Publications by authors named "Philip J Kingsley"

51 Publications

In vivo endocannabinoid dynamics at the timescale of physiological and pathological neural activity.

Neuron 2021 08;109(15):2398-2403.e4

Department of Neurosurgery, Stanford University, Stanford, CA 94305, USA.

The brain's endocannabinoid system is a powerful controller of neurotransmitter release, shaping synaptic communication under physiological and pathological conditions. However, our understanding of endocannabinoid signaling in vivo is limited by the inability to measure their changes at timescales commensurate with the high lability of lipid signals, leaving fundamental questions of whether, how, and which endocannabinoids fluctuate with neural activity unresolved. Using novel imaging approaches in awake behaving mice, we now demonstrate that the endocannabinoid 2-arachidonoylglycerol, not anandamide, is dynamically coupled to hippocampal neural activity with high spatiotemporal specificity. Furthermore, we show that seizures amplify the physiological endocannabinoid increase by orders of magnitude and drive the downstream synthesis of vasoactive prostaglandins that culminate in a prolonged stroke-like event. These results shed new light on normal and pathological endocannabinoid signaling in vivo.
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http://dx.doi.org/10.1016/j.neuron.2021.05.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8351909PMC
August 2021

Targeting diacylglycerol lipase reduces alcohol consumption in preclinical models.

J Clin Invest 2021 Jul 22. Epub 2021 Jul 22.

Department of Psychiatry and Behavioral Sciences, Vanderbilt University School of Medicine, Nashville, United States of America.

Alcohol use disorder (AUD) is associated with substantial morbidity, mortality, and societal cost, and pharmacological treatment options for AUD are limited. The endogenous cannabinoid (eCB) signaling system is critically involved in reward processing and alcohol intake is positively correlated with release of the eCB ligand 2-Arachidonoylglycerol (2-AG) within reward neurocircuitry. Here we show that genetic and pharmacological inhibition of diacylglycerol lipase (DAGL), the rate limiting enzyme in the synthesis of 2-AG, reduces alcohol consumption in a variety of preclinical models ranging from a voluntary free-access model to aversion resistant-drinking and dependence-like drinking induced via chronic intermittent ethanol vapor exposure in mice. DAGL inhibition during either chronic alcohol consumption or protracted withdrawal was devoid of anxiogenic and depressive-like behavioral effects. Lastly, DAGL inhibition also prevented ethanol-induced suppression of GABAergic transmission onto midbrain dopamine neurons, providing mechanistic insight into how DAGL inhibition could affect alcohol reward. These data suggest reducing 2-AG signaling via inhibition of DAGL could represent an effective approach to reduce alcohol consumption across the spectrum of AUD severity.
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http://dx.doi.org/10.1172/JCI146861DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8409586PMC
July 2021

Molecular Imaging of Inflammation in Osteoarthritis Using a Water-Soluble Fluorocoxib.

ACS Med Chem Lett 2020 Oct 24;11(10):1875-1880. Epub 2020 Feb 24.

A. B. Hancock, Jr. Memorial Laboratory for Cancer Research, Departments of Biochemistry, Chemistry, and Pharmacology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, United States.

Clinical imaging approaches to detect inflammatory biomarkers, such as cyclooxygenase-2 (COX-2), may facilitate the diagnosis and therapy of inflammatory diseases. To this end, we report the discovery of -[(rhodamin-X-yl)but-4-yl]-2-[1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1-indol-3-yl]acetamide chloride salt (fluorocoxib D), a hydrophilic analog of fluorocoxib A. Fluorocoxib D inhibits COX-2 selectively in purified enzyme preparations and cells. It exhibits adequate photophysical properties to enable detection of COX-2 in intact cells, in a mouse model of carrageenan-induced acute footpad inflammation and inflammation in a mouse model of osteoarthritis. COX-2-selectivity was verified either by blocking the enzyme's active site with celecoxib or by molecular imaging with nontargeted 5-carboxy-X-rhodamine dye. These data indicate that fluorocoxib D is an ideal candidate for early detection of inflammatory or neoplastic lesions expressing elevated levels of COX-2.
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http://dx.doi.org/10.1021/acsmedchemlett.9b00512DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7549260PMC
October 2020

Aspects of Prostaglandin Glycerol Ester Biology.

Adv Exp Med Biol 2019 ;1161:77-88

A. B. Hancock Memorial Laboratory for Cancer Research, Departments of Biochemistry, Chemistry, and Pharmacology, Vanderbilt Institute of Chemical Biology, Vanderbilt University, Nashville, TN, USA.

The Cyclooxygenase enzymes (COX-1 and COX-2) incorporate 2 molecules of O into arachidonic acid (AA), resulting in an array of bioactive prostaglandins. However, much work has been done showing that COX-2 will perform this reaction on several different AA-containing molecules, most importantly, the endocannabinoid 2-arachidonoylglycerol (2-AG). The products of 2-AG oxygenation, prostaglandin glycerol esters (PG-Gs), are analogous to canonical prostaglandins. This chapter reviews the literature detailing the production, metabolism, and bioactivity of these compounds, as well as their detection in intact animals.
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http://dx.doi.org/10.1007/978-3-030-21735-8_8DOI Listing
October 2019

IRE1α-XBP1 signaling in leukocytes controls prostaglandin biosynthesis and pain.

Science 2019 07;365(6450)

Weill Cornell Graduate School of Medical Sciences, Cornell University. New York, NY 10065, USA.

Inositol-requiring enzyme 1[α] (IRE1[α])-X-box binding protein spliced (XBP1) signaling maintains endoplasmic reticulum (ER) homeostasis while controlling immunometabolic processes. Yet, the physiological consequences of IRE1α-XBP1 activation in leukocytes remain unexplored. We found that induction of prostaglandin-endoperoxide synthase 2 (/Cox-2) and prostaglandin E synthase (/mPGES-1) was compromised in IRE1α-deficient myeloid cells undergoing ER stress or stimulated through pattern recognition receptors. Inducible biosynthesis of prostaglandins, including the pro-algesic mediator prostaglandin E2 (PGE), was decreased in myeloid cells that lack IRE1α or XBP1 but not other ER stress sensors. Functional XBP1 transactivated the human and genes to enable optimal PGE production. Mice that lack IRE1α-XBP1 in leukocytes, or that were treated with IRE1α inhibitors, demonstrated reduced pain behaviors in PGE-dependent models of pain. Thus, IRE1α-XBP1 is a mediator of prostaglandin biosynthesis and a potential target to control pain.
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http://dx.doi.org/10.1126/science.aau6499DOI Listing
July 2019

Discovery of Furanone-Based Radiopharmaceuticals for Diagnostic Targeting of COX-1 in Ovarian Cancer.

ACS Omega 2019 May 24;4(5):9251-9261. Epub 2019 May 24.

A. B. Hancock, Jr., Memorial Laboratory for Cancer Research, Department of Biochemistry, Chemistry and Pharmacology, Vanderbilt Institute of Chemical Biology, Vanderbilt-Ingram Cancer Center, and Department of Radiology and Radiological Sciences, Vanderbilt Institute of Imaging Sciences, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, United States.

In vivo targeting and visualization of cyclooxygenase-1 (COX-1) using multimodal positron emission tomography/computed tomography imaging represents a unique opportunity for early detection and/or therapeutic evaluation of ovarian cancer because overexpression of COX-1 has been characterized as a pathologic hallmark of the initiation and progression of this disease. The furanone core is a common building block of many synthetic and natural products that exhibit a wide range of biological activities. We hypothesize that furanone-based COX-1 inhibitors can be designed as imaging agents for the early detection, delineation of tumor margin, and evaluation of treatment response of ovarian cancer. We report the discovery of 3-(4-fluorophenyl)-5,5-dimethyl-4-(-tolyl)furan-2(5)-one (FDF), a furanone-based novel COX-1-selective inhibitor that exhibits adequate in vivo stability, plasma half-life, and pharmacokinetic properties for use as an imaging agent. We describe a novel synthetic scheme in which a Lewis acid-catalyzed nucleophilic aromatic deiodo[F]fluorination reaction is utilized for the radiosynthesis of [F]FDF. [F]FDF binds efficiently to COX-1 in vivo and enables sensitive detection of ovarian cancer in subcutaneous and peritoneal xenograft models in mice. These results provide the proof of principle for COX-1-targeted imaging of ovarian cancer and identify [F]FDF as a promising lead compound for further preclinical and clinical development.
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http://dx.doi.org/10.1021/acsomega.9b01093DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6545551PMC
May 2019

The association between endogenous opioid function and morphine responsiveness: a moderating role for endocannabinoids.

Pain 2019 Mar;160(3):676-687

Department of Psychiatry, Vanderbilt University Medical Center, Nashville, TN, United States.

We sought to replicate previous findings that low endogenous opioid (EO) function predicts greater morphine analgesia and extended these findings by examining whether circulating endocannabinoids and related lipids moderate EO-related predictive effects. Individuals with chronic low-back pain (n = 46) provided blood samples for endocannabinoid analyses, then underwent separate identical laboratory sessions under 3 drug conditions: saline placebo, intravenous (i.v.) naloxone (opioid antagonist; 12-mg total), and i.v. morphine (0.09-mg/kg total). During each session, participants rated low-back pain intensity, evoked heat pain intensity, and nonpain subjective effects 4 times in sequence after incremental drug dosing. Mean morphine effects (morphine-placebo difference) and opioid blockade effects (naloxone-placebo difference; to index EO function) for each primary outcome (low-back pain intensity, evoked heat pain intensity, and nonpain subjective effects) were derived by averaging across the 4 incremental doses. The association between EO function and morphine-induced back pain relief was significantly moderated by endocannabinoids [2-arachidonoylglycerol (2-AG) and N-arachidonoylethanolamine (AEA)]. Lower EO function predicted greater morphine analgesia only for those with relatively lower endocannabinoids. Endocannabinoids also significantly moderated EO effects on morphine-related changes in visual analog scale-evoked pain intensity (2-AG), drug liking (AEA and 2-AG), and desire to take again (AEA and 2-AG). In the absence of significant interactions, lower EO function predicted significantly greater morphine analgesia (as in past work) and euphoria. Results indicate that EO effects on analgesic and subjective responses to opioid medications are greatest when endocannabinoid levels are low. These findings may help guide development of mechanism-based predictors for personalized pain medicine algorithms.
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http://dx.doi.org/10.1097/j.pain.0000000000001447DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6377294PMC
March 2019

Lysophospholipases cooperate to mediate lipid homeostasis and lysophospholipid signaling.

J Lipid Res 2019 02 27;60(2):360-374. Epub 2018 Nov 27.

A. B. Hancock Jr. Memorial Laboratory for Cancer Research, Departments of Chemistry, Vanderbilt University School of Medicine, Nashville, TN 37232-0146

Lysophospholipids (LysoPLs) are bioactive lipid species involved in cellular signaling processes and the regulation of cell membrane structure. LysoPLs are metabolized through the action of lysophospholipases, including lysophospholipase A1 (LYPLA1) and lysophospholipase A2 (LYPLA2). A new X-ray crystal structure of LYPLA2 compared with a previously published structure of LYPLA1 demonstrated near-identical folding of the two enzymes; however, LYPLA1 and LYPLA2 have displayed distinct substrate specificities in recombinant enzyme assays. To determine how these in vitro substrate preferences translate into a relevant cellular setting and better understand the enzymes' role in LysoPL metabolism, CRISPR-Cas9 technology was utilized to generate stable KOs of and/or in Neuro2a cells. Using these cellular models in combination with a targeted lipidomics approach, LysoPL levels were quantified and compared between cell lines to determine the effect of losing lysophospholipase activity on lipid metabolism. This work suggests that LYPLA1 and LYPLA2 are each able to account for the loss of the other to maintain lipid homeostasis in cells; however, when both are deleted, LysoPL levels are dramatically increased, causing phenotypic and morphological changes to the cells.
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http://dx.doi.org/10.1194/jlr.M087890DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6358295PMC
February 2019

Methylglyoxal-derived posttranslational arginine modifications are abundant histone marks.

Proc Natl Acad Sci U S A 2018 09 27;115(37):9228-9233. Epub 2018 Aug 27.

A.B. Hancock Memorial Laboratory for Cancer Research, Vanderbilt University, Nashville, TN 37232;

Histone posttranslational modifications (PTMs) regulate chromatin dynamics, DNA accessibility, and transcription to expand the genetic code. Many of these PTMs are produced through cellular metabolism to offer both feedback and feedforward regulation. Herein we describe the existence of Lys and Arg modifications on histones by a glycolytic by-product, methylglyoxal (MGO). Our data demonstrate that adduction of histones by MGO is an abundant modification, present at the same order of magnitude as Arg methylation. These modifications were detected on all four core histones at critical residues involved in both nucleosome stability and reader domain binding. In addition, MGO treatment of cells lacking the major detoxifying enzyme, glyoxalase 1, results in marked disruption of H2B acetylation and ubiquitylation without affecting H2A, H3, and H4 modifications. Using RNA sequencing, we show that MGO is capable of altering gene transcription, most notably in cells lacking GLO1. Finally, we show that the deglycase DJ-1 protects histones from adduction by MGO. Collectively, our findings demonstrate the existence of a previously undetected histone modification derived from glycolysis, which may have far-reaching implications for the control of gene expression and protein transcription linked to metabolism.
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http://dx.doi.org/10.1073/pnas.1802901115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6140490PMC
September 2018

Detection of Cyclooxygenase-2-Derived Oxygenation Products of the Endogenous Cannabinoid 2-Arachidonoylglycerol in Mouse Brain.

ACS Chem Neurosci 2018 07 9;9(7):1552-1559. Epub 2018 May 9.

Cyclooxygenase-2 (COX-2) catalyzes the formation of prostaglandins, which are involved in immune regulation, vascular function, and synaptic signaling. COX-2 also inactivates the endogenous cannabinoid (eCB) 2-arachidonoylglycerol (2-AG) via oxygenation of its arachidonic acid backbone to form a variety of prostaglandin glyceryl esters (PG-Gs). Although this oxygenation reaction is readily observed in vitro and in intact cells, detection of COX-2-derived 2-AG oxygenation products has not been previously reported in neuronal tissue. Here we show that 2-AG is metabolized in the brain of transgenic COX-2-overexpressing mice and mice treated with lipopolysaccharide to form multiple species of PG-Gs that are detectable only when monoacylglycerol lipase is concomitantly blocked. Formation of these PG-Gs is prevented by acute pharmacological inhibition of COX-2. These data provide evidence that neuronal COX-2 is capable of oxygenating 2-AG to form a variety PG-Gs in vivo and support further investigation of the physiological functions of PG-Gs.
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http://dx.doi.org/10.1021/acschemneuro.7b00499DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6081739PMC
July 2018

Therapeutic endocannabinoid augmentation for mood and anxiety disorders: comparative profiling of FAAH, MAGL and dual inhibitors.

Transl Psychiatry 2018 04 26;8(1):92. Epub 2018 Apr 26.

Department of Psychiatry and Behavioral Sciences, Vanderbilt University Medical Center, Nashville, TN, USA.

Recent studies have demonstrated anxiolytic potential of pharmacological endocannabinoid (eCB) augmentation approaches in a variety of preclinical models. Pharmacological inhibition of endocannabinoid-degrading enzymes, such as fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), elicit promising anxiolytic effects in rodent models with limited adverse behavioral effects, however, the efficacy of dual FAAH/MAGL inhibition has not been investigated. In the present study, we compared the effects of FAAH (PF-3845), MAGL (JZL184) and dual FAAH/MAGL (JZL195) inhibitors on (1) anxiety-like behaviors under non-stressed and stressed conditions, (2) locomotor activity and body temperature, (3) lipid levels in the brain and (4) cognitive functions. Behavioral analysis showed that PF-3845 or JZL184, but not JZL195, was able to prevent restraint stress-induced anxiety in the light-dark box assay when administered before stress exposure. Moreover, JZL195 treatment was not able to reverse foot shock-induced anxiety-like behavior in the elevated zero maze or light-dark box. JZL195, but not PF-3845 or JZL184, decreased body temperature and increased anxiety-like behavior in the open-field test. Overall, JZL195 did not show anxiolytic efficacy and the effects of JZL184 were more robust than that of PF-3845 in the models examined. These results showed that increasing either endogenous AEA or 2-AG separately produces anti-anxiety effects under stressful conditions but the same effects are not obtained from simultaneously increasing both AEA and 2-AG.
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http://dx.doi.org/10.1038/s41398-018-0141-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5917016PMC
April 2018

Oxidative stress increases M1dG, a major peroxidation-derived DNA adduct, in mitochondrial DNA.

Nucleic Acids Res 2018 04;46(7):3458-3467

A.B. Hancock, Jr., Memorial Laboratory for Cancer Research, Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN, USA.

Reactive oxygen species (ROS) are formed in mitochondria during electron transport and energy generation. Elevated levels of ROS lead to increased amounts of mitochondrial DNA (mtDNA) damage. We report that levels of M1dG, a major endogenous peroxidation-derived DNA adduct, are 50-100-fold higher in mtDNA than in nuclear DNA in several different human cell lines. Treatment of cells with agents that either increase or decrease mitochondrial superoxide levels leads to increased or decreased levels of M1dG in mtDNA, respectively. Sequence analysis of adducted mtDNA suggests that M1dG residues are randomly distributed throughout the mitochondrial genome. Basal levels of M1dG in mtDNA from pulmonary microvascular endothelial cells (PMVECs) from transgenic bone morphogenetic protein receptor 2 mutant mice (BMPR2R899X) (four adducts per 106 dG) are twice as high as adduct levels in wild-type cells. A similar increase was observed in mtDNA from heterozygous null (BMPR2+/-) compared to wild-type PMVECs. Pulmonary arterial hypertension is observed in the presence of BMPR2 signaling disruptions, which are also associated with mitochondrial dysfunction and oxidant injury to endothelial tissue. Persistence of M1dG adducts in mtDNA could have implications for mutagenesis and mitochondrial gene expression, thereby contributing to the role of mitochondrial dysfunction in diseases.
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http://dx.doi.org/10.1093/nar/gky089DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5909422PMC
April 2018

Functional Redundancy Between Canonical Endocannabinoid Signaling Systems in the Modulation of Anxiety.

Biol Psychiatry 2017 Oct 15;82(7):488-499. Epub 2017 Mar 15.

Department of Psychiatry and Behavioral Sciences, Vanderbilt University Medical Center, Nashville, Tennessee; Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee. Electronic address:

Background: Increasing the available repertoire of effective treatments for mood and anxiety disorders represents a critical unmet need. Pharmacological augmentation of endogenous cannabinoid (eCB) signaling has been suggested to represent a novel approach to the treatment of anxiety disorders; however, the functional interactions between two canonical eCB pathways mediated via anandamide (N-arachidonylethanolamine [AEA]) and 2-arachidonoylglycerol (2-AG) in the regulation of anxiety are not well understood.

Methods: We utilized pharmacological augmentation and depletion combined with behavioral and electrophysiological approaches to probe the role of 2-AG signaling in the modulation of stress-induced anxiety and the functional redundancy between AEA and 2-AG signaling in the modulation of anxiety-like behaviors in mice.

Results: Selective 2-AG augmentation reduced anxiety in the light/dark box assay and prevented stress-induced increases in anxiety associated with limbic AEA deficiency. In contrast, acute 2-AG depletion increased anxiety-like behaviors, which was normalized by selective pharmacological augmentation of AEA signaling and via direct cannabinoid receptor 1 stimulation with Δ-tetrahydrocannabinol. Electrophysiological studies revealed 2-AG modulation of amygdala glutamatergic transmission as a key synaptic correlate of the anxiolytic effects of 2-AG augmentation.

Conclusions: Although AEA and 2-AG likely subserve distinct physiological roles, a pharmacological and functional redundancy between these canonical eCB signaling pathways exists in the modulation of anxiety-like behaviors. These data support development of eCB-based treatment approaches for mood and anxiety disorders and suggest a potentially wider therapeutic overlap between AEA and 2-AG augmentation approaches than was previously appreciated.
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http://dx.doi.org/10.1016/j.biopsych.2017.03.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5585044PMC
October 2017

Quantitative Analysis and Discovery of Lysine and Arginine Modifications.

Anal Chem 2017 01 30;89(2):1299-1306. Epub 2016 Dec 30.

Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado , Anschutz Medical Campus, Aurora, Colorado 80045, United States.

Post-translational modifications (PTMs) affect protein function, localization, and stability, yet very little is known about the ratios of these modifications. Here, we describe a novel method to quantitate and assess the relative stoichiometry of Lys and Arg modifications (QuARKMod) in complex biological settings. We demonstrate the versatility of this platform in monitoring recombinant protein modification of peptide substrates, PTMs of individual histones, and the relative abundance of these PTMs as a function of subcellular location. Lastly, we describe a product ion scanning technique that offers the potential to discover unexpected and possibly novel Lys and Arg modifications. In summary, this approach yields accurate quantitation and discovery of protein PTMs in complex biological systems without the requirement of high mass accuracy instrumentation.
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http://dx.doi.org/10.1021/acs.analchem.6b04105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5309163PMC
January 2017

Antitumor Activity of Cytotoxic Cyclooxygenase-2 Inhibitors.

ACS Chem Biol 2016 11 19;11(11):3052-3060. Epub 2016 Sep 19.

Departments of Biochemistry, Chemistry, and Pharmacology, A.B. Hancock Memorial Laboratory for Cancer Research, Vanderbilt Institute of Chemical Biology, Vanderbilt University School of Medicine , 850 RRB, 2220 Pierce Ave., Nashville, Tennessee 37232, United States.

Targeted delivery of chemotherapeutic agents to tumors has been explored as a means to increase the selectivity and potency of cytotoxicity. Most efforts in this area have exploited the molecular recognition of proteins highly expressed on the surface of cancer cells followed by internalization. A related approach that has received less attention is the targeting of intracellular proteins by ligands conjugated to anticancer drugs. An attractive target for this approach is the enzyme cyclooxygenase-2 (COX-2), which is highly expressed in a range of malignant tumors. Herein, we describe the synthesis and evaluation of a series of chemotherapeutic agents targeted to COX-2 by conjugation to indomethacin. Detailed characterization of compound 12, a conjugate of indomethacin with podophyllotoxin, revealed highly potent and selective COX-2 inhibition in vitro and in intact cells. Kinetics and X-ray crystallographic studies demonstrated that compound 12 is a slow, tight-binding inhibitor that likely binds to COX-2's allosteric site with its indomethacin moiety in a conformation similar to that of indomethacin. Compound 12 exhibited cytotoxicity in cell culture similar to that of podophyllotoxin with no evidence of COX-2-dependent selectivity. However, in vivo, compound 12 accumulated selectively in and more effectively inhibited the growth of a COX-2-expressing xenograft compared to a xenograft that did not express COX-2. Compound 12, which we have named chemocoxib A, provides proof-of-concept for the in vivo targeting of chemotherapeutic agents to COX-2 but suggests that COX-2-dependent selectivity may not be evident in cell culture-based assays.
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http://dx.doi.org/10.1021/acschembio.6b00560DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5289892PMC
November 2016

Assay of Endocannabinoid Oxidation by Cyclooxygenase-2.

Methods Mol Biol 2016 ;1412:205-15

A.B. Hancock Jr. Memorial Laboratory for Cancer Research, Vanderbilt Institute of Chemical Biology, Vanderbilt Center in Molecular Toxicology, Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, TN, 37232, USA.

The endocannabinoids, 2-arachidonoylglycerol (2-AG) and arachidonylethanolamide (AEA), are endogenous ligands for the cannabinoid receptors (CB1 and CB2) and are implicated in a wide array of physiological processes. These neutral arachidonic acid (AA) derivatives have been identified as efficient substrates for the second isoform of the cyclooxygenase enzyme (COX-2). A diverse family of prostaglandin glycerol esters (PG-Gs) and prostaglandin ethanolamides (PG-EAs) is generated by the action of COX-2 (and downstream prostaglandin synthases) on 2-AG and AEA. As the biological importance of the endocannabinoid system becomes more apparent, there is a tremendous need for robust, sensitive, and efficient analytical methodology for the endocannabinoids and their metabolites. In this chapter, we describe methodology suitable for carrying out oxygenation of endocannabinoids by COX-2, and analysis of products of endocannabinoid oxygenation by COX-2 and of endocannabinoids themselves from in vitro and cell assays.
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http://dx.doi.org/10.1007/978-1-4939-3539-0_21DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5289390PMC
December 2017

Fluorocoxib A loaded nanoparticles enable targeted visualization of cyclooxygenase-2 in inflammation and cancer.

Biomaterials 2016 Jun 21;92:71-80. Epub 2016 Mar 21.

Department of Biomedical Engineering, Vanderbilt Institute for Nanoscale Science and Engineering, Vanderbilt University School of Engineering, Nashville, TN 37232, USA. Electronic address:

Cyclooxygenase-2 (COX-2) is expressed in virtually all solid tumors and its overexpression is a hallmark of inflammation. Thus, it is a potentially powerful biomarker for the early clinical detection of inflammatory disease and human cancers. We report a reactive oxygen species (ROS) responsive micellar nanoparticle, PPS-b-POEGA, that solubilizes the first fluorescent COX-2-selective inhibitor fluorocoxib A (FA) for COX-2 visualization in vivo. Pharmacokinetics and biodistribution of FA-PPS-b-POEGA nanoparticles (FA-NPs) were assessed after a fully-aqueous intravenous (i.v.) administration in wild-type mice and revealed 4-8 h post-injection as an optimal fluorescent imaging window. Carrageenan-induced inflammation in the rat and mouse footpads and 1483 HNSCC tumor xenografts were successfully visualized by FA-NPs with fluorescence up to 10-fold higher than that of normal tissues. The targeted binding of the FA cargo was blocked by pretreatment with the COX-2 inhibitor indomethacin, confirming COX-2-specific binding and local retention of FA at pathological sites. Our collective data indicate that FA-NPs are the first i.v.-ready FA formulation, provide high signal-to-noise in inflamed, premalignant, and malignant tissues, and will uniquely enable clinical translation of the poorly water-soluble FA compound.
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http://dx.doi.org/10.1016/j.biomaterials.2016.03.028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4833621PMC
June 2016

Cox-2-derived PGE2 induces Id1-dependent radiation resistance and self-renewal in experimental glioblastoma.

Neuro Oncol 2016 10 28;18(10):1379-89. Epub 2016 Mar 28.

Cancer Biology and Genetics Program, Memorial Sloan Kettering Cancer Center, New York, New York (P.J.C., R.T., R.B.); Department of Molecular Biology, Weill Cornell Graduate School of Medical Sciences of Cornell University, New York, New York (R.T.); Departments of Biochemistry, Chemistry, and Pharmacology, A.B. Hancock Jr. Memorial Laboratory for Cancer Research, Vanderbilt Institute of Chemical Biology, Vanderbilt University School of Medicine, Nashville, Tennessee (P.J.K., L.J.M.); Department of Neurosurgery, Memorial Sloan Kettering Cancer Center, New York, New York (F.S., V.S.T); Department of Medicine, Weill Cornell Medical College, New York, (D.C.M, A.J.D.)

Background: In glioblastoma (GBM), Id1 serves as a functional marker for self-renewing cancer stem-like cells. We investigated the mechanism by which cyclooxygenase-2 (Cox-2)-derived prostaglandin E2 (PGE2) induces Id1 and increases GBM self-renewal and radiation resistance.

Methods: Mouse and human GBM cells were stimulated with dimethyl-PGE2 (dmPGE2), a stabilized form of PGE2, to test for Id1 induction. To elucidate the signal transduction pathway governing the increase in Id1, a combination of short interfering RNA knockdown and small molecule inhibitors and activators of PGE2 signaling were used. Western blotting, quantitative real-time (qRT)-PCR, and chromatin immunoprecipitation assays were employed. Sphere formation and radiation resistance were measured in cultured primary cells. Immunohistochemical analyses were carried out to evaluate the Cox-2-Id1 axis in experimental GBM.

Results: In GBM cells, dmPGE2 stimulates the EP4 receptor leading to activation of ERK1/2 MAPK. This leads, in turn, to upregulation of the early growth response1 (Egr1) transcription factor and enhanced Id1 expression. Activation of this pathway increases self-renewal capacity and resistance to radiation-induced DNA damage, which are dependent on Id1.

Conclusions: In GBM, Cox-2-derived PGE2 induces Id1 via EP4-dependent activation of MAPK signaling and the Egr1 transcription factor. PGE2-mediated induction of Id1 is required for optimal tumor cell self-renewal and radiation resistance. Collectively, these findings identify Id1 as a key mediator of PGE2-dependent modulation of radiation response and lend insight into the mechanisms underlying radiation resistance in GBM patients.
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http://dx.doi.org/10.1093/neuonc/now049DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5035519PMC
October 2016

Nuclear Oxidation of a Major Peroxidation DNA Adduct, M1dG, in the Genome.

Chem Res Toxicol 2015 Dec 11;28(12):2334-42. Epub 2015 Nov 11.

A.B. Hancock Jr. Memorial Laboratory for Cancer Research, Departments of †Biochemistry, ‡Chemistry, and §Pharmacology, Vanderbilt Institute of Chemical Biology, Center in Molecular Toxicology, Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine , Nashville, Tennessee 37232-0146, United States.

Chronic inflammation results in increased production of reactive oxygen species (ROS), which can oxidize cellular molecules including lipids and DNA. Our laboratory has shown that 3-(2-deoxy-β-d-erythro-pentofuranosyl)pyrimido[1,2-α]purin-10(3H)-one (M1dG) is the most abundant DNA adduct formed from the lipid peroxidation product, malondialdehyde, or the DNA peroxidation product, base propenal. M1dG is mutagenic in bacterial and mammalian cells and is repaired via the nucleotide excision repair system. Here, we report that M1dG levels in intact DNA were increased from basal levels of 1 adduct per 10(8) nucleotides to 2 adducts per 10(6) nucleotides following adenine propenal treatment of RKO, HEK293, or HepG2 cells. We also found that M1dG in genomic DNA was oxidized in a time-dependent fashion to a single product, 6-oxo-M1dG (to ∼ 5 adducts per 10(7) nucleotides), and that this oxidation correlated with a decline in M1dG levels. Investigations in RAW264.7 macrophages indicate the presence of high basal levels of M1dG (1 adduct per 10(6) nucleotides) and the endogenous formation of 6-oxo-M1dG. This is the first report of the production of 6-oxo-M1dG in genomic DNA in intact cells, and it has significant implications for understanding the role of inflammation in DNA damage, mutagenesis, and repair.
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http://dx.doi.org/10.1021/acs.chemrestox.5b00340DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4993022PMC
December 2015

Multitargeting of selected prostanoid receptors provides agents with enhanced anti-inflammatory activity in macrophages.

FASEB J 2016 Jan 29;30(1):394-404. Epub 2015 Sep 29.

*Department of Biological Sciences, Allergan, Incorporated, Irvine, California, USA; Department of Bioengineering, Imperial College London, London, United Kingdom; Drug Discovery Department, Selcia Limited, Ongar, United Kingdom; and Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee, USA.

A polypharmacologic approach to prostanoid based anti-inflammatory therapeutics was undertaken in order to exploit both the anti- and proinflammatory properties attributed to the various prostanoid receptors. Multitargeting of selected prostanoid receptors yielded a prototype compound, compound 1 (AGN 211377), that antagonizes prostaglandin D2 receptors (DPs) DP1 (49) and DP2 (558), prostaglandin E2 receptors (EPs) EP1 (266) and EP4 (117), prostaglandin F2α receptor (FP) (61), and thromboxane A2 receptor (TP) (11) while sparing EP2, EP3, and prostaglandin I2 receptors (IPs); Kb values (in nanomoles) are given in parentheses. Compound 1 evoked a pronounced inhibition of cytokine/chemokine secretion from lipopolysaccharide or TNF-α stimulated primary human macrophages. These cytokine/chemokines included cluster of designation 40 receptor (CD40), epithelial-derived neutrophil-activating protein 78 (ENA-78), granulocyte colony stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), IL-8, IL-18, monocyte chemotactic protein-1 (CCL2) (MCP-1), tissue plasminogen activator inhibitor (PAI-1), and regulated on activation, normal T cell expressed and secreted (RANTES). In contrast, the inhibitory effects of most antagonists selective for a single receptor were modest or absent, and selective EP2 receptor blockade increased cytokine release in some instances. Compound 1 also showed clear superiority to the cyclooxygenase inhibitors diclofenac and rofecoxib. These findings reveal that blockade of multiple prostanoid receptors, with absent antagonism of EP2 and IP, may provide more effective anti-inflammatory activity than global suppression of prostanoid synthesis or highly selective prostanoid receptor blockade. These investigations demonstrate the first working example of prostanoid receptor polypharmacology for potentially safer and more effective anti-inflammatory therapeutics by blocking multiple proinflammatory receptors while sparing those with anti-inflammatory activity.
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http://dx.doi.org/10.1096/fj.15-275610DOI Listing
January 2016

Targeted imaging of cancer by fluorocoxib C, a near-infrared cyclooxygenase-2 probe.

J Biomed Opt 2015 May;20(5):50502

Cyclooxygenase-2 (COX-2) is a promising target for the imaging of cancer in a range of diagnostic and therapeutic settings. We report a near-infrared COX-2-targeted probe, fluorocoxib C (FC), for visualization of solid tumors by optical imaging. FC exhibits selective and potent COX-2 inhibition in both purified protein and human cancercell lines. In vivo optical imaging shows selective accumulation of FC in COX-2-overexpressing human tumor xenografts [1483 head and neck squamous cell carcinoma (HNSCC)] implanted in nude mice, while minimal uptake is detectable in COX-2-negative tumor xenografts (HCT116)or 1483 HNSCC xenografts preblocked with the COX-2-selective inhibitor celecoxib. Time course imaging studies conducted from 3 h to 7-day post-FC injection revealed a marked reduction in nonspecific fluorescent signals with retention of fluorescence in 1483 HNSCC tumors. Thus, use of FC in a delayed imaging protocol offers an approach to improve imaging signal-to-noise that should improve cancer detection in multiple preclinical and clinical settings.
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http://dx.doi.org/10.1117/1.JBO.20.5.050502DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4430360PMC
May 2015

13-Methylarachidonic acid is a positive allosteric modulator of endocannabinoid oxygenation by cyclooxygenase.

J Biol Chem 2015 Mar 2;290(12):7897-909. Epub 2015 Feb 2.

From the A. B. Hancock Jr. Memorial Laboratory for Cancer Research, Departments of Biochemistry, Vanderbilt Institute of Chemical Biology, Chemistry, and Pharmacology, Vanderbilt Center in Molecular Toxicology, Vanderbilt-Ingram Cancer Center, and Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146,

Cyclooxygenase-2 (COX-2) oxygenates arachidonic acid (AA) and the endocannabinoids 2-arachidonoylglycerol (2-AG) and arachidonylethanolamide to prostaglandins, prostaglandin glyceryl esters, and prostaglandin ethanolamides, respectively. A structural homodimer, COX-2 acts as a conformational heterodimer with a catalytic and an allosteric monomer. Prior studies have demonstrated substrate-selective negative allosteric regulation of 2-AG oxygenation. Here we describe AM-8138 (13(S)-methylarachidonic acid), a substrate-selective allosteric potentiator that augments 2-AG oxygenation by up to 3.5-fold with no effect on AA oxygenation. In the crystal structure of an AM-8138·COX-2 complex, AM-8138 adopts a conformation similar to the unproductive conformation of AA in the substrate binding site. Kinetic analysis suggests that binding of AM-8138 to the allosteric monomer of COX-2 increases 2-AG oxygenation by increasing kcat and preventing inhibitory binding of 2-AG. AM-8138 restored the activity of COX-2 mutants that exhibited very poor 2-AG oxygenating activity and increased the activity of COX-1 toward 2-AG. Competition of AM-8138 for the allosteric site prevented the inhibition of COX-2-dependent 2-AG oxygenation by substrate-selective inhibitors and blocked the inhibition of AA or 2-AG oxygenation by nonselective time-dependent inhibitors. AM-8138 selectively enhanced 2-AG oxygenation in intact RAW264.7 macrophage-like cells. Thus, AM-8138 is an important new tool compound for the exploration of allosteric modulation of COX enzymes and their role in endocannabinoid metabolism.
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http://dx.doi.org/10.1074/jbc.M114.634014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4367288PMC
March 2015

Genetic disruption of 2-arachidonoylglycerol synthesis reveals a key role for endocannabinoid signaling in anxiety modulation.

Cell Rep 2014 Dec 26;9(5):1644-1653. Epub 2014 Nov 26.

Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, TN 37232, USA; Department of Psychiatry, Vanderbilt University School of Medicine, Nashville, TN 37232, USA; Vanderbilt Brain Institute, Vanderbilt University School of Medicine, Nashville, TN 37232, USA; Vanderbilt-Kennedy Center for Research on Human Development, Vanderbilt University School of Medicine, Nashville, TN 37232, USA. Electronic address:

Endocannabinoid (eCB) signaling has been heavily implicated in the modulation of anxiety and depressive behaviors and emotional learning. However, the role of the most-abundant endocannabinoid 2-arachidonoylglycerol (2-AG) in the physiological regulation of affective behaviors is not well understood. Here, we show that genetic deletion of the 2-AG synthetic enzyme diacylglycerol lipase α (DAGLα) in mice reduces brain, but not circulating, 2-AG levels. DAGLα deletion also results in anxiety-like and sex-specific anhedonic phenotypes associated with impaired activity-dependent eCB retrograde signaling at amygdala glutamatergic synapses. Importantly, acute pharmacological normalization of 2-AG levels reverses both phenotypes of DAGLα-deficient mice. These data suggest 2-AG deficiency could contribute to the pathogenesis of affective disorders and that pharmacological normalization of 2-AG signaling could represent an approach for the treatment of mood and anxiety disorders.
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http://dx.doi.org/10.1016/j.celrep.2014.11.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4268380PMC
December 2014

Protein modification by adenine propenal.

Chem Res Toxicol 2014 Oct 24;27(10):1732-42. Epub 2014 Sep 24.

A. B. Hancock Jr. Memorial Laboratory for Cancer Research, Departments of Biochemistry, ‡Chemistry, and §Pharmacology, ∥Mass Spectrometry Research Center, ⊥Center in Molecular Toxicology, #Center for Structural Biology, ∇Department of Medicine, Vanderbilt Institute of Chemical Biology, and Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine , Nashville, Tennessee 37232-0146, United States.

Base propenals are products of the reaction of DNA with oxidants such as peroxynitrite and bleomycin. The most reactive base propenal, adenine propenal, is mutagenic in Escherichia coli and reacts with DNA to form covalent adducts; however, the reaction of adenine propenal with protein has not yet been investigated. A survey of the reaction of adenine propenal with amino acids revealed that lysine and cysteine form adducts, whereas histidine and arginine do not. N(ε)-Oxopropenyllysine, a lysine-lysine cross-link, and S-oxopropenyl cysteine are the major products. Comprehensive profiling of the reaction of adenine propenal with human serum albumin and the DNA repair protein, XPA, revealed that the only stable adduct is N(ε)-oxopropenyllysine. The most reactive sites for modification in human albumin are K190 and K351. Three sites of modification of XPA are in the DNA-binding domain, and two sites are subject to regulatory acetylation. Modification by adenine propenal dramatically reduces XPA's ability to bind to a DNA substrate.
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http://dx.doi.org/10.1021/tx500218gDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4203390PMC
October 2014

Effect of zileuton and celecoxib on urinary LTE4 and PGE-M levels in smokers.

Cancer Prev Res (Phila) 2013 Jul 16;6(7):646-55. Epub 2013 May 16.

Department of Surgery (Head and Neck Service), Memorial Sloan-Kettering Cancer Center, Weill Cornell Medical College, New York, NY 10065, USA.

COX-2 and 5-lipoxygenase (5-LO) use arachidonic acid for the synthesis of eicosanoids that have been implicated in carcinogenesis and cardiovascular disease. The ability of celecoxib, a selective COX-2 inhibitor, to redirect arachidonic acid into the 5-LO pathway can potentially reduce its efficacy as a chemopreventive agent and increase the risk of cardiovascular complications. Levels of urinary prostaglandin E metabolite (PGE-M) and leukotriene E4 (LTE4), biomarkers of the COX and 5-LO pathways, are elevated in smokers. Here, we investigated the effects of zileuton, a 5-LO inhibitor, versus zileuton and celecoxib for 6 ± 1 days on urinary PGE-M and LTE4 levels in smokers. Treatment with zileuton led to an 18% decrease in PGE-M levels (P = 0.03); the combination of zileuton and celecoxib led to a 62% reduction in PGE-M levels (P < 0.001). Levels of LTE4 decreased by 61% in subjects treated with zileuton alone (P < 0.001) and were unaffected by the addition of celecoxib. Although zileuton use was associated with a small overall decrease in PGE-M levels, increased PGE-M levels were found in a subset (19 of 52) of subjects. Notably, the addition of celecoxib to the 5-LO inhibitor protected against the increase in urinary PGE-M levels (P = 0.03). In conclusion, zileuton was an effective inhibitor of 5-LO activity resulting in marked suppression of urinary LTE4 levels and possible redirection of arachidonic acid into the COX-2 pathway in a subset of subjects. Combining celecoxib and zileuton was associated with inhibition of both the COX-2 and 5-LO pathways manifested as reduced levels of urinary PGE-M and LTE4.
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http://dx.doi.org/10.1158/1940-6207.CAPR-13-0083DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3707304PMC
July 2013

Substrate-Selective Inhibition of Cyclooxygenase-2: Development and Evaluation of Achiral Profen Probes.

ACS Med Chem Lett 2012 Sep 15;3(9):759-763. Epub 2012 Aug 15.

A.B. Hancock Jr. Memorial Laboratory for Cancer Research, Departments of Biochemistry, Chemistry, and Pharmacology, Vanderbilt Institute of Chemical Biology, Center in Molecular Toxicology, and Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine , Nashville, Tennessee, United States.

Cyclooxygenase-2 (COX-2) oxygenates arachidonic acid and the endocannabinoids 2-arachidonoylglycerol (2-AG) and arachidonoylethanolamide (AEA). We recently reported that (R)-profens selectively inhibit endocannabinoid oxygenation but not arachidonic acid oxygenation. In this work, we synthesized achiral derivatives of five profen scaffolds and evaluated them for substrate-selective inhibition using in vitro and cellular assays. The size of the substituents dictated the inhibitory strength of the analogs, with smaller substituents enabling greater potency but less selectivity. Inhibitors based on the flurbiprofen scaffold possessed the greatest potency and selectivity, with desmethylflurbiprofen (3a) exhibiting an IC(50) of 0.11 μM for inhibition of 2-AG oxygenation. The crystal structure of desmethylflurbiprofen complexed to mCOX-2 demonstrated a similar binding mode to other profens. Desmethylflurbiprofen exhibited a half-life in mice comparable to that of ibuprofen. The data presented suggest that achiral profens can act as lead molecules toward in vivo probes of substrate-selective COX-2 inhibition.
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http://dx.doi.org/10.1021/ml3001616DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3441040PMC
September 2012

Cyclooxygenase-1-selective inhibitors based on the (E)-2'-des-methyl-sulindac sulfide scaffold.

J Med Chem 2012 Mar 14;55(5):2287-300. Epub 2012 Feb 14.

A. B. Hancock Jr. Memorial Laboratory for Cancer Research, Department of Biochemistry, Vanderbilt Institute of Chemical Biology, Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.

Prostaglandins (PGs) are powerful lipid mediators in many physiological and pathophysiological responses. They are produced by oxidation of arachidonic acid (AA) by cyclooxygenases (COX-1 and COX-2) followed by metabolism of endoperoxide intermediates by terminal PG synthases. PG biosynthesis is inhibited by nonsteroidal anti-inflammatory drugs (NSAIDs). Specific inhibition of COX-2 has been extensively investigated, but relatively few COX-1-selective inhibitors have been described. Recent reports of a possible contribution of COX-1 in analgesia, neuroinflammation, or carcinogenesis suggest that COX-1 is a potential therapeutic target. We designed, synthesized, and evaluated a series of (E)-2'-des-methyl-sulindac sulfide (E-DMSS) analogues for inhibition of COX-1. Several potent and selective inhibitors were discovered, and the most promising compounds were active against COX-1 in intact ovarian carcinoma cells (OVCAR-3). The compounds inhibited tumor cell proliferation but only at concentrations >100-fold higher than the concentrations that inhibit COX-1 activity. E-DMSS analogues may be useful probes of COX-1 biology in vivo and promising leads for COX-1-targeted therapeutic agents.
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http://dx.doi.org/10.1021/jm201528bDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3297362PMC
March 2012

Selection of monoclonal antibodies against 6-oxo-M(1)dG and their use in an LC-MS/MS assay for the presence of 6-oxo-M(1)dG in vivo.

Chem Res Toxicol 2012 Feb 24;25(2):454-61. Epub 2012 Jan 24.

Department of Biochemistry, Vanderbilt Institute of Chemical Biology, Vanderbilt University School of Medicine , Nashville, Tennessee 37232-0146, United States.

Oxidative stress triggers DNA and lipid peroxidation, leading to the formation of electrophiles that react with DNA to form adducts. A product of this pathway, (3-(2'-deoxy-β-d-erythro-pentofuranosyl)-pyrimido[1,2-α]purine-10(3H)-one), or M(1)dG, is mutagenic in bacterial and mammalian cells and is repaired by the nucleotide excision repair pathway. In vivo, M(1)dG is oxidized to a primary metabolite, (3-(2-deoxy-β-d-erythro-pentofuranosyl)-pyrimido[1,2-α]purine-6,10(3H,5H)-dione, or 6-oxo-M(1)dG, which is excreted in urine, bile, and feces. We have developed a specific monoclonal antibody against 6-oxo-M(1)dG and have incorporated this antibody into a procedure for the immunoaffinity isolation of 6-oxo-M(1)dG from biological matrices. The purified analyte is quantified by LC-MS/MS using a stable isotope-labeled analogue ([(15)N(5)]-6-oxo-M(1)dG) as an internal standard. Healthy male Sprague-Dawley rats excreted 6-oxo-M(1)dG at a rate of 350-1893 fmol/kg·d in feces. This is the first report of the presence of the major metabolite of M(1)dG in rodents without exogenous introduction of M(1)dG.
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http://dx.doi.org/10.1021/tx200494hDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3285145PMC
February 2012

Fluorinated COX-2 inhibitors as agents in PET imaging of inflammation and cancer.

Cancer Prev Res (Phila) 2011 Oct 7;4(10):1536-45. Epub 2011 Sep 7.

A.B. Hancock, Jr., Memorial Laboratory for Cancer Research, Department of Biochemistry, Chemistry and Pharmacology, Vanderbilt Institute of Chemical Biology, Center for Molecular Toxicology, TN, USA.

COX-2 is a major contributor to the inflammatory response and cancer progression so it is an important target for prevention and therapy. COX-2 is absent or expressed at low levels in most epithelial cells but is found at high levels in inflammatory lesions, and many premalignant and malignant tumors. Thus, it is an attractive target for molecular imaging. We report a series of novel fluorinated imaging agents, derived from indomethacin or celecoxib that selectively inhibit COX-2. The most promising lead, compound 7, was a fluorinated derivative of celecoxib. Kinetic analysis revealed that this fluorinated compound is a slow, tight-binding inhibitor of COX-2 and exhibits minimal inhibitory activity against COX-1. Efficient incorporation of (18)F into compound 7 by radiochemical synthesis and intravenous injection provided sufficient signal for in vivo positron emission tomography (PET) imaging. Selective uptake of (18)F-7 was observed in inflamed rat paws compared with the noninflamed contralateral paws and uptake was blocked by pretreatment with the COX-2 inhibitor, celecoxib. Uptake of (18)F-7 was not observed when inflammation was induced in COX-2-null mice. In nude mice bearing both a COX-2-expressing human tumor xenograft (1483) and a COX-2-negative xenograft (HCT116), (18)F-7 selectively accumulated in the COX-2-expressing tumor. Accumulation was blocked by pretreatment of the animals with celecoxib. The in vitro and in vivo properties of compound 7 suggest it will be a useful probe for early detection of cancer and for evaluation of the COX-2 status of premalignant and malignant tumors.
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http://dx.doi.org/10.1158/1940-6207.CAPR-11-0120DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3214660PMC
October 2011

The role of NAG-1/GDF15 in the inhibition of intestinal polyps in APC/Min mice by sulindac.

Cancer Prev Res (Phila) 2011 Jan;4(1):150-60

Laboratory of Molecular Carcinogenesis, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina, USA.

The antitumor effects of nonsteroidal anti-inflammatory drugs (NSAID) are assumed to be due to the inhibition of COX activity, but COX-independent mechanisms may also play an important role. NSAID-activated gene (NAG-1/GDF15) is induced by NSAIDs and has antitumorigenic activities. To determine the contribution of COX-2 inhibition and NAG-1/GDF15 expression to the prevention of colon carcinogenesis by NSAIDs, we evaluated several sulindac derivatives [des-methyl (DM)-sulindac sulfide and its prodrug DM-sulindac] that do not inhibit COX-2 activity. Sulindac sulfide and DM-sulindac induced the expression of NAG-1/GDF15 in HCT116 cells as determined by quantitative real-time PCR and Western blot. We fed APC/Min mice with 320 ppm of sulindac and doses of DM-sulindac. Only sulindac significantly inhibited tumor formation inAPC/Min mice. To determine the pharmacokinetic properties of sulindac and DM-sulindac in vivo, wild-type C57/B6 mice were fed with sulindac and DM-sulindac at 80, 160, and 320 ppm. High-performance liquid chromatography analysis revealed that the conversion of DM-sulindac to DM-sulindac sulfide (active form) was less efficient than the conversion of sulindac to sulindac sulfide (active form) in the mice. Lower levels of DM-sulindac sulfide accumulated in intestinal and colon tissues in comparison with sulindac sulfide. In addition, NAG-1/GDF15 was induced in the liver of sulindac-fed mice but not in the DM-sulindac-fed mice. Collectively, our results suggest that the tumor-inhibitory effects of sulindac in APC/Min mice may be due to, in part, NAG-1/GDF15 induction in the liver. Our study also suggests that pharmacologic properties should be carefully evaluated when developing drug candidates.
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http://dx.doi.org/10.1158/1940-6207.CAPR-10-0196DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3970176PMC
January 2011
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