Organic cation transporters (OCTs) play a pivotal role in the absorption, tissue distribution, and excretion of a diverse array of substances, and currently the nature of the biochemical interactions between substrate and OCTs are unknown. Therefore, identifying which amino acid residues are critical for OCT-substrate interactions is of central importance to understanding and predicting interactions between drugs and OCTs. A three-dimensional (3-D) homology model of human OCT3 was generated using the crystal structure of a high affinity phosphate transporter from Piriformospora indica (PiPT) as template, and putative binding pocket for the prototypical hOCT3 ligand 1-methyl-4-phenylpyridinium (MPP+) was identified through docking studies. Five residues, Phe36, Val40, Trp358, Glu451 and Asp478, were identified as potentially mediating hOCT3-MPP+ interactions, and confirmed through in vitro studies. Additionally, 3-D homology modeling of the functional hOCT3 mutant Val40Leu, and all non-functional hOCT3 mutants, indicated changes in binding pocket architecture consistent with weakening of ligand-transporter interactions. Docking of structurally divergent hOCT3 substrates indicated binding interactions in the same general region as that identified for MPP+, albeit with mostly unique residues. Interspecies differences were explored by generating 3-D homology models for rat and murine Oct3. Results from docking studies using compounds exhibiting vastly different binding affinities (Km or IC50) towards the OCT3/Oct3 orthologs were consistent with varying strength in ligand-transporter binding pocket interactions. Finally, a series of novel compounds exhibiting anti-depressant-like activity was screened for OCT interaction in vitro, and demonstrated significant inhibitory effects on OCTs for many of the compounds.

The Dukat laboratory developed 2-amino-6-chloro-3,4-dihydroquinazoline (A6CDQ) as a 5-HT3 receptor ligand. A6CDQ and one of its positional isomers, the 7-chloro analog A7CDQ, produced antidepressant-like effects in the mouse tail suspension test (TST). We investigated and systematically ruled out a solely 5-HT3 receptor or hSERT mediated mechanism of antidepressant-like effect for both A6CDQ and A7CDQ. The role of organic cation transporter 3 (OCT3) as an alternative mechanism in the regulation of neurotransmitters including serotonin (5-HT) and the therapeutic potential of targeting hOCT3 to achieve antidepressant effects has been established. By virtue of possessing protonatable nitrogen atoms, 2-aminodihyroquinazolines could potentially exhibit activity at OCT3. A major goal of our present study was to explore the non-serotonergic mechanism of antidepressant-like effects and to study the as yet unexplored structure-activity-relationships (SARs) at OCT3. We examined the role of i) the chloro group, ii) the methylene bridge and iii) electronic/lipophilic effects at the 6-position. We developed the first 3-D homology models of both the human and mouse orthologs of OCT3, conducted docking studies and HINT analysis, and identified critical amino acid residues interacting with 2-aminodihydroquinazoline analogs at hOCT3 and mOCT3. Retention of antidepressant-like activity in the mouse and potential locomotor stimulant effects for TST-active doses were thoroughly investigated. We have successfully investigated initial SAR of 2-aminodihydroquinazolines at hOCT3 and generated the first 3-D homology models of hOCT3 and mOCT3. Highly potent and selective compounds could potentially be developed as radioligands to probe the binding site of OCT3 and as a mechanistically novel class of antidepressants.

Proteomic techniques were used to analyze the protein expression profile of the early-stage differentiation of pig embryonic stem cells (ES cells). The pig ES cells were induced to develop to neuronal cells by all-trans retinoic acid (ATRA) in vitro by Tainan Livestock Research Institute. The ES cells were cultured with ATRA and collected at time intervals of 0, 1, 2, 4, 8 and 10 days. The cell lysates were analyzed by two-dimensional electrophoresis, and the differentially expressed proteins are identified by MALDI-TOF. Our data shows that the expression profile of pig ES cells is similar to other mammalian models but with some differences. Preliminary pig ES cells 2D database was set up. Six spots each with up or down-regulation in neurogenesis were identified by MS. These proteins may become the good markers of pig ES cells into neural cells by RA. Among those proteins, vimentin, prohibitin and annexin A10 were up-regulated, zinc finger protein 482 (ZNF482), fyn-related kinase (FRK) and annexin A1 were down-regulated during differentiation of pig ES cells to neural cells. Addtionally, we ultilized RT-PCR technique to investigate mRNA expression during neurogenesis, vimentin and prohibitin was up-regulated, anxa1(annexin A1) was slightly down-regulated, neuroD1 and neurogenin 2 were high expression on day 10, beta-catenin was high expression on day 8 to 10.