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John O Miners - One of the best experts on this subject based on the ideXlab platform.
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effects of amino acid substitutions at positions 33 and 37 on udp glucuronosyltransferase 1a9 ugt1a9 activity and substrate selectivity
Biochemical Pharmacology, 2012Co-Authors: Porntipa Korprasertthaworn, Krongtong Yoovathaworn, Benjamin C Lewis, Peter I. Mackenzie, Andrew Rowland, John O MinersAbstract:Abstract UGT1A9 contributes to the glucuronidation of numerous drugs and xenobiotics. There is evidence to suggest that the Met33Thr substitution, as occurs in the polymorphic variant UGT1A9*3, variably affects xenobiotic glucuronidation. The equivalent position in UGT1A4 is also known to influence enzyme activity, whilst an N-terminal domain histidine (His37 in UGT1A9) is believed to function as the catalytic base in most UGT enzymes. To elucidate the roles of key amino acids and characterise structure–function relationships, we determined the effects of amino acid substitutions at positions 33 and 37 of UGT1A9 on the kinetics of 4-methylumbelliferone (4-MU), mycophenolic acid (MPA), propofol (PRO), Sulfinpyrazone (SFZ), frusemide (FSM), ( S )-naproxen (NAP) and retigabine (RTB) glucuronidation, compounds that undergo glucuronidation at either a phenolic (4-MU, MPA, PRO), carboxylate (FSM, NAP), acidic carbon (SFZ) or amine (RTB) function. Substitution of Met33 with Val, Ile, Thr, and Gln, as occur in UGT1A1, UGT1A3, UGT1A4 and UGT1A6 respectively, variably affected kinetics and catalytic efficiency. Whilst K m values were generally higher and V max and CL int values were generally lower than for wild-type UGT1A9 with most substrate-mutant pairs, the pattern and the magnitude of the changes in each parameter differed substantially. Moreover, exceptions occurred; CL int values for MPA and FSM glucuronidation by the position-33 mutants were the same as or higher than that of UGT1A9. Mutation of His37 abolished activity towards all substrates, except RTB N-glucuronidation. The data confirm the importance of single amino acids for UGT enzyme activity and substrate selectivity, and support a pivotal role for residue-33 in facilitating substrate binding to UGT1A9.
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Characterization of the Binding of Drugs to Human Intestinal Fatty Acid Binding Protein (IFABP): Potential Role of IFABP as an Alternative to Albumin for in Vitro-in Vivo Extrapolation of Drug Kinetic Parameters□S
2009Co-Authors: Andrew Rowl, Peter I. Mackenzie, Kathleen M. Knights, John O MinersAbstract:This work characterized for the first time the binding of acidic, neutral, and basic drugs to human intestinal fatty acid binding protein (IFABP) and, for comparison, to bovine serum albumin (BSA). In addition, the study investigated whether IFABP can sub-stitute for BSA as a constituent in incubations of human liver microsomes (HLMs) in in vitro-in vivo extrapolation (IV-IVE) stud-ies. Each molecule of purified IFABP bound a single molecule of the fluorescent probe 1-anilino-8-naphthalene sulfonate or arachi-donic acid with Kd values similar to those reported for rat IFABP. Basic drugs bound negligibly to IFABP. Based on fraction unbound (fu) at a protein concentration of 0.5 % (w/v), binding of acidic and neutral drugs ranged from minor (fu> 0.8) to moderate (fu 0.5–0.8). Of the compounds screened, highest binding to IFABP was ob-served for Sulfinpyrazone (an acid) and -estradiol (a neutral com-pound). However, binding to IFABP was lower than to BSA for al
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selectivity of substrate trifluoperazine and inhibitor amitriptyline androsterone canrenoic acid hecogenin phenylbutazone quinidine quinine and Sulfinpyrazone probes for human udp glucuronosyltransferases
Drug Metabolism and Disposition, 2006Co-Authors: Verawan Uchaipichat, Peter I. Mackenzie, David J Elliot, John O MinersAbstract:Relatively few selective substrate and inhibitor probes have been identified for human UDP-glucuronosyltransferases (UGTs). This work investigated the selectivity of trifluoperazine (TFP), as a substrate, and amitriptyline, androsterone, canrenoic acid, hecogenin, phenylbutazone, quinidine, quinine, and Sulfinpyrazone, as inhibitors, for human UGTs. Selectivity was assessed using UGTs 1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B7, and 2B15 expressed in HEK293 cells. TFP was confirmed as a highly selective substrate for UGT1A4. However, TFP bound extensively to both HEK293 lysate and human liver microsomes in a concentration-dependent manner (fuinc 0.20-0.59). When corrected for nonspecific binding, Km values for TFP glucuronidation were similar for both UGT1A4 (4.1 microM) and human liver microsomes (6.1+/-1.2 microM) as the enzyme sources. Of the compounds screened as inhibitors, hecogenin, alone, was selective; significant inhibition was observed only for UGT1A4 (IC50 1.5 microM). Using phenylbutazone and quinine as "models," inhibition kinetics were variously described by competitive and noncompetitive mechanisms. Inhibition of UGT2B7 by quinidine was also investigated further, because the effects of this compound on morphine pharmacokinetics (a known UGT2B7 substrate) have been ascribed to inhibition of P-glycoprotein. Quinidine inhibited human liver microsomal and recombinant UGT2B7, with respective Ki values of 335+/-128 microM and 186 microM. In conclusion, TFP and hecogenin represent selective substrate and inhibitor probes for UGT1A4, although the extensive nonselective binding of the former should be taken into account in kinetic studies. Amitriptyline, androsterone, canrenoic acid, hecogenin, phenylbutazone, quinidine, quinine, and Sulfinpyrazone are nonselective UGT inhibitors.
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Selectivity of substrate (trifluoperazine) and inhibitor (amitriptyline, androsterone, canrenoic acid, hecogenin, phenylbutazone, quinidine, quinine, and Sulfinpyrazone) "probes" for human udpglucuronosyltransferases. Drug Metab Dispos
2006Co-Authors: Verawan Uchaipichat, Peter I. Mackenzie, David J Elliot, John O MinersAbstract:Relatively few selective substrate and inhibitor probes have been identified for human UDP-glucuronosyltransferases (UGTs). This work investigated the selectivity of trifluoperazine (TFP), as a sub-strate, and amitriptyline, androsterone, canrenoic acid, hecogenin, phenylbutazone, quinidine, quinine, and Sulfinpyrazone, as inhibi-tors, for human UGTs. Selectivity was assessed using UGTs 1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B7, and 2B15 expressed in HEK293 cells. TFP was confirmed as a highly selective substrate for UGT1A4. However, TFP bound extensively to both HEK293 lysate and human liver microsomes in a concentration-dependent manner (fuinc 0.20–0.59). When corrected for nonspecific binding, Km values for TFP glucuronidation were similar for both UGT1A4 (4.1 M) and human liver microsomes (6.1 1.2 M) as the enzyme sources. Of the compounds screened as inhibitors, hecogenin, alone, was selective; significant inhibition was observed only fo
Frans G M Russel - One of the best experts on this subject based on the ideXlab platform.
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effect of hypouricaemic and hyperuricaemic drugs on the renal urate efflux transporter multidrug resistance protein 4
British Journal of Pharmacology, 2009Co-Authors: Azza A K Elsheikh, J J M W Van Den Heuvel, Jan B Koenderink, Frans G M RusselAbstract:BACKGROUND AND PURPOSE: The xanthine oxidase inhibitors allopurinol and oxypurinol are used to treat hyperuricaemia, whereas loop and thiazide diuretics can cause iatrogenic hyperuricaemia. Some uricosuric drugs and salicylate have a bimodal action on urate renal excretion. The mechanisms of action of these hypo- and hyperuricaemic drugs on the handling of urate in renal tubules have not been fully elucidated. Recently, we identified the multidrug resistance protein (MRP) 4 as a luminal efflux transporter for urate in the proximal tubule. EXPERIMENTAL APPROACH: Here, we studied the effect of these drugs on [(14)C]urate transport using human embryonic kidney 293 cells overexpressing human MRP4 and in membrane vesicles isolated from these cells. KEY RESULTS: Allopurinol stimulated MRP4-mediated cellular urate efflux and allopurinol and oxypurinol both markedly stimulated urate transport by MRP4 in membrane vesicles. Bumetanide and torasemide had no effect, whereas furosemide, chlorothiazide, hydrochlorothiazide, salicylate, benzbromarone and Sulfinpyrazone inhibited urate transport, at concentrations ranging from nanomolar up to millimolar. Probenecid stimulated urate transport at 0.1 microM and inhibited transport at higher concentrations. CONCLUSIONS AND IMPLICATIONS: These data suggest that inhibition of MRP4-mediated urate efflux by furosemide and thiazide diuretics could have an important function in their hyperuricaemic mechanisms. Furthermore, stimulation of MRP4-mediated renal urate efflux could be a new mechanism in the hypouricaemic action of allopurinol and oxypurinol. In conclusion, MRP4 may provide a potential target for drugs affecting urate homoeostasis, which needs to be further evaluated in vivo.
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contribution of multidrug resistance protein 2 mrp2 abcc2 to the renal excretion of p aminohippurate pah and identification of mrp4 abcc4 as a novel pah transporter
Journal of The American Society of Nephrology, 2004Co-Authors: Pascal H E Smeets, Remon A M H Van Aubel, Alfons C Wouterse, Jeroen J M W Van Den Heuvel, Frans G M RusselAbstract:p-Aminohippurate (PAH) is the classical substrate used in the characterization of organic anion transport in renal proximal tubular cells. Although basolateral transporters for PAH uptake from blood into the cell have been well characterized, there is still little knowledge on the apical urinary efflux transporters. The multidrug resistance protein 2 (MRP2/ABCC2) is localized to the apical membrane and mediates ATP-dependent PAH transport, but its contribution to urinary PAH excretion is not known. In this report, we show that renal excretion of PAH in isolated perfused kidneys from wild-type and Mrp2-deficient (TR(-)) rats is not significantly different. Uptake of [(14)C]PAH in membrane vesicles expressing two different MRP2 clones isolated from Sf9 and MDCKII cells exhibited a low affinity for PAH (Sf9, 5 +/- 2 mM; MDCKII, 2.1 +/- 0.6 mM). Human MRP4 (ABCC4), which has recently been localized to the apical membrane, expressed in Sf9 cells had a much higher affinity for PAH (K(m) = 160 +/- 50 microM). Various inhibitors of MRP2-mediated PAH transport also inhibited MRP4. Probenecid stimulated MRP2 at low concentrations but had no effect on MRP4; but at high probenecid concentrations, both MRP2 and MRP4 were inhibited. Sulfinpyrazone only stimulated MRP2, but inhibited MRP4. Real-time PCR and Western blot analysis showed that renal cortical expression of MRP4 is approximately fivefold higher as compared with MRP2. MRP4 is a novel PAH transporter that has higher affinity for PAH and is expressed more highly in kidney than MRP2, and may therefore be more important in renal PAH excretion.
Peter I. Mackenzie - One of the best experts on this subject based on the ideXlab platform.
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effects of amino acid substitutions at positions 33 and 37 on udp glucuronosyltransferase 1a9 ugt1a9 activity and substrate selectivity
Biochemical Pharmacology, 2012Co-Authors: Porntipa Korprasertthaworn, Krongtong Yoovathaworn, Benjamin C Lewis, Peter I. Mackenzie, Andrew Rowland, John O MinersAbstract:Abstract UGT1A9 contributes to the glucuronidation of numerous drugs and xenobiotics. There is evidence to suggest that the Met33Thr substitution, as occurs in the polymorphic variant UGT1A9*3, variably affects xenobiotic glucuronidation. The equivalent position in UGT1A4 is also known to influence enzyme activity, whilst an N-terminal domain histidine (His37 in UGT1A9) is believed to function as the catalytic base in most UGT enzymes. To elucidate the roles of key amino acids and characterise structure–function relationships, we determined the effects of amino acid substitutions at positions 33 and 37 of UGT1A9 on the kinetics of 4-methylumbelliferone (4-MU), mycophenolic acid (MPA), propofol (PRO), Sulfinpyrazone (SFZ), frusemide (FSM), ( S )-naproxen (NAP) and retigabine (RTB) glucuronidation, compounds that undergo glucuronidation at either a phenolic (4-MU, MPA, PRO), carboxylate (FSM, NAP), acidic carbon (SFZ) or amine (RTB) function. Substitution of Met33 with Val, Ile, Thr, and Gln, as occur in UGT1A1, UGT1A3, UGT1A4 and UGT1A6 respectively, variably affected kinetics and catalytic efficiency. Whilst K m values were generally higher and V max and CL int values were generally lower than for wild-type UGT1A9 with most substrate-mutant pairs, the pattern and the magnitude of the changes in each parameter differed substantially. Moreover, exceptions occurred; CL int values for MPA and FSM glucuronidation by the position-33 mutants were the same as or higher than that of UGT1A9. Mutation of His37 abolished activity towards all substrates, except RTB N-glucuronidation. The data confirm the importance of single amino acids for UGT enzyme activity and substrate selectivity, and support a pivotal role for residue-33 in facilitating substrate binding to UGT1A9.
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Characterization of the Binding of Drugs to Human Intestinal Fatty Acid Binding Protein (IFABP): Potential Role of IFABP as an Alternative to Albumin for in Vitro-in Vivo Extrapolation of Drug Kinetic Parameters□S
2009Co-Authors: Andrew Rowl, Peter I. Mackenzie, Kathleen M. Knights, John O MinersAbstract:This work characterized for the first time the binding of acidic, neutral, and basic drugs to human intestinal fatty acid binding protein (IFABP) and, for comparison, to bovine serum albumin (BSA). In addition, the study investigated whether IFABP can sub-stitute for BSA as a constituent in incubations of human liver microsomes (HLMs) in in vitro-in vivo extrapolation (IV-IVE) stud-ies. Each molecule of purified IFABP bound a single molecule of the fluorescent probe 1-anilino-8-naphthalene sulfonate or arachi-donic acid with Kd values similar to those reported for rat IFABP. Basic drugs bound negligibly to IFABP. Based on fraction unbound (fu) at a protein concentration of 0.5 % (w/v), binding of acidic and neutral drugs ranged from minor (fu> 0.8) to moderate (fu 0.5–0.8). Of the compounds screened, highest binding to IFABP was ob-served for Sulfinpyrazone (an acid) and -estradiol (a neutral com-pound). However, binding to IFABP was lower than to BSA for al
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selectivity of substrate trifluoperazine and inhibitor amitriptyline androsterone canrenoic acid hecogenin phenylbutazone quinidine quinine and Sulfinpyrazone probes for human udp glucuronosyltransferases
Drug Metabolism and Disposition, 2006Co-Authors: Verawan Uchaipichat, Peter I. Mackenzie, David J Elliot, John O MinersAbstract:Relatively few selective substrate and inhibitor probes have been identified for human UDP-glucuronosyltransferases (UGTs). This work investigated the selectivity of trifluoperazine (TFP), as a substrate, and amitriptyline, androsterone, canrenoic acid, hecogenin, phenylbutazone, quinidine, quinine, and Sulfinpyrazone, as inhibitors, for human UGTs. Selectivity was assessed using UGTs 1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B7, and 2B15 expressed in HEK293 cells. TFP was confirmed as a highly selective substrate for UGT1A4. However, TFP bound extensively to both HEK293 lysate and human liver microsomes in a concentration-dependent manner (fuinc 0.20-0.59). When corrected for nonspecific binding, Km values for TFP glucuronidation were similar for both UGT1A4 (4.1 microM) and human liver microsomes (6.1+/-1.2 microM) as the enzyme sources. Of the compounds screened as inhibitors, hecogenin, alone, was selective; significant inhibition was observed only for UGT1A4 (IC50 1.5 microM). Using phenylbutazone and quinine as "models," inhibition kinetics were variously described by competitive and noncompetitive mechanisms. Inhibition of UGT2B7 by quinidine was also investigated further, because the effects of this compound on morphine pharmacokinetics (a known UGT2B7 substrate) have been ascribed to inhibition of P-glycoprotein. Quinidine inhibited human liver microsomal and recombinant UGT2B7, with respective Ki values of 335+/-128 microM and 186 microM. In conclusion, TFP and hecogenin represent selective substrate and inhibitor probes for UGT1A4, although the extensive nonselective binding of the former should be taken into account in kinetic studies. Amitriptyline, androsterone, canrenoic acid, hecogenin, phenylbutazone, quinidine, quinine, and Sulfinpyrazone are nonselective UGT inhibitors.
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Selectivity of substrate (trifluoperazine) and inhibitor (amitriptyline, androsterone, canrenoic acid, hecogenin, phenylbutazone, quinidine, quinine, and Sulfinpyrazone) "probes" for human udpglucuronosyltransferases. Drug Metab Dispos
2006Co-Authors: Verawan Uchaipichat, Peter I. Mackenzie, David J Elliot, John O MinersAbstract:Relatively few selective substrate and inhibitor probes have been identified for human UDP-glucuronosyltransferases (UGTs). This work investigated the selectivity of trifluoperazine (TFP), as a sub-strate, and amitriptyline, androsterone, canrenoic acid, hecogenin, phenylbutazone, quinidine, quinine, and Sulfinpyrazone, as inhibi-tors, for human UGTs. Selectivity was assessed using UGTs 1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B7, and 2B15 expressed in HEK293 cells. TFP was confirmed as a highly selective substrate for UGT1A4. However, TFP bound extensively to both HEK293 lysate and human liver microsomes in a concentration-dependent manner (fuinc 0.20–0.59). When corrected for nonspecific binding, Km values for TFP glucuronidation were similar for both UGT1A4 (4.1 M) and human liver microsomes (6.1 1.2 M) as the enzyme sources. Of the compounds screened as inhibitors, hecogenin, alone, was selective; significant inhibition was observed only fo
Piet Borst - One of the best experts on this subject based on the ideXlab platform.
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vinblastine and Sulfinpyrazone export by the multidrug resistance protein mrp2 is associated with glutathione export
British Journal of Cancer, 2000Co-Authors: Raymond Evers, M De Haas, Rolf W Sparidans, Jos H Beijnen, Pieter Wielinga, J Lankelma, Piet BorstAbstract:The multidrug resistance proteins MRP1 and MRP2 are members of the same subfamily of ATP-binding cassette transporters. Besides organic molecules conjugated to negatively charged ligands, these proteins also transport cytotoxic drugs for which no negatively charged conjugates are known to exist. In polarized MDCKII cells, MRP1 routes to the lateral plasma membrane, and MRP2 to the apical plasma membrane. In these cells MRP1 transports daunorubicin, and MRP2 vinblastine; both transporters export reduced glutathione (GSH) into the medium. We demonstrate that glutathione transport in MDCKII-MRP1 cells is inhibited by the inhibitors of organic anion transporters Sulfinpyrazone, indomethacin, probenecid and benzbromarone. In MDCKII-MRP2 cells, GSH export is stimulated by low concentrations of Sulfinpyrazone or indomethacin, whereas export is inhibited down to control levels at high concentrations. We find that unmodified Sulfinpyrazone is a substrate for MRP2, also at concentrations where GSH export is inhibited. We also show that GSH export in MDCKII-MRP2 cells increases in the presence of vinblastine, and that the stochiometry between drug and GSH exported is between two and three. Our data indicate that transport of Sulfinpyrazone and vinblastine is associated with GSH export. However, at high Sulfinpyrazone concentrations this compound is transported without GSH. Models of MRP action are discussed that could explain these results. © 2000 Cancer Research Campaign
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transport of the glutathione conjugate of ethacrynic acid by the human multidrug resistance protein mrp
FEBS Letters, 1996Co-Authors: Guido J R Zaman, Nicole H P Cnubben, Peter J Van Bladeren, Raymond Evers, Piet BorstAbstract:The multidrug resistance protein MRP has been shown to mediate the transport of glutathione S-conjugates across membranes. In this study we demonstrate that the glutathione S-conjugate of the diuretic drug ethacrynic acid, which is an efficient inhibitor of glutathione S-transferases, is a high-affinity substrate and inhibitor of the glutathione S-conjugate pump associated with MRP. This implies that ethacrynic acid may modulate drug resistance of tumor cells not only by inhibiting glutathione S-transferase activity, but also by inhibiting the export of drug conjugates from the cell by MRP. Chemicals/CAS: Adenosine Monophosphate, 61-19-8; ATP-Binding Cassette Transporters; Ethacrynic Acid, 58-54-8; Glutathione, 70-18-8; Multidrug Resistance-Associated Proteins; Recombinant Proteins; Sulfinpyrazone, 57-96-5
Verawan Uchaipichat - One of the best experts on this subject based on the ideXlab platform.
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selectivity of substrate trifluoperazine and inhibitor amitriptyline androsterone canrenoic acid hecogenin phenylbutazone quinidine quinine and Sulfinpyrazone probes for human udp glucuronosyltransferases
Drug Metabolism and Disposition, 2006Co-Authors: Verawan Uchaipichat, Peter I. Mackenzie, David J Elliot, John O MinersAbstract:Relatively few selective substrate and inhibitor probes have been identified for human UDP-glucuronosyltransferases (UGTs). This work investigated the selectivity of trifluoperazine (TFP), as a substrate, and amitriptyline, androsterone, canrenoic acid, hecogenin, phenylbutazone, quinidine, quinine, and Sulfinpyrazone, as inhibitors, for human UGTs. Selectivity was assessed using UGTs 1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B7, and 2B15 expressed in HEK293 cells. TFP was confirmed as a highly selective substrate for UGT1A4. However, TFP bound extensively to both HEK293 lysate and human liver microsomes in a concentration-dependent manner (fuinc 0.20-0.59). When corrected for nonspecific binding, Km values for TFP glucuronidation were similar for both UGT1A4 (4.1 microM) and human liver microsomes (6.1+/-1.2 microM) as the enzyme sources. Of the compounds screened as inhibitors, hecogenin, alone, was selective; significant inhibition was observed only for UGT1A4 (IC50 1.5 microM). Using phenylbutazone and quinine as "models," inhibition kinetics were variously described by competitive and noncompetitive mechanisms. Inhibition of UGT2B7 by quinidine was also investigated further, because the effects of this compound on morphine pharmacokinetics (a known UGT2B7 substrate) have been ascribed to inhibition of P-glycoprotein. Quinidine inhibited human liver microsomal and recombinant UGT2B7, with respective Ki values of 335+/-128 microM and 186 microM. In conclusion, TFP and hecogenin represent selective substrate and inhibitor probes for UGT1A4, although the extensive nonselective binding of the former should be taken into account in kinetic studies. Amitriptyline, androsterone, canrenoic acid, hecogenin, phenylbutazone, quinidine, quinine, and Sulfinpyrazone are nonselective UGT inhibitors.
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Selectivity of substrate (trifluoperazine) and inhibitor (amitriptyline, androsterone, canrenoic acid, hecogenin, phenylbutazone, quinidine, quinine, and Sulfinpyrazone) "probes" for human udpglucuronosyltransferases. Drug Metab Dispos
2006Co-Authors: Verawan Uchaipichat, Peter I. Mackenzie, David J Elliot, John O MinersAbstract:Relatively few selective substrate and inhibitor probes have been identified for human UDP-glucuronosyltransferases (UGTs). This work investigated the selectivity of trifluoperazine (TFP), as a sub-strate, and amitriptyline, androsterone, canrenoic acid, hecogenin, phenylbutazone, quinidine, quinine, and Sulfinpyrazone, as inhibi-tors, for human UGTs. Selectivity was assessed using UGTs 1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B7, and 2B15 expressed in HEK293 cells. TFP was confirmed as a highly selective substrate for UGT1A4. However, TFP bound extensively to both HEK293 lysate and human liver microsomes in a concentration-dependent manner (fuinc 0.20–0.59). When corrected for nonspecific binding, Km values for TFP glucuronidation were similar for both UGT1A4 (4.1 M) and human liver microsomes (6.1 1.2 M) as the enzyme sources. Of the compounds screened as inhibitors, hecogenin, alone, was selective; significant inhibition was observed only fo
