Kin Sing Stephen Lee

Epoxy metabolites of docosahexaenoic acid (DHA) inhibit angiogenesis, tumor growth, and metastasis

Epidemiological and preclinical evidence supports that omega-3 dietary fatty acids (fish oil) reduce the risks of macular degeneration and cancers, but the mechanisms by which these omega-3 lipids inhibit angiogenesis and tumorigenesis are poorly understood. Here we show that epoxydocosapentaenoic acids (EDPs), which are lipid mediators produced by cytochrome P450 epoxygenases from omega-3 fatty acid docosahexaenoic acid, inhibit VEGF- and fibroblast growth factor 2-induced angiogenesis in vivo, and suppress endothelial cell migration and protease production in vitro via a VEGF receptor 2-dependent mechanism. When EDPs (0.05 mg⋅kg−1⋅d−1) are coadministered with a low-dose soluble epoxide hydrolase inhibitor, EDPs are stabilized in circulation, causing ∼70% inhibition of primary tumor growth and metastasis. Contrary to the effects of EDPs, the corresponding metabolites derived from omega-6 arachidonic acid, epoxyeicosatrienoic acids, increase angiogenesis and tumor progression. These results designate epoxyeicosatrienoic acids and EDPs as unique endogenous mediators of an angiogenic switch to regulate tumorigenesis and implicate a unique mechanistic linkage between omega-3 and omega-6 fatty acids and cancers.

Tuning the electronic absorption of protein-embedded all-trans-retinal.

Seeing the Light Rhodopsins respond to a range of electromagnetic radiation—allowing visual perception over a broad wavelength range in animals and facilitating light-driven ion transport and phototaxis in microorganisms. All rhodopsins contain an embedded retinal chromophore in which absorbance is tuned by the protein environment. To gain insight into how the protein tunes absorbance, Wang et al. (p. 1340; see the Perspective by Sakmar) turned to a smaller soluble protein, cellular retinol binding protein II. They engineered the protein to fully encapsulate and covalently bind all-trans-retinal as a Schiff base. From this starting point, they used rational mutagenesis to vary the absorption maximum over a range of more than 200 nanometers by altering the electrostatic environment of the protein-binding pocket. Opsin-based light absorption was tuned over a 200-nanometer range by rationally engineering retinol-binding protein. Protein-chromophore interactions are a central component of a wide variety of critical biological processes such as color vision and photosynthesis. To understand the fundamental elements that contribute to spectral tuning of a chromophore inside the protein cavity, we redesigned human cellular retinol binding protein II (hCRBPII) to fully encapsulate all-trans-retinal and form a covalent bond as a protonated Schiff base. This system, using rational mutagenesis designed to alter the electrostatic environment within the binding pocket of the host protein, enabled regulation of the absorption maximum of the pigment in the range of 425 to 644 nanometers. With only nine point mutations, the hCRBPII mutants induced a systematic shift in the absorption profile of all-trans-retinal of more than 200 nanometers across the visible spectrum.

Unique mechanistic insights into the beneficial effects of soluble epoxide hydrolase inhibitors in the prevention of cardiac fibrosis

Tissue fibrosis represents one of the largest groups of diseases for which there are very few effective therapies. In the heart, myocardial infarction (MI) resulting in the loss of cardiac myocytes can culminate in adverse cardiac remodeling leading to eventual heart failure. Adverse cardiac remodeling includes myocyte hypertrophy, fibrosis, and electrical remodeling. We have previously demonstrated the beneficial effects of several potent soluble epoxide hydrolase inhibitors (sEHIs) in different models of cardiac hypertrophy and failure. Here, we directly determine the molecular mechanisms underlying the beneficial effects of sEHIs in cardiac remodeling post-MI. Treatment with a potent sEHI, 1-trifluoromethoxyphenyl-3-(1-propionylpiperidine-4-yl)urea (TPPU), which was started 1 wk post-MI in a murine model, results in a significant improvement in cardiac function. Importantly, treatment with TPPU results in a decrease in cardiac fibrosis as quantified using histological and immunostaining techniques. Moreover, single-cell–based assays demonstrate that treatment with TPPU results in a significant decrease not only in the percentages but also the proliferative capacity of different populations of cardiac fibroblasts as well as a reduction in the migration of fibroblasts into the heart from the bone marrow. Our study provides evidence for a possible unique therapeutic strategy to reduce cardiac fibrosis and improve cardiac function post-MI.

An omega-3 epoxide of docosahexaenoic acid lowers blood pressure in angiotensin-II-dependent hypertension.

Abstract: Mediators of antihypertensive actions of docosahexaenoic acid (DHA) are largely unknown. The omega-3 epoxide of DHA, 19, 20-EDP (epoxy docosapentaenoic acid), is metabolized by soluble epoxide hydrolase (sEH), which also metabolizes the anti-inflammatory and antihypertensive arachidonic acid epoxides, epoxyeicosatrienoic acids (EETs). Based in part on plasma levels of EDPs after a DHA-rich diet, we hypothesized that 19, 20-EDP contributes to the antihypertensive actions of DHA in angiotensin-II (Ang-II)–dependent hypertension. Treatment individually with 19, 20-EDP and a potent sEH inhibitor TPPU (1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea) significantly lowered blood pressure (BP) as compared with Ang-II–infused animals. The largest reduction in BP was obtained with the combination of 19, 20-EDP and TPPU, which was more efficacious than the combination of 14, 15-EET and TPPU. Oxylipin profiling revealed that 19, 20-EDP and 14, 15-EET infusion affected not only most metabolites of the P450 pathway but also renal levels of prostaglandin-E2. Our findings suggest that 19, 20-EDP is a mediator of the antihypertensive effects of DHA in Ang-II–dependent hypertension. It seems that 19, 20-EDP requires metabolic stabilization with a sEH inhibitor to be most effective in lowering BP, although both TPPU and 19, 20-EDP are so effective on their own that demonstrating additive or synergistic interactions is difficult.

Epoxy Fatty Acids and Inhibition of the Soluble Epoxide Hydrolase Selectively Modulate GABA Mediated Neurotransmission to Delay Onset of Seizures

In the brain, seizures lead to release of large amounts of polyunsaturated fatty acids including arachidonic acid (ARA). ARA is a substrate for three major enzymatic routes of metabolism by cyclooxygenase, lipoxygenase and cytochrome P450 enzymes. These enzymes convert ARA to potent lipid mediators including prostanoids, leukotrienes and epoxyeicosatrienoic acids (EETs). The prostanoids and leukotrienes are largely pro-inflammatory molecules that sensitize neurons whereas EETs are anti-inflammatory and reduce the excitability of neurons. Recent evidence suggests a GABA-related mode of action potentially mediated by neurosteroids. Here we tested this hypothesis using models of chemically induced seizures. The level of EETs in the brain was modulated by inhibiting the soluble epoxide hydrolase (sEH), the major enzyme that metabolizes EETs to inactive molecules, by genetic deletion of sEH and by direct administration of EETs into the brain. All three approaches delayed onset of seizures instigated by GABA antagonists but not seizures through other mechanisms. Inhibition of neurosteroid synthesis by finasteride partially blocked the anticonvulsant effects of sEH inhibitors while the efficacy of an inactive dose of neurosteroid allopregnanolone was enhanced by sEH inhibition. Consistent with earlier findings, levels of prostanoids in the brain were elevated. In contrast, levels of bioactive EpFAs were decreased following seizures. Overall these results demonstrate that EETs are natural molecules which suppress the tonic component of seizure related excitability through modulating the GABA activity and that exploration of the EET mediated signaling in the brain could yield alternative approaches to treat convulsive disorders.

Endoplasmic reticulum stress in the peripheral nervous system is a significant driver of neuropathic pain.

Significance Here we define the causative role of endoplasmic reticulum (ER) stress on selective modulation of pain signaling. High levels of ER stress and neuropathic pain in diabetic animals are reduced using ER stress blockers. In healthy animals, turning on the ER stress signal transduction cascade generates an immediate but lasting and site restricted painful phenotype, which is reversible by ER stress blockers. This previously unnoticed mechanism explains the broad lack of efficacy of available analgesics and should ignite the discovery of a new generation of therapeutics that do not directly quell ion channel or neurotransmitter activity. Despite intensive effort and resulting gains in understanding the mechanisms underlying neuropathic pain, limited success in therapeutic approaches have been attained. A recently identified, nonchannel, nonneurotransmitter therapeutic target for pain is the enzyme soluble epoxide hydrolase (sEH). The sEH degrades natural analgesic lipid mediators, epoxy fatty acids (EpFAs), therefore its inhibition stabilizes these bioactive mediators. Here we demonstrate the effects of EpFAs on diabetes induced neuropathic pain and define a previously unknown mechanism of pain, regulated by endoplasmic reticulum (ER) stress. The activation of ER stress is first quantified in the peripheral nervous system of type I diabetic rats. We demonstrate that both pain and markers of ER stress are reversed by a chemical chaperone. Next, we identify the EpFAs as upstream modulators of ER stress pathways. Chemical inducers of ER stress invariably lead to pain behavior that is reversed by a chemical chaperone and an inhibitor of sEH. The rapid occurrence of pain behavior with inducers, equally rapid reversal by blockers and natural incidence of ER stress in diabetic peripheral nervous system (PNS) argue for a major role of the ER stress pathways in regulating the excitability of the nociceptive system. Understanding the role of ER stress in generation and maintenance of pain opens routes to exploit this system for therapeutic purposes.

Rational Design of a Colorimetric pH Sensor from a Soluble Retinoic Acid Chaperone

Reengineering of cellular retinoic acid binding protein II (CRABPII) to be capable of binding retinal as a protonated Schiff base is described. Through rational alterations of the binding pocket, electrostatic perturbations of the embedded retinylidene chromophore that favor delocalization of the iminium charge lead to exquisite control in the regulation of chromophoric absorption properties, spanning the visible spectrum (474-640 nm). The pKa of the retinylidene protonated Schiff base was modulated from 2.4 to 8.1, giving rise to a set of proteins of varying colors and pH sensitivities. These proteins were used to demonstrate a concentration-independent, ratiometric pH sensor.

“Turn-On” Protein Fluorescence: In Situ Formation of Cyanine Dyes

Protein reengineering of cellular retinoic acid binding protein II (CRABPII) has yielded a genetically addressable system, capable of binding a profluorophoric chromophore that results in fluorescent protein/chromophore complexes. These complexes exhibit far-red emission, with high quantum efficiencies and brightness and also exhibit excellent pH stability spanning the range of 2–11. In the course of this study, it became evident that single mutations of L121E and R59W were most effective in improving the fluorescent characteristics of CRABPII mutants as well as the kinetics of complex formation. The readily crystallizable nature of these proteins was invaluable to provide clues for the observed spectroscopic behavior that results from single mutation of key residues.

Optimized Inhibitors of Soluble Epoxide Hydrolase Improve in Vitro Target Residence Time and in Vivo Efficacy

Diabetes is affecting the life of millions of people. A large proportion of diabetic patients suffer from severe complications such as neuropathic pain, and current treatments for these complications have deleterious side effects. Thus, alternate therapeutic strategies are needed. Recently, the elevation of epoxy-fatty acids through inhibition of soluble epoxide hydrolase (sEH) was shown to reduce diabetic neuropathic pain in rodents. In this report, we describe a series of newly synthesized sEH inhibitors with at least 5-fold higher potency and doubled residence time inside both the human and rodent sEH enzyme than previously reported inhibitors. These inhibitors also have better physical properties and optimized pharmacokinetic profiles. The optimized inhibitor selected from this new series displayed improved efficacy of almost 10-fold in relieving pain perception in diabetic neuropathic rats as compared to the approved drug, gabapentin, and previously published sEH inhibitors. Therefore, these new sEH inhibitors could be an attractive alternative to treat diabetic neuropathy in humans.

Symmetric adamantyl-diureas as soluble epoxide hydrolase inhibitors

A series of inhibitors of the soluble epoxide hydrolase (sEH) containing two urea groups has been developed. Inhibition potency of the described compounds ranges from 2.0 μM to 0.4 nM. 1,6-(Hexamethylene)bis[(adamant-1-yl)urea] (3b) was found to be a potent slow tight binding inhibitor (IC50=0.5 nM) with a strong binding to sEH (Ki=3.1 nM) and a moderately long residence time on the enzyme (koff=1.05 × 10(-3) s(-1); t1/2=11 min).