Cloning and expression of canine serotonin and noradrenaline reuptake transporters: comparison of SRI, NRI and SNRI binding affinities at human transporters

Clare Christy, Caroline Tolley, Helen Wightman, Rachel McCoy & Stephen Phillips (introduced by Wesley Miner) Pfizer Global Research and Development, Ramsgate Road, Sandwich, Kent CT13 9NJ.

Serotonin and noradrenaline reuptake transporters (SERT and NET) play a fundamental role in limiting synaptic transmission in the central and peripheral nervous systems. Serotonin and/ or noradrenaline reuptake inhibitors (SRIs, NRIs, SNRIs) are currently used to treat depression, anxiety, ADHD, stress urinary incontinence and neuropathic pain. However, in order to develop safer, more efficacious drugs to modulate monoamine neurotransmission, it is important to understand the translation of compound pharmacology between human and pre-clinical species. Thus, the aim of the present study was to clone and express canine SERT and NET (cSERT and cNET) cDNAs in a recombinant system to enable comparison of SRI, NRI and SNRI binding affinities between the canine and human transporters.

cSERT and cNET were identified and cloned de novo from placental RNA using 5’and 3’ rapid amplification of cDNA ends and RT-PCR. Stable recombinant HEK293 cell lines expressing cSERT and cNET were generated and functional expression was confirmed by specific uptake of [3H]-serotonin or [3H]-noradrenaline respectively. Subsequently, membranes were prepared from these cells and HEK293 cells expressing recombinant human SERT or NET (hSERT or hNET; obtained from RD Blakely), scintillation proximity assays (SPA) were developed and compound affinity for each of the transporters was determined by their ability to compete with radioligand binding using [3H]-citalopram or [3H]-nisoxetine. Compound affinity was calculated by determination of the IC50 value, with subsequent conversion to Ki using the method described by Cheng and Prusoff (1973). Data shown are expressed as geometric mean IC50 or Ki (nM) with lower and upper 95% confidence intervals. cSERT and cNET cDNAs encoded 630 and 619 amino acid proteins respectively which shared ~93% homology with the human transporters. Stable, recombinant expression of the transporters was confirmed by duloxetine- or reboxetine-mediated inhibition of binding/ uptake of [3H]-serotonin or [3H]-noradrenaline by whole cells (IC50 7.4nM (3.5, 15.7), n=2, and 15.2nM (11.7, 19.9), n=3 respectively). Subsequently, SRI, NRI and SNRI binding affinities at each transporter were determined by SPA (n=4-5). Rank order of compound affinity at cNET and hNET was identical (reboxetine>desipramine >nisoxetine>duloxetine>nomifensine>paroxetine>fluoxetine>sertraline>venlafaxine), whilst K i values showed an overall 1.2 fold shift in affinity from hNET to cNET when looking at the complete data set. Similarly, rank order of compound affinity at cSERT and hSERT was identical (paroxetine>duloxetine>sertraline>fluoxetine>venlafaxine >desipramine>nisoextine> reboxetine>nomifensine), with a 1.15 fold shift in affinity.

In summary, cSERT and cNET cDNAs have been cloned and are highly homologous with the human transporters. The transporter binding affinities for SRIs, NRIs and SNRIs from distinct chemical series correlate well with those obtained with the corresponding human transporters, therefore allowing translation of pharmacological effects seen with these compounds across the species.

Cheng Y.C.and Prussoff W.H. (1973). Biochem. Pharmacol, 22: 3099-3108.