Two transport binding sites of P-glycoprotein are unequal yet contingent: initial rate kinetic analysis by ATP hydrolysis demonstrates intersite dependence

Wang, Er-jia; Casciano, Christopher N.; Clement, Robert P.; Johnson, William W.

doi:10.1016/s0167-4838(00)00125-4

Cited by 62 publications

(57 citation statements)

References 47 publications

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“…1B, itraconazole had no significant effect on the transport of Rho (another fluorescent marker). This result is likely due to the distinct binding sites on P-gp that have often been described (35,36). Because these cell lines overexpress the respective transporter enzymes, the IC 50 would be expected to be higher in these evaluations than under in vivo conditions, where far fewer copies of the enzyme would be contained per cell.…”

Section: Resultsmentioning

confidence: 99%

“…As ATP is consumed at a purported rate of about one or two molecules per transport event, the hydrolysis of ATP represents the transport activity or the transport function (1,6,28,30,35). Itraconazole caused a concentrationdependent increase in the rate of ATP hydrolysis relative to the baseline rate, indicating that as a substrate for P-gp it causes comparatively rapid hydrolysis (Fig.…”

Section: Resultsmentioning

confidence: 99%

Section: Chemicalsmentioning

confidence: 99%

“…The consumption of ATP was quantified by determining the amount of inorganic orthophosphate, which forms a color complex with molybdate, that was liberated (4,35). We have modified an ATP hydrolysis assay, based on determination of the amount of phosphate released, using membrane microsome preparations; the assay was carried out in a 96-well microplate (35). The microsomes were thawed on ice prior to dilution to 3.5 g of protein per well in ice-cold ATPase buffer (3 mM sodium ATP, 50 mM KCl, 10 mM MgSO 4 , 3 mM dithiothreitol, 50 mM Tris-HCl [pH 7.0]) containing 0.5 mM EGTA (to inhibit Ca-ATPase), 0.5 mM ouabain (to inhibit the Na-and K-ATPases), and 3 mM sodium azide (to inhibit the mitochondrial ATPase).…”

Section: Chemicalsmentioning

confidence: 99%

“…The substrates enter from the lipid bilayer (27) and can bind to two (or more) nonidentical sites (35). Moreover, allosteric and perhaps synergistic effects have been observed for certain substrate combinations and conditions (36).…”

mentioning

confidence: 99%

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Interaction of Common Azole Antifungals with P Glycoprotein

Wang¹,

Lew²,

Casciano³

et al. 2002

Antimicrob Agents Chemother

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213

151

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Both eucaryotic and procaryotic cells are resistant to a large number of antibiotics because of the activities of export transporters. The most studied transporter in the mammalian ATP-binding cassette transporter superfamily, P glycoprotein (P-gp), ejects many structurally unrelated amphiphilic and lipophilic xenobiotics. Observed clinical interactions and some in vitro studies suggest that azole antifungals may interact with P-gp. Such an interaction could both affect the disposition and exposure to azole antifungal therapeutics and partially explain the clinical drug interactions observed with some antifungals. Using a whole-cell assay in which the retention of a marker substrate is evaluated and quantified, we studied the abilities of the most widely prescribed orally administered azole antifungals to inhibit the function of this transporter. In a cell line presenting an overexpressed amount of the human P-gp transporter, itraconazole and ketoconazole inhibited P-gp function with 50% inhibitory concentrations (IC 50 s) of ϳ2 and ϳ6 M, respectively. Cyclosporin A was inhibitory with an IC 50 of 1.4 M in this system. Uniquely, fluconazole had no effect in this assay, a result consistent with known clinical interactions. The effects of these azole antifungals on ATP consumption by P-gp (representing transport activity) were also assessed, and the K m values were congruent with the IC 50 s. Therefore, exposure of tissue to the azole antifungals may be modulated by human P-gp, and the clinical interactions of azole antifungals with other drugs may be due, in part, to inhibition of P-gp transport.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Chemicalsmentioning

confidence: 99%

Section: Chemicalsmentioning

confidence: 99%

mentioning

confidence: 99%

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Interaction of Common Azole Antifungals with P Glycoprotein

Wang¹,

Lew²,

Casciano³

et al. 2002

Antimicrob Agents Chemother

Self Cite

213

151

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Membrane Transport Proteins and Drug Transport

Swaan

2003

Burger's Medicinal Chemistry and Drug Discovery

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To exert their intended therapeutic goal, drugs need to cross epithelial barriers to reach their site of action. Transport proteins have critical physiological roles in nutrient absorption and transport of endogenous compounds. These systems can serve as useful pharmacological targets and may be used as a mechanism to increase drug absorption. However, we have little understanding of these proteins at the molecular level because of the absence of high resolution crystal structures. Numerous efforts have been made to characterize the P‐glycoprotein efflux pump, the peptide transporter PepT1, and the apical sodium‐dependent transporter, which are important not only for their native transporter function but also as drug targets to increase absorption and bioactivity. In vitro and computational approaches have been applied to gain some insight into these transporters with some success. This represents an opportunity for optimizing molecules as substrates for the solute transporters and providing a further screening system for drug discovery. Clearly the future growth in knowledge of transporter function will be led by integrated in vitro and in silico approaches.

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Drug Transport and Membrane Transport Proteins

Swaan

2010

Burger's Medicinal Chemistry and Drug Discovery

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In order to exert their intended therapeutic goal, drugs need to cross epithelial barriers to reach their site of action. Transport proteins have critical physiological roles in nutrient absorption and transport of endogenous compounds. These systems can serve as useful pharmacological targets and may be utilized as a mechanism to increase drug absorption. However, there is little understanding of these proteins at the molecular level due to their resistance to crystallization. Numerous efforts have been made to characterize the P‐glycoprotein efflux pump, the peptide transporter (PepT1), and the apical sodium dependent transporter (ASBT), which are important not only for their native transporter function but also as drug targets to increase absorption and bioactivity. In vitro and computational approaches have been applied to gain some insight into these transporters and their substrate specificities. This represents an opportunity for optimizing molecules as substrates for solute transporters and providing a further screening system for drug discovery. This chapter will demonstrates that future growth in knowledge of transporter function will be lead by integrated in vitro and in silico approaches.

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Two transport binding sites of P-glycoprotein are unequal yet contingent: initial rate kinetic analysis by ATP hydrolysis demonstrates intersite dependence

Cited by 62 publications

References 47 publications

Interaction of Common Azole Antifungals with P Glycoprotein

Interaction of Common Azole Antifungals with P Glycoprotein

Membrane Transport Proteins and Drug Transport

Drug Transport and Membrane Transport Proteins

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