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PubMed1985

Properties of a mutant lactose carrier of Escherichia coli with a Cys148----Ser148 substitution.

The cysteine residue at position 148 in the lactose carrier protein of Escherichia coli has been replaced by serine using oligonucleotide-directed, site-specific mutagenesis of the lac Y gene. The mutant carrier is incorporated into the cytoplasmic membrane to the same extent as the wild-type carrier, confers a lactose-positive phenotype on cells, and actively transports lactose and other galactosides. However, the maximum rate of transport for several substrates is reduced by a factor of 6-10 while the apparent affinity is reduced by a factor of 2-4. Carrier activity in the mutant is much less sensitive to sulfhydryl reagents (HgCl2, p-(chloromercuri)benzenesulfonate and N-ethylmaleimide) than in the wild type, and beta-D-galactosyl 1-thio-beta-D-galactoside does not protect the mutant carrier against slow inactivation by N-ethylmaleimide. It is concluded that the Cys148 residue is not essential for carrier-catalyzed galactoside: proton symport and that its alkylation presumbly prohibits access of the substrate to the binding site by steric hindrance. A serine residue at position 148 in the amino acid sequence appears to alter the protein structure in such a way that one or more sulfhydryl groups elsewhere in the protein become accessible to alkylating agents thereby inhibiting transport. Recently, Trumble et al. [(1984) Biochem. Biophys. Res. Commun. 119, 860-867] arrived at similar conclusions by investigating a mutant carrier with a Cys148----Gly148 replacement.

Amino Acid Sequence↗
PubMed1985

Implication of proteases in the respiration dependent inactivation of the lactose permease of E. coli.

The lactose permease of E. coli becomes irreversibly inactivated during lactose transport under conditions of high respiratory activity. This inactivation is characterized by a decrease in the steady state of lactose accumulation, a decrease in the influx rate of lactose, and a decrease in the transmembrane electrical potential. We report here that inhibitors of serine proteases (phenylmethylsulfonyl fluoride and N-alpha-P-tosyl-L-lysine chloromethyl ketone) prevent this inactivation, thus implicating proteases in this process.

Escherichia coli↗
PubMed2005

A live-cell high-throughput screening assay for identification of fatty acid uptake inhibitors.

We developed a live-cell high-throughput assay system using the baker's yeast Saccharomyces cerevisiae to screen for chemical compounds that will inhibit fatty acid uptake. The target for the inhibitors is a mammalian fatty acid transport protein (mmFATP2), which is involved in the fatty acid transport and activation pathway. The mmFATP2 was expressed in a S. cerevisiae mutant strain deficient in Fat1p-dependent fatty acid uptake and reduced in long-chain fatty acid activation, fat1Deltafaa1Delta. To detect fatty acid import, a fluorescent fatty acid analog, 4,4-difluoro-5-methyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoic acid (C1-BODIPY-C12), was incubated with cells expressing FATP2 in a 96-well plate. The mmFATP2-dependent C1-BODIPY-C12 uptake was monitored by measuring intracellular C1-BODIPY-C12 fluorescence on a microtiter plate reader, whereas extracellular fluorescence was quenched by a cell viability dye, trypan blue. Using this high-throughput screening method, we demonstrate that the uptake of the fluorescent fatty acid ligand was effectively competed by the natural fatty acid oleate. Inhibition of uptake was also demonstrated to occur when cells were pretreated with sodium azide or Triacsin C. This yeast live-cell-based assay is rapid to execute, inexpensive to implement, and has adequate sensitivity for high-throughput screening. The assay basis and limitations are discussed.

Biological Transport↗
PubMed2004

Identification and characterisation of a new class of highly specific and potent inhibitors of the mitochondrial pyruvate carrier.

Two novel thiazolidine compounds, GW604714X and GW450863X, were found to be potent inhibitors of mitochondrial respiration supported by pyruvate but not other substrates. Direct measurement of pyruvate transport into rat liver and yeast mitochondria confirmed that these agents inhibited the mitochondrial pyruvate carrier (MPC) with K(i) values <0.1 muM. Inhibitor titrations of pyruvate-dependent respiration by heart mitochondria gave values (+/-S.E.) for the concentration of inhibitor binding sites (pmol per mg protein) and their K(i) (nM) of 56.0+/-0.9 and 0.057+/-0.010 nM for the more hydrophobic GW604714X; for GW450863X the values were 59.9+/-4.6 and 0.60+/-0.12 nM. [(3)H]-methoxy-GW450863X binding was also used to determine the MPC content of the heart, kidney, liver and brain mitochondria giving values of 56, 40, 26 and 20 pmol per mg protein respectively. Binding to yeast mitochondria was <10% of that in rat liver mitochondria, consistent with the slow rate of pyruvate transport into yeast mitochondria. [(3)H]-methoxy-GW450863X binding was inhibited by GW604714X and by the established MPC inhibitor, UK5099. The absorbance spectra of GW450863X and GW604714X were markedly changed by the addition of beta-mercaptoethanol suggesting that the novel inhibitors, like alpha-cyanocinnamate, possess an activated double bond that attacks a critical cysteine residue on the MPC. However, no labelled protein was detected following SDS-PAGE suggesting that the covalent modification is reversible. GW604714X and GW450863X inhibited l-lactate transport by the plasma membrane monocarboxylate transporter MCT1, but at concentrations more than four orders of magnitude greater than the MPC.

Animals↗
PubMed2004

Quantitative structure activity relationship studies on the flavonoid mediated inhibition of multidrug resistance proteins 1 and 2.

In the present study, the effects of a large series of flavonoids on multidrug resistance proteins (MRPs) were studied in MRP1 and MRP2 transfected MDCKII cells. The results were used to define the structural requirements of flavonoids necessary for potent inhibition of MRP1- and MRP2-mediated calcein transport in a cellular model. Several of the methoxylated flavonoids are among the best MRP1 inhibitors (IC(50) values, ranging between 2.7 and 14.3 microM) followed by robinetin, myricetin and quercetin (IC(50) values ranging between 13.6 and 21.8 microM). Regarding inhibition of MRP2 activity especially robinetin and myricetin appeared to be good inhibitors (IC(50) values of 15.0 and 22.2 microM, respectively). Kinetic characterization revealed that the two transporters differ marginally in the apparent K(m) for the substrate calcein. For one flavonoid, robinetin, the kinetics of inhibition were studied in more detail and revealed competitive inhibition with respect to calcein, with apparent inhibition constants of 5.0 microM for MRP1 and 8.5 microM for MRP2. For inhibition of MRP1, a quantitative structure activity relationship (QSAR) was obtained that indicates three structural characteristics to be of major importance for MRP1 inhibition by flavonoids: the total number of methoxylated moieties, the total number of hydroxyl groups and the dihedral angle between the B- and C-ring. Regarding MRP2 mediated calcein efflux inhibition, only the presence of a flavonol B-ring pyrogallol group seems to be an important structural characteristic. Overall, this study provides insight in the structural characteristics involved in MRP inhibition and explores the differences between inhibitors of these two transporters, MRP1 and MRP2. Ultimately, MRP2 displays higher selectivity for flavonoid type inhibition than MRP1.

Animals↗
PubMed2005

Physiological, pharmacological and clinical features of the multidrug resistance protein 2.

Multidrug resistance protein 2 (MRP2, ABCC2) is a drug efflux pump belonging to the ATP-binding cassette (ABC) transporter superfamily. MRP2 is present predominantly at the biliary pole of hepatocytes and is also expressed in the kidney and intestine. It plays a major role in hepato-biliary elimination of many structurally diverse xenobiotics, including organic anions and drug conjugates, and therefore most likely contributes to pharmacokinetic parameters of these compounds. MRP2 also handles endogenous molecules such as bilirubin, and its overexpression has been shown to confer a multidrug resistance phenotype to tumoral cells. MRP2 expression can be regulated by endogenous substances such as inflammatory cytokines and biliary acids. The MRP2 levels and activity can also be affected by a large panel of xenobiotics, including chemopreventive agents and ligands of the pregnane X receptor, which may be a potential source of drug-drug interactions and drug adverse effects. MRP2 appears therefore as one of the major drug efflux pumps of the organism, whose functional and regulatory features are important to consider, notably for drug disposition.

Animals↗
PubMed2005

The slow and long-lasting blockade of dopamine transporters in human brain induced by the new antidepressant drug radafaxine predict poor reinforcing effects.

BACKGROUND: (2S,3S)-2-(3-Chlorophenyl)-3,5,5,-trimethyl-2-morpholinol hydrochloride (radafaxine) is a new antidepressant that blocks dopamine transporters (DAT). A concern with drugs that block (DAT) is their potential reinforcing effects and abuse liability. Using positron emission tomography (PET) we have shown that for DAT-blocking drugs to produce reinforcing effects they must induce >50% DAT blockade and the blockade has to be fast (within 15 minutes). This study measures the potency and kinetics for DAT blockade by radafaxine in human brain. METHODS: PET and [11C]cocaine were used to estimate DAT blockade at 1, 4, 8, and 24 hours after radafaxine (40 mg p.o.) in 8 controls. Plasma pharmacokinetics and behavioral and cardiovascular effects were measured in parallel. RESULTS: DAT blockade by radafaxine was slow, and at 1 hour, it was 11%. Peak blockade occurred at about 4 hours and was 22%. Blockade was long lasting: at 8 hours 17%, and at 24 hours 15%. Peak plasma concentration occurred about 4 to 8 hours. No behavioral or cardiovascular effects were observed. CONCLUSIONS: The relatively low potency of radafaxine in blocking DAT and its slow blockade suggests that it is unlikely to have reinforcing effects. This is consistent with preclinical studies showing no self-administration. This is the first utilization of PET to predict abuse liability of a new antidepressant in humans based on DAT occupancy and pharmacokinetics.

Adult↗
PubMed2005

Serotonin transporter availability in patients with schizophrenia: a positron emission tomography imaging study with [11C]DASB.

BACKGROUND: Postmortem studies have reported several alterations in serotonin transporter (SERT) binding parameters in patients with schizophrenia. The aim of this study was to compare SERT availability in vivo in patients with schizophrenia and matched control subjects. METHODS: Ten medication-free patients with schizophrenia and 10 healthy subjects underwent positron emission tomography (PET) scans for 90 min after 11C-3-amino-4-(2-dimethylaminomethylphenylthio)benzonitrile ([11C]DASB) injection. Metabolite-corrected arterial input function was measured. Regional distribution volumes (mL/g) were derived with a two tissue compartment kinetic model. Outcome measures for SERT availability included binding potential (BP) and the specific-to-nonspecific equilibrium partition coefficient (V3''). Ten brain regions with high density of SERT and where SERT availability can be reliably quantified with [11C]DASB were included in the analysis. RESULTS: No significant differences were observed in regional BP or V3'' between patients and control subjects. No significant relationships were observed between regional SERT availability and severity of positive, negative, and depressive symptoms. CONCLUSIONS: This study failed to detect alterations of SERT availability in patients with schizophrenia; however, this study does not rule out the possibility that schizophrenia might be associated with alterations of SERT density in the cortical regions, where the [11C]DASB-specific binding signal is too low for reliable quantification of SERT.

Adult↗
PubMed2004

The anandamide membrane transporter. Structure-activity relationships of anandamide and oleoylethanolamine analogs with phenyl rings in the polar head group region.

A new series of anandamide and N-oleoylethanolamine analogs, most of which with aromatic moieties in the head group region, has been synthesized and evaluated as inhibitors of anandamide uptake. Some of them efficaciously inhibit the uptake process with K(i) values in the low micromolar range (2.4-21.2microM). Strict structural requisites are needed to observe a significant inhibition and in no case inhibition of fatty acid amidohydrolase overlaps with inhibition of anandamide uptake.

Arachidonic Acids↗
PubMed2004

Structural requirements for 2,4- and 3,6-disubstituted pyran biomimetics of cis-(6-benzhydryl-piperidin-3-yl)-benzylamine compounds to interact with monoamine transporters.

In our effort to delineate novel pharmacophoric configuration of bioisosteric pyran versions of cis-(6-benzhydryl-piperidin-3-yl)-benzylamine derivatives in interacting with the monoamine transporter, further structure-activity relationship study was carried out. Both cis and trans 2,4- and 3,6-disubstituted derivatives were synthesized to determine the positional importance of N-substitution on affinity for monoamine transporters, that is the dopamine transporter (DAT), the serotonin transporter (SERT), and the norepinephrine transporter (NET) in rat brain. For that purpose, the potency of compounds was determined in competing for the binding of [(3)H]WIN 35,428, [(3)H]citalopram, and [(3)H]nisoxetine, respectively. Selected compounds were also evaluated for their activity in inhibiting the uptake of [(3)H]DA by DAT. Our binding results demonstrated potency in 3,6-disubstituted derivatives while 2,4-disubstituted derivatives failed to exhibit any appreciable binding affinity. Further structural exploration of the exocyclic N-atom in 3,6-disubstituted derivatives produced compounds potent at both DAT and NET. Compounds 16h and 16o with hydroxyl and amino groups in the phenyl moiety of the benzyl group produced the highest activity for the NET. In this regard, compound 16e with a methoxy substituent produced weak affinity at NET, which upon conversion into a hydroxyl functionality as in 16h produced potent affinity for the NET. Various indole derivatives displayed different interactions; the 5-substituted indole derivative 16n exerted potent affinity for NET, confirming the bioisosteric equivalence between this indole moiety and the phenyl-4-hydroxy group in 16h.

Animals↗
PubMed2005

Synthesis, structural identification, and ligand binding of tropane ring analogs of paroxetine and an unexpected aza-bicyclo[3.2.2]nonane rearrangement product.

The structural requirements for high affinity at the serotonin transporter (5-HTT) have been investigated through the preparation of rigid paroxetine analogs. Tropane-derived analogs (4a-i) of paroxetine (2) were designed and synthesized as potential inhibitors of serotonin reuptake based on the structural and biological similarity between the two compound classes. Overall, the affinity of tropane-derived analogs at the 5-HTT was found to be at least an order of magnitude lower than that of paroxetine and ranged from 2-400nM. The reduced affinity at the 5-HTT may be attributed to the inability of the rigid tropane-derived analogs to adopt conformations favored by the 5-HTT. Within the series of tropane analogs, the 2beta,3beta- and 2beta,3alpha-isomers, 4a and 4d, were the most potent at the DAT and NET and are also significantly more potent than paroxetine (2) suggesting that their reduced conformational flexibility maximizes residence time in conformations favored by these transporters. Examination of the previously published preparation and structural assignment of 4a by additional NMR and X-ray crystallographic data has established that nucleophilic addition to the intermediate 2beta-methanesulfonyloxymethyl-3beta-(4-fluorophenyl)tropane unexpectedly provided the aza-bicyclo[3.2.2]nonane derivative 10a.

Alkanes↗
PubMed2005

Lobelane analogues as novel ligands for the vesicular monoamine transporter-2.

A series of lobelane analogues has been synthesized and their structure-activity relationships at the vesicular monoamine transporter-2 (VMAT2) have been evaluated. The most potent analogues in this series were the cis-2,6-piperidino analogues, 25b, 27b, 28b, and 30b, with K(i) values ranging from 430 to 580 nM.

Animals↗
PubMed2004

MexAB-OprM specific efflux pump inhibitors in Pseudomonas aeruginosa. Part 3: Optimization of potency in the pyridopyrimidine series through the application of a pharmacophore model.

The addition of substituents to the pyridopyrimidine scaffold of MexAB-OprM specific efflux pump inhibitors was explored. As predicted by a pharmacophore model, the incorporation substituents at the 2-position improved potency. Piperidines were found to be optimal, and further introduction of polar groups without compromising the activity was shown to be feasible. Careful positioning of the essential acidic moiety of the pharmacophore relative to the scaffold led to the discovery of vinyl tetrazoles with still greater potency.

Bacterial Outer Membrane Proteins↗
PubMed2004

Novel halogenated nitrobenzylthioinosine analogs as es nucleoside transporter inhibitors.

Nucleoside transporter inhibitors have potential therapeutic applications as anticancer, antiviral, cardioprotective, and neuroprotective agents. We have synthesized and flow cytometrically evaluated the binding affinity of a series of novel halogenated nitrobenzylthioinosine analogs at the human es nucleoside transporter. Structure-activity relationships indicate the importance of hydrophobicity and electron withdrawing capacity of substituents at the para-position of the 6-position benzyl substituent. All of the compounds showed high binding affinity as shown by their ability to displace the fluorescent es transporter ligand, SAENTA-X8-fluorescein. Compound 16 (6-S-(para-iodobenzyl)-6-thioinosine) was the most tightly bound within the series with a K(i) of 3.88 nM (NBMPR exhibited a K(i) of 0.70 nM). This compound has higher affinity than the widely used nonnucleoside, nucleoside transport inhibitor, dipyridamole (K(i) = 8.79 nM), and may serve as a new lead compound.

Flow Cytometry↗
PubMed2004

MexAB-OprM specific efflux pump inhibitors in Pseudomonas aeruginosa. Part 4: Addressing the problem of poor stability due to photoisomerization of an acrylic acid moiety.

Exchange of the ethylene tether in a series of pyridopyrimidine-based MexAB-OprM specific efflux pump inhibitors to an amide bond stabilized the olefin of the acrylic acid moiety, preventing facile photoisomerization to the Z-isomer. Furthermore, the activity was drastically improved in the amide tether variants, providing extremely potent acrylic acid and vinyl tetrazole analogues.

Acrylates↗
PubMed2004

Quinazolinone-based fungal efflux pump inhibitors. Part 1: Discovery of an (N-methylpiperazine)-containing derivative with activity in clinically relevant Candida spp.

The discovery of a series of quinazolinone-based fungal efflux pump inhibitors by high-throughput screening for potentiation of fluconazole in C. albicans is described. Attempts to improve the aqueous solubility of screening hits led to the discovery of an analog with greatly improved physical properties and activity against clinically-relevant Candida spp.

ATP-Binding Cassette Transporters↗