Systematic Assessment of Fragment Identification for Multitarget Drug Design.

Designed multitarget ligands are a popular approach to generating efficient and safe drugs, and fragment-based strategies have been postulated as a versatile avenue to discover multitarget ligand leads. To systematically probe the potential of fragment-based multiple ligand discovery, we have employed a large fragment library for comprehensive screening on five targets chosen from proteins for which multitarget ligands have been successfully developed previously (soluble epoxide hydrolase, leukotriene A4 hydrolase, 5-lipoxygenase, retinoid X receptor, farnesoid X receptor). Differential scanning fluorimetry served as primary screening method before fragments hitting at least two targets were validated in orthogonal assays. Thereby, we obtained valuable fragment leads with dual-target engagement for six out of ten target combinations. Our results demonstrate the applicability of fragment-based approaches to identify starting points for polypharmacological compound development with certain limitations.

Leukotriene A4 hydrolase deficiency protects mice from diet-induced obesity by increasing energy expenditure through neuroendocrine axis.

Obesity is a health problem worldwide, and brown adipose tissue (BAT) is important for energy expenditure. Here, we explored the role of leukotriene A4 hydrolase (LTA4 H), a key enzyme in the synthesis of the lipid mediator leukotriene B4 (LTB4 ), in diet-induced obesity. LTA4 H-deficient (LTA4 H-KO) mice fed a high-fat diet (HFD) showed a lean phenotype, and bone-marrow transplantation studies revealed that LTA4 H-deficiency in non-hematopoietic cells was responsible for this lean phenotype. LTA4 H-KO mice exhibited greater energy expenditure, but similar food intake and fecal energy loss. LTA4 H-KO BAT showed higher expression of thermogenesis-related genes. In addition, the plasma thyroid-stimulating hormone and thyroid hormone concentrations, as well as HFD-induced catecholamine secretion, were higher in LTA4 H-KO mice. In contrast, LTB4 receptor (BLT1)-deficient mice did not show a lean phenotype, implying that the phenotype of LTA4 H-KO mice is independent of the LTB4 /BLT1 axis. These results indicate that LTA4 H mediates the diet-induced obesity by reducing catecholamine and thyroid hormone secretion.

Assessment of the Effect of Sorafenib on Omega-6 and Omega-3 Epoxyeicosanoid Formation in Patients with Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is a leading cause of cancer death. The multikinase inhibitor sorafenib is widely used for systemic therapy in advanced HCC. Sorafenib might affect epoxyeicosanoids, as it is also a potent inhibitor of the soluble epoxide hydrolase (sEH), which catalyzes the conversion of epoxides derived from long-chain polyunsaturated fatty acids (PUFAs), such as arachidonic acid (AA) and omega-3 docosahexaenoic acid (DHA), into their corresponding diols. Experimental studies with AA-derived epoxyeicosatrienoic acids (EETs) showed that they can promote tumor growth and metastasis, while DHA-derived 19,20-epoxydocosapentaenoic acid (19,20-EDP) was shown to have anti-tumor activity in mice. In this pilot study, we assessed the effect of sorafenib treatment on the presence of lipid mediators, such as EETs, in blood of the patients with HCC using the lipidomics technology. We found a significant increase in 11,12-EET and 14,15-EET levels in HCC patients treated with sorafenib. Furthermore, while not significant in this small sample set, the data presented indicate that sorafenib can also increase the level of omega-3 DHA-derived 19,20-EDP. While the effect on EETs might hamper the anti-tumor effect of sorafenib, we hypothesize that supplementation of DHA in sorafenib-treated HCC patients could increase the level of 19,20-EDP and thereby enhance its anti-tumor effect.

Assessment of soluble epoxide hydrolase activity in vivo: A metabolomic approach.

Soluble epoxide hydrolase (sEH) converts several FFA epoxides to corresponding diols. As many as 15 FFA epoxide-diol ratios are measured to infer sEH activity from their ratios. Using previous data, we assessed if individual epoxide-diol ratios all behave similarly to reflect changes in sEH activity, and whether analyzing these ratios together increases the power to detect changes in in-vivo sEH activity. We demonstrated that epoxide-diol ratios correlated strongly with each other (P < 0.05), suggesting these ratios all reflect changes in sEH activity. Furthermore, we developed a modeling approach to analyze all epoxide-diol ratios simultaneously to infer global sEH activity, named SAMI (Simultaneous Analysis of Multiple Indices). SAMI improved power in detecting changes in sEH activity in animals and humans when compared to individual ratio estimates. Thus, we introduce a new powerful method to infer sEH activity by combining metabolomic determination and simultaneous analysis of all measurable epoxide-diol pairs.

Effect of fungal mycelia on the HPLC-UV and UV-vis spectrophotometric assessment of mycelium-bound epoxide hydrolase using glycidyl phenyl ether.

The use of mycelia as biocatalysts has technical and economic advantages. However, there are several difficulties in obtaining accurate results in mycelium-catalysed reactions. Firstly, sample extraction, indispensable because of the presence of mycelia, can bring into the extract components with a similar structure to that of the analyte of interest; secondly, mycelia can influence the recovery of the analyte. We prepared calibration standards of 3-phenoxy-1,2-propanediol (PPD) in the pure solvent and in the presence of mycelia (spiked before or after extraction) from five fungi (Aspergillus niger, Aspergillus tubingensis, Penicillium aurantiogriseum, Penicillium sp. and Aspergillus terreus). The quantification of PPD was carried out by HPLC-UV and UV-vis spectrophotometry. The manuscript shows that the last method is as accurate as the HPLC method. However, the colorimetric method led to a higher data throughput, which allowed the study of more samples in a shorter time. Matrix effects were evaluated visually from the plotted calibration data and statistically by simultaneously comparing the intercept and slope of calibration curves performed with solvent, post-extraction spiked standards and pre-extraction spiked standards. Significant differences were found between the post- and pre-extraction spiked matrix-matched functions. Pre-extraction spiked matrix-matched functions based on A. tubingensis mycelia, selected as the reference, were validated and used to compensate for low recoveries. These validated functions were successfully applied to the quantification of PPD achieved during the hydrolysis of glycidyl phenyl ether by mycelium-bound epoxide hydrolases and equivalent hydrolysis yields were determined by HPLC-UV and UV-vis spectrophotometry. This study may serve as starting point to implement matrix effects evaluation when mycelium-bound epoxide hydrolases are studied.

Diverse ways of perturbing the human arachidonic acid metabolic network to control inflammation.

Inflammation and other common disorders including diabetes, cardiovascular disease, and cancer are often the result of several molecular abnormalities and are not likely to be resolved by a traditional single-target drug discovery approach. Though inflammation is a normal bodily reaction, uncontrolled and misdirected inflammation can cause inflammatory diseases such as rheumatoid arthritis and asthma. Nonsteroidal anti-inflammatory drugs including aspirin, ibuprofen, naproxen, or celecoxib are commonly used to relieve aches and pains, but often these drugs have undesirable and sometimes even fatal side effects. To facilitate safer and more effective anti-inflammatory drug discovery, a balanced treatment strategy should be developed at the biological network level. In this Account, we focus on our recent progress in modeling the inflammation-related arachidonic acid (AA) metabolic network and subsequent multiple drug design. We first constructed a mathematical model of inflammation based on experimental data and then applied the model to simulate the effects of commonly used anti-inflammatory drugs. Our results indicated that the model correctly reproduced the established bleeding and cardiovascular side effects. Multitarget optimal intervention (MTOI), a Monte Carlo simulated annealing based computational scheme, was then developed to identify key targets and optimal solutions for controlling inflammation. A number of optimal multitarget strategies were discovered that were both effective and safe and had minimal associated side effects. Experimental studies were performed to evaluate these multitarget control solutions further using different combinations of inhibitors to perturb the network. Consequently, simultaneous control of cyclooxygenase-1 and -2 and leukotriene A4 hydrolase, as well as 5-lipoxygenase and prostaglandin E2 synthase were found to be among the best solutions. A single compound that can bind multiple targets presents advantages including low risk of drug-drug interactions and robustness regarding concentration fluctuations. Thus, we developed strategies for multiple-target drug design and successfully discovered several series of multiple-target inhibitors. Optimal solutions for a disease network often involve mild but simultaneous interventions of multiple targets, which is in accord with the philosophy of traditional Chinese medicine (TCM). To this end, our AA network model can aptly explain TCM anti-inflammatory herbs and formulas at the molecular level. We also aimed to identify activators for several enzymes that appeared to have increased activity based on MTOI outcomes. Strategies were then developed to predict potential allosteric sites and to discover enzyme activators based on our hypothesis that combined treatment with the projected activators and inhibitors could balance different AA network pathways, control inflammation, and reduce associated adverse effects. Our work demonstrates that the integration of network modeling and drug discovery can provide novel solutions for disease control, which also calls for new developments in drug design concepts and methodologies. With the rapid accumulation of quantitative data and knowledge of the molecular networks of disease, we can expect an increase in the development and use of quantitative disease models to facilitate efficient and safe drug discovery.

Overview of recent drug discovery approaches for new generation leukotriene A4 hydrolase inhibitors.

INTRODUCTION: LTA(4)H is a bifunctional enzyme with hydrolase and aminopeptidase activities. The hydrolase function of this enzyme specifically catalyzes the rate-limiting step in the conversion of LTA(4) to LTB(4), one of the most potent chemoattractant and activator of neutrophils. The wealth of in vitro and in vivo data favors in support of LTA(4)H as an appealing target for the discovery and development of anti-inflammatory drugs. AREAS COVERED: The authors provide an overview of the recent advances on LTA(4)H inhibitors since 2000. The review details the medicinal chemistry efforts leading to the generation of novel inhibitor chemotypes with desirable drug-like properties as well as the advantages and disadvantages of LTA(4)H as a desirable therapeutic target. EXPERT OPINION: Most of the LTA(4)H inhibitors block pro-inflammatory LTB(4) biosynthesis by concomitant inhibition of both the hydrolase and aminopeptidase activities of LTA(4)H. However, the degradation of another endogenous chemoattractant substrate (PGP) by aminopeptidase function of LTA(4)H was shown, introducing a new anti-inflammatory mission for this pro-inflammatory enzyme. LTA(4)H inhibitors were also shown to maintain anti-inflammatory lipoxin formation. Hence, the data on new LTA(4)H inhibitors should be cautiously interpreted with regard to potential repercussions of preventing PGP degradation as well as for the clinical benefits of concomitant lipoxin formation.

DNA damage and susceptibility assessment in industrial workers exposed to styrene.

Styrene is a widely used chemical in the manufacture of synthetic rubber, resins, polyesters, and plastics. The highest levels of human exposure to styrene occur during the production of reinforced plastic products. The objective of this study was to examine occupational exposure to styrene in a multistage approach, in order to integrate the following endpoints: styrene in workplace air, mandelic and phenylglyoxylic acids (MA + PGA) in urine, sister chromatid exchanges (SCE), micronuclei (MN), DNA damage (comet assay), and genetic polymorphisms of metabolizing enzymes (CYP2E1, EPHX1, GSTM1, GSTT1, and GSTP1). Seventy-five workers from a fiberglass-reinforced plastics factory and 77 unexposed controls took part in the study. The mean air concentration of styrene in the breathing zone of workers (30.4 ppm) and the mean concentration of urinary metabolites (MA + PGA = 443 ± 44 mg/g creatinine) exceeded the threshold limit value (TLV) and the biological exposure index (BEI). Significantly higher SCE frequency rate and DNA damage were observed in exposed workers, but MN frequency was not markedly modified by exposure. With respect to the effect of genetic polymorphisms on different exposure and effect biomarkers studied, an increase in SCE levels with elevated microsomal epoxide hydrolase activity was noted in exposed workers, suggesting a possible exposure-genotype interaction.

Approaches to acrylamide physiologically based toxicokinetic modeling for exploring child-adult dosimetry differences.

Dietary exposure to acrylamide is common as a result of its formation during the cooking of carbohydrate foods. This leads to widespread human exposure in adults and children alike. Acrylamide is neurotoxic and is metabolized by cytochrome P-450 (CYP) 2E1 to a mutagenic epoxide, glycidamide. This article describes a modeling framework for assessing acrylamide and glycidamide dosimetry in rats and human adults and children. The challenges in building a physiologically based toxicokinetic (PBTK) model that is compatible with existing rat and human data are described, with an emphasis on calibration against the hemoglobin adduct database. This exploratory PBTK model was adapted to children by incorporating life-stage-specific parameters consistent with children's changing physiology and metabolic capacity for processes involved in acrylamide disposition in terms of CYP2E1, glutathione conjugation, and epoxide hydrolase. Monte Carlo analysis was used to simulate the distribution of internal doses to gain an initial understanding of the range of child/adult differences possible. This analysis suggests modest dosimetry differences between children and adults, with area-under-the-curve (AUC) doses for the 99th percentile child up to fivefold greater than the median adult for both acrylamide and glycidamide. Early life immaturities tended to exert a greater effect on acrylamide than glycidamide dosimetry because immaturities in CYP2E1 and glutathione counteract one another for glycidamide AUC, but both lead to greater acrylamide dose. The analysis points toward glutathione conjugation parameters as being particularly influential and uncertain in early life, making this a key area for future research.

Optimizing bioconversion pathways through systems analysis and metabolic engineering.

We demonstrate a general approach for metabolic engineering of biocatalytic systems comprising the uses of a chemostat for strain improvement and radioisotopic tracers for the quantification of pathway fluxes. Flux determination allows the identification of target pathways for modification as validated by subsequent overexpression of the corresponding gene. We demonstrate this method in the indene bioconversion network of Rhodococcus modified for the overproduction of 1,2-indandiol, a key precursor for the AIDS drug Crixivan.

Species differences in the metabolism of olefins: implications for risk assessment.

Olefinic compounds are commercially valuable because they form useful polymeric substances. The same chemical property (presence of double bonds) that makes the olefins useful may also cause them to be toxic in the body. The double bonds of olefins can be oxidized by cytochrome P450 enzymes to epoxides, which are electrophiles that can react with DNA and may cause alterations in the genetic information carried by that macromolecule. Epoxides can be rendered inactive toward DNA by binding to proteins, by hydrolysis to diols through epoxide hydrolase enzymes (EHs), or by forming conjugates with glutathione via glutathione S-transferase (GST) activities. The balance between the oxidizing enzymatic activities and the hydrolyzing or conjugating enzymatic activities in the livers of different species can influence the potential toxicity of the olefins. The location of the enzymes and the potential for concerted reactions in which epoxides are inactivated immediately after formation will also influence the potential toxicity of the olefins. Cytochrome P450 enzymes and EHs are in microsomes located in the rough endoplasmic reticulum surrounding the nucleus where the DNA is located. GST is in the cytoplasm of the cell. In the case of 1,3-butadiene (BD), such enzymatic differences may strongly influence the toxicity in different species. The mouse, in which BD is a potent multi-site carcinogen, has the lowest microsomal EH activity of any species. This allows the monoepoxides formed in the microsomes by cytochrome P450 enzymes to be further oxidized to the highly genotoxic diepoxide (DEB), and both epoxides can either be released into the blood for distribution throughout the body or can react with DNA in the nucleus. The rat, in which BD is a weak carcinogen, has much higher levels of microsomal EH, and only trace amounts of DEB enter the bloodstream. Major BD metabolites in primates suggest that the hydrolysis pathway is even more prominent in primates than in rats. Data suggest that BD will be much less toxic in primates than in mice. Considering these quantitative differences in metabolism may help to reduce the uncertainties in extrapolating animal data on olefin toxicity to health risk assessments for humans exposed to the compounds.

Assessment of possible protective roles of selenium, zinc, and cis-stilbene oxide against acute T-2 toxin poisoning: a preliminary report.

The efficacy of two free radical scavengers, selenium and zinc, and a microsomal epoxide hydrolase-inducing agent, cis-stilbene oxide on the acute toxicity of T-2 toxin, a potent cytotoxic trichothecene, was investigated. Mice were pretreated daily for 3 consecutive days with either zinc sulfate (4.4 mg/kg, intraperitoneally [i.p.]), sodium selenite (1, 2, and 3 mg/kg i.p.) or cis-stilbene oxide (50 mg/kg i.p.). A full 24-hr after the final dosing with these agents, mice were given T-2 toxin (2, 2.5, or 3 mg/kg i.p.). The acute lethal toxicity of T-2 toxin (2.5 mg/kg) was reduced by administration of only sodium selenite (3 mg/kg) and cis-stilbene oxide (50 mg/kg). No significant effect on weight gain was observed.

Immunohistochemical assessment of human microsomal epoxide hydrolase in primary and secondary liver neoplasm: a quantitative approach.

1. Epoxide hydrolases form an enzyme family involved in the metabolism of a variety of xenobiotics including cytostatic drugs and carcinogens. Whether human microsomal epoxide hydrolase--one of the main members of the epoxide hydrolase family--is expressed in neoplasia of the liver has been the subject of a controversial discussion. 2. We therefore developed a quantitative immunohistochemical assay and monitored epoxide hydrolase expression in hepatocellular carcinomas (HCC, n = 20), cholangio-cellular carcinomas (CCC, n = 2) and liver metastases (n = 57) of tumours of various origins, and compared the expression intensities and patterns to normal liver tissue. 3. In normal liver tissue microsomal epoxide hydrolase displays expression of the constitutive type with non-zonal staining of all hepatocytes. 4. When using a quantitative immunohistochemical approach statistically significant differences in microsomal epoxide hydrolase expression were observed between normal tissue, hepatocellular carcinoma and liver metastases (mean optical density 2.35, 1.63 and 0.21 respectively, p = 2.9, 6.3 and 18.9). These data indicate differential expression in different types of liver neoplasm. 5. As microsomal epoxide hydrolase is involved in metabolism of different xenobiotics our findings may have implications for tumour progression.