Progesterone photoaffinity labels P-glycoprotein in multidrug-resistant human leukemic lymphoblasts

We show for the first time that [3H]progesterone ([3H]PRG) can directly photoaffinity label membrane proteins prepared from a multidrug-resistant human leukemic lymphoblastic cell line CEM/VLB5K. A 170-kDa protein in CEM/VLB5K cell membranes was specifically labeled by [3H]PRG, which we identified as P-glycoprotein (Pgp) by immunoprecipitation with monoclonal antibody C219. The anticancer drug vinblastine and multidrug resistance reversing agent verapamil as well as several steroidal hormones were examined for their ability to interfere with [3H]PRG binding to Pgp. We found that 200-fold molar excess of vinblastine strongly inhibited (93%) the binding of [3H]PRG to Pgp compared with verapamil (80%), progesterone (78%), testosterone (46%), dexamethasone (25%), and aldosterone (56%). The results of this study provide direct evidence that progesterone can bind to Pgp and support the hypothesis that under physiological conditions Pgp may play a role in the excretion of progesterone from certain cells. Importantly, our results show that under our conditions vinblastine and verapamil are better able to compete with [3H]PRG for binding to Pgp than are other steroids, including testosterone, corticosteroids, and mineralocorticoids.     

The alpha 1-adrenergic photoaffinity probe [125I]arylazidoprazosin binds to a specific peptide of P-glycoprotein in multidrug-resistant cells

Much evidence suggests that P-glycoprotein (P-gp) confers multidrug-resistance (MDR) in tumor cells by energy-dependent efflux of hydrophobic cytotoxic agents. In this study, we have used the alpha 1-adrenergic photoaffinity probe, [125I]arylazidoprazosin ([125I]AAP), and identified P-gp as a specific acceptor for prazosin. Drugs to which MDR cells are resistant, including vincristine, vinblastine, doxorubicin, actinomycin D and colchicine as well as agents reversing MDR, including verapamil, nicardipine, prenylamine, diltiazem, trifluoperazine, dibucaine, reserpine, monensin, and progesterone, differentially reduced [125I]AAP photolabeling of P-gp. We also analyzed the influence of alpha 2-adrenergic drugs and dopaminergic drugs on [125I]AAP photolabeling of P-gp. Limited proteolysis of [125I]AAP photolabeled P-gp with Staphylococcus aureus V8 protease revealed that prazosin binds to a single 8 kDa fragment of P-gp.     

Reversal of P-glycoprotein-mediated multidrug resistance by cholesterol derived from low density lipoprotein in a vinblastine-resistant human lymphoblastic leukemia cell line.

P-glycoprotein (P-gp) is believed to be one of the most common causes of multidrug resistance (MDR) in chemotherapy. Studies have shown that the biosynthesis of cholesterol and cholesterol esters interfere with the function of P-gp. Since low density lipoprotein (LDL) carries a large amount of cholesterol, we investigated the effect of cholesterol derived from LDL on a line of human lymphoblastic leukemia MDR cells, CEM/VLB. Our results demonstrated that, in addition to increased cytotoxicity, the uptake of vinblastine in CEM/VLB cells increased, and LDL subsequently increased the intracellular vinblastine concentrations retained by CEM/VLB cells. The cholesterol levels in the membrane of the MDR cells were restored, while LDL significantly decreased the P-gp-associated ATPase activity. Current studies have shown that LDL leads to the resensitization of CEM/VLB cells to cytotoxic agents, likely through the restoration of cholesterol and reduction of P-gp-associated ATPase in the cell membrane.     

Biomimetic one-pot synthesis of vinblastine: NAD(P)H-mediated vinblastine synthesis from the product of FMN-mediated vindoline–catharanthine coupling under near-ultraviolet light

Vinblastine (VLB) is an antineoplastic agent contained in leaves of Catharanthus roseus. One-pot synthesis of VLB from vindoline and catharanthine, biosynthetic precursors in the plant, was achieved under biomimetic conditions. FMN-mediated coupling of vindoline and catharanthine under irradiation with near-ultraviolet light was followed by NAD(P)H-mediated direct conversion of the coupling product to VLB in the dark. The yield of VLB was governed by the level of NAD(P)H added, and the highest yield was obtained by incubation with 10 mM NAD(P)H for 5 h. It is postulated, as one concept of VLB biosynthesis, that similar non-enzymic VLB synthesis may occur in the plant leaves.     

3H-azidopine photoaffinity labeling of high molecular weight proteins in chloroquine resistant falciparum malaria

Using 3H-azidopine, we have succeeded in labeling proteins from chloroquine resistant (CR) human falciparum malaria parasites in the molecular weight range of 155-170 kd. Vinblastine does not compete, but azidopine blocks the labeling using 3H-azidopine. Relatively little or no labeling of the 155-170 kd protein is seen in the chloroquine sensitive strain using 3H-azidopine. Further competition can be seen with nicardipine and reserpine (71%) respectively and verapamil (61%), chloroquine (48%), quinacrine (56%), trifluoperazine (32%) and chlorpromazine (33%). We speculate that this may be the glycoprotein responsible for the resistance to chloroquine in falciparum malaria.     

Cross-resistance to anti-HIV nucleoside analogs in multidrug-resistant human cells

Human multidrug-resistant K562/ADM cells showed 12-fold and 31-fold resistance to AZT (3'-azido-2', 3'dideoxythymidine) and DDC (2', 3'-dideoxycytidine), respectively. Other multidrug-resistant human cells CEM/VLB100 and AdrRMCF-7 also showed resistance to these nucleoside analogs. However, verapamil (10 microM) failed to reverse the resistance to the nucleoside analogs. Accumulation of [3H]AZT in human multidrug-resistant K562/ADM, CEM/VLB100 and AdrRMCF-7 cells decreased by 23, 35, and 42% respectively, as compared to their parental cells. These results suggest that anti-HIV nucleoside analogs including AZT, DDC could be transported by outward drug-transport system in the multidrug-resistant cells.     

In vitro activity of novel dual action MDR anthranilamide modulators with inhibitory activity on CYP-450 (Part 2)

Synthesis and in vitro cytotoxicity assays of new anthranilamide MDR modulators have been performed to assess their inhibition potency on the P-glycoprotein (P-gp) transporter. Previous studies showed that the replacement of the aromatic spacer group between nitrogen atoms (N(1) and N(2)) in the P-gp inhibitor XR9576 with ethyl or propyl chain is optimal for P-gp inhibition potency. To confirm that observation, the ethyl or the propyl linker arm was replaced with a pyrrolidine or an alicyclic group such as cyclohexyl. In addition, an arylpiperazinyl group and two methoxyl groups onto the anthranilic part were introduced to assess their effect on the anti P-gp activity. Five molecules were prepared and evaluated on CEM/VLB500. All new anthranilamides were more potent than verapamil, most of them exhibited a lower cytotoxicity than XR9576. Compound 5 was the most potent and its inhibition activity was similar to XR9576. Interestingly, in vitro biotransformation studies of compounds 4 and 5 using human CYP-450 isoforms revealed, that conversely to XR9576, compounds 4 and 5 inhibited CYP3A4, an enzyme that colocalizes with P-gp in the intestine and contributes to tumor cell chemoresistance by enhancing the biodisposition of numerous drugs, notably paclitaxel. In that context, 5 might be suitable for further drug development.     

Synthesis and chemical characterization of 2-methoxy-N(10)-substituted acridones needed to reverse vinblastine resistance in multidrug resistant (MDR) cancer cells

In an attempt to find clinically useful modulators of multidrug resistance (MDR), a series of 19 N(10)-substituted-2-methoxyacridone analogues has been synthesized. 2-Methoxyacridone and its derivatives (1-19) were synthesized. Compound 1 was prepared by the Ullmann condensation of o-chlorobenzoic acid and p-anisidine followed by cyclization using polyphosphoric acid. This compound undergoes N-alkylation in the presence of phase transfer catalyst (PTC). Stirring of 2-methoxy acridone with 1-bromo-3-chloropropane or 1-bromo-4-chlorobutane in a two-phase system consisting of organic phase (tetrahydrofuran) and 6N potassium hydroxide in the presence of tetrabutylammonium bromide leads to the formation of compounds 2 and 11 in good yield. N-(omega-Chloroalkyl) analogues were found to undergo iodide catalyzed nucleophilic substitution reaction with various secondary amines. Products were characterized by UV, IR, 1H and 13C NMR, mass-spectral data and elemental analysis. The lipophilicity expressed in log(10) P and pK(a) of compounds have been determined. All compounds were examined for their ability to increase the uptake of vinblastine (VLB) in MDR KBCh(R)-8-5 cells and the results showed that the compounds 7, 10, 12, and 15-19 at 100 microM caused a 1.05- to 1.7-fold greater accumulation of vinblastine than did a similar concentration of the standard modulator, verapamil (VRP). However, the effects on VLB uptake were specific because these derivatives had little effect in the parental drug sensitive line KB-3-1. Steady state accumulation of VLB, a substrate for P-glycoprotein (P-gp) mediated efflux, was studied in the MDR cell line KBCh(R)-8-5 in the presence and absence of novel MDR modulators. Results of the efflux experiment showed that VRP and each of the modulators (1-19) significantly inhibited the efflux of VLB, suggesting that they may be competitors for P-gp. From among the compounds examined, 14 except 1, 2, 4, 8, and 11, exhibited greater efflux inhibiting activity than VRP. All the 19 compounds effectively compete with [(3)H] azidopine for binding to P-gp, pointed out this transport membrane protein as their likely site of action. Cytotoxicity has been determined and the IC(50) values lie in the range 8.00-18.50 microM for propyl and 4-15 microM for butyl derivatives against KBCh(R)-8-5 cells suggesting that the antiproliferative activity increases as chain length increases from 3 to 4 carbons at N(10)-position. Compounds at IC(10) were evaluated for their efficacy to modulate the cytotoxicity of VLB in KBCh(R)-8-5 cells and found that the modulators enhanced the cytotoxicity of VLB by 5- to 35-fold. Modulators 12, 14-16, and 19 like VRP, were able to completely reverse the 24-fold resistance of KBCh(R)-8-5 cells to VLB. Examination of the relationship between lipophilicity and antagonism of MDR showed a reasonable correlation suggesting that hydrophobicity is one of the determinants of potency for anti-MDR activity of 2-methoxyacridones.     

Theoretical insight into the structural mechanism for the binding of vinblastine with tubulin

Vinblastine (VLB) is one of vinca alkaloids with high cytotoxicity toward cancer cells approved for clinical use. However, because of drug resistance, toxicity, and other side effects caused from the use of VLB, new vinca alkaloids with higher cytotoxicity toward cancer cells and other good qualities need to develop. One strategy is to further study and better understand the essence why VLB possesses the high cytotoxicity toward cancer cells. In present work, by using molecular simulation, molecular docking, density functional calculation, and the crystal structure of α,β-tubulin complex, we find two modes labeled in catharanthine moiety (CM) and vindoline moiety (VM) modes of VLB bound with the interface of α,β-tubulin to probe the essence why VLB has the high cytotoxicity toward cancer cells. In the CM mode, nine key residues B-Ser178, B-Asp179, B-Glu183, B-Tyr210, B-Asp226, C-Lys326, C-Asp327, C-Lys336, and C-Lys352 from the α,β-tubulin complex are determined as the active sites for the interaction of VLB with α,β-tubulin. Some of them such as B-Ser178, B-Glu183, B-Tyr210, B-Asp226, C-Lys326, C-Asp327, and C-Lys336 are newly identified as the active sites in present work. The affinity between VLB and the active pocket within the interface of α,β-tubulin is ?60.8 kJ mol^?1 in the CM mode. In the VM mode, that is a new mode established in present paper, nine similar key residues B-Lys176, B-Ser178, B-Asp179, B-Glu183, B-Tyr210, B-Asp226, C-Lys326, C-Asp327, and C-Lys336 from the α,β-tubulin complex are found as the active sites for the interaction with VLB. The difference is from one key residue C-Lys352 in the CM mode changed to the key residue B-Lys176 in the VM mode. The affinity between VLB and the active pocket within the interface of α,β-tubulin is ?96.3 kJ mol^?1 in the VM mode. Based on the results obtained in present work, and because VLB looks like two faces, composed of CM and VM both to have similar polar active groups, to interact with the active sites, we suggest double-faces sticking mechanism for the binding of VLB to the interface of α,β-tubulin. The double-faces sticking mechanism can be used to qualitatively explain high cytotoxicity toward cancer cells of vinca alkaloids including vinblastine, vincristine, vindestine, and vinorelbine approved for clinical use and vinflunine still in a phase III clinical trial. Furthermore, this mechanism will be applied to develop novel vinca alkaloids with much higher cytotoxicity toward cancer cells.     

Reversal of multidrug resistance by calcium channel blocker SR33557 without photoaffinity labeling of P-glycoprotein

The altered pharmacology of drugs in multidrug-resistant cells (decreased accumulation and retention) appears to be mediated by a high molecular weight integral membrane protein, called P-glycogprotein (P-gp). Agents known to reverse this pleiotropic drug resistance (chemosensitizers) have been shown to interact with P-gp; and as such, the inhibition of photoaffinity labeling by P-gp probes (such as [3H]azidopine) has been proposed as a basis for mass screening of chemosensitizers. In this study, we provide direct evidence that a novel calcium channel blocker (SR33557), which was 4.5 times more potent in sensitizing P388/ADR cells to doxorubicin as compared to verapamil (while inducing a similar increase in uptake and decrease in efflux of [14C]doxorubicin, did not compete for the [3H]azidopine-binding site on P-gp, whereas verapamil did. Moreover, SR33557, which is inherently photoactivable, did not photolabel P-gp, but a 65-kDa protein did appear to be an acceptor; and this binding was displaced by diltiazem and nifedipine, but not by verapamil. Finally, the implication for the participation of a sphingomyelin/sphingosine cycle (as a potential lipid second messenger system) in the chemosensitization of P388/ADR cells was investigated. 30 microM SR33557 induced a 72% inhibition in acid lysosomal sphingomyelinase activity, a 5-fold increase in sphingosine levels, and a 75% inhibition in intracellular protein kinase C activity. Although no direct link is established between these observations and P-gp activity, further studies on a possible sphingosine-mediated regulation of P-gp may yield information on the involvement of this second messenger system in the action of SR33557.     

Multiple Drugs Compete for Transport via the Plasmodium falciparum Chloroquine Resistance Transporter at Distinct but Interdependent Sites

Mutations in the “chloroquine resistance transporter” (PfCRT) are a major determinant of drug resistance in the malaria parasite Plasmodium falciparum. We have previously shown that mutant PfCRT transports the antimalarial drug chloroquine away from its target, whereas the wild-type form of PfCRT does not. However, little is understood about the transport of other drugs via PfCRT or the mechanism by which PfCRT recognizes different substrates. Here we show that mutant PfCRT also transports quinine, quinidine, and verapamil, indicating that the protein behaves as a multidrug resistance carrier. Detailed kinetic analyses revealed that chloroquine and quinine compete for transport via PfCRT in a manner that is consistent with mixed-type inhibition. Moreover, our analyses suggest that PfCRT accepts chloroquine and quinine at distinct but antagonistically interacting sites. We also found verapamil to be a partial mixed-type inhibitor of chloroquine transport via PfCRT, further supporting the idea that PfCRT possesses multiple substrate-binding sites. Our findings provide new mechanistic insights into the workings of PfCRT, which could be exploited to design potent inhibitors of this key mediator of drug resistance.     

Evidence for a substrate specific and inhibitable drug efflux system in chloroquine resistant Plasmodium falciparum strains

The mechanism underpinning chloroquine drug resistance in the human malarial parasite Plasmodium falciparum remains controversial. By investigating the kinetics of chloroquine accumulation under varying-trans conditions, we recently presented evidence for a saturable and energy-dependent chloroquine efflux system present in chloroquine resistant P. falciparum strains. Here, we further characterize the putative chloroquine efflux system by investigating its substrate specificity using a broad range of different antimalarial drugs. Our data show that preloading cells with amodiaquine, primaquine, quinacrine, quinine, and quinidine stimulates labeled chloroquine accumulation under varying-trans conditions, while mefloquine, halofantrine, artemisinin, and pyrimethamine do not induce this effect. In the reverse of the varying-trans procedure, we show that preloaded cold chloroquine can stimulate quinine accumulation. On the basis of these findings, we propose that the putative chloroquine efflux system is capable of transporting, in addition to chloroquine, structurally related quinoline and methoxyacridine antimalarial drugs. Verapamil and the calcium/calmodulin antagonist W7 abrogate stimulated chloroquine accumulation and energy-dependent chloroquine extrusion. Our data are consistent with a substrate specific and inhibitible drug efflux system being present in chloroquine resistant P. falciparum strains.     

Mechanism of interaction of vinca alkaloids with tubulin: catharanthine and vindoline

The interactions of the vinca alkaloid drugs catharanthine and vindoline with tubulin have been investigated and compared with those of vinblastine and vincristine. Both drugs were found to be less effective in bringing about the inhibition of tubulin self-assembly into microtubules than vincristine and vinblastine, the drug to protein molar ratio required being 3 orders of magnitude greater. An analytical ultracentrifuge study has shown that catharanthine can induce the self-association of tubulin into linear indefinite polymers with an efficacy that is 75% that of vinblastine or vincristine, the intrinsic dimerization constant for the liganded protein being K2 congruent to 1 x 10(5) M-1. The effect of vindoline was marginally detectable. Binding studies of catharanthine using the gel batch and fluorescence perturbation techniques showed a polymerization-linked binding of one catharanthine molecule per tubulin alpha-beta dimer with a binding constant of (2.8 +/- 0.4) x 10(3) M-1. For vindoline, binding to tubulin was marginally detectable by fluorescence spectroscopy, although addition of vindoline to tubulin did generate a difference spectrum. It was concluded that the binding of vinblastine/vincristine to tubulin and its consequences are determined by the interaction of the indole part of catharanthine with tubulin, the role of vindoline being that of an anchor.     

Acridones circumvent P-glycoprotein-associated multidrug resistance (MDR) in cancer cells

Multidrug resistance (MDR) mediated by overexpression of MDR1 P-glycoprotein (P-gp) is one of the best characterized transporter-mediated barriers to successful chemotherapy in cancer patients. Chemosensitizers are the agents that increase the sensitivity of multidrug-resistant cells to the toxic influence of previously less effective drugs. In an attempt to find such vital chemosensitizers, a series of N(10)-substituted-2-chloroacridone analogous (1-17) have been synthesized. Compound 1 was prepared by the Ullmann condensation of o-chlorobenzoic acid and p-chloroaniline followed by cyclization. The N-(omega-chloroalkyl) analogues were found to undergo iodide catalyzed nucleophilic substitution reaction with secondary amines and the resultant products were characterized by spectral methods. The lipophilicity expressed in log(10)P and pK(a) of compounds has been determined. All compounds were examined for their ability to increase the uptake of vinblastine (VLB) in MDR KBCh(R)-8-5 cells and the results showed that the compounds 6, 8, 11-14, 16, and 17 at their respective IC(50) concentrations caused a 1.0- to 1.7-fold greater accumulation of VLB than did a similar concentration of the standard modulator, verapamil (VRP). Results of the efflux experiment showed that VRP and each of the modulators significantly inhibited the efflux of VLB, suggesting that they may be competitors for P-gp. All modulators effectively competing with [(3)H]azidopine for binding to P-gp pointed out this transport membrane protein as their likely site of action. Compounds at IC(10) were evaluated for their efficacy to modulate the cytotoxicity of VLB and the results showed that modulators 11, 13, 14, 16, and 17 were able to completely reverse the 25-fold resistance of KBCh(R)-8-5 cells to VLB. Examination of the relationship between lipophilicity and antagonism of MDR showed a reasonable correlation suggesting that hydrophobicity is one of the determinants of potency for anti-MDR activity of 2-chloroacridones. The results allowed us to draw preliminary conclusions about structural features of 2-chloroacridones important for MDR modulation.     

Uptake of L-[propyl-2,3-3H]dihydroalprenolol by intact HeLa cells

This report describes the uptake of L-[propyl-2,3-3H]dihydroalprenolol, a beta-adrenergic antagonist, by HeLa (human adenocarcinoma) cells. [3H]Dihydroalprenolol binds to sites of high capacity and low affinity in intact HeLa cells. The binding achieves equilibrium rapidly and is rapidly reversible. Bound [3H]dihydroalprenolol is displaceable by beta-adrenergic antagonists in a nonstereoselective fashion, but is not displaceable by isoproterenol, an adrenergic agonist. Phentolamine, an alpha-adrenergic antagonist, and chloroquine, a lysosomotropic amine, also compete for [3H]dihydroalprenolol binding sites. [3H]Dihydroalprenolol binding is inhibited by metabolic inhibitors, but not by cytoskeletal blocking agents. The binding is sensitive to extracellular pH (less binding at lower pH) and is temperature-sensitive (less binding at lower temperatures). The bound radioligand is rapidly reversed following hypotonic lysis of the cells. These [3H]dihydroalprenolol binding sites in intact HeLa cells therefore do not have the characteristics expected for beta-adrenergic receptors. Further studies showed that beta-adrenergic receptors could be detected in a HeLa membrane preparation using [125I]iodohydroxybenzylpindolol, and that chloroquine had very low affinity for these receptors. We conclude that [3H]dihydroalprenolol diffuses across the plasma membrane of intact HeLa cells and accumulates in acidic intracellular compartments.     

Isopetasin and S-isopetasin as novel P-glycoprotein inhibitors against multidrug-resistant cancer cells

BackgroundA major problem of cancer treatment is the development of multidrug resistance (MDR) to chemotherapy. MDR is caused by different mechanisms such as the expression of the ABC-transporters P-glycoprotein (P-gp, MDR1, ABCB1) and breast cancer resistance protein (BCRP, ABCG2). These transporters efflux xenobiotic toxins, including chemotherapeutics, and they were found to be overexpressed in different cancer types.PurposeIdentification of novel molecules that overcome MDR by targeting ABC-transporters.MethodsResazurin reduction assay was used for cytotoxicity test. AutoDock 4.2. was used for molecular docking. The function of P-gp and BCRP was tested using a doxorubicin uptake assay and an ATPase assay. ROS generation was detected using flow cytometry for the measurement of H₂DCFH-DA fluorescence. Annexin/PI staining was applied for the detection of apoptosis. Bioinformatic analyses were performed using LigandScout 3.12. software and DataWarrior software.ResultsIn our search for new molecules that selectively act against resistant phenotypes, we identified isopetasin and S-isopetasin, which are bioactive natural products from Petasites formosanus. They exerted collateral sensitivity towards leukemia cells with high P-gp expression in CEM/ADR5000 cells, compared to sensitive wild-type CCRF-CEM leukemia cells. Also, they revealed considerable activity towards breast cancer cells overexpressing breast cancer resistance protein, MDA-MB-231-BCRP clone 23. This motivated us to investigate whether the function of P-gp was inhibited. In-silico results showed the compounds bound with high affinity and interacted with key amino acid residues in P-gp . Then, we found that the two compounds increased doxorubicin accumulation in P-gp overexpressing CEM/ADR5000 by three-fold compared to cells without inhibitor. P-gp-mediated drug efflux was ATP-dependent. Isopetasin and S-isopetasin increased the ATPase activity of human P-gp in a comparable fashion as verapamil used as control P-gp inhibitor. As isopetasin and S-isopetasin exerted dual roles, first as cytotoxic compounds and then as P-gp inhibitors, we suggested that their P-gp inhibition is part of a larger complex of mechanisms to induce cell death in cancer patients. P-gp dysfunction induces mitochondrial stress to generate ATP. Upon continuing stress by P-gp inhibition, the mitochondria generate reactive oxygen species (ROS). Initially established for verapamil, this theory was validated in the present study for isopetasin and S-isopetasin, as treatment with the two candidates increased ROS levels in CEM/ADR5000 cells followed by apoptosis.ConclusionOur study highlights the importance of isopetasin and S-isopetasin as novel ROS-generating and apoptosis-inducing P-gp inhibitors.     

Specific derivatization of the vesicle monoamine transporter with novel carrier-free radioiodinated reserpine and tetrabenazine photoaffinity labels

Two iodophenylazide derivatives of reserpine and one iodophenylazide derivative of tetrabenazine have been synthesized and characterized as photoaffinity labels of the vesicle monoamine transporter (VMAT2). These compounds are 18-O-[3-(3'-iodo-4'-azidophenyl)-propionyl]methyl reserpate (AIPPMER), 18-O-[N-(3'-iodo-4'-azidophenethyl)glycyl]methyl reserpate (IAPEGlyMER), and 2-N-[(3'-iodo-4'-azidophenyl)-propionyl]tetrabenazine (TBZ-AIPP). Inhibition of [3H]dopamine uptake into purified chromaffin granule ghosts showed IC50 values of approximately 37 nM for reserpine, 83 nM for AIPPMER, 200 nM for IAPEGlyMER, and 2.1 microM for TBZ-AIPP. Carrier-free radioiodinated [125I]IAPEGlyMER and [125I]TBZ-AIPP were synthesized and used to photoaffinity label chromaffin granule membranes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed specific [125I]IAPEGlyMER labeling of a polypeptide that migrated as a broad band (approximately 55-90 kDa), with the majority of the label located between 70 and 80 kDa. The labeling by [125I]IAPEGlyMER was blocked by 100 nM reserpine, 10 microM tetrabenazine, 1 mM serotonin, and 10 mM (-)-norepinephrine and dopamine. Analysis of [125I]TBZ-AIPP-labeled chromaffin granule membranes by SDS-PAGE and autoradiography demonstrated specific labeling of a similar polypeptide, which was blocked by 1 microM reserpine and 10 microM tetrabenazine. Incubation of [125I]TBZ-AIPP-photolabeled chromaffin granule membranes in the presence of the glycosidase N-glycanase shifted the apparent molecular weight of VMAT2 to approximately 51 kDa. These data indicate that [125I]IAPEGlyMER and [125I]TBZ-AIPP are effective photoaffinity labels for VMAT2.     

Uptake and Efflux of Quinacrine, a Candidate for the Treatment of Prion Diseases, at the Blood-Brain Barrier

1. A clinical trial of quinacrine in patients with Creutzfeldt-Jakob disease is now in progress. The permeability of drugs through the blood-brain barrier (BBB) is a determinant of their therapeutic efficacy for prion diseases. The mechanism of quinacrine transport across the BBB was investigated using mouse brain endothelial cells (MBEC4). 2. The permeability of quinacrine through MBEC4 cells was lower than that of sodium fluorescein, a BBB-impermeable marker. The basolateral-to-apical transport of quinacrine was greater than its apical-to-basolateral transport. In the presence of P-glycoprotein (P-gp) inhibitor, cyclosporine or verapamil, the apical-to-basolateral transport of quinacrine increased. The uptake of quinacrine by MBEC4 cells was enhanced in the presence of cyclosporine or verapamil. 3. Quinacrine uptake was highly concentrative, this event being carried out by a saturable and carrier-mediated system with an apparent Km of 52.1 microM. Quinacrine uptake was insensitive to Na+-depletion and changes in the membrane potential and sensitive to changes in pH. This uptake was decreased by tetraethylammonium and cimetidine, a substrate and an inhibitor of organic cation transporters, respectively. 4. These findings suggest that quinacrine transport at the BBB is mediated by the efflux system (P-gp) and the influx system (organic cation transporter-like machinery).     

Mitochondrial matrix granules in soft tissues. II. Isolation and initial characterization of a calcium-precipitable, soluble lipoprotein subfraction from brown fat and liver mitochondria

Degradation of ¹²⁵I-labelled HDL ([¹²⁵I]HDL) was measured in isolated rat hepatocytes that had been preincubated with [¹²⁵I]HDL and then reincubated in fresh medium without [¹²⁵I]HDL. About 5 % of the [¹²⁵I]HDL associated with the cells in advance were degraded per hour at 37 °C. This in vitro degradation was inhibited about 50% by lysosomal inhibitors such as chloroquine, ammonia and leupeptin. Depolymerization of microtubuli by colchicine inhibited the degradation of [¹²⁵I]HDL to about 65–75 % of the control cells. Cytochalasin B (CB), a destabilizer of microfilaments, had a less marked effect on the degradation in vitro. Degradation of [¹²⁵I]HDL associated with cells in vivo after intravenous injection was also studied in isolated cells. About 8.5% of the [¹²⁵I]HDL associated with the cells in vivo were degraded per hour in the isolated cells. The effects of ammonia, chloroquine, leupeptin and colchicine on HDL degradation were similar for [¹²⁵I]HDL taken up in vivo and in vitro. Subcellular fractionation by centrifugation in sucrose gradients indicated that [¹²⁵I]HDL associated with hepatocytes in vivo are primarily accumulated in lysosomes. [¹²⁵I]HDL associated with the cells in vitro are located in organelles whose distribution coincides with that of 5′-nucleotidase. These organelles may be endocytic vesicles. It is concluded that the internalization of [¹²⁵I]HDL in rat hepatocytes is relatively slow. The intracellular degradation of the apoproteins of HDL is at least partly lysosomal.     

Interaction of quinoline antimalarial drugs with ferriprotoporphyrin IX, a solid state spectroscopy study

To investigate the nature of binding of quinoline antimalarial drugs to heme and to extract experimental evidence for this binding, the interaction of ferriprotoporphyrin IX (FP) with chloroquine and quinacrine (both of which have a similar side chain) and quinoline methanol antimalarials quinine and mefloquine has been studied using IR and NIR-Raman spectroscopy in the solid state. Attenuated total reflectance infrared spectroscopic data clearly show that heme in chloroquine-FP complex is not μ-oxo dimeric indicating that the hypothesis that chloroquine binds to FP μ-oxo dimer with a stoichiometry of 1 chloroquine:2 μ-oxo dimers is not valid in the solid state. Moreover, the first vibrational spectroscopy evidence is presented for the formation of hydrogen bonding between a propionate group of heme and the tertiary amino nitrogen of chloroquine and quinacrine. Raman spectroscopy data does not provide any evidence to support the formation of a similar salt bridge in the complexes of FP with quinine and mefloquine; however, it suggests that the interaction of these drugs with FP happens through coordination of the Fe(III) center of the porphyrin to the 9-hydroxy group of the drug.