title: [3H]R75231--a new radioligand for the nitrobenzylthioinosine sensitive nucleoside transport proteins. Characterization of (+/-)-[3H]R75231 binding to calf lung membranes, stereospecificity of its two stereoisomers, and comparison with [3H]nitrobenzylthioinosine binding.
abstract: The tritiated analogue of R75231 ((+-)-2-(aminocarbonyl)-N-(4-amino-2,6-dichlorophenyl)-4-[5,5-bis (4-fluorophenyl)pentyl]-1-piperazineacetamide) has been examined as a new radioligand for (nitrobenzylthioinosine sensitive) nucleoside transport proteins. [3H]R75231 was prepared in two steps from R69064 ((+-)-4-[5,5-bis[4-fluorophenyl)-4-pentenyl]-2-piperazinecarboxamide+ ++ dihydrochloride) with a specific activity of 0.23 TBq/mmol (6.3 Ci/mmol). [3H]R75231 bound in a pseudo-irreversible and saturable manner to a membrane preparation of calf lung tissue. The new radioligand displayed high affinity (KD = 0.32 +/- 0.06 nmol/l at 25 degrees C) and capacity (Bmax = 6.1 +/- 0.3 pmol/mg protein). Specific [3H]R75231 binding could be fully displaced by both structural analogues and reference inhibitors such as dipyridamole, NBI, dilazep and hexobendine, as well as by various nucleosides. The two stereoisomers of R75231, R88016 ((+)-R75231) and R88021 ((-)-R-75231), potently displaced specific [3H]R75231 and [3H]NBI binding, R88021 being 30-fold more active than R88016. Pseudo-Hill coefficients derived from the shape of all the [3H]R75231 displacement curves were approximately unity. In contrast, R75231 and most of its analogues displaced specific [3H]NBI binding with pseudo-Hill coefficients consistently larger than unity under identical experimental conditions. This latter finding is suggestive for the existence of two distinct binding sites for the two radioligands, which may or may not overlap to some extent.
authors.affiliation: Division of Medicinal Chemistry, Center for Bio-Pharmaceutical Sciences, Leiden, The Netherlands.
authors.forename: A P
authors.initials: AP
authors.lastname: IJzerman
authors.forename: M
authors.initials: M
authors.lastname: Kruidering
authors.forename: A
authors.initials: A
authors.lastname: van Weert
authors.forename: H
authors.initials: H
authors.lastname: van Belle
authors.forename: C
authors.initials: C
authors.lastname: Janssen
mesh_terms: D000345:Affinity Labels; D000818:Animals; D002417:Cattle; D066298:In Vitro Techniques; D008168:Lung / Q000378:metabolism*; D008566:Membranes / Q000378:metabolism; D010879:Piperazines / Q000378:metabolism*; D011869:Radioligand Assay; D013237:Stereoisomerism; D013868:Thioinosine / Q000031:analogs & derivatives* / Q000378:metabolism; D014316:Tritium
chemical_list: D000345:Affinity Labels; D010879:Piperazines; C072040:draflazine; D014316:Tritium; D013868:Thioinosine; C001789:4-nitrobenzylthioinosine