that MgADP, either by direct binding to NBF2 or by hydrolysis of bound MgATP at NBF2, stabilizes prebound 8-azido-ATP binding at NBF1. The sulfonylurea glibenclamide caused release of prebound 8-azido-[alpha-32P]ATP from SUR1 in the presence of MgADP or MgATP in a concentration-dependent manner. This direct biochemical evidence of cooperative interaction in nucleotide binding of the two NBFs of SUR1 suggests that glibenclamide both blocks this cooperative binding of ATP and MgADP and, in cooperation with the MgADP bound at NBF2, causes ATP to be released from NBF1.

Activation of NMDA receptors requires the presence of glycine as a coagonist which binds to a site that is allosterically linked to the glutamate binding site. To identify the protein constituents of the glycine binding site in situ the photoaffinity label [3H]CGP 61594 was synthesized. In reversible binding assays using crude rat brain membranes, [3H]CGP 61594 labeled with high affinity (K(D) = 23 nM) the glycine site of the NMDA receptor. This was evident from the Scatchard analysis, the displacing potencies of various glycine site ligands and the allosteric modulation of [3H]CGP 61594 binding by ligands of the glutamate and polyamine sites. Electrophysiological experiments in a neocortical slice preparation identified CGP 61594 as a glycine antagonist. Upon UV-irradiation, a protein band of 115 kDa was specifically photolabeled by [3H]CGP 61594 in brain membrane preparations. The photolabeled protein was identified as the NR1 subunit of the NMDA receptor by NR1 subunit-specific immunoaffinity chromatography. Thus, [3H]CGP 61594 is the first photoaffinity label for the glycine site of NMDA receptors. It will serve as a tool for the identification of structural elements that are involved in the formation of the glycine binding domain of NMDA receptors in situ and will thereby complement the mutational analysis of recombinant receptors.