Comparative inhibition by substrate analogues 3-methoxy- and 3-hydroxydesaminokynurenine and an improved 3 step purification of recombinant human kynureninase.

BACKGROUND: Kynureninase is a key enzyme on the kynurenine pathway of tryptophan metabolism. One of the end products of the pathway is the neurotoxin quinolinic acid which appears to be responsible for neuronal cell death in a number of important neurological diseases. This makes kynureninase a possible therapeutic target for diseases such as Huntington's, Alzheimer's and AIDS related dementia, and the development of potent inhibitors an important research aim. RESULTS: Two new kynurenine analogues, 3-hydroxydesaminokynurenine and 3-methoxydesaminokynurenine, were synthesised as inhibitors of kynureninase and tested on the tryptophan-induced bacterial enzyme from Pseudomonas fluorescens, the recombinant human enzyme and the rat hepatic enzyme. They were found to be mixed inhibitors of all three enzymes displaying both competitive and non competitive inhibition. The 3-hydroxy derivative gave low K(i )values of 5, 40 and 100 nM respectively. An improved 3-step purification scheme for recombinant human kynureninase was also developed. CONCLUSION: For kynureninase from all three species the 2-amino group was found to be crucial for activity whilst the 3-hydroxyl group played a fundamental role in binding at the active site presumably via hydrogen bonding. The potency of the various inhibitors was found to be species specific. The 3-hydroxylated inhibitor had a greater affinity for the human enzyme, consistent with its specificity for 3-hydroxykynurenine as substrate, whilst the methoxylated version yielded no significant difference between bacterial and human kynureninase. The modified purification described is relatively quick, simple and cost effective

1-(5-Chloro-2,4-dihydroxy­phen­yl)-2-(4-ethoxy­phen­yl)ethanone

The structure of the title compound, C16H15ClO4, contains aryl rings which are inclined by 75.6 (1)° to each other. It displays intra­molecular O—H⋯O hydrogen bonding between the 2-hydr­oxy and carbonyl groups, forming a six-membered ring. Furthermore, the 4-hydr­oxy group, acting as a hydrogen-bond donor, is bound to the O atom of the 2-hydr­oxy group of another mol­ecule

Human metabolism and elimination of the anthocyanin, cyanidin-3-glucoside: a 13C-tracer study

BACKGROUND: Evidence suggests that the consumption of anthocyanin-rich foods beneficially affects cardiovascular health; however, the absorption, distribution, metabolism, and elimination (ADME) of anthocyanin-rich foods are relatively unknown. OBJECTIVE: We investigated the ADME of a (13)C5-labeled anthocyanin in humans. DESIGN: Eight male participants consumed 500 mg isotopically labeled cyanidin-3-glucoside (6,8,10,3',5'-(13)C5-C3G). Biological samples were collected over 48 h, and (13)C and (13)C-labeled metabolite concentrations were measured by using isotope-ratio mass spectrometry and liquid chromatography-tandem mass spectrometry. RESULTS: The mean +/- SE percentage of (13)C recovered in urine, breath, and feces was 43.9 +/- 25.9% (range: 15.1-99.3% across participants). The relative bioavailability was 12.38 +/- 1.38% (5.37 +/- 0.67% excreted in urine and 6.91 +/- 1.59% in breath). Maximum rates of (13)C elimination were achieved 30 min after ingestion (32.53 +/- 14.24 mug(13)C/h), whereas (13)C-labeled metabolites peaked (maximum serum concentration: 5.97 +/- 2.14 mumol/L) at 10.25 +/- 4.14 h. The half-life for (13)C-labeled metabolites ranged between 12.44 +/- 4.22 and 51.62 +/- 22.55 h. (13)C elimination was greatest between 0 and 1 h for urine (90.30 +/- 15.28 mug/h), at 6 h for breath (132.87 +/- 32.23 mug/h), and between 6 and 24 h for feces (557.28 +/- 247.88 mug/h), whereas the highest concentrations of (13)C-labeled metabolites were identified in urine (10.77 +/- 4.52 mumol/L) and fecal samples (43.16 +/- 18.00 mumol/L) collected between 6 and 24 h. Metabolites were identified as degradation products, phenolic, hippuric, phenylacetic, and phenylpropenoic acids. CONCLUSION: Anthocyanins are more bioavailable than previously perceived, and their metabolites are present in the circulation fo

Single-cell multi-omics analysis of the immune response in COVID-19

Funder: Lister Institute of Preventive Medicine; doi: https://doi.org/10.13039/501100001255Funder: University College London, Birkbeck MRC Doctoral Training ProgrammeFunder: The Jikei University School of MedicineFunder: Action Medical Research (GN2779)Funder: NIHR Clinical Lectureship (CL-2017-01-004)Funder: NIHR (ACF-2018-01-004) and the BMA FoundationFunder: Chan Zuckerberg Initiative (grant 2017-174169) and from Wellcome (WT211276/Z/18/Z and Sanger core grant WT206194)Funder: UKRI Innovation/Rutherford Fund Fellowship allocated by the MRC and the UK Regenerative Medicine Platform (MR/5005579/1 to M.Z.N.). M.Z.N. and K.B.M. have been funded by the Rosetrees Trust (M944)Funder: Barbour FoundationFunder: ERC Consolidator and EU MRG-Grammar awardsFunder: Versus Arthritis Cure Challenge Research Grant (21777), and an NIHR Research Professorship (RP-2017-08-ST2-002)Funder: European Molecular Biology Laboratory (EMBL)Abstract: Analysis of human blood immune cells provides insights into the coordinated response to viral infections such as severe acute respiratory syndrome coronavirus 2, which causes coronavirus disease 2019 (COVID-19). We performed single-cell transcriptome, surface proteome and T and B lymphocyte antigen receptor analyses of over 780,000 peripheral blood mononuclear cells from a cross-sectional cohort of 130 patients with varying severities of COVID-19. We identified expansion of nonclassical monocytes expressing complement transcripts (CD16+C1QA/B/C+) that sequester platelets and were predicted to replenish the alveolar macrophage pool in COVID-19. Early, uncommitted CD34+ hematopoietic stem/progenitor cells were primed toward megakaryopoiesis, accompanied by expanded megakaryocyte-committed progenitors and increased platelet activation. Clonally expanded CD8+ T cells and an increased ratio of CD8+ effector T cells to effector memory T cells characterized severe disease, while circulating follicular helper T cells accompanied mild disease. We observed a relative loss of IgA2 in symptomatic disease despite an overall expansion of plasmablasts and plasma cells. Our study highlights the coordinated immune response that contributes to COVID-19 pathogenesis and reveals discrete cellular components that can be targeted for therapy

A new class of NO-donor pro-drugs triggered by γ-glutamyl transpeptidase with potential for reno-selective vasodilatation

The authors are grateful to the Wellcome Trust (Catalyst Biomedica Development Award 063729/Z/01/Z) for financial support.This communication describes the synthesis of a new class of N-hydroxyguanidine (NHG) pro-drugs which release nitric oxide (NO), triggered by the action of gamma-glutamyl transpeptidase (gamma-GT), and have potential for the treatment of acute renal injury/failure (ARI/ARF).Publisher PDFPeer reviewe

Synthesis of deuterium labelled desulfoglucosinolates as internal standards for LC-MS analysis

The syntheses of three deuterium labelled desulfoglucosinolates are described. These are the phenethyl, 1-methoxyindolyl and 4-methoxyindolyl derivatives. The compounds were prepared as internal standards for use in the quantitative LC-MS analysis of glucosinolates to improve the sensitivity of the analytical procedure. (C) 1999 Elsevier Science Ltd. All rights reserved.</p

N-hydroxyguanidines and related compounds as nitric oxide donors

The design of new, improved NO-donor drugs is an important pharmacological objective due to the biological importance of nitric oxide. N-Hydroxyguanidines represent a useful class of NO donors where the mechanism of action is based on the biosynthetic pathway for NO. Thirty new N-arylalkyl-N’-hydroxyguanidines were synthesized and their vasodilatation activity examined by myography in rat aortic rings. The observed relaxations were reversed by ODQ, which is an inhibitor of the guanylate cyclase, implying that this was an NO dependent vasodilatation. The most active compounds were also tested in the isolated perfused kidney (IPK) giving the vasodilatation properties. Preliminary results indicated that N-phenyl-N’- hydroxyguanidine showed the best pharmacological profile with EC₅₀= 19.9 μM and ca. 100% reversibility with ODQ. A series of N-phenylalkyl-N’-hydroxyguanidines were synthesised. NO donor activity was found to be fairly constant up to three methylene groups, and then decreased. Substitutions in the benzene ring of N-phenylethyl-N’-hydroxyguanidine demonstrated that various electron-withdrawing and electron-donating groups in the para position did not significantly affect the NO donor activity of this series of analogues. The nitro and trifluoromethyl substituted compounds gave the best biological profiles. Additionally, a novel heterocyclic, N–furfuryl-N’–hydroxyguanidine possessed very promising vasodilatation properties. In general, almost all the N-arylalkyl-N’-hydroxyguanidines behaved as potent NO donors in the rat aorta assay. In order to establish the influence of the free NH₂ group in the hydroxyguanidine functionality on the vasodilatation properties, N,N-dimethyl and N-methyl-N’- hydroxyguanidines were successfully synthesised. Unfortunately, they have not been tested yet in the biological assay. However, their NMR spectra showed some unusual features and their detailed analysis and X-ray data are presented herein. In addition a series of hydroxamic acids was synthesised and the NO donor activity investigated using the same biological methodology. It was found that the 3-phenylpropionohydroxamic acid was the most potent compound with EC₅₀ = 6 μM and ODQ = 96%. However, behavior in the IPK indicated that hydroxamic acids did not undergo the same biological pathway as in the rat aorta. Two different types of enzyme-activated pro-drugs were designed using N-hydroxyguanidines as the NO donating molecule. Synthetic studies towards these targets were carried out using various synthetic approaches. The desired molecules have not yet been synthesised but the chemistry explored so far has indicated potentially more successful approaches that could be attempted.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Novel methodology for the synthesis of ¹³C-Labelled phenols and its application to the total synthesis of polyphenols

The base-catalysed reaction of 4H-pyran-4-one with a range of nucleophiles, namely diethyl malonate, ethyl acetoacetate, nitromethane, acetylacetone and ethyl cyanoacetate, was developed as a reliable, high yielding method for the preparation of para-substituted phenols. The methodology was extended to include the use of the substituted pyranones, maltol, 2,6-dimethyl-4H-pyran-4-one and diethyl chelidonate. Reactions were studied using conventional heating methods and microwave irradiation. Microwave irradiation had definite beneficial effects, with improved yields, reduced reaction times and cleaner reaction profiles. The potential of this methodology was examined for the regioselective placement of ¹³C-atoms into benzene rings using ¹³C-labelled nucleophiles or ¹³C-labelled 4H-pyran-4-ones. [3,5-13C₂]4H-Pyran-4-one and [2,6-13C₂]4H-pyran-4-one were prepared from various ¹³C-labelled versions of triethyl orthoformate and acetone. This methodology was applied to the synthesis of [1,3,5-¹³C₃]gallic acid, via the base-catalysed reaction of [3,5-¹³C₂]4H-pyran-4-one with diethyl [2-¹³C]malonate, followed by subsequent transformations to yield [1,3,5-¹³C₃]gallic acid. The preparation of [2-¹³C]phloroglucinol was carried out via [2-¹³C]resorcinol, with regioselective placement of a single ¹³C-atom into the aromatic ring. This was accomplished from non-aromatic precursors, with the source of the ¹³C-atom being [¹³C]methyl iodide. The key step in this synthesis was the introduction of the third hydroxyl group, which was achieved using a modified iridium-catalysed C-H activation/borylation/oxidation procedure. The scope of an existing C-H activation/borylation reaction was modified and expanded to include a range of protected resorcinol derivatives. A catalyst system was developed which allowed high conversion to the intermediate arylboronic acids, followed by oxidation using aqueous Oxone® to yield the corresponding phenols. Finally, to demonstrate the potential of these new methods for application in the synthesis of isotopically labelled natural products and polyphenols, the syntheses of ¹³C-labelled anthocyanins were studied. A route was developed that could be applied to the synthesis of either cyanidin-3-glucoside or delphinidin-3-glucoside. Only the final coupling/cyclisation step to yield the desired anthocyanin targets remains to be carried out.EThOS - Electronic Theses Online ServiceGlaxoSmithKlineBiotechnology and Biological Sciences Research CouncilEaStCHEMGBUnited Kingdo

The synthesis of isotopically labelled glucoraphanin for metabolic studies

The first synthesis of a stable isotopically labelled derivative of the glucosinolate glucoraphanin, namely [10-C-13, 11,12-H-2(5)]glucoraphanin, is described. This also represents the first total chemical synthesis of glucoraphanin itself. (c) 2007 Published by Elsevier Ltd.</p

A facile synthesis of isoflavone 7-O-glucuronides

An efficient method is presented for the synthesis of isoflavone 7-glucuronides using a N-(4-methoxyphenyl)-trifluoroacetimidate glucuronsyl donor. A 4-hexanoyl derivative of the isoflavone is used in the coupling reaction, both for protection and to improve solubility. These glucuronides are the human metabolites of estrogenic dietary isotlavones, but their biological activity and pharmacokinetics have yet to be established as until now there were no good methods for their synthesis. (c) 2006 Elsevier Ltd. All rights reserved.</p