Abstract
Cholinergic hypofunction is associated with decreased attention and cognitive deficits in the central nervous system in addition to compromised motor function. Consequently, stimulation of cholinergic neurotransmission is a rational therapeutic approach for the potential treatment of a variety of neurological conditions. High affinity choline uptake (HACU) into acetylcholine (ACh)-synthesizing neurons is critically mediated by the sodium- and pH-dependent high-affinity choline transporter (CHT, encoded by the SLC5A7 gene). This transporter is comparatively well-characterized but otherwise unexplored as a potential drug target. We therefore sought to identify small molecules that would enable testing of the hypothesis that positive modulation of CHT mediated transport would enhance activity-dependent cholinergic signaling. We utilized existing and novel screening techniques for their ability to reveal both positive and negative modulation of CHT using literature tools. A screening campaign was initiated with a bespoke compound library comprising both the Pfizer Chemogenomic Library (CGL) of 2,753 molecules designed specifically to help enable the elucidation of new mechanisms in phenotypic screens and 887 compounds from a virtual screening campaign to select molecules with field-based similarities to reported negative and positive allosteric modulators. We identified a number of previously unknown active and structurally distinct molecules that could be used as tools to further explore CHT biology or as a starting point for further medicinal chemistry.
Highlights
Cholinergic neurons are responsible for transmitting signals to a wide range of tissues within the peripheral and central nervous systems
There is no differential expression of other molecules required for acetylcholine synthesis (CHAT), transport (SLC18A3/vAChT) or hydrolysis (ACHE); nor in muscarinic and nicotinic acetylcholine receptor subunits (Supplementary Figures 2B,C)
Parallel experiments were performed on the SH-SY5Y stable cell lines which overexpressed the codon-optimized choline transporter (CHT)-GFP fusion transcript in addition to some basal SLC5A7 expression in parental cells in addition to ACHE, SLC18A3, and CHAT expression (Supplementary Figures 2D–F)
Summary
Cholinergic neurons are responsible for transmitting signals to a wide range of tissues within the peripheral and central nervous systems. Additional regulation likely includes specific protein-protein interactions; the complex partners are not well-defined (Kar et al, 1998; Ribeiro et al, 2003; Parikh et al, 2006, 2014; Brock et al, 2007; Misawa et al, 2008; Pinthong et al, 2008; Cuddy et al, 2012, 2014, 2015; Kristofikova et al, 2013; Fishwick and Rylett, 2015) This raises the possibility of modulating CHT surface localization to impact on transport Vmax in addition to direct modulation of transport function through affinity (Km) or rate
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