title: Chiral recognition of amethopterin enantiomers by the reduced folate carrier in Caco-2 cells.
abstract: Stereoselectivity of the human reduced folate carrier (RFC1) in Caco-2 cells was examined using methotrexate (L-amethopterin, L-MTX) and its antipode (D-amethopterin, D-MTX) as model substrates. The initial uptake rate of L-MTX into Caco-2 cells followed Michaelis-Menten kinetics with a Km value of approximately 1 microM. The Eadie-Hofstee plot of the RFC1-mediated L-MTX uptake showed that it was mediated by a single transport system, RFC1. Dixon plots revealed that L-MTX uptake was inhibited competitively by folic acid (FA), L-MTX and D-MTX, with Ki values of approximately 0.8, 1.5 and 180 microM, respectively. The results showed that the affinities of FA and L-MTX to RFC1 were approximately 120-fold greater than that of D-MTX. The uptake of L- and D-MTX into Caco-2 cells was also measured using LC-MS/MS analysis, which revealed that the L-MTX uptake was at least 7-fold greater than that of D-MTX. The present study revealed significant stereoselectivity of RFC1 toward amethopterin enantiomers with the L-isomer being much more favored.
authors.affiliation: School of Pharmacy, Kitasato University, Tokyo, Japan.
authors.forename: Tomoya
authors.initials: T
authors.lastname: Narawa
authors.forename: Yasuyuki
authors.initials: Y
authors.lastname: Tsuda
authors.forename: Tomoo
authors.initials: T
authors.lastname: Itoh
mesh_terms: D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D000465:Algorithms; D018938:Caco-2 Cells; D002853:Chromatography, Liquid; D005492:Folic Acid / Q000378:metabolism / Q000494:pharmacology; D005493:Folic Acid Antagonists / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D013058:Mass Spectrometry; D008727:Methotrexate / Q000737:chemistry* / Q000378:metabolism*; D011339:Probenecid / Q000494:pharmacology; D011506:Proteins / Q000378:metabolism; D058978:Reduced Folate Carrier Protein / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013237:Stereoisomerism; D014528:Uricosuric Agents / Q000494:pharmacology
chemical_list: D005493:Folic Acid Antagonists; D011506:Proteins; D058978:Reduced Folate Carrier Protein; C548699:SLC19A1 protein, human; D014528:Uricosuric Agents; D005492:Folic Acid; D011339:Probenecid; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D008727:Methotrexate