title: Design, synthesis, and evaluation of 2-diethanolamino-4,8-diheptamethyleneimino-2-(N-aminoethyl-N-ethanolamino)-6-(N,N-diethanolamino)pyrimido[5,4-d]pyrimidine-fluorescein conjugate (8MDP-fluor), as a novel equilibrative nucleoside transporter probe.
abstract: Nucleoside transporters are integral membrane glycoproteins that play critical roles in physiological nucleoside and nucleobase fluxes, and influence the efficacy of many nucleoside chemotherapy drugs. Fluorescent reporter ligands/substrates have been shown to be useful in the analysis of nucleoside transporter (NT) protein expression and discovery of new NT inhibitors. In this study, we have developed a novel dipyridamole (DP)-based equilibrative nucleoside transporter 1 (ENT1) fluorescent probe. The potent ENT1 and ENT2 inhibitor analogue of dipyridamole, 2,6-bis(diethanolamino)-4,8-diheptamethyleneiminopyrimido[5,4-d]pyrimidine (4, 8MDP), was modified to replace one β-hydroxyethyl group of the amino substituent at the 2-position with a β-aminoethyl group and then conjugated through the amino group to 6-(fluorescein-5-carboxamido)hexanoyl moiety to obtain a new fluorescent molecule, 2-diethanolamino-4,8-diheptamethyleneimino-2-(N-aminoethyl-N-ethanolamino)-6-(N,N-diethanolamino)pyrimido[5,4-d]pyrimidine-fluorescein conjugate, designated 8MDP-fluorescein (8MDP-fluor, 6). The binding affinities of 8MDP-fluor at ENT1 and ENT2 are reflected by the uridine uptake inhibitory K(i) values of 52.1 nM and 285 nM, respectively. 8MDP-fluor was successfully demonstrated to be a flow cytometric probe for ENT1 comparable to the nitrobenzylmercaptopurine riboside (NBMPR) analogue ENT1 fluorescent probe SAENTA-X8-fluorescein (SAENTA-fluor, 1). This is the first reported dipyridamole-based ENT1 fluorescent probe, which adds a novel tool for probing ENT1, and possibly ENT2.
authors.affiliation: Department of Pharmaceutical Sciences, College of Pharmacy, University of Tennessee Health Sciences Center, Memphis, Tennessee 38163, United States.
authors.forename: Wenwei
authors.initials: W
authors.lastname: Lin
authors.forename: John K
authors.initials: JK
authors.lastname: Buolamwini
mesh_terms: D015195:Drug Design; D033721:Equilibrative Nucleoside Transporter 1 / Q000037:antagonists & inhibitors* / Q000096:biosynthesis; D019793:Fluorescein / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D005452:Fluoresceins / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D006801:Humans; D020014:K562 Cells; D008024:Ligands; D015394:Molecular Structure; D011743:Pyrimidines / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D013329:Structure-Activity Relationship
chemical_list: C562067:2-diethanolamino-4,8-diheptamethyleneimino-6-((N-6-(fluorescein-6-carboxamido)hexanoylaminoethyl)ethanolamino)pyrimido(5,4-d)pyrimidine; D033721:Equilibrative Nucleoside Transporter 1; D005452:Fluoresceins; D005456:Fluorescent Dyes; D008024:Ligands; D011743:Pyrimidines; D019793:Fluorescein