USE OF IDENTICAL ASSAY CONDITIONS FOR COCAINE ANALOG BINDING AND DOPAMINE UPTAKE TO IDENTIFY POTENTIAL COCAINE ANTAGONISTS

The addictive and euphorogenic properties of cocaine are thought to re sult from inhibition of the dopamine transporter (DAT). Recent evidenc e suggests that dopamine and cocaine bind to distinct sites on the tra nsporter protein. Therefore it should be possible to design drugs whic h specifically inhibit cocaine recognition by the DAT while permitting the transporter to maintain its function of accumulating dopamine. On e way to monitor such activity is to compare the inhibition constants of test agents for inhibition of radiolabelled dopamine uptake (K-iupt ake) and inhibition of the binding of a cocaine ligand such as [H-3]2 beta-carbomethoxy 3 beta-(fluorophenyl)tropane (CFT; K-ibind) and sele ct for compounds with K-iuptake/K-ibind ratios greater than unity. Bec ause others have shown that compounds can exhibit K-iuptake/K-ibind ra tios greater than unity when the assays are performed under non-identi cal conditions, we have established these assays under identical condi tions of time, temperature and buffer using a Chinese hamster ovary (C HO) cell line which stably expresses the human DAT. Kinetic and satura tion analyses were performed on both assays and over 200 structurally diverse compounds were screened. Using identical assay parameters, sev eral series of compounds having K-iuptake/K-ibind ratios significantly greater than unity were identified. Such compounds include local anes thetics (procaine, dibucaine, tolperisone, dyclonine, diperodone), ant ipsychotic agents (10-(diethylaminopropionyl)phenothiazine antidepress ants (desipramine, imipramine, protriptyline), a diuretic (5-N-methyl- N-isobutyl-amilioride), an anticholinergic agent (prindinol), a PKC in hibitor (H-8), a calcium channel antagonist (loperamide) and an antima larial compound (chloroquine). To our knowledge, even though these com pounds exhibit low binding affinities (3-24 mu M), they represent some of the most cocaine site-selective compounds identified to date using identical assay parameters. (C) 1997 Elsevier Science Ireland Ltd.