Molecular Dynamics Analysis of Apolipoprotein-D - Lipid Hydroperoxide Interactions: Mechanism for Selective Oxidation of Met-93

Background: Recent studies suggest reduction of radical-propagating fatty acid hydroperoxides to inert hydroxides by interaction with apolipoprotein-D (apoD) Met93 may represent an antioxidant function for apoD. The nature and structural consequences of this selective interaction are unknown. Methodology/Principal Findings: Herein we used molecular dynamics (MD) analysis to address these issues. Longtimescale simulations of apoD suggest lipid molecules are bound flexibly, with the molecules free to explore multiple conformations in a binding site at the entrance to the classical lipocalin ligand-binding pocket. Models of 5s- 12s- and 15s hydroperoxyeicosatetraenoic acids were created and the lipids found to wrap around Met93 thus providing a plausible mechanism by which eicosatetraenoic acids bearing hydroperoxides on different carbon atoms can interact with Met93 to yield Met93 sulfoxide (Met93SO). Simulations of glycosylated apoD indicated that a second solvent exposed Met at position 49 was shielded by a triantennerary N-glycan attached to Asn45 thereby precluding lipid interactions. MD simulations of apoD showed B-factors of the loop containing Met93SO were higher in the oxidized protein, indicating increased flexibility that is predicted to destabilize the protein and promote self-association. Conclusions/Significance: These studies provide novel insights into the mechanisms that may contribute to the antioxidant function of apoD and the structural consequences that result if Met93SO is not redox-cycled back to its native state

Modeling and Molecular Dynamics Simulations on the in situ Murine Cytochrome P450 4F System

Cytochrome P450s are major participants in the maintenance and well-being of cellular function and have important roles in the health and disease of living creatures. The ω-hydroxylation, catalyzed by CYP4 family members, has been observed to be an important metabolic pathway for the homeostasis of mammalian cells as it regulates inflammatory processes with the eicosanoid cascade of metabolites of the ω-6 polyunsaturated fatty acid, arachidonic acid. Many human CYP4F and murine Cyp4f subfamily members have recently gained interest for their usage as potential cancer biomarkers as the expression of these proteins are modified in tumor cells. 20-HETE, the ω-hydroxylated product of arachidonic acid, has gained attention for being the chief metabolic product of interest in vascular function, tumor progression and propagation. Whether or not individual Cyp4f isoforms are responsible for the production of this metabolite is of great interest to medicine as such insight could provide researchers with new avenues of study in the fight against cancer. One particular Cyp4f isozyme, Cyp4f13, has received relatively little study until only very recently and is the focus of the work presented in this thesis, as it has not fully had its role in eicosanoid metabolism understood. Using a combination of computational chemistry approaches, this study focuses on exploring the murine cytochrome P450 4f13 system and its active site using all-atomistic Molecular Dynamics Simulation of a homology model. With the embedded protein solvated and in situ environment replicated, the resting state of the substrate-free Cyp4f13 system was generated. Solvation of the active site was performed to explore the inner active cavity of the P450 system, with subsequent molecular docking and mutation of active site residues performed in order to gain insight into the interactions present in the protein-substrate complex. Protonation state changes were observed to have significant effects on both protein structure and arachidonate binding through electrostatic interactions. Leu137, Arg237, and Gly327 were modified and displayed drastic effects on predicted regiospecificity on the P450 substrate. With the insights obtained, we hope to further the understanding of murine Cyp4f13-catalyzed ω-hydroxylation of arachidonic acid

Chemical probes for studying cyclooxygenase-2 and nitric oxide in living systems

Molecular imaging enables the direct detection of analytes and biomolecular species within their native biological environment. Although the field derives from diagnostic biomedical imaging, there has been a significant shift over the past couple decades towards using imaging to evaluate and discover biology. In general, molecular imaging relies on the development of chemical or biochemical tools that accumulate at the site of interest or under undergo a selective, observable change following target engagement. Activity-based sensing is a powerful expansion of molecular imaging because it measures chemical reactivity rather than concentration. Chapter 1 serves as an introduction to molecular imaging with a historical tone. It also defines and highlights key examples of binding-based and activity-based sensing probes to contextualize the following chapters. Chapter 2 discusses the design and validation of a fluorescent probe for detecting cyclooxygenase-2 activity with live cells, as well as the discovery of oxygen-dependent regulation that is not observed on the protein expression level. Chapter 3 summarizes our progress towards the development of photoacoustic probes for imaging nitric oxide within live animals. Topics include the preparation of a photoacoustic probe for imaging nitric oxide in a small animal model of inflammation, the optimization of the aza-BODIPY dye platform to detect cancer-derived nitric oxide, and progress towards a multimodal dye platform for photoacoustic and fluorescence imaging

Cytochrome P450 2J2 mediated metabolism of omega-6 and omega-3 endocannabinoids

Cytochrome P450 2J2 (CYP2J2) is membrane bound enzyme that is highly expressed in the cardiovascular system and brain where it mediates the metabolism of polyunsaturated fatty acids into oxygenated bioactive metabolites. This enzyme is most well-known for the conversion of arachidonic acid into epoxyeicosatrienoic acids (EETs) regioisomers, 14,15-, 11,12-, 8,9-, and 5,6-EET that mediate vasodilatory, pro-angiogenic and anti-inflammatory effects. More recently, CYP2J2 has been identified as a key enzyme for the metabolism of omega-3 fatty acids, such as eicosapentanoic acid (EPA) and docosahexaenoic acid (DHA), producing epoxygenated metabolites that provide cardioprotective benefits. The biochemical study of the CYP2J2 mediated metabolism of xenobiotics and endogenous fatty acids have been difficult due to challenges with protein expression and solubilization. To circumvent these issues, a series of N-terminal modifications were introduced in the CYP2J2 N-terminus that enabled heterologous expression in E. coli. Importantly, to address the propensity of CYP2J2 to aggregate in solution, each construct was incorporated into the native membrane bilayers of nanodiscs for rigorous spectroscopic and functional studies. Notably, all N-terminal constructs were stably bound within the nanodisc bilayers, which prompted efforts to elucidate additional motifs that impart membrane binding. These observations were further explored in a study using molecular dynamics simulations which revealed CYP2J2 membrane insertion at the F-G loop, through hydrophobic residues Trp-235, Ille-236, and Phe-239. To explore the role of these residues, three F-G loop mutants were prepared from the truncated CYP2J2 construct (Δ34), which included Δ34-I236D, Δ34-F239H and Δ34-I236D/F239H. Interestingly, the expression of the CYP2J2 F-G loop mutants in E. coli were shown to be localized to the cytosolic fraction at a greater percentage relative to construct Δ34 while functional characterization revealed that the double mutant, Δ34-I236D/F239H, exhibited the greatest solubility while maintaining native-like enzymatic activity. Further, the membrane insertion characteristics were examined by monitoring CYP2J2 Trp-quenching fluorescence spectroscopy upon binding nanodiscs containing pyrene phospholipids. Relative to the Δ34 construct, all three F-G loop mutants exhibited lower Trp quenching indicating reduced membrane insertion. The original CYP2J2 expression and characterization studies enabled metabolic screening of endogenous polyunsaturated fatty acid (PUFA) derived substrates that included derivatives of AA, EPA and DHA. Notably, these efforts resulted in the discovery of the CYP2J2-nanodisc mediated oxygenation of omega-6 derived endocannabinoids, thereby revealing a previously unknown metabolic pathway. The endocannabinoids, anandamide (AEA) and 2-arachidonylglycerol (2-AG), are endogenous lipid mediators that are agonists of cannabinoid receptor-1 (CB1) and cannabinoid receptor-2 (CB2) with effects similar to the active ingredient of marijuana. Reactions of CYP2J2 with AEA formed four AEA-epoxyeicosatrienoic acids, whereas incubations with 2-AG yielded detectable levels of only two 2-AG epoxides. Additionally, 2-AG was shown to undergo enzymatic oxidative cleavage to form AA through a NADPH-dependent reaction with CYP2J2 and cytochrome P450 reductase. The demonstrated ability of CYP2J2 to oxygenate AEA and 2-AG raised questions whether CYP2J2 could also accommodate the omega-3 derived endocannabinoids, eicosapentaenoic ethanolamide (EPEA) and docosahexaenoic ethanolamide (DHEA), as substrates. Indeed, subsequent metabolism studies of CYP2J2 with EPEA/DHEA revealed the production of a new class of ω-3 derived metabolites that we termed the omega-3 endocannabinoid epoxides – epoxyeicosatetraenoic-ethanolamide (EEQ-EA) and epoxydocosapentaenoic-ethanolamide (EDP-EA). These newly discovered endogenous metabolites share structural similarity to both their endocannabinoid and epoxide parent compounds, with potential to exert physiological effects mediated through both signaling pathways. Both EEQ-EAs and EDP-EAs were endogenously present in rat brain and peripheral organs as determined via LC-MS/MS with 19,20-EDP-EA also detectable in pooled human plasma. Using LC-MS/MS we demonstrated the direct production of these lipids by recombinant human CYP2J2 epoxygenase, rat brain microsomes and activated BV-2 microglia cells when supplemented with the parent EPEA or DHEA lipids. The discovery of these metabolites prompted a panel of in vitro assays to provide a first ever examination of their biological effects. Most notably, upon LPS stimulation of BV-2 microglia cells, both 17,18-EEQ-EA and 19,20-EDP-EA decreased pro-inflammatory IL-6 and nitric oxide whilst increasing anti-inflammatory IL-10. Additional studies revealed that these metabolites exerted anti-angiogenic effects in HMVEC cells, vasodilatory properties in bovine coronary arteries and reciprocally regulated platelet aggregation in washed human platelets. Taken together, the following thesis details new methods for the study of CYP2J2 within the membrane bilayers of nanodiscs. These techniques were used to reveal a new role for CYP2J2 in the metabolism of endocannabinoids into novel endocannabinoid metabolites with important physiological functions in the heart and brain

Atomic-level characterization of protein-lipid interactions using molecular dynamics simulations

Peripheral membrane proteins are structurally diverse proteins that are involved in fundamental cellular processes. Their activity of these proteins is frequently modulated through their interaction with cellular membranes, and as a result techniques to study the interfacial interaction between peripheral proteins and the membrane are in high demand. Due to the fluid nature of the membrane and the reversibility of protein–membrane interactions, the experimental study of these systems remains a challenging task. Molecular dynamics (MD) simulations offer a suitable approach to study protein–lipid interactions with high spatial and temporal resolution. Here, we present a summary of recent applications of MD simulations to study the interaction of different classes of membrane proteins with lipid bilayers at the atomic level. Specific systems studied include membrane-bound cytochrome P450 (CYP) enzymes, a class membrane proteins involved in the metabolism of a wide range of molecules, the hemagglutinin fusion peptide (HAfp), a small peptide that mediates the fusion process of the influenza virus to a host cell, and the T-cell immunoglobulin and mucin domain (Tim) proteins, involved in the mechanism of lipid recognition by T-cells

Cellular Oxidative Stress

This book collects 17 original research papers and 9 reviews that are part of the Special Issue “Cellular Oxidative Stress”, published in the journal Antioxidants. Oxidative stress on a cellular level affects the function of tissues and organs and may eventually lead to disease. Therefore, a precise understanding of how oxidative stress develops and can be counteracted is of utmost importance. The scope of the book is to emphasize the latest findings on the cellular targets of oxidative stress and the potential beneficial effect of antioxidants on human health

IN SILICO METHODS FOR DRUG DESIGN AND DISCOVERY

Computer-aided drug design (CADD) methodologies are playing an ever-increasing role in drug discovery that are critical in the cost-effective identification of promising drug candidates. These computational methods are relevant in limiting the use of animal models in pharmacological research, for aiding the rational design of novel and safe drug candidates, and for repositioning marketed drugs, supporting medicinal chemists and pharmacologists during the drug discovery trajectory.Within this field of research, we launched a Research Topic in Frontiers in Chemistry in March 2019 entitled “In silico Methods for Drug Design and Discovery,” which involved two sections of the journal: Medicinal and Pharmaceutical Chemistry and Theoretical and Computational Chemistry. For the reasons mentioned, this Research Topic attracted the attention of scientists and received a large number of submitted manuscripts. Among them 27 Original Research articles, five Review articles, and two Perspective articles have been published within the Research Topic. The Original Research articles cover most of the topics in CADD, reporting advanced in silico methods in drug discovery, while the Review articles offer a point of view of some computer-driven techniques applied to drug research. Finally, the Perspective articles provide a vision of specific computational approaches with an outlook in the modern era of CADD

Continuing professional development - challenge for professional organization

Professions, as one of key sectors of social systems, bear a leading role in the existing social work organization. Free professions take up a special place and significance, all the way from Roman artes liberales to our times. Pharmaceutical profession, as one of the oldest, led by ethical principles, is regulated by postulates accepted by the profession members, and in modern times established through legislations. Typical determinants of the regulated professions, which also refer to pharmacists, as chamber members, are as follows: following ethical principles, specific skills and knowledge, professional development, autonomy at work, continuing improvement, competencies development, professional associations, licensing