Discovery of Competitive and Noncompetitive Ligands of the Organic Cation Transporter 1 (OCT1; SLC22A1)

Organic cation transporter 1 (OCT1) plays a critical role in the hepatocellular uptake of structurally diverse endogenous compounds and xenobiotics. Here we identified competitive and noncompetitive OCT1-interacting ligands in a library of 1780 prescription drugs by combining in silico and in vitro methods. Ligands were predicted by docking against a comparative model based on a eukaryotic homologue. In parallel, high-throughput screening (HTS) was conducted using the fluorescent probe substrate ASP⁺ in cells overexpressing human OCT1. Thirty competitive OCT1 ligands, defined as ligands predicted in silico as well as found by HTS, were identified. Of the 167 ligands identified by HTS, five were predicted to potentially cause clinical drug interactions. Finally, virtual screening of 29 332 metabolites predicted 146 competitive OCT1 ligands, of which an endogenous neurotoxin, 1-benzyl-1,2,3,4-tetrahydroisoquinoline, was experimentally validated. In conclusion, by combining docking and in vitro HTS, competitive and noncompetitive ligands of OCT1 can be predicted

Targeted Disruption in Mice of a Neural Stem Cell-Maintaining, KRAB-Zn Finger-Encoding Gene That Has Rapidly Evolved in the Human Lineage

<div><p>Understanding the genetic basis of the physical and behavioral traits that separate humans from other primates is a challenging but intriguing topic. The adaptive functions of the expansion and/or reduction in human brain size have long been explored. From a brain transcriptome project we have identified a KRAB-Zn finger protein-encoding gene (M003-A06) that has rapidly evolved since the human-chimpanzee separation. Quantitative RT-PCR analysis of different human tissues indicates that M003-A06 expression is enriched in the human fetal brain in addition to the fetal heart. Furthermore, analysis with use of immunofluorescence staining, neurosphere culturing and Western blotting indicates that the mouse ortholog of M003-A06, Zfp568, is expressed mainly in the embryonic stem (ES) cells and fetal as well as adult neural stem cells (NSCs). Conditional gene knockout experiments in mice demonstrates that Zfp568 is both an NSC maintaining- and a brain size-regulating gene. Significantly, molecular genetic analyses show that human M003-A06 consists of 2 equilibrated allelic types, H and C, one of which (H) is human-specific. Combined contemporary genotyping and database mining have revealed interesting genetic associations between the different genotypes of M003-A06 and the human head sizes. We propose that M003-A06 is likely one of the genes contributing to the uniqueness of the human brain in comparison to other higher primates.</p> </div

The Ka/Ks Ratios between Human and Mouse for Genes Expressed in Brain, Liver, and/or Muscle

<p>The number of genes is given in parentheses above each Ka/Ks value; the 95% confidence interval is given in parentheses below each Ka/Ks value. Expression data for brain and liver were from Enard et al. [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0050013#pbio-0050013-b005" target="_blank">5</a>], and data fro muscle were from Public Expression Profiling Resource (<a href="http://pepr.cnmcresearch.org" target="_blank">http://pepr.cnmcresearch.org</a>).</p

Lineage-Specific Ka/Ks Ratios for Brain-Expressed cDNAs

<div><p>The Ka and Ks values along each branch were calculated by the PAML method [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0050013#pbio-0050013-b018" target="_blank">18</a>], and the Ka/Ks ratios are given.</p> <p>(A) There were 1,469 brain-expressed genes common to human, OWM, and mouse.</p> <p>(B) There were 1,668 brain-expressed genes common to human, chimpanzee, and OWM.</p> <p>For details, see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0050013#pbio-0050013-t002" target="_blank">Tables 2</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0050013#pbio-0050013-t003" target="_blank">3</a>.</p></div

Metformin Is a Substrate and Inhibitor of the Human Thiamine Transporter, THTR‑2 (SLC19A3)

The biguanide metformin is widely used as first-line therapy for the treatment of type 2 diabetes. Predominately a cation at physiological pH’s, metformin is transported by membrane transporters, which play major roles in its absorption and disposition. Recently, our laboratory demonstrated that organic cation transporter 1, OCT1, the major hepatic uptake transporter for metformin, was also the primary hepatic uptake transporter for thiamine, vitamin B1. In this study, we tested the reverse, i.e., that metformin is a substrate of thiamine transporters (THTR-1, SLC19A2, and THTR-2, SLC19A3). Our study demonstrated that human THTR-2 (hTHTR-2), SLC19A3, which is highly expressed in the small intestine, but not hTHTR-1, transports metformin (<i>K</i>_m = 1.15 ± 0.2 mM) and other cationic compounds (MPP⁺ and famotidine). The uptake mechanism for hTHTR-2 was pH and electrochemical gradient sensitive. Furthermore, metformin as well as other drugs including phenformin, chloroquine, verapamil, famotidine, and amprolium inhibited hTHTR-2 mediated uptake of both thiamine and metformin. Species differences in the substrate specificity of THTR-2 between human and mouse orthologues were observed. Taken together, our data suggest that hTHTR-2 may play a role in the intestinal absorption and tissue distribution of metformin and other organic cations and that the transporter may be a target for drug–drug and drug–nutrient interactions

Physical maps of M003-A06 of the human and chimpanzee.

<p>Top, chromosomal location of M003-A06 with its flanking genes indicated. Below the top map are the exon-intron gene organization and protein structure of M003-A06. The protein-coding sequences of the exons are in black. The positions of the non-synonymous nucleotides of the human H, C1 and C2 alleles in comparison to the chimpanzee homolog are indicated by the stars. The arrow head and the two arrows indicate the non-synonymous nucleotide substitutions that create the stop codon in the human H allele and the alternative splicing sites in the human C1 and C2 alleles, respectively. The synonymous nucleotide substitution in the chimpanzee gene is indicated by the vertical line.</p

Associations between different genotypes of M003-A06 and the relative head sizes.

<p>Associations with the relative head sizes of Chinese at Taiwan. The DNA samples of 1,244 Chinese children at Taiwan were collected at birth and 6 month of age. After genotyping, the combined C allele frequencies (C1+C2) and the relative head sizes were calculated as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047481#s4" target="_blank">Materials and Methods</a> and compared (Numbers of the HH, HC and CC are 653, 490 and 101, respectively). Note the significantly larger relative head size for the CC genotype than for either HH or HC at birth (p = 0.0018, left panel), but not at the age of six months (p = 0.8, right panel). *p<0.05, *** p<0.001, NS: not significant.</p

Summary of the sequence variations in the M003-A06 genes in human populations and chimpanzee.

<p>The sequence variations among the H, C1, C2 alleles of human M003-A06 and the chimpanzee M003-A06 gene are listed. The amino acid changes at the non-synonymous SNPs are indicated in the parentheses. The single synonymous nucleotide difference between the human and chimpanzee is indicated by the star.</p>*<p>The synonymous nucleotide difference between human and chimpanzee.</p>a<p>The locations of SNPs relative to A (+1) of the start codon (ATG).</p>b<p>The amino acids at the non-synonymous sites.</p

Allele frequencies and genotype frequencies of M003-A06 in different human ethnic groups.

<p>The frequencies of M003-A06 among different ethnic groups except for the Taiwanese are derived from the Hapmap Phase 3 data (<a href="http://hapmap.ncbi.nlm.nih.gov" target="_blank">http://hapmap.ncbi.nlm.nih.gov</a>). The genotype frequencies expected from the allele frequencies are listed in the parentheses, and all of them are close to the actual genotype frequencies suggesting a Hardy-Weinberg equilibrium (see text). Taiwanese, data from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047481#pone-0047481-g006" target="_blank"><b>Figure 6</b></a>; JPT, Japanese in Tokyo, Japan; CHB, Han Chinese in Beijing, China; CEU, Utah residents with Northern and Western European ancestry from the CEPH collection; ASW, African ancestry in Southwest USA; GIH, Gujarati Indians in Houston, Texas; YRI, Yoruba in Ibadan, Nigeria.</p

Plot of the excess of nonsynonymous substitutions between human and chimpanzee against Ka.

<p>The Ka values (X-axis) and the numbers of the excess nonsynonymous substitutions (Y-axis) between human and chimpanzee for 1,668 brain expressed genes were estimated by the maximum likelihood method implemented in PAML and plotted. The excess was calculated as [(number of changes in human) - (number of changes in chimpanzee)]. Thus, a positive value indicates more changes in the human lineage than in the chimpanzee lineage and a negative value means more changes in the chimpanzee lineage. The arrow points to the gene M003-A06, which has the highest number of excess nonsynonymous substitutions in the human lineage.</p