Characteristic distribution of the study population.

<p>¹ Chi-square test or Fisher’s exact test if any number in a cell is less than five.</p><p>Characteristic distribution of the study population.</p

Pros and Cons of the Tuberculosis Drugome Approach – An Empirical Analysis

<div><p>Drug-resistant <i>Mycobacterium tuberculosis</i> (MTB), the causative pathogen of tuberculosis (TB), has become a serious threat to global public health. Yet the development of novel drugs against MTB has been lagging. One potentially powerful approach to drug development is computation-aided repositioning of current drugs. However, the effectiveness of this approach has rarely been examined. Here we select the “TB drugome” approach – a protein structure-based method for drug repositioning for tuberculosis treatment – to (1) experimentally validate the efficacy of the identified drug candidates for inhibiting MTB growth, and (2) computationally examine how consistently drug candidates are prioritized, considering changes in input data. Twenty three drugs in the TB drugome were tested. Of them, only two drugs (tamoxifen and 4-hydroxytamoxifen) effectively suppressed MTB growth at relatively high concentrations. Both drugs significantly enhanced the inhibitory effects of three first-line anti-TB drugs (rifampin, isoniazid, and ethambutol). However, tamoxifen is not a top-listed drug in the TB drugome, and 4-hydroxytamoxifen is not approved for use in humans. Computational re-examination of the TB drugome indicated that the rankings were subject to technical and data-related biases. Thus, although our results support the effectiveness of the TB drugome approach for identifying drugs that can potentially be repositioned for stand-alone applications or for combination treatments for TB, the approach requires further refinements via incorporation of additional biological information. Our findings can also be extended to other structure-based drug repositioning methods.</p></div

List of the drugs tested in this study.

a<p>Ranking was based on homology-based structural predictions and experimentally determined structures of MTB proteins.</p>b<p>Ranking was based solely on experimentally determined structures of MTB proteins.</p>c<p>Chemical Abstracts Service.</p>d<p>4-hydroxytamoxifen is not an FDA-approved human drug.</p>e<p>Plasma concentrations of a drug may vary significantly between experiments because of biological variations and differences in dosing scheme, experimental design, and drug-detection technology. For simplicity, here we selected only one reference for each drug. For hormone replacement therapies (e.g., estradiol, levothyroxine, liothyronine, and progesterone), the plasma concentration may indicate the concentration of the hormone of interest with or without administration of the therapy. Also, concentrations in the reference studies may have been reported in units other than mg/L (e.g., nmol/L). In such cases, the units were converted to mg/L.</p

Disease-Related Growth Factor and Embryonic Signaling Pathways Modulate an Enhancer of <i>TCF21</i> Expression at the 6q23.2 Coronary Heart Disease Locus

<div><p>Coronary heart disease (CHD) is the leading cause of mortality in both developed and developing countries worldwide. Genome-wide association studies (GWAS) have now identified 46 independent susceptibility loci for CHD, however, the biological and disease-relevant mechanisms for these associations remain elusive. The large-scale meta-analysis of GWAS recently identified in Caucasians a CHD-associated locus at chromosome 6q23.2, a region containing the transcription factor <i>TCF21</i> gene. TCF21 (Capsulin/Pod1/Epicardin) is a member of the basic-helix-loop-helix (bHLH) transcription factor family, and regulates cell fate decisions and differentiation in the developing coronary vasculature. Herein, we characterize a <i>cis</i>-regulatory mechanism by which the lead polymorphism rs12190287 disrupts an atypical activator protein 1 (AP-1) element, as demonstrated by allele-specific transcriptional regulation, transcription factor binding, and chromatin organization, leading to altered <i>TCF21</i> expression. Further, this element is shown to mediate signaling through platelet-derived growth factor receptor beta (PDGFR-β) and Wilms tumor 1 (WT1) pathways. A second disease allele identified in East Asians also appears to disrupt an AP-1-like element. Thus, both disease-related growth factor and embryonic signaling pathways may regulate CHD risk through two independent alleles at <i>TCF21</i>.</p></div

Allele-specific transcriptional activity at rs12190287.

<p>(<b>a</b>) Transcriptional activity of rs12190287-C and G variants were determined in heterozygous primary human coronary artery smooth muscle (HCASM), rat aortic smooth muscle cells (RASM) and HEK, HepG2, and A7r5 cell lines. pLuc-MCS vector containing the putative enhancer region for each rs12190287 variant (C-Luc and G-Luc) was transfected for 24 hours and the ratio of firefly and <i>Renilla</i> luciferase activities were normalized to empty reporter (pLuc). *P<0.01 versus G-Luc for each condition. (<b>b</b>) Dual-luciferase assay of wildtype rs12190287 enhancer or mutants, DelT or T/A (as shown), transfected in A7r5 as described above. *P<0.001 versus G-Luc for each condition. (<b>c</b>) Electrophoretic mobility shift assays (EMSA) showing protein binding to [γ³²P]ATP-labeled rs12190287 C/G probes incubated with nuclear extract (NE) from various cell types, along with 100× excess C, G, or negative control (C, G, or Ctrl comp) unlabeled probe as competitor. Arrows and bar-headed lines represent specific and non-specific shifted complexes, respectively. (<b>d</b>) Dual-luciferase assay of rs12190287 C/G enhancer co-transfected with empty vector (Empty) or constitutively active protein kinase A (PKA) or mitogen-activated protein kinase (MEKK) in A7r5 using consensus CRE and AP-1 reporters as positive controls. *P<0.001 versus C-Luc+Empty or CRE-Luc+Veh or AP1-Luc+Veh where indicated. (<b>e</b>) EMSA of rs12190287 C/G with respective competition showing protein binding using control A7r5 or PMA treated A7r5 NE. Values are mean ± SD from triplicates. Similar results were observed from three independent experiments.</p

Predicted model of AP-1 dependent regulation of <i>TCF21</i> at rs12190287 and rs12524865.

<p>Individuals carrying risk alleles for rs12190287 or rs12524865 at 6q23.2 are expected to have increased <i>TCF21</i> expression upon stimulation of PDGFR-β by PDGF-BB in coronary artery smooth muscle cells, due to increased enrichment of active histone modifications (represented by closed and open diamonds) leading to an open chromatin conformation, allowing binding of an active AP-1 TF complex containing various combinations of c-Jun, JunD, and ATF3. WT1 functions as a transrepressor of this active complex at rs12190287, whereby WT1 may fine-tune the spatial and temporal activation of <i>TCF21</i> expression. Multiple kinases other than mitogen-activated kinase (MEKK) may be involved in the activation and recruitment of AP-1 complexes to these binding sites.</p

WT1 regulation of rs12190287 <i>in vitro</i> and <i>in vivo</i>.

<p>(<b>a</b>) Dual-luciferase assay of rs12190287 C/G enhancer transfected with human WT1-B (−KTS), WT1-D (+KTS) expression constructs in A7r5 cells, measured after 24 hours. *P<0.01 versus C-Luc or G-Luc+Empty. (<b>b</b>) Dual-luciferase assay of rs12190287 C/G enhancer co-transfected with human c-JUN and WT1-B or WT1-D expression constructs in A7r5 cells. AP1-Luc reporter was used as a positive control. *P<0.05 versus C-Luc or G-Luc+cJun. (<b>c</b>) Dual-luciferase assay of rs12190287 C/G enhancer transfected in heterozygous HCASMC with siRNA against <i>WT1</i> (−/+KTS) compared to negative control (Neg si). *P<0.05 versus C-Luc or G-luc+Neg si. (<b>d</b>) TaqMan based qRT-PCR results showing relative human <i>WT1</i> mRNA expression levels in heterozygous HCASMC treated with TGF-β1 or PDGF-BB for the indicated times. (<b>e</b>) Total enrichment of WT1 at rs12190287 enhancer, <i>FOSB</i> or <i>MYOG</i> promoter regions determined by chromatin immunoprecipitation (ChIP) in heterozygous HCASMC treated with PDGF-BB for 6 hrs. Values represent fold change relative to enrichment with IgG control. *P<0.01 versus Control WT1. (<b>f</b>) Allele-specific enrichment of WT1 at rs12190287 determined by HaploChIP in heterozygous HCASMC treated with PDGF-BB, shown as normalized allelic-ratio C/G. *P<0.0005 versus Control WT1. Values are mean ± SD from triplicates. Similar results were observed from three independent experiments.</p

<i>In silico</i> allele-specific transcription factor binding to rs12190287.

<p>Predicted transcription factor binding site searches were performed using five programs: TRANSFAC, PROMO, MatInspector, TFSearch, and JASPAR. The SNP is shown in boldface within the predicted binding sequence. Core binding sequence shown in capital letters for TRANSFAC and MatInspector generated sequences. The scores represent matrix sequence similarity for each program. A minimum threshold of 85.0 was used for each program, and lowered to 75.0 for allelic comparison if necessary.</p