Karine Auclair - Academia.edu (original) (raw)

Papers by Karine Auclair

Antimicrobial resistance is a global health crisis offering little reprieve. The situation urgent... more Antimicrobial resistance is a global health crisis offering little reprieve. The situation urgently calls for new drug targets and therapies for infections. We have previously suggested a different approach to treat infections, termed bacterio-modulation, in which a compound modulates the bacterial response to the host immune defense. Herein we show that monocytes infected with Salmonella enterica spp. Typhimurium can be cured using non-antimicrobial compounds that resensitize the bacterium to itaconate, a macrophage-derived antimicrobial metabolite. We propose that this represents a novel strategy to treat infections.

The production of itaconate by macrophages was only discovered in 2011. A rapidly increasing numb... more The production of itaconate by macrophages was only discovered in 2011. A rapidly increasing number of studies have since revealed essential biological roles for itaconate, ranging from antimicrobial to immunomodulator. Itaconate has been estimated to reach low-millimolar concentrations in activated macrophages, including those within infected lungs and brains, whereas itaconate’s MIC towards several bacterial strains were measured to be in the low-to-mid-millimolar range, casting some doubts on the antibacterial role of itaconate in vivo. Several of these investigations, in particular those measuring MIC values of itaconate or itaconic acid, have however tended to ignore the high acidity of this small diacid (pKas 3.85 and 5.45), thereby potentially biasing the MIC measurements. We report herein that: 1) at high concentration, itaconic acid can significantly reduce the pH of growth media; 2) the antibacterial activity of itaconate increases in a synergistic manner with acidity; 3) ...

ABSTRACTThe Plasmodium parasites that cause malaria are adept at developing resistance to antimal... more ABSTRACTThe Plasmodium parasites that cause malaria are adept at developing resistance to antimalarial drugs, necessitating the search for new antiplasmodials. Although several amide analogs of pantothenate (pantothenamides) show potent antiplasmodial activity, hydrolysis by pantetheinases (or vanins) present in blood rapidly inactivates them. We report herein the facile synthesis and biological activity of a small library of pantothenamide analogs in which the labile amide group is replaced with a variety of heteroaromatic rings. Several of the new analogs display antiplasmodial activity in the nanomolar range against P. falciparum and/or P. knowlesi in the presence of pantetheinase. A previously reported triazole and an isoxazole derivative presented here were further characterized and found to possess high selectivity indices, medium or high Caco-2 permeability, and medium or low microsomal clearance in vitro. Although we show here that the two compounds fail to suppress prolifer...

Lignocellulosic material is an abundant renewable resource with the potential to replace petroleu... more Lignocellulosic material is an abundant renewable resource with the potential to replace petroleum as a feedstock for the production of fuels and chemicals. The large scale deployment of biomass saccharification is, however, hampered by the necessity to use aggressive reagents and conditions, formation of side-products, and the difficulty to reach elevated monosaccharide concentrations in the crude product. Herein we report the high efficacy of Reactive Aging (or Raging, a technique where enzymatic reaction mixtures, without any bulk aqueous or organic solvent, are treated to multiple cycles of milling and aging) for gram-scale saccharification of raw lignocellulosic biomass samples from different agricultural sources (corn stover, wheat straw, and sugarcane bagasse). The solvent-free enzymatic conversion of lignocellulosic biomass was found to proceed in excellent yields (ca. 90%) at protein loadings as low as 2% w/w, without the need for any prior chemical pre-treatment or high te...

Although cellulose has been identified as the foremost candidate for the replacement of fossil fu... more Although cellulose has been identified as the foremost candidate for the replacement of fossil fuels, its recalcitrant nature prevents the full deployment of technologies based on its saccharification. We recently reported a possible strategy to resolve this conundrum: using cellulases under “RAging” - a solvent-free process that utilizes enzymes under mechanochemical conditions - to achieve fast, efficient hydrolytic depolymerization of cellulosic materials into glucose. β-Glucosidases catalyze the last and often limiting step of this process, i.e. the formation of glucose from cellobiose. Here, we reveal the high sensitivity of β-glucosidases to ball milling, as well as an unexpected stabilization effect of inert surfaces, enabling the protection of β-glucosidases under mechanochemical treatment. This approach provides an unexpected strategy to control the reactivity of enzymes under mechanochemical conditions. Finally, our results also provide the very first demonstration of enzy...

Bioconjugate Chemistry, 2017

Human cytochrome P450 3A4 (CYP3A4) is responsible for the metabolism of the majority of drugs. As... more Human cytochrome P450 3A4 (CYP3A4) is responsible for the metabolism of the majority of drugs. As such, it is implicated in many adverse drug-drug and food-drug interactions, and is of significant interest to the pharmaceutical industry. This enzyme is known to simultaneously bind multiple ligands and display atypical enzyme kinetics, suggestive of allostery and cooperativity. As well, evidence of a postulated peripheral allosteric binding site has provoked debate around its significance and location. We report the use of bioconjugation to study the significance of substrate binding at the proposed allosteric site and its effect on CYP3A4 activity. CYP3A4 mutants were created and covalently modified with various small molecules including progesterone. The labeled mutants displayed enhanced kinetic stability and improved activity in testosterone and 7-benzyloxy-(4-trifluoromethyl)coumarin oxidation assays. Our work applies a new strategy to study cytochrome P450 allostery and supports the hypothesis that substrate binding at the postulated allosteric site of CYP3A4 may induce functional cooperativity.

Antimicrobial agents and chemotherapy, Dec 19, 2016

The biosynthesis of CoA from pantothenate and the utilization of CoA in essential biochemical pat... more The biosynthesis of CoA from pantothenate and the utilization of CoA in essential biochemical pathways represent promising antimalarial drug targets. Pantothenamides, amide-bearing pantothenate analogues, have potential as antimalarials, but a serum enzyme called pantetheinase degrades pantothenamides, rendering them inactive in vivo In this study we characterize a series of 19 compounds that mimic pantothenamides with a stable triazole group instead of the labile amide. Two of these pantothenamides are active against the intraerythrocytic stage parasite with IC50 values of ∼50 nM and three others have sub-micromolar IC50 values. We show that the compounds target CoA biosynthesis and/or utilization. We investigated one of the compounds for its ability to interact with the Plasmodium falciparum pantothenate kinase, the first enzyme involved in the conversion of pantothenate to CoA and show that the compound inhibits the phosphorylation of [(14)C]pantothenate by the P. falciparum pant...

Mycology, 2004

17 Molecular Genetics of Lovastatin and Compactin Biosynthesis C. RICHARD HUTCHINSON and JONATHAN... more 17 Molecular Genetics of Lovastatin and Compactin Biosynthesis C. RICHARD HUTCHINSON and JONATHAN KENNEDY Kosan Biosciences, Inc., Hayward, California, USA CHEONSEOK PARK Department of Food Science and Technology, Kyunghee University, Yongin, South ...

Protein Expression and Purification, 2003

The YLR205c gene of Saccharomyces cerevisiae does not show significant sequence identity to any k... more The YLR205c gene of Saccharomyces cerevisiae does not show significant sequence identity to any known gene, except for heme oxygenase (22% to human HO-1). The YLR205 ORF was cloned and overexpressed in both Escherichia coli and S. cerevisiae. Both expression systems yielded proteins that bound heme tightly. The isolated YLR205c protein underwent reduction in the presence of either NADPH-cytochrome P450 reductase or NADH-putidaredoxin-putidaredoxin reductase but did not exhibit heme oxygenase activity. The protein exhibited modest H 2 O 2-dependent peroxidase activities with guaiacol, potassium iodide, and 2,2 0-azino-bis(3ethylbenzothiazoline-6-sulfonic acid (ABTS). Thus, YLR205c codes for a hemoprotein of unkown physiological function that exhibits peroxidase activity.

Bioorganic & Medicinal Chemistry Letters, 2014

Pantothenamides are N-substituted pantothenate derivatives which are known to exert antimicrobial... more Pantothenamides are N-substituted pantothenate derivatives which are known to exert antimicrobial activity through interference with coenzyme A (CoA) biosynthesis or downstream CoA-utilizing proteins. A previous report has shown that replacement of the ProR methyl group of the benchmark N-pentylpantothenamide with an allyl group (R-anti configuration) yielded one of the most potent antibacterial pantothenamides reported so far (MIC of 3.2 μM for both sensitive and resistant Staphylococcus aureus). We describe herein a synthetic route for accessing the corresponding R-syn diastereomer using a key diastereoselective reduction with Baker's yeast, and report on the scope of this reaction for modified systems. Interestingly, whilst the R-anti diastereomer is the only one to show antibacterial activity, the R-syn isomer proved to be significantly more potent against the malaria parasite (IC 50 of 2.4 ± 0.2 μM). Our research underlines the striking influence that stereochemistry has on the biological activity of pantothenamides, and may find utility in the study of various CoA-utilizing systems.

Antonie van Leeuwenhoek, International Journal of General and Molecular Microbiology, 2000

Lovastatin biosynthesis in Aspergillus terreus involves two unusual type I multifunctional polyke... more Lovastatin biosynthesis in Aspergillus terreus involves two unusual type I multifunctional polyketide syntheses (PKSs). Lovastatin nonaketide synthase (LNKS), the product of the lovB gene, is an iterative PKS that interacts with LovC, a putative enoyl reductase, to catalyze the 35 separate reactions in the biosynthesis of dihydromonacolin L, a lovastatin precursor. LNKS also displays Diels-Alderase activity in vitro. Lovastatin diketide synthase (LDKS) made by lovF, in contrast, acts non-iteratively like the bacterial modular PKSs to make (2R)-2-methylbutyric acid. Then, like LNKS, LDKS interacts closely with another protein, the LovD transesterase enzyme that catalyzes attachment of the 2-methylbutyric acid to monacolin J in the final step of the lovastatin pathway. Key features of the genes for these four enzymes and others, plus the regulatory and self-resistance factors involved in lovastatin production, are also described.

Journal of the American Chemical Society, 2002

Discrete, nonligated water molecules are often found in the crystal structure active sites of met... more Discrete, nonligated water molecules are often found in the crystal structure active sites of metalloproteins in general, and hemoproteins in particular. 1 Their roles can range from simple impediments to rapid ligation, as in the globins, 2 to participation in important hydrogen-bonding networks in the catalytic cycles of enzymes such as the cytochromes P450. 3 1 H NMR spectroscopy can identify water molecules within an enzyme by the detection of an NOE 4 between the water molecule and specific enzyme protons. 5 Even buried water molecules, however, exchange too fast with the bulk water (∼10 3 s-1) to yield discrete 1 H NMR signals. 5 Nevertheless, water molecules localized within a protein can still be identified by 1 H NMR through NOEs between the bulk water signal and individual protein resonances. 5 For nonlabile protons such NOEs are difficult to assign because of severe resolution problems. Enzyme labile protons are easier to assign, but it must be shown that chemical exchange does not contribute to magnetization transfer. 5 Direct (water-enzyme contact), versus indirect NOEs (NOE to a nearby, rapidly exchanging enzyme labile proton), can be differentiated solely on the basis of a molecular structure. 5 Heme oxygenase-1, 6 HO-1, is a ∼32-kDa membrane-bound enzyme that employes heme as substrate and cofactor in the conversion 7 of heme to iron, CO, and biliverdin IXR. The net reaction consumes three O 2 , seven electrons, and nine protons. Recombinant, truncated, soluble, and fully active HO-1 has been shown to act via a hydroperoxy intermediate 8,9 rather than the more common ferryl form. The initial crystal structures of human HO-1, 10 hHO-1, and rat HO-1 11 did not provide information as to how such a novel hydroperoxy intermediate is stabilized. However, the loss of activity upon mutating the conserved Asp140 12,13 and the X-ray detection of a water molecule localized near Asp140 12 lead to the proposal that this water molecule provides the weak H-bond required to stabilize the ligated hydroperoxy species. We have demonstrated 14-16 by 1 H NMR that the cyanide-inhibited, substratebound hHO-1 complex exhibits 14 an active-site molecular structure that is, for the most part, indistinguishable from that revealed in the earlier crystal structure. 10 However, an extensive H-bond network with very strong H-bonds (labile protons with chemical shift 9-17 ppm) was identified 16 which was not recognizable in the crystal structure. This network involves the catalytically critical Asp140 and extends from the distal pocket through the opposite end of the enzyme, with the NHs of Arg85, Lys86, Ala165, and Phe166 and the side-chain labile protons of Tyr58, Trp96, His132, and Arg136, serving as donors to four carboxylates (Glu62, Asp92, Asp140, Glu202) and one carbonyl (Gly163). 16 We demonstrate

Journal of the American Chemical Society, 2000

The fungal metabolite lovastatin (1) 1 and its derivatives are cholesterol-lowering drugs that ac... more The fungal metabolite lovastatin (1) 1 and its derivatives are cholesterol-lowering drugs that act as potent inhibitors of (3S)hydroxy-3-methylglutaryl-coenzyme A reductase. 2 Although 1 and compactin 3 have attracted attention from synthetic chemists, 4 these drugs and some analogues (e.g., simvastatin, pravastatin) which are used in humans are manufactured by fermentation, either directly or with subsequent chemical or microbial modification. Studies on the biosynthesis of 1 in Aspergillus terreus indicate that it is formed by a polyketide pathway. 5-7 Of special interest is the proposal of an enzyme-catalyzed Diels-Alder cyclization of the intermediate hexaketide triene to generate the decalin system (Figure 1). 7,8 This idea is supported by the formation of dihydromonacolin L (2) 9 by a heterologous host, A. nidulans containing the loVB and loVC genes from A. terreus. 10,11 Corresponding heterologous expression of the loVB protein (lovastatin nonaketide synthase, LNKS) without loVC leads to truncated pyrones 3 and 4, formed due to failure of enoyl reduction at the tetraketide stage. 10 There are proposals that enzyme-catalyzed Diels Alder reactions may occur during biosynthesis of many secondary metabolites, 12 but the demonstrated ability of pure biological macromolecules to promote this process has been limited to catalytic antibodies generated from synthetic haptens 13 and to synthetic RNA fragments that bind metals. 14 There is also a report of a crude cellfree preparation from the fungus Alternaria solani that oxidizes an achiral allylic alcohol, prosolanopyrone II, to a conjugated triene aldehyde, thereby triggering intramolecular Diels-Alder cyclization to an optically active product, solanopyrone A. 15 We now report that purified LNKS catalyzes intramolecular Diels-Alder endo closure of a substrate analogue, (E,E,E)-(R)-6-methyldodecatri-2,8,10-enoic acid N-acetylcysteamine (NAC) thioester (5), to a bicyclic system with the same ring stereochemistry as 2, which is different from that obtained in nonenzymatic cyclization.

Journal of the American Chemical Society, 2011

Tomlinson Foundation, and the Winnipeg Police Association. We also thank the Center in Green Chem... more Tomlinson Foundation, and the Winnipeg Police Association. We also thank the Center in Green Chemistry and Catalysis for its support and A. B. Charette (Universit e de Montr eal, Canada) for suggestions.

Journal of Biotechnology, 2007

P450 enzymes are of great interest for drug metabolism and as potential biocatalysts. Like most P... more P450 enzymes are of great interest for drug metabolism and as potential biocatalysts. Like most P450s, purified CYP3A4 is normally handled and stored in solution because lyophilization greatly reduces its activity. We show here that colyophilization of this enzyme with sucrose or trehalose, but not mannitol, crown ethers or cyclodextrins, allow recovery of full enzymatic activity after rehydration. Sorbitol was almost as efficient, with 85% retention of the original activity. We also show that similar protection is observed through colyophilization of CYP2D6 with trehalose. This procedure should greatly facilitate handling, storage, or use of these enzymes in anhydrous media.

ChemBioChem, 2007

P450 reductase OR263 plasmid was kindly donated to us by Dr. Charles B. Kasper from the Universit... more P450 reductase OR263 plasmid was kindly donated to us by Dr. Charles B. Kasper from the University of Wisconsin. The culture media ingredients yeast extract, tryptone, peptone, as well as the CYP2D6 substrate AMMC (3-[2-(N,N-diethyl-N-methylammonium)ethyl]-7-methoxy-4-methylcoumarin) and the product AHMC (3-[2-(diethylamino)ethyl]-7hydroxy-4-methylcoumarin hydrochloride) were purchased from BD Biosciences (San Jose, CA). DH5a supercompetent cells and agarose were from Invitrogen (Carlsbad, CA). The 2',5'-ADP Sepharose 4B and DEAE Sepharose resins were from Amersham Bioscience (Baie d'Urfé, QC, Canada). The His-Select TM Nickel Affinity Gel was purchased from Sigma (St. Louis, US). The CYP3A4 substrate testosterone (4-androsten-17β-ol-3-one) was a king gift from Dr. Eisenberg in our department and the metabolites 6β-hydroxytestosterone (4-androsten-6β,17β-diol-3-one) and 6α-hydroxytestosterone (4

Biotechnology and Bioengineering, 2009

P450 enzymes are of high interest for synthetic applications due to their ability to catalyze hyd... more P450 enzymes are of high interest for synthetic applications due to their ability to catalyze hydroxylation reactions at inactivated C-H bonds. The low solubility of many substrates in buffer, however, is limiting the applications of P450s. Our recent demonstration that the P450 enzymes CYP2D6 and CYP3A4 can function very well in biphasic solvent systems is one step towards overcoming this drawback, but is not practical when substrates or products are unstable in water, or with water-soluble products. An alternative strategy, which also facilitates enzyme recycling, is to directly resuspend lyophilized enzyme into nearly anhydrous organic solvents. Interestingly, we report here that CYP2D6 colyophilized with trehalose and suspended in n-decane shows higher activity than in aqueous buffer. This study demonstrates the unexpected high tolerance of CYP2D6 to some low water organic solvents and provides an alternative strategy to facilitate the use of this enzyme in synthesis.