Triazole Substitution of a Labile Amide Bond Stabilizes Pantothenamides and Improves Their Antiplasmodial Potency

The biosynthesis of coenzyme A (CoA) from pantothenate and the utilization of CoA in essential biochemical pathways represent promising antimalarial drug targets. Pantothenamides, amide derivatives of pantothenate, 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 50% inhibitory concentrations (IC50s) of ∼50 nM, and three others have submicromolar IC50s. 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 [14C]pantothenate by the P. falciparum pantothenate kinase, but the inhibition does not correlate with antiplasmodial activity. Furthermore, the compounds are not toxic to human cells and, importantly, are not degraded by pantetheinase

Itaconate: an antimicrobial metabolite of macrophages

Itaconate is a conjugated 1,4-dicarboxylate produced by macrophages. This small molecule has recently received increasing attention due to its role in modulating the immune response of macrophages upon exposure to pathogens. Itaconate has also been proposed to play an antimicrobial function; however, this has not been explored as intensively. Consistent with the latter, itaconate is known to show antibacterial activity in vitro and was reported to inhibit isocitrate lyase, an enzyme required for survival of bacterial pathogens in mammalian systems. Recent studies have revealed bacterial growth inhibition under biologically relevant conditions. In addition, an antimicrobial role for itaconate is substantiated by the high concentration of itaconate found in bacteria-containing vacuoles, and by the production of itaconate-degrading enzymes in pathogens such as Salmonella enterica ser. Typhimurium, Pseudomonas aeruginosa, and Yersinia pestis. This review describes the current state of literature in understanding the role of itaconate as an antimicrobial agent in host-pathogen interactions.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author

Use of bioconjugation with cytochrome P450 enzymes

Bioconjugation, defined as chemical modification of biomolecules, is widely employed in biological and biophysical studies. It can expand functional diversity and enable applications ranging from biocatalysis, biosensing and even therapy. This review summarizes how chemical modifications of cytochrome P450 enzymes (P450s or CYPs) have contributed to improving our understanding of these enzymes. Genetic modifications of P450s have also proven very useful but are not covered in this review. Bioconjugation has served to gain structural information and investigate the mechanism of P450s via photoaffinity labeling, mechanism-based inhibition (MBI) and fluorescence studies. P450 surface acetylation and protein cross-linking have contributed to the investigation of protein complexes formation involving P450 and its redox partner or other P450 enzymes. Finally, covalent immobilization on polymer surfaces or electrodes has benefited the areas of biocatalysis and biosensor design. This article is part of a Special Issue entitled: Cytochrome P450 biodiversity and biotechnology, edited by Erika Plettner, Gianfranco Gilardi, Luet Wong, Vlada Urlacher, Jared Goldstone

Controlling substrate specificity and product regio- andstereo-selectivities of P450 enzymes without mutagenesis

P450 enzymes (P450s) are well known for their ability to oxidize unactivated CAH bonds with high regioandstereoselectivity. Hence, there is emerging interest in exploiting P450s as potential biocatalysts.Although bacterial P450s typically show higher activity than their mammalian counterparts, they tendto be more substrate selective. Most drug-metabolizing P450s on the other hand, display remarkablesubstrate promiscuity, yet product prediction remains challenging. Protein engineering is one establishedstrategy to overcome these issues. A less explored, yet promising alternative involves substrateengineering. This review discusses the use of small molecules for controlling the substrate specificityand product selectivity of P450s. The focus is on two approaches, one taking advantage of non-covalentdecoy molecules, and the other involving covalent substrate modifications

Allosteric Activation of Cytochrome P450 3A4 via Progesterone Bioconjugation

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

Contribution of language studies to the understanding of cognitive impairment and its progression over time in Parkinson’s disease

Parkinson's disease is a frequent neurodegenerative disease that is mostly known for its motor symptoms. However, cognitive impairment is now recognised as an important part of the disease. Studies of cognitive impairment in Parkinson's disease reveal considerable heterogeneity in terms of which cognitive domains are impaired, and of how cognitive impairment progresses over time. In parallel, a growing body of research reports language difficulties in Parkinson's disease, more specifically in the domains of sentence processing and lexical-semantic processing. In this review, the performance of patients with Parkinson's disease in these domains of language will be reviewed with a focus on the links that they have with the rest of cognition and on how they could contribute to the earlier and more precise characterization and prediction of cognitive impairment in Parkinson's disease. More specifically, the potential for modulation of complexity and sensitivity of language tasks to mild deficits and difficulties that are predictive of further decline will be emphasized. Other motivations for studying language difficulties in this disease will also be discussed

Steroid bioconjugation to a CYP3A4 allosteric site and its effect on substrate binding and coupling efficiency.

Human cytochrome P450 3A4 (CYP3A4) is an important drug metabolizing enzyme involved in a number of drug-drug and food-drug interactions. As such, much effort has been devoted into investigating its mechanism of interaction with ligands. CYP3A4 has one of the highest levels of substrate promiscuity for an enzyme, and can even bind multiple ligands simultaneously. The location and orientation of these ligands depend on the chemical structure and stoichiometry, and are generally poorly understood. In the case of the steroid testosterone, up to three copies of the molecule can associate with the enzyme at once, likely two in the active site and one at a postulated allosteric site. Recently, we demonstrated that steroid bioconjugation at the allosteric site results in an increase in activity of CYP3A4 toward testosterone and 7-benzyloxy-4-trifluoromethylcoumarin oxidation. Here, using the established bioconjugation methodology, we show how steroid bioconjugation at the allosteric site affects the heme spin state, the binding affinity (KS) of CYP3A4 for testosterone, as well as the enzyme coupling efficiency

Global ITC fitting methods in studies of protein allostery

Allostery is a nearly ubiquitous feature of biological systems in which ligand binding or covalent modification at one site alters the activities of distant sites in a macromolecule or macromolecular complex. The molecular mechanisms underlying this phenomenon have been studied for decades. Nevertheless there are many aspects that remain poorly understood. ITC yields detailed information on the thermodynamics of biomacromolecular interactions and their coupling to additional equilibria, therefore in principle it is a powerful tool for better understanding how allostery is achieved. A particularly powerful approach involves simultaneously fitting multiple ITC data sets together with those of complementary techniques, especially nuclear magnetic resonance and circular dichroism spectroscopies. In this review, we describe several group-fitting methods for discriminating between different binding models and for improving the accuracy of thermodynamic parameters extracted from variable-temperature ITC data. The techniques were applied to the antibiotic resistance-causing enzyme aminoglycoside-6'-acetyltransferase Ii, uncovering the existence of competition between opposing mechanisms and ligand-dependent switching of the underlying mechanism. These novel observations underline the potential of combining ITC and spectroscopic techniques to study allostery

Controlling the Reactivity of Enzymes in Mechanochemistry: Inert Surfaces Protect β-Glucosidase Activity During Ball Milling

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 enzymatic equilibrium under mechanochemical conditions

Structural Dynamics of Cytochrome P450 3A4 in the Presence of Substrates and Cytochrome P450 Reductase.

Cytochrome P450 3A4 (CYP3A4) is the most important drug-metabolizing enzyme in humans and has been associated with harmful drug interactions. The activity of CYP3A4 is known to be modulated by several compounds and by the electron transfer partner, cytochrome P450 reductase (CPR). The underlying mechanism of these effects, however, is poorly understood. We have used hydrogen-deuterium exchange mass spectrometry to investigate the impact of binding of CPR and of three different substrates (7-benzyloxy-4-trifluoromethyl-coumarin, testosterone, and progesterone) on the conformational dynamics of CYP3A4. Here, we report that interaction of CYP3A4 with substrates or with the oxidized or reduced forms of CPR leads to a global rigidification of the CYP3A4 structure. This was evident from the suppression of deuterium exchange in several regions of CYP3A4, including regions known to be involved in protein-protein interactions (helix C) and substrate binding and specificity (helices B and E, and loop K/β1). Furthermore, the bimodal isotopic distributions observed for some CYP3A4-derived peptides were drastically impacted upon binding to CPR and/or substrates, suggesting the existence of stable CYP3A4 conformational populations that are perturbed by ligand/CPR binding. The results have implications for understanding the mechanisms of ligand binding, allostery, and catalysis in CYP enzymes