Carboxylesterases are well known for their role in the metabolism of xenobiotics. However, recent studies have also implicated carboxylesterases in regulating a number of physiological processes including metabolic homeostasis and macrophage development, underlying the need to quantify them individually. Unfortunately, current methods for selectively measuring the catalytic activity of individual carboxylesterases are not sufficiently sensitive to support many biological studies. In order to develop a more sensitive and selective method to measure the activity of human carboxylesterase 1 (hCE1), we generated and tested novel substrates with a fluorescent aminopyridine leaving group. hCE1 showed at least a 10-fold higher preference for the optimized substrate 4-MOMMP than the 13 other esterases tested. Because of the high stability of 4-MOMMP and its hydrolysis product, this substrate can be used to measure esterase activity over extended incubation periods yielding a low picogram (femtomol) limit of detection. This sensitivity is comparable to current ELISA methods; however, the new assay quantifies only the catalytically active enzyme facilitating direct correlation to biological processes. The method described herein may allow hCE1 activity to be used as a biomarker for predicting drug pharmacokinetics, early detection of hepatocellular carcinoma, and other disease states where the activity of hCE1 is altered.

The virulence factor cystic fibrosis transmembrane conductance regulator (CFTR) inhibitory factor (Cif) is secreted by Pseudomonas aeruginosa and is the founding member of a distinct class of epoxide hydrolases (EHs) that triggers the catalysis-dependent degradation of the CFTR. We describe here the development of a series of potent and selective Cif inhibitors by structure-based drug design. Initial screening revealed 1a (KB2115), a thyroid hormone analog, as a lead compound with low micromolar potency. Structural requirements for potency were systematically probed, and interactions between Cif and 1a were characterized by X-ray crystallography. On the basis of these data, new compounds were designed to yield additional hydrogen bonding with residues of the Cif active site. From this effort, three compounds were identified that are 10-fold more potent toward Cif than our first-generation inhibitors and have no detectable thyroid hormone-like activity. These inhibitors will be useful tools to study the pathological role of Cif and have the potential for clinical application.

In vertebrates, soluble epoxide hydrolase (sEH) hydrolyzes natural epoxy-fatty acids (EpFAs), which are chemical mediators modulating inflammation, pain, and angiogenesis. Chick embryos are used to study angiogenesis, particularly its role in cardiovascular biology and pathology. To find potent and bio-stable inhibitors of the chicken sEH (chxEH) a library of human sEH inhibitors was screened. Derivatives of 1(adamantan-1-yl)-3-(trans-4-phenoxycyclohexyl) urea were found to be very potent tight binding inhibitors (KI <150 pM) of chxEH while being relatively stable in chicken liver microsomes, suggesting their usefulness to study the role of EpFAs in chickens.

The soluble epoxide hydrolase (sEH) is a key enzyme in the metabolism of epoxy-fatty acids, signaling molecules involved in numerous biologies. Toward finding novel inhibitors of sEH, a library of known drugs was tested for inhibition of sEH. We found that fulvestrant, an anticancer agent, is a potent (KI = 26 nM) competitive inhibitor of sEH. From this observation, we found that alkyl-sulfoxides represent a new kind of pharmacophore for the inhibition of sEH.

The EPXH2 gene encodes soluble epoxide hydrolase (sEH), which has two distinct enzyme activities: epoxide hydrolase (Cterm-EH) and phosphatase (Nterm-phos). The Cterm-EH is involved in the metabolism of arachidonic acid epoxides that play important roles in blood pressure, cell growth, inflammation, and pain. While recent findings suggested complementary biological roles for Nterm-phos, research is limited by the lack of potent bioavailable inhibitors of this phosphatase activity. Also, a potent bioavailable inhibitor of this activity could be important in the development of therapy for cardiovascular diseases. We report herein the development of an HTS enzyme-based assay for Nterm-phos (Z′ > 0.9) using AttoPhos as the substrate. This assay was used to screen a wide variety of chemical entities, including a library of known drugs that have reached through clinical evaluation (Pharmakon 1600), as well as a library of pesticides and environmental toxins. We discovered that ebselen inhibits sEH phosphatase activity. Ebselen binds to the N-terminal domain of sEH (KI = 550 nM) and chemically reacts with the enzyme to quickly and irreversibly inhibit Nterm-phos, and subsequently Cterm-EH, and thus represents a new class of sEH inhibitor.

Adamantyl ureas were previously identified as a group of compounds active against Mycobacterium tuberculosis in culture with minimum inhibitor concentrations (MICs) below 0.1 μg/ml. These compounds have been shown to target MmpL3, a protein involved in secretion of trehalose mono-mycolate. They also inhibit both human soluble epoxide hydrolase (hsEH) and M. tuberculosis epoxide hydrolases. However, active compounds to date have high c Log P’s and are poorly soluble, leading to low bioavailability and thus limiting any therapeutic application. In this study, a library of 1600 ureas (mostly adamantyl ureas), which were synthesized for the purpose of increasing the bioavailability of inhibitors of hsEH, was screened for activity against M. tuberculosis. 1-Adamantyl-3-phenyl ureas with a polar para substituent were found to retain moderate activity against M. tuberculosis and one of these compounds was shown to be present in serum after oral administration to mice. However, neither it, nor a closely related analog, reduced M. tuberculosis infection in mice. No correlation between in vitro potency against M. tuberculosis and the hsEH inhibition were found supporting the concept that activity against hsEH and M. tuberculosis can be separated. Also there was a lack of correlation with c Log P and inhibition of the growth of M. tuberculosis. Finally, members of two classes of adamantyl ureas that contained polar components to increase their bioavailability, but lacked efficacy against growing M. tuberculosis, were found to taken up by the bacterium as effectively as a highly active apolar urea suggesting that these modifications to increase bioavailability affected the interaction of the urea against its target rather than making them unable to enter the bacterium.A library of 1600 ureas was screened in vitro for activity against Mycobacterium tuberculosis.

The microsomal epoxide hydrolase (mEH) plays a significant role in the metabolism of numerous xenobiotics. In addition, it has a potential role in sexual development and bile acid transport, and it is associated with a number of diseases such as emphysema, spontaneous abortion, eclampsia, and several forms of cancer. Toward developing chemical tools to study the biological role of mEH, we designed and synthesized a series of absorbent and fluorescent substrates. The highest activity for both rat and human mEH was obtained with the fluorescent substrate cyano(6-methoxy-naphthalen-2-yl)methyl glycidyl carbonate (11). An in vitro inhibition assay using this substrate ranked a series of known inhibitors similarly to the assay that used radioactive cis-stilbene oxide but with a greater discrimination between inhibitors. These results demonstrate that the new fluorescence-based assay is a useful tool for the discovery of structure–activity relationships among mEH inhibitors. Furthermore, this substrate could also be used for the screening chemical library with high accuracy and with a Z′ value of approximately 0.7. This new assay permits a significant decrease in labor and cost and also offers the advantage of a continuous readout. However, it should not be used with crude enzyme preparations due to interfering reactions.

l-Leucine aminopeptidases (LAPs) are implicated in the progress of many pathological disorders and play some regulatory roles in tumor cell proliferation, invasion, and/or angiogenesis. Thus, LAPs not only could become new diagnostic or prognostic biomarkers but also may have potential as novel molecular targets for the treatment of several cancers. Highly sensitive assays are critical for early detection of changes in LAP activity and for screening potent LAP inhibitors. In this study, we developed a novel and highly sensitive fluorescent assay for LAPs based on substituted aminopyridines as fluorescent reporters. This assay was at least 100- and 20-fold more sensitive than commercial colorimetric and fluorescent LAP substrates, respectively. We also showed that this assay was a useful tool for monitoring LAP activities in extracts from cancer cell lines, as well as for the high-throughput screening of inhibitors, which could lead to new cancer treatments.

A method for pharmacokinetic studies using cassette dosing associated with serial bleeding in mice is described. PK profiles of four soluble epoxide hydrolase inhibitors were determined following oral, subcutaneous or intraperitoneal administration individually or in cassette dosing. Parent analyses were performed on only 5 μL of whole blood from serial bleeds (up to 10 per animal), by LC/MS/MS. An accuracy (88–100%) and precision (<10% RSD) were observed, leading to reliable datum points for PK calculation. PK profiles, Tmax, Cmax and half-life values after cassette dosing were similar to the individual PK results. This method dramatically increases speed of data collection while dramatically reducing cost and animal usage. The results presented here clearly indicate that this proposed method could be applicable to high-throughput PK studies.