James A. Wilkinson

A novel role for small molecule glycomimetics in the protection against lipid-induced endothelial dysfunction: Involvement of Akt/eNOS and Nrf2/ARE signaling

BACKGROUND Glycomimetics are a diverse array of saccharide-inspired compounds, designed to mimic the bioactive functions of glycosaminoglycans. Therefore, glycomimetics represent a unique source of novel therapies to target aberrant signaling and protein interactions in a wide range of diseases. We investigated the protective effects of four newly synthesized small molecule glycomimetics against lipid-induced endothelial dysfunction, with an emphasis on nitric oxide (NO) and oxidative stress. METHODS Four aromatic sugar mimetics were synthesized by the stepwise transformation of 2,5-dihydroxybenzoic acid to derivatives (C1-C4) incorporating sulfate groups to mimic the structure of heparan sulfate. RESULTS Glycomimetic-treated human umbilical vein endothelial cells (HUVECs) were exposed to palmitic acid to model lipid-induced oxidative stress. Palmitate-induced impairment of NO production was restored by the glycomimetics, through activation of Akt/eNOS signaling. Furthermore, C1-C4 significantly inhibited palmitate-induced reactive oxygen species (ROS) production, lipid peroxidation, and activity and expression of NADPH oxidase. These effects were attributed to activation of the Nrf2/ARE pathway and downstream activation of cellular antioxidant and cytoprotective proteins. In ex vivo vascular reactivity studies, the glycomimetics (C1-C4) also demonstrated a significant improvement in endothelium-dependent relaxation and decreased ROS production and NADPH oxidase activity in isolated mouse thoracic aortic rings exposed to palmitate. CONCLUSIONS The small molecule glycomimetics, C1-C4, protect against lipid-induced endothelial dysfunction through up-regulation of Akt/eNOS and Nrf2/ARE signaling pathways. Thus, carbohydrate-derived therapeutics are a new class of glycomimetic drugs targeting endothelial dysfunction, regarded as the first line of defense against vascular complications in cardiovascular disease.

Stereoselective cycloadditions of chiral acyl-nitroso compounds; hydrolytic reactions of a clyclopentadiene adduct

Treatment of the adduct between the acyl nitroso intermediate derived from (R)-α-hydroxyphenylacetohydroxamic acid and cyclopentadiene, 1, with dilute aqueous acid provides a high yield of the cyclopentene 2 hydrochloride, suitable for further, synthetically useful transformations.

Stereoselective cycloadditions of chiral acyl-nitroso compounds; unexpected formation of an oxazolidinone

Abstract On treatment with either N -phenyltrifluoromethanesulphonimide or p -toluenesulphonyl chloride and base, hydroxamic acid 3 provides the oxazolidinone 4 in moderate yield, probably by a pathway involving fragmentation; Boc derivative 8 is suitable substrate for Pd o catalyzed nucleophilic displacement.

Generation of superoxide and singlet oxygen from α-tocopherolquinone and analogues

Three potential routes to generation of reactive oxygen species (ROS) from α-tocopherolquinone (α-TQ) have been identified. The quinone of the water-soluble vitamin E analogue Trolox C (Trol-Q) is reduced by hydrated electron and isopropanol α-hydroxyalkyl radical, and the resulting semiquinone reacts with molecular oxygen to form superoxide with a second order rate constant of 1.3 × 108 dm3/mol/s, illustrating the potential for redox cycling. Illumination (UV-A, 355 nm) of the quinone of 2,2,5,7,8-pentamethyl-6-hydroxychromanol (PMHC-Q) leads to a reactive short-lived (ca. 10− 6 s) triplet state, able to oxidise tryptophan with a second order rate constant greater than 109 dm3/mol/s. The triplet states of these quinones sensitize singlet oxygen formation with quantum yields of about 0.8. Such potentially damaging reactions of α-TQ may in part account for the recent findings that high levels of dietary vitamin E supplementation lack any beneficial effect and may lead to slightly enhanced levels of overall mortality.

Stereoselective cycloadditions of chiral acyl-nitroso compounds; palladium(0) catalyzed allylic displacement with concomitant loss of the chiral auxiliary

The methylated diacetate 4, derived from the readily available hydroxamic acid 1, undergoes Pdo catalyzed nucleophilic allylic displacement under conditions which also result in the removal of the chiral auxiliary and its replacement with an easily removed acetyl group.

204 Glycomimetics; A Novel Class of Drugs to Protect Against Free Fatty Acid-Induced Endothelial Dysfunction

Background Endothelial dysfunction is a key player in cardiovascular disease (CVD) complications and novel drugs are required to treat this pathological process. Glycosaminoglycans (GAGs) are key molecules that regulate signalling in many biological processes and drugs that mimic their structure could be a novel source of therapeutics to target specific CVD pathways. Purpose We have synthesised a set of four glycomimetic compounds and our objective was to determine whether they could activate protective pathways in endothelial cells subjected to fatty acid-induced endothelial dysfunction. Methods Glycomimetics, C1-C4, were synthesised by the stepwise transformation of 2,5-dihydroxybenzoic acid to a range of 2,5-substituted benzoic acid derivatives, incorporating the key sulphate groups to mimic heparan sulphate. Human Umbilical Vein Endothelial Cells (HUVECs) were treated with glycomimetics (1µM) in the presence or absence of the free fatty acid, palmitate. DAF-2 and H2DCF-DA assays were used to determine NO and reactive oxygen species (ROS) production, respectively. Lipid peroxidation colorimetric and antioxidant enzyme activity ssays were also carried out. RT-PCR and western blotting were utilised to measure Akt, eNOS, Nrf-2, NQO-1 and HO-1 expression. Endothelial function was determined ex vivo using acetylcholine-induced endothelium-dependent relaxation in mouse thoracic aortic rings by wire myography. Results All four glycomimetics protected against palmitate-induced oxidative stress and enhanced NO production in vitro via upregulation of Akt/eNOS signalling, activation of the Nrf2/ARE pathway and down-regulation of ROS-induced lipid peroxidation. Under palmitate-induced oxidative stress, ex vivo endothelium-dependent relaxation was significantly enhanced by all four glycomimetics. Furthermore, the glycomimetics did not induce HUVEC activation, as determined by lack of ICAM-1 protein. Conclusion We have developed a new set of small molecule glycomimetics that do not activate ECs and protect against free fatty acid-induced endothelial dysfunction both in vitro and ex vivo. Future work will focus on developing the glycomimetics into drug-like therapies that target endothelial damage.

43 A novel role for small molecule glycomimetics in the protection against lipid-induced endothelial dysfunction

Glycomimetics are molecules that mimic the structure of carbohydrates involved in important biological processes. Small molecule glycomimetics are an untapped source of novel therapies for endothelial dysfunction, a hallmark of cardiovascular complications associated with diabetes. The current study aims to investigate the possible protective effects of newly synthesised small molecule glycomimetics against lipid-induced endothelial dysfunction, with an emphasis on nitric oxide (NO) and induced oxidative stress. Glycomimetics were synthesised by the stepwise transformation of 2,5-dihydroxybenzoic acid to a range of 2,5-substituted benzoic acid derivatives incorporating the key sulfate groups to mimic the interactions of heparan sulfate. Acetylcholine-induced endothelium-dependent relaxation in mouse thoracic aortic rings was measured using wire myography, and human umbilical vein endothelial cells (HUVECs) function was assessed in the presence or absence of palmitate, with or without the test glycomimetics. NO and reactive oxygen species (ROS) production was measured using DAF-2 and H2DCF-DA, respectively. Colorimetric assays were used to determine lipid peroxidation and activity of the antioxidant enzymes. Expression of Akt, eNOS, Nrf-2, NQO-1 and HO-1 were assessed using RT-PCR and western blotting. At 1 µM concentration, the synthesised glycomimetics significantly improved endothelium-dependent relaxation ex vivo and protected HUVECs against palmitate-induced oxidative stress and reduced NO production. Pre-incubation of HUVECs with all compounds upregulated Akt/eNOS signalling, activated Nrf2/ARE pathway, and suppressed ROS-induced lipid peroxidation. In conclusion, our newly synthesised small molecule glycomimetics protect against lipid-induced endothelial dysfunction. These novel cytoprotective effects open the door to a new class of therapeutic drugs to target endothelial dysfunction.