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Molecular Determinants for the Interaction of the Valvulopathic Anorexigen Norfenfluramine with the 5-HT_2B Receptor

Departments of Biochemistry (V.S., B.L.R.), Psychiatry (B.L.R.), and Neurosciences (B.L.R.), Case Western Reserve University School of Medicine, Cleveland, Ohio; and Department of Medicinal Chemistry, School of Pharmacy, Virginia Commonwealth University, Richmond, Virginia (M.D., R.A.G.)

S-(+)-Norfenfluramine (SNF)—an active metabolite of the now-banned anorexigen fenfluramine—has been implicated in the drug's appetite-suppressing actions and its life-threatening cardiovascular side effects. SNF reduces appetite through serotonin 5-HT_2C receptor activation; it causes cardiopulmonary side effects through 5-HT_2B receptor activation. Thus, we attempted to identify molecular determinants of SNF binding to 5-HT_2B receptors distinct from those underlying SNF-5-HT_2C/2A receptor interactions. Mutagenesis implicated Val2.53 in SNF binding to 5-HT_2B receptors. Ligand docking simulations suggested both Val2.53 -methyl groups form stabilizing van der Waals' (vdW) interactions with the -methyl group of SNF. A V2.53L mutation induced a 17-fold decrease in affinity; molecular dynamics (MD) simulations suggested that this decrease resulted from the loss of one 2.53--methyl group vdW interaction. Supporting this, 1) the binding of norfenfluramine (NF) analogs lacking an S-(+) -methyl group (RNF and -desmethyl-NF) was less sensitive to the V2.53L mutation, and 2) a V2.53A mutation decreased SNF affinity 190-fold, but decreased RNF and -desmethyl-NF affinities only 16- and 45-fold, respectively. We next addressed whether the -methyl group of SNF contributes to 5-HT_2C/2A receptor affinity. Removal of the -methyl group (RNF and -desmethyl-NF), which reduced 5-HT_2B receptor binding 3-fold, did not affect 5-HT_2C/2A receptor binding. An -ethyl substituent (-ethyl-NF), which decreased 5-HT_2B receptor affinity 46-fold, reduced 5-HT_2C and 5-HT_2A receptor binding by 14- and 5-fold, respectively. Finally, we determined that residue 2.53 affects SNF potency and efficacy at 5-HT_2B receptors but not at 5-HT_2C and 5-HT_2A receptors. In conclusion, vdW interactions between residue 2.53 and the -methyl group of SNF contribute to the ligand's 5-HT₂ receptor subtype-selective pharmacology.

Received for publication November 12, 2004.

Accepted for publication April 14, 2005.

Address correspondence to: Bryan L. Roth, Case Western Reserve University School of Medicine, Dept. of Biochemistry, Rm. W441, 2109 Adelbert Rd., Cleveland, OH 44106-4935. E-mail: bryan.roth{at}case.edu

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