Received for publication October 17, 2007.
Revised February 6, 2008.
Accepted for publication February 25, 2008.

Me-talnetant and osanetant interact within overlapping but not identical binding pockets in the human tachykinin NK₃ receptor transmembrane domains

Abstract

Recent clinical trials have indicated that neurokinin 3 receptor antagonists osanetant and talnetant may treat symptoms of schizophrenia. Using site-directed mutagenesis, rhodopsin-based modeling, [³H]Me-talnetant and [³H]osanetant binding and functional Schild analyses, we have demonstrated the important molecular determinants of NKB, Me-talnetant and osanetant binding pockets. The residues N138^2.57, N142^2.61, L232^45.49, Y315^6.51, F342^7.39 and M346^7.43 were found to be crucial for the NKB-binding site. We observed that the M134^2.53A, V169^3.36M, F342^7.39M and S341^7.38I/F342^7.39M mutations resulted in the complete loss of [³H]Me-talnetant and [³H]osanetant binding affinities and also abolished their functional potencies in an NKB-evoked accumulation of [³H]IP assay, while the mutations V95^1.42A, N142^2.61A, Y315^6.51F and M346^7.43A behaved differently between two antagonists’ interacting modes. V95^1.42A and M346^7.43A significantly decreased Me-talnetant's affinity and potency. Y315^6.51F, while not affecting Me-talnetant, led to a significant decrease in affinity and potency of osanetant. The mutation N142^2.61A, which abolished osanetant's potency and affinity, led to a significant increase in Me-talnetant's affinity and potency. The proposed docking mode was further validated using RO4908594, from another chemical class. Interestingly, the mutation F342^7.39A caused a 80-fold gain of RO4908594 binding affinity, but the same mutation resulted in the complete loss of Me-talnetant's and partial loss of osanetant's affinity. These observations show that the binding pocket of Me-talnetant and osanetant are overlapping, but not identical. Taken together, our data are consistent with the proposed docking modes where Me-talnetant reaches deeply into the pocket formed by TM1, -2 and -7, whereas osanetant fills the pocket TM3, -5 and -6 with its phenyl-piperidine moiety.

Key words: Neuropeptides, Tachykinin, Gq/11 family, Structure determinations, Mutagenesis/Chimeric approaches, Receptor binding studies, Anti-psychotics