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  • To this end we evaluated novel D analogs


    To this end, we evaluated novel D22 analogs for selectivity to inhibit substrate transport in OCT2, OCT3, and PMAT heterologous cell trans-AUCB synthesis systems, and in mouse hippocampal and striatal preparations. Chosen analogs were based upon availability of essential chemical precursors (e.g. 6-substituted-2-methyl quinolines). Five analogs contained varying isocyanine ring substituents (halogen and methoxy groups), two were based upon the structurally related cyanine heterocyclic ring system (Table 1). Initial characterization showed that the analogs were significantly less potent inhibitors at alpha-1 adrenergic receptors (compounds 2, 5, 6 & 7), giving these analogs potential for lesser in vivo off-target effects than D22 (Krause-Heuer et al., 2017). Moreover, some analogs were less potent inhibitors of spontaneous locomotor activity (compounds 6 & 7), and independently, showed antidepressant-like activity (i.e. reduced time spent immobile) in the mouse tail suspension test (compounds 5 & 6) (Krause-Heuer et al., 2017). This independent antidepressant-like effect is unique given our previous studies showed D22 to produce antidepressant-like effects in wildtype mice only when given with a sub-effective dose of a selective serotonin reuptake inhibitor (SSRI) (Horton et al., 2013), or when given to mice with genetically reduced SERT expression (Baganz et al., 2008). However, it remains unclear if D22, or its analogs, have any activity at the high-affinity/low-capacity transporters DAT, NET, and SERT. Likewise, which of the low-affinity/high-capacity transporter(s) mediate the antidepressant-like effects of D22, and certain analogs, remains unclear. We found that these analogs are not superior to D22 in discriminating among OCTs/PMAT. However, importantly, we found that analogs with stand-alone antidepressant-like activity (previously reported (Krause-Heuer et al., 2017)) support the development of compounds with combined ability to inhibit both low-affinity/high-capacity transporters, such as OCT3, and high-affinity/low-capacity transporters, such as SERT, as therapeutics with potentially improved efficacy for treatment of psychiatric disorders.
    Materials and methods
    Discussion We previously characterized antidepressant-like effects of seven D22 analogs (Krause-Heuer et al., 2017). Here, we tested their binding specificity at high-affinity/low-capacity (SERT, DAT, NET) and low-affinity/high-capacity (OCT2, OCT3, PMAT) transporters to understand the relationship between their affinity and ability to produce antidepressant-like behavioral effects in mice. All analogs bound with low affinity to mSERT, mDAT and mNET, determined by [3H]citalopram, [3H]WIN 35428, or [3H]nisoxetine binding displacement, respectively (Fig. 1, Fig. 2, Fig. 3). D22 and a subset of the analogs (1, 2, 4, 6, 7) showed higher affinity for mDAT than the endogenous neurotransmitter dopamine (Table 2, DAT column). Likewise, a smaller subset of the analogs (4, 6, 7) showed higher affinity for mSERT than the endogenous neurotransmitter 5-HT (Table 2-SERT column). Consistent with D22, all analogs were more potent inhibitors of [3H]MPP+ uptake via hOCT3 than hOCT2 or hPMAT (Fig. 6, Table 4). Together with our previous behavioral characterization of these D22 analogs (Krause-Heuer et al., 2017), and published findings (Horton et al., 2013), our results suggest that multimodal inhibition at both low- and high-affinity transporter families may be a strategy for compound development with improved therapeutic efficacy for disorders like depression. Several investigations have elegantly demonstrated the contribution of corticosterone- or D22-sensitive transporters to monoamine clearance (Daws, 2009, Feng et al., 2009; Gasser et al., 2006 ; Hill et al., 2011, Shaskan and Snyder, 1970, Takeda et al., 2002, Yoshikawa et al., 2013; Zwart et al., 2001 ). Mouse models are an essential preclinical resource to study the action of low-affinity transporters. Functional similarities have been observed between the uptake kinetics of substrates like dopamine, MPP+, and serotonin by human and mouse PMAT (Miura et al., 2017, Shirasaka et al., 2016). On the other hand, hOCT3 and mOCT3 were found to share similar affinities for MPP+, but not serotonin (Massmann et al., 2014). Others have found corticosterone to either have enhanced potency for hOCT3 (Gründemann et al., 2002) or equipotent blockade at hOCT3 and mOCT3 (Massmann et al., 2014). Of the few studies that have evaluated species-specific transporter functions, observed differences can depend on the competing ligand, as well as experimental conditions (e.g. temperature, type of radioligand). Our experiments in mouse tissue and high-affinity transporter expressing cell lines help expand functional information of these transporters both in a commonly used model of preclinical relevance, and commonly used cell line.