Publications by authors named "Olivier Boutaud"

64 Publications

Discovery and optimization of a novel CNS penetrant series of mGlu PAMs based on a 1,4-thiazepane core with in vivo efficacy in a preclinical Parkinsonian model.

Bioorg Med Chem Lett 2021 Feb 5;37:127838. Epub 2021 Feb 5.

Warren Center for Neuroscience Drug Discovery, Vanderbilt University, Nashville, TN 37232, USA; Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA; Department of Chemistry, Vanderbilt University, Nashville, TN 37232, USA; Vanderbilt Institute of Chemical Biology, Vanderbilt University, Nashville, TN 37232, USA. Electronic address:

A high throughput screen (HTS) identified a novel, but weak (EC = 6.2 μM, 97% Glu Max) mGlu PAM chemotype based on a 1,4-thiazepane core, VU0544412. Reaction development and chemical optimization delivered a potent mGlu PAM VU6022296 (EC = 32.8 nM, 108% Glu Max) with good CNS penetration (K = 0.45, K = 0.70) and enantiopreference. Finally, VU6022296 displayed robust, dose-dependent efficacy in reversing Haloperidol-Induced Catalepsy (HIC), a rodent preclinical Parkinson's disease model.
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http://dx.doi.org/10.1016/j.bmcl.2021.127838DOI Listing
February 2021

Lead optimization of the VU0486321 series of mGlu PAMs. Part 4: SAR reveals positive cooperativity across multiple mGlu receptor subtypes leading to subtype unselective PAMs.

Bioorg Med Chem Lett 2021 Jan 27;32:127724. Epub 2020 Nov 27.

Warren Center for Neuroscience Drug Discovery, Vanderbilt University, Nashville, TN 37232, USA; Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA; Department of Chemistry, Vanderbilt University, Nashville, TN 37232, USA; Vanderbilt Institute of Chemical Biology, Vanderbilt University, Nashville, TN 37232, USA. Electronic address:

Further optimization of the VU0486321 series of highly selective and CNS-penetrant mGlu PAMs identified unique 'molecular switches' on the central aromatic ring that engendered positive cooperativity with multiple mGlu subtypes across the receptor family, resulting in compounds with comparable activity at Group I (mGlu) and Group III (mGlu) mGlu receptors, receptors. These exciting data suggests this PAM chemotype appears to bind to multiple mGlu receptors, and that subtype selectivity is dictated by the degree of cooperativity, not a subtype selective, unique allosteric binding site. Moreover, there is interesting therapeutic potential for mGlu PAMs, as well as the first report of a GPCR allosteric 'privileged structure'.
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http://dx.doi.org/10.1016/j.bmcl.2020.127724DOI Listing
January 2021

Dicarbonyl Electrophiles Mediate Inflammation-Induced Gastrointestinal Carcinogenesis.

Gastroenterology 2020 Nov 13. Epub 2020 Nov 13.

Division of Gastroenterology, Hepatology, and Nutrition, Department of Medicine, Vanderbilt University Medical Center, Nashville, Tennessee; Center for Mucosal Inflammation and Cancer, Vanderbilt University Medical Center, Nashville, Tennessee; Department of Pathology, Microbiology and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee; Veterans Affairs Tennessee Valley Healthcare System, Nashville, Tennessee.

Background & Aims: Inflammation in the gastrointestinal tract may lead to the development of cancer. Dicarbonyl electrophiles, such as isolevuglandins (isoLGs), are generated from lipid peroxidation during the inflammatory response and form covalent adducts with amine-containing macromolecules. Thus, we sought to determine the role of dicarbonyl electrophiles in inflammation-associated carcinogenesis.

Methods: The formation of isoLG adducts was analyzed in the gastric tissues of patients infected with Helicobacter pylori from gastritis to precancerous intestinal metaplasia, in human gastric organoids, and in patients with colitis and colitis-associated carcinoma (CAC). The effect on cancer development of a potent scavenger of dicarbonyl electrophiles, 5-ethyl-2-hydroxybenzylamine (EtHOBA), was determined in transgenic FVB/N insulin-gastrin (INS-GAS) mice and Mongolian gerbils as models of H pylori-induced carcinogenesis and in C57BL/6 mice treated with azoxymethane-dextran sulfate sodium as a model of CAC. The effect of EtHOBA on mutations in gastric epithelial cells of H pylori-infected INS-GAS mice was assessed by whole-exome sequencing.

Results: We show increased isoLG adducts in gastric epithelial cell nuclei in patients with gastritis and intestinal metaplasia and in human gastric organoids infected with H pylori. EtHOBA inhibited gastric carcinoma in infected INS-GAS mice and gerbils and attenuated isoLG adducts, DNA damage, and somatic mutation frequency. Additionally, isoLG adducts were elevated in tissues from patients with colitis, colitis-associated dysplasia, and CAC as well as in dysplastic tumors of C57BL/6 mice treated with azoxymethane-dextran sulfate sodium. In this model, EtHOBA significantly reduced adduct formation, tumorigenesis, and dysplasia severity.

Conclusions: Dicarbonyl electrophiles represent a link between inflammation and somatic genomic alterations and are thus key targets for cancer chemoprevention.
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http://dx.doi.org/10.1053/j.gastro.2020.11.006DOI Listing
November 2020

Mitochondrial Isolevuglandins Contribute to Vascular Oxidative Stress and Mitochondria-Targeted Scavenger of Isolevuglandins Reduces Mitochondrial Dysfunction and Hypertension.

Hypertension 2020 Dec 5;76(6):1980-1991. Epub 2020 Oct 5.

From the Vanderbilt University Medical Center, Nashville, TN (A.D., L.X., V.A., I.Z.-I., A.V., M.A., V.Y., R.R.N., O.B., M.G.L., F.T.B., S. Davies, L.J.R., D.G.H., S. Dikalov).

Hypertension remains a major health problem in Western Societies, and blood pressure is poorly controlled in a third of patients despite use of multiple drugs. Mitochondrial dysfunction contributes to hypertension, and mitochondria-targeted agents can potentially improve treatment of hypertension. We have proposed that mitochondrial oxidative stress produces reactive dicarbonyl lipid peroxidation products, isolevuglandins, and that scavenging of mitochondrial isolevuglandins improves vascular function and reduces hypertension. To test this hypothesis, we have studied the accumulation of mitochondrial isolevuglandins-protein adducts in patients with essential hypertension and Ang II (angiotensin II) model of hypertension using mass spectrometry and Western blot analysis. The therapeutic potential of targeting mitochondrial isolevuglandins was tested by the novel mitochondria-targeted isolevuglandin scavenger, mito2HOBA. Mitochondrial isolevuglandins in arterioles from hypertensive patients were 250% greater than in arterioles from normotensive subjects, and ex vivo mito2HOBA treatment of arterioles from hypertensive subjects increased deacetylation of a key mitochondrial antioxidant, SOD2 (superoxide dismutase 2). In human aortic endothelial cells stimulated with Ang II plus TNF (tumor necrosis factor)-α, mito2HOBA reduced mitochondrial superoxide and cardiolipin oxidation, a specific marker of mitochondrial oxidative stress. In Ang II-infused mice, mito2HOBA diminished mitochondrial isolevuglandins-protein adducts, raised Sirt3 (sirtuin 3) mitochondrial deacetylase activity, reduced vascular superoxide, increased endothelial nitric oxide, improved endothelium-dependent relaxation, and attenuated hypertension. Mito2HOBA preserved mitochondrial respiration, protected ATP production, and reduced mitochondrial permeability pore opening in Ang II-infused mice. These data support the role of mitochondrial isolevuglandins in endothelial dysfunction and hypertension. We conclude that scavenging of mitochondrial isolevuglandins may have therapeutic potential in treatment of vascular dysfunction and hypertension.
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http://dx.doi.org/10.1161/HYPERTENSIONAHA.120.15236DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7666054PMC
December 2020

Scavenging of reactive dicarbonyls with 2-hydroxybenzylamine reduces atherosclerosis in hypercholesterolemic Ldlr mice.

Nat Commun 2020 08 14;11(1):4084. Epub 2020 Aug 14.

Department of Medicine, Division of Cardiovascular Medicine, Atherosclerosis Research Unit, Vanderbilt University School of Medicine, Nashville, TN, 37232, USA.

Lipid peroxidation generates reactive dicarbonyls including isolevuglandins (IsoLGs) and malondialdehyde (MDA) that covalently modify proteins. Humans with familial hypercholesterolemia (FH) have increased lipoprotein dicarbonyl adducts and dysfunctional HDL. We investigate the impact of the dicarbonyl scavenger, 2-hydroxybenzylamine (2-HOBA) on HDL function and atherosclerosis in Ldlr mice, a model of FH. Compared to hypercholesterolemic Ldlr mice treated with vehicle or 4-HOBA, a nonreactive analogue, 2-HOBA decreases atherosclerosis by 60% in en face aortas, without changing plasma cholesterol. Ldlr mice treated with 2-HOBA have reduced MDA-LDL and MDA-HDL levels, and their HDL display increased capacity to reduce macrophage cholesterol. Importantly, 2-HOBA reduces the MDA- and IsoLG-lysyl content in atherosclerotic aortas versus 4-HOBA. Furthermore, 2-HOBA reduces inflammation and plaque apoptotic cells and promotes efferocytosis and features of stable plaques. Dicarbonyl scavenging with 2-HOBA has multiple atheroprotective effects in a murine FH model, supporting its potential as a therapeutic approach for atherosclerotic cardiovascular disease.
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http://dx.doi.org/10.1038/s41467-020-17915-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7429830PMC
August 2020

Highly Reactive Isolevuglandins Promote Atrial Fibrillation Caused by Hypertension.

JACC Basic Transl Sci 2020 Jun 27;5(6):602-615. Epub 2020 May 27.

Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee.

Oxidative damage is implicated in atrial fibrillation (AF), but antioxidants are ineffective therapeutically. The authors tested the hypothesis that highly reactive lipid dicarbonyl metabolites, or isolevuglandins (IsoLGs), are principal drivers of AF during hypertension. In a hypertensive murine model and stretched atriomyocytes, the dicarbonyl scavenger 2-hydroxybenzylamine (2-HOBA) prevented IsoLG adducts and preamyloid oligomers (PAOs), and AF susceptibility, whereas the ineffective analog 4-hydroxybenzylamine (4-HOBA) had minimal effect. Natriuretic peptides generated cytotoxic oligomers, a process accelerated by IsoLGs, contributing to atrial PAO formation. These findings support the concept of pre-emptively scavenging reactive downstream oxidative stress mediators as a potential therapeutic approach to prevent AF.
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http://dx.doi.org/10.1016/j.jacbts.2020.04.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7315188PMC
June 2020

Safety, tolerability, and pharmacokinetics of repeated oral doses of 2-hydroxybenzylamine acetate in healthy volunteers: a double-blind, randomized, placebo-controlled clinical trial.

BMC Pharmacol Toxicol 2020 01 6;21(1). Epub 2020 Jan 6.

Department of Pharmacology, Division of Clinical Pharmacology, Vanderbilt University, Nashville, TN, 37232, USA.

Background: 2-Hydroxybenzylamine (2-HOBA) is a selective dicarbonyl electrophile scavenger being developed as a nutritional supplement to help protect against the development of conditions associated with dicarbonyl electrophile formation, such as the cognitive decline observed with Mild Cognitive Impairment or Alzheimer's disease.

Methods: This study evaluated the safety, tolerability, and pharmacokinetics of repeated oral doses of 2-HOBA acetate (500 or 750 mg) administered to healthy volunteers every eight hours for two weeks. The effects of 2-HOBA on cyclooxygenase function and cerebrospinal fluid penetrance of 2-HOBA were also investigated.

Results: Repeated oral administration of 2-HOBA was found to be safe and well-tolerated up to 750 mg TID for 15 days. 2-HOBA was absorbed within 2 h of administration, had a half-life of 2.10-3.27 h, and an accumulation ratio of 1.38-1.52. 2-HOBA did not interfere with cyclooxygenase function and was found to be present in cerebrospinal fluid 90 min after dosing.

Conclusions: Repeated oral administration of 2-HOBA was found to be safe and well-tolerated. These results support continued development of 2-HOBA as a nutritional supplement.

Trial Registration: Studies are registered at ClinicalTrials.gov (NCT03555682 Registered 13 June 2018, NCT03554096 Registered 12 June 18).
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http://dx.doi.org/10.1186/s40360-020-0382-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6945443PMC
January 2020

Isolevuglandins as mediators of disease and the development of dicarbonyl scavengers as pharmaceutical interventions.

Pharmacol Ther 2020 01 16;205:107418. Epub 2019 Oct 16.

Division of Clinical Pharmacology and Departments of Pharmacology and Medicine, Vanderbilt University, Nashville, TN, United States.

Products of lipid peroxidation include a number of reactive lipid aldehydes such as malondialdehyde, 4-hydroxy-nonenal, 4-oxo-nonenal, and isolevuglandins (IsoLGs). Although these all contribute to disease processes, the most reactive are the IsoLGs, which rapidly adduct to lysine and other cellular primary amines, leading to changes in protein function, cross-linking and immunogenicity. Their rapid reactivity means that only IsoLG adducts, and not the unreacted aldehyde, can be readily measured. This high reactivity also makes it challenging for standard cellular defense mechanisms such as aldehyde reductases and oxidases to dispose of them before they react with proteins and other cellular amines. This led us to seek small molecule primary amines that might trap and inactivate IsoLGs before they could modify cellular proteins or other endogenous cellular amines such as phosphatidylethanolamines to cause disease. Our studies identified 2-aminomethylphenols including 2-hydroxybenzylamine as IsoLG scavengers. Subsequent studies showed that they also trap other lipid dicarbonyls that react with primary amines such as 4-oxo-nonenal and malondialdehyde, but not hydroxyalkenals like 4-hydroxy-nonenal that preferentially react with soft nucleophiles. This review describes the use of these 2-aminomethylphenols as dicarbonyl scavengers to assess the contribution of IsoLGs and other amine-reactive lipid dicarbonyls to disease and as therapeutic agents.
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http://dx.doi.org/10.1016/j.pharmthera.2019.107418DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7495735PMC
January 2020

Gastroesophageal Reflux Induces Protein Adducts in the Esophagus.

Cell Mol Gastroenterol Hepatol 2019 8;7(2):480-482.e7. Epub 2018 Nov 8.

Department of Surgery, University of Miami, Miami, Florida; Department of Surgery, Vanderbilt University Medical Center, Nashville, Tennessee; Department of Veterans Affairs, Miami VA Healthcare System, Miami, Florida. Electronic address:

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http://dx.doi.org/10.1016/j.jcmgh.2018.10.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6410348PMC
May 2019

First-in-human study assessing safety, tolerability, and pharmacokinetics of 2-hydroxybenzylamine acetate, a selective dicarbonyl electrophile scavenger, in healthy volunteers.

BMC Pharmacol Toxicol 2019 Jan 5;20(1). Epub 2019 Jan 5.

Department of Pharmacology, Division of Clinical Pharmacology, Vanderbilt University, Nashville, TN, 37232, USA.

Background: 2-Hydroxybenzylamine (2-HOBA) is a selective scavenger of dicarbonyl electrophiles that protects proteins and lipids from being modified by these electrophiles. It is currently being developed for use as a nutritional supplement to help maintain good health and protect against the development of conditions associated with dicarbonyl electrophile formation, such as the cognitive decline associated with Mild Cognitive Impairment and Alzheimer's disease.

Methods: In this first-in-human study, the safety, tolerability, and pharmacokinetics of six ascending single oral doses of 2-HOBA acetate were tested in eighteen healthy human volunteers.

Results: Reported adverse events were mild and considered unlikely to be related to 2-HOBA. There were no clinically significant changes in vital signs, ECG recordings, or clinical laboratory parameters. 2-HOBA was fairly rapidly absorbed, with a t of 1-2 h, and eliminated, with a t of approximately 2 h. Both t and t were independent of dose level, while C and AUC increased proportionally with dose level.

Conclusions: 2-HOBA acetate was safe and well-tolerated at doses up to 825 mg in healthy human volunteers, positioning it as a good candidate for continued development as a nutritional supplement.

Trial Registration: This study is registered at ClinicalTrials.gov (NCT03176940).
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http://dx.doi.org/10.1186/s40360-018-0281-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6321651PMC
January 2019

Tobacco smoking induces cardiovascular mitochondrial oxidative stress, promotes endothelial dysfunction, and enhances hypertension.

Am J Physiol Heart Circ Physiol 2019 03 4;316(3):H639-H646. Epub 2019 Jan 4.

Division of Clinical Pharmacology, Vanderbilt University Medical Center , Nashville, Tennessee.

Tobacco smoking is a major risk factor for cardiovascular disease and hypertension. It is associated with the oxidative stress and induces metabolic reprogramming, altering mitochondrial function. We hypothesized that cigarette smoke induces cardiovascular mitochondrial oxidative stress, which contributes to endothelial dysfunction and hypertension. To test this hypothesis, we studied whether the scavenging of mitochondrial HO in transgenic mice expressing mitochondria-targeted catalase (mCAT) attenuates the development of cigarette smoke/angiotensin II-induced mitochondrial oxidative stress and hypertension compared with wild-type mice. Two weeks of exposure of wild-type mice with cigarette smoke increased systolic blood pressure by 17 mmHg, which was similar to the effect of a subpresssor dose of angiotensin II (0.2 mg·kg·day), leading to a moderate increase to the prehypertensive level. Cigarette smoke exposure and a low dose of angiotensin II cooperatively induced severe hypertension in wild-type mice, but the scavenging of mitochondrial HO in mCAT mice completely prevented the development of hypertension. Cigarette smoke and angiotensin II cooperatively induced oxidation of cardiolipin (a specific biomarker of mitochondrial oxidative stress) in wild-type mice, which was abolished in mCAT mice. Cigarette smoke and angiotensin II impaired endothelium-dependent relaxation and induced superoxide overproduction, which was diminished in mCAT mice. To mimic the tobacco smoke exposure, we used cigarette smoke condensate, which induced mitochondrial superoxide overproduction and reduced endothelial nitric oxide (a hallmark of endothelial dysfunction in hypertension). Western blot experiments indicated that tobacco smoke and angiotensin II reduce the mitochondrial deacetylase sirtuin-3 level and cause hyperacetylation of a key mitochondrial antioxidant, SOD2, which promotes mitochondrial oxidative stress. NEW & NOTEWORTHY This work demonstrates tobacco smoking-induced mitochondrial oxidative stress, which contributes to endothelial dysfunction and development of hypertension. We suggest that the targeting of mitochondrial oxidative stress can be beneficial for treatment of pathological conditions associated with tobacco smoking, such as endothelial dysfunction, hypertension, and cardiovascular diseases.
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http://dx.doi.org/10.1152/ajpheart.00595.2018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6459311PMC
March 2019

Bid maintains mitochondrial cristae structure and function and protects against cardiac disease in an integrative genomics study.

Elife 2018 10 3;7. Epub 2018 Oct 3.

Department of Cell and Developmental Biology, Vanderbilt University, Nashville, United States.

Bcl-2 family proteins reorganize mitochondrial membranes during apoptosis, to form pores and rearrange cristae. In vitro and in vivo analysis integrated with human genetics reveals a novel homeostatic mitochondrial function for Bcl-2 family protein Bid. Loss of full-length Bid results in apoptosis-independent, irregular cristae with decreased respiration. mice display stress-induced myocardial dysfunction and damage. A gene-based approach applied to a biobank, validated in two independent GWAS studies, reveals that decreased genetically determined BID expression associates with myocardial infarction (MI) susceptibility. Patients in the bottom 5% of the expression distribution exhibit >4 fold increased MI risk. Carrier status with nonsynonymous variation in Bid's membrane binding domain, Bid, associates with MI predisposition. Furthermore, Bid but not Bid associates with Mcl-1, previously implicated in cristae stability; decreased MCL-1 expression associates with MI. Our results identify a role for Bid in homeostatic mitochondrial cristae reorganization, that we link to human cardiac disease.
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http://dx.doi.org/10.7554/eLife.40907DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6234033PMC
October 2018

In vitro safety pharmacology evaluation of 2-hydroxybenzylamine acetate.

Food Chem Toxicol 2018 Nov 22;121:541-548. Epub 2018 Sep 22.

Metabolic Technologies, Inc., Ames, IA, 50010, USA; Department of Animal Science, Iowa State University, Ames, IA, 50010, USA. Electronic address:

2-hydroxybenzylamine (2-HOBA), a compound found in buckwheat, is a potent scavenger of reactive γ-ketoaldehydes, which are increased in diseases associated with inflammation and oxidative stress. While the potential of 2-HOBA is promising, studies were needed to characterize the safety of the compound before clinical trials. In a series of experiments, the risks of 2-HOBA-mediated mutagenicity and cardio-toxicity were assessed in vitro. The effects of 2-HOBA on the mRNA expression of select cytochrome P450 (CYP) enzymes were also assessed in cryopreserved human hepatocytes. Further, the distribution and metabolism of 2-HOBA in blood were determined. Our results indicate that 2-HOBA is not cytotoxic or mutagenic in vitro and does not induce the expression of CYP1A2, CYP2B6, or CYP3A4 in human hepatocytes. The results of the hERG testing showed a low risk of cardiac QT wave prolongation. Plasma protein binding and red blood cell distribution characteristics indicate low protein binding and no preferential distribution into erythrocytes. The major metabolites identified were salicylic acid and the glycoside conjugate of 2-HOBA. Together, these findings support development of 2-HOBA as a nutritional supplement and provide important information for the design of further preclinical safety studies in animals as well as for human clinical trials with 2-HOBA.
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http://dx.doi.org/10.1016/j.fct.2018.09.047DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6220894PMC
November 2018

Residual cyclooxygenase activity of aspirin-acetylated COX-2 forms 15 R-prostaglandins that inhibit platelet aggregation.

FASEB J 2019 01 10;33(1):1033-1041. Epub 2018 Aug 10.

Division of Clinical Pharmacology, Department of Pharmacology, Vanderbilt University Medical School, Nashville, Tennessee, USA.

Aspirin (acetylsalicylic acid) inhibits prostaglandin (PG) synthesis by transfer of its acetyl group to a serine residue in the cyclooxygenase (COX) active site. Acetylation of Ser530 inhibits catalysis by preventing access of arachidonic acid substrate in the COX-1 isoenzyme. Acetylated COX-2, in contrast, gains a new catalytic activity and forms 15 R hydroxy-eicosatetraenoic acid (15 R-HETE) as alternate product. Here we show that acetylated COX-2 also retains COX activity, forming predominantly 15 R-configuration PGs (70 or 62% 15 R, respectively, determined using radiolabeled substrate or LC-MS analysis). Although the K of arachidonic acid for acetylated COX-2 was ∼3-fold lower than for uninhibited COX-2, the catalytic efficiency for PG formation by the acetylated enzyme was reduced 10-fold due to a concomitant decrease in V. Aspirin increased 15 R-PGD but not 15 R-PGE in isolated human leukocytes activated with LPS to induce COX-2. 15 R-PGD inhibited human platelet aggregation induced by the thromboxane receptor agonist U46,619, and this effect was abrogated by an antagonist of the DP1 prostanoid receptor. We conclude that acetylation of Ser530 in COX-2 not only triggers formation of 15 R-HETE but also allows oxygenation and cyclization of arachidonic acid to a 15 R-PG endoperoxide. 15 R-PGs are novel products of aspirin therapy via acetylation of COX-2 and may contribute to its antiplatelet and other pharmacologic effects.-Giménez-Bastida, J. A., Boeglin, W. E., Boutaud, O., Malkowski, M. G., Schneider, C. Residual cyclooxygenase activity of aspirin-acetylated COX-2 forms 15 R-prostaglandins that inhibit platelet aggregation.
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http://dx.doi.org/10.1096/fj.201801018RDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6355089PMC
January 2019

Total Synthesis and Biological Activity of the Arachidonic Acid Metabolite Hemiketal E.

Org Lett 2018 07 19;20(13):4020-4022. Epub 2018 Jun 19.

Department of Chemistry , Vanderbilt University, Vanderbilt Institute of Chemical Biology Nashville , Tennessee 37232 , United States.

The total synthesis of hemiketal E (HKE) has been accomplished using a gold(I)-mediated cycloisomerization followed by oxidation of the enol ether product to introduce a unique keto-hemiketal, the core structure of HKE. Synthetic hemiketal E reproduced biosynthetically derived HKE in the inhibition of human platelet aggregation.
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http://dx.doi.org/10.1021/acs.orglett.8b01578DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6425725PMC
July 2018

Inhibition of the Biosynthesis of Prostaglandin E2 By Low-Dose Aspirin: Implications for Adenocarcinoma Metastasis.

Cancer Prev Res (Phila) 2016 Nov 23;9(11):855-865. Epub 2016 Aug 23.

Department of Pharmacology, School of Medicine, Vanderbilt University, Nashville, Tennessee.

Meta-analyses have demonstrated that low-dose aspirin reduces the risk of developing adenocarcinoma metastasis, and when colon cancer is detected during aspirin treatment, there is a remarkable 83% reduction in risk of metastasis. As platelets participate in the metastatic process, the antiplatelet action of low-dose aspirin likely contributes to its antimetastatic effect. Cycloxooxygenase-2 (COX-2)-derived prostaglandin E (PGE) also contributes to metastasis, and we addressed the hypothesis that low-dose aspirin also inhibits PGE biosynthesis. We show that low-dose aspirin inhibits systemic PGE biosynthesis by 45% in healthy volunteers (P < 0.0001). Aspirin is found to be more potent in colon adenocarcinoma cells than in the platelet, and in lung adenocarcinoma cells, its inhibition is equivalent to that in the platelet. Inhibition of COX by aspirin in colon cancer cells is in the context of the metastasis of colon cancer primarily to the liver, the organ exposed to the same high concentrations of aspirin as the platelet. We find that the interaction of activated platelets with lung adenocarcinoma cells upregulates COX-2 expression and PGE biosynthesis, and inhibition of platelet COX-1 by aspirin inhibits PGE production by the platelet-tumor cell aggregates. In conclusion, low-dose aspirin has a significant effect on extraplatelet cyclooxygenase and potently inhibits COX-2 in lung and colon adenocarcinoma cells. This supports a hypothesis that the remarkable prevention of metastasis from adenocarcinomas, and particularly from colon adenocarcinomas, by low-dose aspirin results from its effect on platelet COX-1 combined with inhibition of PGE biosynthesis in metastasizing tumor cells. Cancer Prev Res; 9(11); 855-65. ©2016 AACR.
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http://dx.doi.org/10.1158/1940-6207.CAPR-16-0094DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5093073PMC
November 2016

Cyclooxygenase inhibition targets neurons to prevent early behavioural decline in Alzheimer's disease model mice.

Brain 2016 07 13;139(Pt 7):2063-81. Epub 2016 May 13.

1 Department of Neurology and Neurological Sciences, Stanford University School of Medicine, Stanford, CA 94305, USA

Identifying preventive targets for Alzheimer's disease is a central challenge of modern medicine. Non-steroidal anti-inflammatory drugs, which inhibit the cyclooxygenase enzymes COX-1 and COX-2, reduce the risk of developing Alzheimer's disease in normal ageing populations. This preventive effect coincides with an extended preclinical phase that spans years to decades before onset of cognitive decline. In the brain, COX-2 is induced in neurons in response to excitatory synaptic activity and in glial cells in response to inflammation. To identify mechanisms underlying prevention of cognitive decline by anti-inflammatory drugs, we first identified an early object memory deficit in APPSwe-PS1ΔE9 mice that preceded previously identified spatial memory deficits in this model. We modelled prevention of this memory deficit with ibuprofen, and found that ibuprofen prevented memory impairment without producing any measurable changes in amyloid-β accumulation or glial inflammation. Instead, ibuprofen modulated hippocampal gene expression in pathways involved in neuronal plasticity and increased levels of norepinephrine and dopamine. The gene most highly downregulated by ibuprofen was neuronal tryptophan 2,3-dioxygenase (Tdo2), which encodes an enzyme that metabolizes tryptophan to kynurenine. TDO2 expression was increased by neuronal COX-2 activity, and overexpression of hippocampal TDO2 produced behavioural deficits. Moreover, pharmacological TDO2 inhibition prevented behavioural deficits in APPSwe-PS1ΔE9 mice. Taken together, these data demonstrate broad effects of cyclooxygenase inhibition on multiple neuronal pathways that counteract the neurotoxic effects of early accumulating amyloid-β oligomers.
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http://dx.doi.org/10.1093/brain/aww117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4939702PMC
July 2016

Cardiolipin fatty acid remodeling regulates mitochondrial function by modifying the electron entry point in the respiratory chain.

Mitochondrion 2016 05 13;28:88-95. Epub 2016 Apr 13.

Department of Pharmacology, Vanderbilt University Medical Center, 536 Robinson Research Building, Nashville, TN 37232-6602, United States. Electronic address:

Modifications of cardiolipin (CL) levels or compositions are associated with changes in mitochondrial function in a wide range of pathologies. We have made the discovery that acetaminophen remodels CL fatty acids composition from tetralinoleoyl to linoleoyltrioleoyl-CL, a remodeling that is associated with decreased mitochondrial respiration. Our data show that CL remodeling causes a shift in electron entry from complex II to the β-oxidation electron transfer flavoprotein quinone oxidoreductase (ETF/QOR) pathway. These data demonstrate that electron entry in the respiratory chain is regulated by CL fatty acid composition and provide proof-of-concept that pharmacological intervention can be used to modify CL composition.
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http://dx.doi.org/10.1016/j.mito.2016.04.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4860904PMC
May 2016

Modification of platelet proteins by malondialdehyde: prevention by dicarbonyl scavengers.

J Lipid Res 2015 Nov 16;56(11):2196-205. Epub 2015 Sep 16.

Department of Pharmacology, Vanderbilt University, Nashville, TN.

The thromboxane synthase converts prostaglandin H(2) to thromboxane A(2) and malondialdehyde (MDA) in approximately equimolar amounts. A reactive dicarbonyl, MDA forms covalent adducts of amino groups, including the ε-amine of lysine, but the importance of this reaction in platelets was unknown. Utilizing a novel LC/MS/MS method for analysis of one of the MDA adducts, the dilysyl-MDA cross-link, we demonstrated that dilysyl-MDA cross-links in human platelets are formed following platelet activation via the cyclooxygenase (COX)-1/thromboxane synthase pathway. Salicylamine and analogs of salicylamine were shown to react with MDA preferentially, thereby preventing formation of lysine adducts. Dilysyl-MDA cross-links were measured in two diseases known to be associated with increased platelet activation. Levels of platelet dilysyl-MDA cross-links were increased by 2-fold in metabolic syndrome relative to healthy subjects, and by 1.9-fold in sickle cell disease (SCD). In patients with SCD, the reduction of platelet dilysyl-MDA cross-links following administration of nonsteroidal anti-inflammatory drug provided evidence that MDA modifications of platelet proteins in this disease are derived from the COX pathway. In summary, MDA adducts of platelet proteins that cross-link lysines are formed on platelet activation and are increased in diseases associated with platelet activation. These protein modifications can be prevented by salicylamine-related scavengers.
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http://dx.doi.org/10.1194/jlr.P063271DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4617406PMC
November 2015

Understanding the role of prostaglandin E2 in regulating human platelet activity in health and disease.

Thromb Res 2015 Sep 28;136(3):493-503. Epub 2015 May 28.

Department of Pharmacology, Vanderbilt University, Nashville, TN 37232. Electronic address:

The platelet thrombus is the major pathologic entity in acute coronary syndromes, and antiplatelet agents are a mainstay of therapy. However, individual patient responsiveness to current antiplatelet drugs is variable, and all drugs carry a risk of bleeding. An understanding of the complex role of Prostaglandin E2 (PGE2) in regulating thrombosis offers opportunities for the development of novel individualized antiplatelet treatment. However, deciphering the platelet regulatory function of PGE2 has long been confounded by non-standardized experimental conditions, extrapolation of murine data to humans, and phenotypic differences in PGE2 response. This review synthesizes past and current knowledge about PGE2 effects on platelet biology, presents a rationale for standardization of experimental protocols, and provides insight into a molecular mechanism by which PGE2-activated pathways could be targeted for new personalized antiplatelet therapy to inhibit pathologic thrombosis without affecting hemostasis.
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http://dx.doi.org/10.1016/j.thromres.2015.05.027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4553088PMC
September 2015

Reactive γ-ketoaldehydes promote protein misfolding and preamyloid oligomer formation in rapidly-activated atrial cells.

J Mol Cell Cardiol 2015 Feb 18;79:295-302. Epub 2014 Nov 18.

Departments of Medicine and Pharmacology, Vanderbilt University School of Medicine, Nashville, TN, USA. Electronic address:

Rapid activation causes remodeling of atrial myocytes resembling that which occurs in experimental and human atrial fibrillation (AF). Using this cellular model, we previously observed transcriptional upregulation of proteins implicated in protein misfolding and amyloidosis. For organ-specific amyloidoses such as Alzheimer's disease, preamyloid oligomers (PAOs) are now recognized to be the primary cytotoxic species. In the setting of oxidative stress, highly-reactive lipid-derived mediators known as γ-ketoaldehydes (γ-KAs) have been identified that rapidly adduct proteins and cause PAO formation for amyloid β1-42 implicated in Alzheimer's. We hypothesized that rapid activation of atrial cells triggers oxidative stress with lipid peroxidation and formation of γ-KAs, which then rapidly crosslink proteins to generate PAOs. To investigate this hypothesis, rapidly-paced and control, spontaneously-beating atrial HL-1 cells were probed with a conformation-specific antibody recognizing PAOs. Rapid stimulation of atrial cells caused the generation of cytosolic PAOs along with a myocyte stress response (e.g., transcriptional upregulation of Nppa and Hspa1a), both of which were absent in control, unpaced cells. Rapid activation also caused the formation of superoxide and γ-KA adducts in atriomyocytes, while direct exposure of cells to γ-KAs resulted in PAO production. Increased cytosolic atrial natriuretic peptide (ANP), and the generation of ANP oligomers with exposure to γ-KAs and rapid atrial HL-1 cell stimulation, strongly suggest a role for ANP in PAO formation. Salicylamine (SA) is a small molecule scavenger of γ-KAs that can protect proteins from modification by these reactive compounds. PAO formation and transcriptional remodeling were inhibited when cells were stimulated in the presence of SA, but not with the antioxidant curcumin, which is incapable of scavenging γ-KAs. These results demonstrate that γ-KAs promote protein misfolding and PAO formation as a component of the atrial cell stress response to rapid activation, and they provide a potential mechanistic link between oxidative stress and atrial cell injury.
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http://dx.doi.org/10.1016/j.yjmcc.2014.11.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4302000PMC
February 2015

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

Suppression of Alzheimer-associated inflammation by microglial prostaglandin-E2 EP4 receptor signaling.

J Neurosci 2014 Apr;34(17):5882-94

Department of Neurology and Neurological Sciences, Stanford University School of Medicine, Stanford, California 94305, Neurosciences Graduate Program, Stanford University, Stanford, California 94305, and Department of Pharmacology, Vanderbilt University, Nashville, Tennessee 37232.

A persistent and nonresolving inflammatory response to accumulating Aβ peptide species is a cardinal feature in the development of Alzheimer's disease (AD). In response to accumulating Aβ peptide species, microglia, the innate immune cells of the brain, generate a toxic inflammatory response that accelerates synaptic and neuronal injury. Many proinflammatory signaling pathways are linked to progression of neurodegeneration. However, endogenous anti-inflammatory pathways capable of suppressing Aβ-induced inflammation represent a relatively unexplored area. Here we report that signaling through the prostaglandin-E2 (PGE2) EP4 receptor potently suppresses microglial inflammatory responses to Aβ42 peptides. In cultured microglial cells, EP4 stimulation attenuated levels of Aβ42-induced inflammatory factors and potentiated phagocytosis of Aβ42. Microarray analysis demonstrated that EP4 stimulation broadly opposed Aβ42-driven gene expression changes in microglia, with enrichment for targets of IRF1, IRF7, and NF-κB transcription factors. In vivo, conditional deletion of microglial EP4 in APPSwe-PS1ΔE9 (APP-PS1) mice conversely increased inflammatory gene expression, oxidative protein modification, and Aβ deposition in brain at early stages of pathology, but not at later stages, suggesting an early anti-inflammatory function of microglial EP4 signaling in the APP-PS1 model. Finally, EP4 receptor levels decreased significantly in human cortex with progression from normal to AD states, suggesting that early loss of this beneficial signaling system in preclinical AD development may contribute to subsequent progression of pathology.
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http://dx.doi.org/10.1523/JNEUROSCI.0410-14.2014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3996215PMC
April 2014

Levuglandin forms adducts with histone h4 in a cyclooxygenase-2-dependent manner, altering its interaction with DNA.

Biochemistry 2014 Apr 11;53(15):2436-41. Epub 2014 Apr 11.

Departments of †Pharmacology, ‡Pathology, and §Medicine, Vanderbilt University , Nashville, Tennessee 37232, United States.

Inflammation and subsequent cyclooxygenase-2 (COX-2) activity has long been linked with the development of cancer, although little is known about any epigenetic effects of COX-2. A product of COX-2 activation, levuglandin (LG) quickly forms covalent bonds with nearby primary amines, such as those in lysine, which leads to LG-protein adducts. Here, we demonstrate that COX-2 activity causes LG-histone adducts in cultured cells and liver tissue, detectable through LC-MS, with the highest incidence in histone H4. Adduction is blocked by a γ-ketoaldehyde scavenger, which has no effect on COX-2 activity as measured by PGE2 production. Formation of the LG-histone adduct is associated with an increased histone solubility in NaCl, indicating destabilization of the nucleosome structure; this is also reversed with scavenger treatment. These data demonstrate that COX-2 activity can cause histone adduction and loosening of the nucleosome complex, which could lead to altered transcription and contribute to carcinogenesis.
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http://dx.doi.org/10.1021/bi401673bDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4004227PMC
April 2014

Rational Design of Novel Pyridinol-Fused Ring Acetaminophen Analogues.

ACS Med Chem Lett 2013 Aug;4(8):710-714

Department of Pharmacology, Division of Clinical Pharmacology, Vanderbilt University Medical Center, 536 Robinson Research Building, Nashville, Tennessee 37232-6602, United States.

Acetaminophen (ApAP) is an electron donor capable of reducing radicals generated by redox cycling of hemeproteins. It acts on the prostaglandin H synthases (cyclooxygenases; COXs) to reduce the protoporphyrin radical cation in the peroxidase site of the enzyme, thus preventing the intra-molecular electron transfer that generates the Tyr385 radical required for abstraction of a hydrogen from arachidonic acid to initiate prostaglandin synthesis. Unrelated to this pharmacological action, metabolism of ApAP by CYPs yields an iminoquinone electrophile that is responsible for the hepatotoxicity, which results from high doses of the drug. We synthesized novel heterocyclic phenols predicted to be electron donors. Two of these inhibited the oxygenation of arachidonic acid by PGHS-1 and myoglobin and also were shown to be more metabolically stable and exhibited less direct cytotoxicity than acetaminophen. They are leading candidates for studies to determine whether they are free of the metabolism-based hepatotoxicity produced by acetaminophen.
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http://dx.doi.org/10.1021/ml4000904DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3904457PMC
August 2013

Acetaminophen inhibits cytochrome c redox cycling induced lipid peroxidation.

Biochem Biophys Res Commun 2012 Jun 23;423(2):224-8. Epub 2012 May 23.

Department of Medicine, Division of Clinical Pharmacology, Vanderbilt University School of Medicine, TN 37232, USA.

Cytochrome (cyt) c can uncouple from the respiratory chain following mitochondrial stress and catalyze lipid peroxidation. Accumulating evidence shows that this phenomenon impairs mitochondrial respiratory function and also initiates the apoptotic cascade. Therefore, under certain conditions a pharmacological approach that can inhibit cyt c catalyzed lipid peroxidation may be beneficial. We recently showed that acetaminophen (ApAP) at normal pharmacologic concentrations can prevent hemoprotein-catalyzed lipid peroxidation in vitro and in vivo by reducing ferryl heme to its ferric state. We report here, for the first time, that ApAP inhibits cytochrome c-catalyzed oxidation of unsaturated free fatty acids and also the mitochondrial phospholipid, cardiolipin. Using isolated mitochondria, we also showed that ApAP inhibits cardiolipin oxidation induced by the pro-apoptotic protein, tBid. We found that the IC(50) of the inhibition of cardiolipin oxidation by ApAP is similar in both intact isolated mitochondria and cardiolipin liposomes, suggesting that ApAP penetrates well into the mitochondria. Together with our previous results, the findings presented herein suggest that ApAP is a pleiotropic inhibitor of peroxidase catalyzed lipid peroxidation. Our study also provides a potentially novel pharmacological approach for inhibiting the cascade of events that can result from redox cycling of cyt c.
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http://dx.doi.org/10.1016/j.bbrc.2012.05.058DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3389218PMC
June 2012

Suboptimal inhibition of platelet cyclooxygenase-1 by aspirin in metabolic syndrome.

Hypertension 2012 Mar 6;59(3):719-25. Epub 2012 Feb 6.

Department of Medicine, Vanderbilt University Medical Center, Nashville, TN, USA.

Interindividual variation in the ability of aspirin to inhibit platelet cyclooxygenase-1 (COX-1) could account for some on-treatment cardiovascular events. Here, we sought to determine whether there are clinical phenotypes that are associated with a suboptimal pharmacological effect of aspirin. In a prospective, 2-week study, we evaluated the effect of aspirin (81 mg) on platelet COX-1 in 135 patients with stable coronary artery disease by measuring serum thromboxane B(2) (sTxB(2)) as an indicator of inhibition of platelet COX-1. A nested randomized study compared enteric-coated with immediate-release formulations of aspirin. We found that sTxB(2) was systematically higher among the 83 patients with metabolic syndrome than among the 52 patients without (median: 4.0 versus 3.02 ng/mL; P=0.013). Twelve patients (14%) with metabolic syndrome, but none without metabolic syndrome, had sTxB(2) levels consistent with inadequate inhibition of COX (sTxB(2) ≥13 ng/mL). In linear regression models, metabolic syndrome (but none of its individual components) significantly associated with higher levels of log-transformed sTxB(2) (P=0.006). Higher levels of sTxB(2) associated with greater residual platelet function measured by aggregometry-based methods. Among the randomized subset, sTxB(2) levels were systematically higher among patients receiving enteric-coated aspirin. Last, urinary 11-dehydro thromboxane B(2) did not correlate with sTxB(2), suggesting that the former should not be used to quantitate aspirin's pharmacological effect on platelets. In conclusion, metabolic syndrome, which places patients at high risk for thrombotic cardiovascular events, strongly and uniquely associates with less effective inhibition of platelet COX-1 by aspirin.
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http://dx.doi.org/10.1161/HYPERTENSIONAHA.111.181404DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3418792PMC
March 2012

Protein kinase C regulation of 12-lipoxygenase-mediated human platelet activation.

Mol Pharmacol 2012 Mar 9;81(3):420-30. Epub 2011 Dec 9.

Department of Medicine, Cardeza Foundation for Hematologic Research, Thomas Jefferson University, Philadelphia, PA 19107, USA.

Platelet activation is important in the regulation of hemostasis and thrombosis. Uncontrolled activation of platelets may lead to arterial thrombosis, which is a major cause of myocardial infarction and stroke. After activation, metabolism of arachidonic acid (AA) by 12-lipoxygenase (12-LOX) may play a significant role in regulating the degree and stability of platelet activation because inhibition of 12-LOX significantly attenuates platelet aggregation in response to various agonists. Protein kinase C (PKC) activation is also known to be an important regulator of platelet activity. Using a newly developed selective inhibitor for 12-LOX and a pan-PKC inhibitor, we investigated the role of PKC in 12-LOX-mediated regulation of agonist signaling in the platelet. To determine the role of PKC within the 12-LOX pathway, a number of biochemical endpoints were measured, including platelet aggregation, calcium mobilization, and integrin activation. Inhibition of 12-LOX or PKC resulted in inhibition of dense granule secretion and attenuation of both aggregation and αIIbβ(3) activation. However, activation of PKC downstream of 12-LOX inhibition rescued agonist-induced aggregation and integrin activation. Furthermore, inhibition of 12-LOX had no effect on PKC-mediated aggregation, indicating that 12-LOX is upstream of PKC. These studies support an essential role for PKC downstream of 12-LOX activation in human platelets and suggest 12-LOX as a possible target for antiplatelet therapy.
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http://dx.doi.org/10.1124/mol.111.075630DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3286293PMC
March 2012

On-tissue chemical derivatization of 3-methoxysalicylamine for MALDI-imaging mass spectrometry.

J Mass Spectrom 2011 Aug;46(8):840-6

Department of Pharmacology, Vanderbilt University, Nashville, TN 37232-6602, USA.

MALDI-imaging mass spectrometry (IMS) has been shown to be a powerful tool to study drug distributions in organ tissue as well as whole animal bodies. Nevertheless, not all drugs are amenable to MALDI while others may be limited by poor sensitivity poor sensitivity. The use of chemical derivatization to improve detection of small molecules by mass spectrometry techniques is well documented. To our knowledge, however, this approach has not been applied to direct tissue analysis of small organic molecules. In this manuscript, we demonstrate the use of on-tissue chemical derivatization of a small organic molecule, 3-methoxysalicylamine (3-MoSA) a scavenger of γ-ketoaldehydes. Derivatization of 3-MoSA with 1,1'-thiocarbonyldiimidazole (TCDI) results in an oxothiazolidine derivative which is detected with much greater sensitivity by MALDI than 3-MoSA itself. TCDI treatment of tissue from mice dosed with 3-MoSA allowed images to be obtained showing its spatial distribution as well as its pharmacokinetic profile in different organs. These images correlated well with results obtained from HPLC-MS/MS analyses of the same tissues. These results provide proof-of-concept that on-tissue chemical derivatization can be used to improve detection of a small organic molecule by MALDI-IMS.
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http://dx.doi.org/10.1002/jms.1958DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3174490PMC
August 2011

Determination of 3-methoxysalicylamine levels in mouse plasma and tissue by liquid chromatography-tandem mass spectrometry: application to in vivo pharmacokinetics studies.

J Chromatogr B Analyt Technol Biomed Life Sci 2011 May 21;879(15-16):1098-104. Epub 2011 Mar 21.

Department of Pharmacology, Vanderbilt University, Nashville, TN 37232-6602, USA.

We report the development of a sensitive liquid chromatography-tandem mass spectrometric assay to quantitate 3-methoxysalicylamine (3-MoSA) in biological samples. Derivatization with 1,1'-thiocarbonyldiimidazole followed by C(18) reverse-phase chromatography allowed the detection of both analyte and internal standard (hexylsalicylamine) using electrospray ionization and selected reaction monitoring (SRM) in positive ion mode. We monitored the transitions from m/z 196.7 to 65.1 and from m/z 250.1 to 77.1 for 3-MoSA and HxSA, respectively. The method is validated with respect to linearity (r(2)=0.995), precision (<17% RSD), recovery (100% for 3-MoSA and HxSA), and stability (77% after storage up to 7 month at -80°C). The LOD and LOQ were 16.12 and 48.87 μg/l, respectively and the LLOQ of 1 pg/ml. In addition, we used this assay to analyze the pharmacokinetics of 3-MoSA in mouse plasma and tissues following both intraperitoneal and oral administration, providing new information regarding the distribution of this compound in vivo.
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http://dx.doi.org/10.1016/j.jchromb.2011.03.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3091354PMC
May 2011