医薬品リード化合物の標的タンパク質に対する作用機序に関する研究

学位の種別: 論文博士審査委員会委員 : （主査）東京大学教授 大西 康夫, 東京大学教授 清水 謙多郎, 東京大学教授 吉田 稔, 東京大学教授 伏信 進矢, 東京大学講師 勝山 陽平University of Tokyo(東京大学

Synthesis of Unnatural Flavonoids and Stilbenes by Exploiting the Plant Biosynthetic Pathway in Escherichia coli

SummaryFlavonoids and stilbenes have attracted much attention as potential targets for nutraceuticals, cosmetics, and pharmaceuticals. We have developed a system for producing “unnatural” flavonoids and stilbenes in Escherichia coli. The artificial biosynthetic pathway included three steps. These included a substrate synthesis step for CoA esters synthesis from carboxylic acids by 4-coumarate:CoA ligase, a polyketide synthesis step for conversion of the CoA esters into flavanones by chalcone synthase and chalcone isomerase, and into stilbenes by stilbene synthase, and a modification step for modification of the flavanones by flavone synthase, flavanone 3β-hydroxylase and flavonol synthase. Incubation of the recombinant E. coli with exogenously supplied carboxylic acids led to production of 87 polyketides, including 36 unnatural flavonoids and stilbenes. This system is promising for construction of a larger library by employing other polyketide synthases and modification enzymes

Universal and Quantitative Method To Evaluate Inhibitor Potency for Cysteinome Proteins Using a Nonspecific Activity-Based Protein Profiling Probe

Recently, there have been a limited number of new, validated targets for small-molecule drug discovery in the pharmaceutical industry. Although there are approximately 30 000 genes in the human genome, only 2% are targeted by currently approved small-molecule drugs. One reason that many targets remain neglected by drug discovery programs is the absence of biochemical assays enabling evaluation of the potency of inhibitors in a quantitative and high-throughput manner. To overcome this issue, we developed a biochemical assay to evaluate the potency of both reversible and irreversible inhibitors using a nonspecific thiol-labeling fluorescent probe. The assay can be applied to any targets with a cysteine residue in a pocket that can accommodate small-molecule ligands. By constructing a mathematical model, we showed that the potency of compounds can be quantitatively evaluated by performing an activity-based protein profiling assay. In addition, the validity of the theory was confirmed experimentally using epidermal growth factor receptor kinase as a model target. This approach provides an assay system for targets for which biochemical assays cannot be developed. Our approach can potentially not only expand the number of exploitable targets but also accelerate the lead optimization process by providing quantitative structure–activity relationship information

A Novel Selective Inhibitor of Delta-5 Desaturase Lowers Insulin Resistance and Reduces Body Weight in Diet-Induced Obese C57BL/6J Mice

<div><p>Obesity is now recognized as a state of chronic low-grade inflammation and is called as metabolic inflammation. Delta-5 desaturase (D5D) is an enzyme that metabolizes dihomo-γ-linolenic acid (DGLA) to arachidonic acid (AA). Thus, D5D inhibition increases DGLA (precursor to anti-inflammatory eicosanoids) while decreasing AA (precursor to pro-inflammatory eicosanoids), and could result in synergistic improvement in the low-grade inflammatory state. Here, we demonstrate reduced insulin resistance and the anti-obesity effect of a D5D selective inhibitor (compound-326), an orally active small-molecule, in a high-fat diet-induced obese (DIO) mouse model. <i>In vivo</i> D5D inhibition was confirmed by determining changes in blood AA/DGLA profiles. In DIO mice, chronic treatment with compound-326 lowered insulin resistance and caused body weight loss without significant impact on cumulative calorie intake. Decreased macrophage infiltration into adipose tissue was expected from mRNA analysis. Increased daily energy expenditure was also observed following administration of compound-326, in line with sustained body weight loss. These data indicate that the novel D5D selective inhibitor, compound-326, will be a new class of drug for the treatment of obese and diabetic patients.</p></div

Structure of compound-326.

<p>Structure of compound-326.</p

Effect of chronic treatment with compound-326 on energy expenditure in DIO mice.

<p>Daily energy expenditure (DEE) was monitored on days 1 (<b>A</b>), 8 (<b>B</b>), 15 (<b>C</b>), 21 (<b>D</b>), and 56 (<b>E</b>) during the study indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166198#pone.0166198.g005" target="_blank">Fig 5</a>.</p

Effects of chronic treatment with compound-326 on BW, calorie intake, and blood PD markers in DIO mice.

<p>DIO mice (initial BW; 31.1±1.6 g) were treated with compound-326 (0.1, 1, and 10 mg/kg), 30 mg/kg sibutramine, or vehicle p.o. for 6 weeks. (<b>A</b>) Changes in BW from initial value. (<b>B</b>) Daily calorie intake during the study. (<b>C</b>) Changes in blood AA and DGLA concentrations after 6-week treatment. (<b>D</b>) Changes in the blood AA to DGLA ratio after 6-week treatment. Data are expressed as mean ± <i>SE</i> (n = 7). #<i>p</i>≤ 0.025 vs. DIO vehicle by Williams' test. §<i>p</i>≤ 0.025 vs. DIO vehicle by Shirley-Williams test. *<i>p</i>≤ 0.05, **<i>p</i>≤ 0.01 vs. DIO vehicle by Aspin-Welch test. ¶<i>p</i>≤ 0.05, ¶¶<i>p</i>≤ 0.01 vs. DIO vehicle by Student's t-test.</p

Effects of chronic treatment with compound-326 on BW, calorie intake, and fat mass in DIO mice.

<p>DIO mice were treated with 10 mg/kg compound-326, p.o., for 11 weeks. (<b>A</b>) Changes in BW. (<b>B</b>) Changes in daily calorie intake. (<b>C</b>) Weights of different body fat depots after 11-week treatment. Data are expressed as mean ± <i>SE</i> (n = 7). *<i>p</i>≤ 0.05, **<i>p</i>≤ 0.01 vs. DIO vehicle by Aspin-Welch test. ¶<i>p</i>≤ 0.05, ¶¶<i>p</i>≤ 0.01 vs. DIO vehicle by Student's t-test.</p