Automated Extraction of Information on Chemical–P-glycoprotein Interactions from the Literature

Knowledge of the interactions between drugs and transporters is important for drug discovery and development as well as for the evaluation of their clinical safety. We recently developed a text-mining system for the automatic extraction of information on chemical–CYP3A4 interactions from the literature. This system is based on natural language processing and can extract chemical names and their interaction patterns according to sentence context. The present study aimed to extend this system to the extraction of information regarding chemical–transporter interactions. For this purpose, the key verb list designed for cytochrome P450 enzymes was replaced with that for known drug transporters. The performance of the system was then tested by examining the accuracy of information on chemical–P-glycoprotein (P-gp) interactions extracted from randomly selected PubMed abstracts. The system achieved 89.8% recall and 84.2% precision for the identification of chemical names and 71.7% recall and 78.6% precision for the extraction of chemical–P-gp interactions

Automated Extraction of Information on Chemical–P-glycoprotein Interactions from the Literature

Knowledge of the interactions between drugs and transporters is important for drug discovery and development as well as for the evaluation of their clinical safety. We recently developed a text-mining system for the automatic extraction of information on chemical–CYP3A4 interactions from the literature. This system is based on natural language processing and can extract chemical names and their interaction patterns according to sentence context. The present study aimed to extend this system to the extraction of information regarding chemical–transporter interactions. For this purpose, the key verb list designed for cytochrome P450 enzymes was replaced with that for known drug transporters. The performance of the system was then tested by examining the accuracy of information on chemical–P-glycoprotein (P-gp) interactions extracted from randomly selected PubMed abstracts. The system achieved 89.8% recall and 84.2% precision for the identification of chemical names and 71.7% recall and 78.6% precision for the extraction of chemical–P-gp interactions

Inhibition of the H3K4 methyltransferase SET7/9 ameliorates peritoneal fibrosis

<div><p>Transforming growth factor-β1 (TGF-β1) is a major mediator of peritoneal fibrosis and reportedly affects expression of the H3K4 methyltransferase, SET7/9. SET7/9-induced H3K4 mono-methylation (H3K4me1) critically activates transcription of fibrosis-related genes. In this study, we examined the effect of SET7/9 inhibition on peritoneal fibrosis in mice and in human peritoneal mesothelial cells (HPMCs). We also examined SET7/9 expression in nonadherent cells isolated from the effluent of peritoneal dialysis (PD) patients. Murine peritoneal fibrosis was induced by intraperitoneal injection of methylglyoxal (MGO) into male C57/BL6 mice over 21 days. Sinefungin, a SET7/9 inhibitor, was administered subcutaneously just before MGO injection (10 mg/kg). SET7/9 expression was elevated in both MGO-injected mice and nonadherent cells isolated from the effluent of PD patients. SET7/9 expression was positively correlated with dialysate/plasma ratio of creatinine in PD patients. Sinefungin was shown immunohistochemically to suppress expression of mesenchymal cells and collagen deposition, accompanied by decreased H3K4me1 levels. Peritoneal equilibration tests showed that sinefungin attenuated the urea nitrogen transport rate from plasma and the glucose absorption rate from the dialysate. <i>In vitro</i>, sinefungin suppressed TGF-β1-induced expression of fibrotic markers and inhibited H3K4me1. These findings suggest that inhibiting the H3K4 methyltransferase SET7/9 ameliorates peritoneal fibrosis.</p></div

Sinefungin inhibited the expression of H3K4me1 but not that of TGF-β1 in mice with peritoneal fibrosis.

<p>(A) Typical H3K4me1 levels in peritoneal tissue of control mice, MGO-injected mice treated with vehicle only, and MGO-injected mice treated with sinefungin (immunohistochemical [IHC] stain, ×200). (B) Numbers of H3K4me1-positive (H3K4me1^<b>+</b>) cells in the 3 groups of mice. (C) Typical TGF-β1 expression in peritoneal tissue of control mice, MGO-injected mice treated with vehicle only and MGO-injected mice treated with sinefungin (IHC stain, ×200). (D) Numbers of TGF-β1-positive (TGF-β1^<b>+</b>) cells in the 3 groups of mice. The quantitative data are presented as dot plots in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0196844#pone.0196844.s003" target="_blank">S3 Fig</a>. (E) Two-color immunohistochemical staining showing localization of H3K4me1 (blue-gray) and collagens I (brown). (F) The concentration of TGF-β1 protein in mouse PD effluent was quantitated by ELISA. Scale Bar = 200 μm. Data are means ± S.D. *, <i>P</i> < 0.05 (one-way ANOVA followed by <i>post hoc</i> test using <i>t</i> test with Bonferroni correction; <i>n</i> = 5 mice per group).</p

BIX01294 reduces collagen I and III expression in mice with peritoneal fibrosis.

<p>(A) Immunohistochemical staining shows typical collagen I expression in peritoneal tissue of control mice, MGO-injected mice, and MGO-injected mice treated with BIX01294 (original magnification, ×200). (B) Graph indicates the number of collagen I-positive pixels in the three groups of mice. (C) Immunohistochemical staining shows typical collagen III expression in the peritoneal tissue of control mice, MGO-injected mice, and MGO-injected mice treated with BIX01294 (original magnification, ×200). (D) Graph indicates the number of collagen III-positive pixels in the three groups of mice. Data are expressed as the mean ± SE. Statistical analysis was performed by analysis of variance followed by Tukey’s post-hoc test. *<i>P</i> < 0.05, n = 5 mice per group.</p

G9a expression is regulated by TGF-β1 in HPMCs, and BIX01294 represses TGF-β1-induced fibrotic changes.

<p>(A) Representative western blot analysis showing the levels of G9a protein expression in TGF-β1 (5 ng/mL)-stimulated HPMCs at various time points. Quantification is shown in the lower panel. (B) Representative western blot analysis of the expression of α-SMA (C) zonula occludens-1 (ZO-1), and (D) secreted fibronectin of HPMCs. Quantification is shown in the lower panel. (E) Representative western blot analysis showing the levels of H3K9me1 in TGF-β1-stimulated in HPMCs. Quantification is shown in the lower panel. Data are expressed as the mean ± SE. Statistical analysis was performed by analysis of variance followed by Tukey’s post-hoc test. *<i>P</i> < 0.05, n = 5 samples per group.</p

BIX01294 suppresses peritoneal thickening and cell density in MGO-injected mice.

<p>(A) Masson’s trichrome staining shows the typical thickness of peritoneal tissue in control mice, MGO-injected mice, and MGO-injected mice treated with BIX01294 (original magnification, ×200). (B) Graph indicates quantification of the peritoneal thickness in the three groups of mice. (C) Hematoxylin-eosin staining shows typical cellularity of peritoneal tissue in control mice, MGO-injected mice, and MGO-injected mice treated with BIX01294 (original magnification, ×200). (D) Graph indicates quantification of cell density in the three groups of mice. Data are expressed as the mean ± SE. Statistical analysis was performed by analysis of variance followed by Tukey’s post-hoc test. *<i>P</i> < 0.05, n = 5 mice per group.</p

Sinefungin suppressed expression of extracellular matrix (ECM)-associated genes and H3K4me1 level at <i>Col1A2</i> promoters.

<p>Quantitative real-time polymerase chain reaction (PCR) analysis of mRNA expression of (A) <i>ACTA2</i> (<i>α-SMA</i>), (B) <i>Col1A2</i>, (C) <i>CTGF</i> and (D) <i>PAI-1</i> in HPMCs (standardized to glyceraldehyde 3-phosphate dehydrogenase [<i>GAPDH</i>]). (E) Representative chromatin immunoprecipitation (ChIP) assay of the binding of the H3K4me1 protein (H3K4me1-Ab) to <i>Col1A2</i> promoters in HPMCs. Negative control: mouse immunoglobulin G (IgG). Full-length gels are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0196844#pone.0196844.s005" target="_blank">S5 Fig</a>. Data are means ± S.D. *, <i>P</i> < 0.05 (one-way ANOVA followed by the <i>post hoc</i> test using <i>t</i> test with Bonferroni correction; <i>n</i> = 5 samples per group).</p

Sinefungin inhibited TGF-β1-induced fibrotic markers and H3K4me1 in HPMCs.

<p>Representative Western blotting results for the expression of (A) α-SMA (B) secreted fibronectin and (C) zonula occludens-1 (ZO-1) of HPMCs. α-tubulin was used as an internal control. Lower panel: quantification. (D) Representative Western blotting analysis showing level of H3K4me1 in HPMCs stimulated by TGF-β1. H3 was used as the internal control. Lower panel: quantification. Full-length blots are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0196844#pone.0196844.s004" target="_blank">S4 Fig</a>. Data are means ± S.D. *, <i>P</i> < 0.05 (one-way ANOVA followed by the <i>post hoc</i> test using <i>t</i> test with Bonferroni correction; <i>n</i> = 5 samples per group).</p

Investigation of Fluorescein Derivatives as Substrates of Organic Anion Transporting Polypeptide (OATP) 1B1 To Develop Sensitive Fluorescence-Based OATP1B1 Inhibition Assays

Organic anion transporting polypeptide (OATP) 1B1 plays an important role in the hepatic uptake of various drugs. Because OATP1B1 is a site of drug–drug interactions (DDIs), evaluating the inhibitory potential of drug candidates on OATP1B1 is required during drug development. For establishing a highly sensitive, high-throughput fluorescence-based OATP1B1 inhibition assay system, the present study focused on fluorescein (FL) and its derivatives and evaluated their uptake via OATP1B1 as well as OATP1B3 and OATP2B1 using the transporter-expressing human embryonic kidney 293 cells. We identified 2′,7′-dichlorofluorescein (DCF), 4′,5′-dibromofluorescein (DBF), and Oregon green (OG) as good OATP1B1 substrates with <i>K</i>_m values of 5.29, 4.16, and 54.1 μM and <i>V</i>_max values of 87.9, 48.1, and 187 pmol/min/mg protein, respectively. In addition to FL, fluo-3, and 8-fluorescein-cAMP, OG, and DBF were identified as OATP1B3 substrates. FL, OG, DCF, and DBF were identified as OATP2B1 substrates. Among the FL derivatives, DCF displayed the highest OATP1B1-mediated uptake. The <i>K</i>_i values of 14 compounds on OATP1B1 determined with DCF as a probe exhibited good agreement with those obtained using [³H]­estradiol-17β-glucuronide (E₂G) as a substrate, whereas [³H]­estrone-3-sulfate and [³H]­sulfobromophthalein yielded higher <i>K</i>_i values for all inhibitors than DCF. Mutually competitive inhibition observed between DCF and E₂G suggested that they share the same binding site on OATP1B1. Therefore, DCF as well as E₂G can be used as sensitive probes for in vitro OATP1B1 inhibition assays, which will help mitigate the risk of false-negative DDI predictions potentially caused by substrate-dependent <i>K</i>_i variations