Structural basis of TRPV1 modulation by endogenous bioactive lipids.

TRP ion channels are modulated by phosphoinositide lipids, but the underlying structural mechanisms remain unclear. The capsaicin- and heat-activated receptor, TRPV1, has served as a model for deciphering lipid modulation, which is relevant to understanding how pro-algesic agents enhance channel activity in the setting of inflammatory pain. Identification of a pocket within the TRPV1 transmembrane core has provided initial clues as to how phosphoinositide lipids bind to and regulate the channel. Here we show that this regulatory pocket in rat TRPV1 can accommodate diverse lipid species, including the inflammatory lipid lysophosphatidic acid, whose actions are determined by their specific modes of binding. Furthermore, we show that an empty-pocket channel lacking an endogenous phosphoinositide lipid assumes an agonist-like state, even at low temperature, substantiating the concept that phosphoinositide lipids serve as negative TRPV1 modulators whose ejection from the binding pocket is a critical step toward activation by thermal or chemical stimuli.

Structural basis of TRPV1 modulation by endogenous bioactive lipids.

TRP ion channels are modulated by phosphoinositide lipids, but the underlying structural mechanisms remain unclear. The capsaicin- and heat-activated receptor, TRPV1, has served as a model for deciphering lipid modulation, which is relevant to understanding how pro-algesic agents enhance channel activity in the setting of inflammatory pain. Identification of a pocket within the TRPV1 transmembrane core has provided initial clues as to how phosphoinositide lipids bind to and regulate the channel. Here we show that this regulatory pocket can accommodate diverse lipid species, including the inflammatory lipid lysophosphatidic acid (LPA), whose actions are determined by their specific modes of binding. Furthermore, we show that an 'empty pocket' channel lacking an endogenous phosphoinositide lipid assumes an agonist-like state, even at low temperature, substantiating the concept that phosphoinositide lipids serve as negative TRPV1 modulators whose ejection from the binding pocket is a critical step towards activation by thermal or chemical stimuli.

Anthracycline derivatives inhibit cardiac CYP2J2.

Anthracycline chemotherapeutics are highly effective, but their clinical usefulness is hampered by adverse side effects such as cardiotoxicity. Cytochrome P450 2J2 (CYP2J2) is a cytochrome P450 epoxygenase in human cardiomyocytes that converts arachidonic acid (AA) to cardioprotective epoxyeicosatrienoic acid (EET) regioisomers. Herein, we performed biochemical studies to understand the interaction of anthracycline derivatives (daunorubicin, doxorubicin, epirubicin, idarubicin, 5-iminodaunorubicin, zorubicin, valrubicin, and aclarubicin) with CYP2J2. We utilized fluorescence polarization (FP) to assess whether anthracyclines bind to CYP2J2. We found that aclarubicin bound the strongest to CYP2J2 despite it having large bulky groups. We determined that ebastine competitively inhibits anthracycline binding, suggesting that ebastine and anthracyclines may share the same binding site. Molecular dynamics and ensemble docking revealed electrostatic interactions between the anthracyclines and CYP2J2, contributing to binding stability. In particular, the glycosamine groups in anthracyclines are stabilized by binding to glutamate and aspartate residues in CYP2J2 forming salt bridge interactions. Furthermore, we used iterative ensemble docking schemes to gauge anthracycline influence on EET regioisomer production and anthracycline inhibition on AA metabolism. This was followed by experimental validation of CYP2J2-mediated metabolism of anthracycline derivatives using liquid chromatography tandem mass spectrometry fragmentation analysis and inhibition of CYP2J2-mediated AA metabolism by these derivatives. Taken together, we use both experimental and theoretical methodologies to unveil the interactions of anthracycline derivatives with CYP2J2. These studies will help identify alternative mechanisms of how anthracycline cardiotoxicity may be mediated through the inhibition of cardiac P450, which will aid in the design of new anthracycline derivatives with lower toxicity.

Classifying liganded states in heterogeneous single-particle cryo-EM datasets.

A powerful aspect of single-particle cryogenic electron microscopy is its ability to determine high-resolution structures from samples containing heterogeneous mixtures of the same macromolecule in different conformational or compositional states. Beyond determining structures at higher resolutions, one outstanding question is if macromolecules with only subtle conformation differences, such as the same protein bound with different ligands in the same binding pocket, can be separated reliably, and if information concerning binding kinetics can be derived from the particle distributions of different conformations obtained in classification. In this study, we address these questions by assessing the classification of synthetic heterogeneous datasets of Transient Receptor Potential Vanilloid 1 generated by combining different homogeneous experimental datasets. Our results indicate that classification can isolate highly homogeneous subsets of particle for calculating high-resolution structures containing individual ligands, but with limitations.

Anti-inflammatory dopamine- and serotonin-based endocannabinoid epoxides reciprocally regulate cannabinoid receptors and the TRPV1 channel.

The endocannabinoid system is a promising target to mitigate pain as the endocannabinoids are endogenous ligands of the pain-mediating receptors-cannabinoid receptors 1 and 2 (CB1 and CB2) and TRPV1. Herein, we report on a class of lipids formed by the epoxidation of N-arachidonoyl-dopamine (NADA) and N-arachidonoyl-serotonin (NA5HT) by epoxygenases. EpoNADA and epoNA5HT are dual-functional rheostat modulators of the endocannabinoid-TRPV1 axis. EpoNADA and epoNA5HT are stronger modulators of TRPV1 than either NADA or NA5HT, and epoNA5HT displays a significantly stronger inhibition on TRPV1-mediated responses in primary afferent neurons. Moreover, epoNA5HT is a full CB1 agonist. These epoxides reduce the pro-inflammatory biomarkers IL-6, IL-1ß, TNF-α and nitrous oxide and raise anti-inflammatory IL-10 cytokine in activated microglial cells. The epoxides are spontaneously generated by activated microglia cells and their formation is potentiated in the presence of anandamide. Detailed kinetics and molecular dynamics simulation studies provide evidence for this potentiation using the epoxygenase human CYP2J2. Taken together, inflammation leads to an increase in the metabolism of NADA, NA5HT and other eCBs by epoxygenases to form the corresponding epoxides. The epoxide metabolites are bioactive lipids that are potent, multi-faceted molecules, capable of influencing the activity of CB1, CB2 and TRPV1 receptors.

CYP2J2 Molecular Recognition: A New Axis for Therapeutic Design.

Cytochrome P450 (CYP) epoxygenases are a special subset of heme-containing CYP enzymes capable of performing the epoxidation of polyunsaturated fatty acids (PUFA) and the metabolism of xenobiotics. This dual functionality positions epoxygenases along a metabolic crossroad. Therefore, structure-function studies are critical for understanding their role in bioactive oxy-lipid synthesis, drug-PUFA interactions, and for designing therapeutics that directly target the epoxygenases. To better exploit CYP epoxygenases as therapeutic targets, there is a need for improved understanding of epoxygenase structure-function. Of the characterized epoxygenases, human CYP2J2 stands out as a potential target because of its role in cardiovascular physiology. In this review, the early research on the discovery and activity of epoxygenases is contextualized to more recent advances in CYP epoxygenase enzymology with respect to PUFA and drug metabolism. Additionally, this review employs CYP2J2 epoxygenase as a model system to highlight both the seminal works and recent advances in epoxygenase enzymology. Herein we cover CYP2J2's interactions with PUFAs and xenobiotics, its tissue-specific physiological roles in diseased states, and its structural features that enable epoxygenase function. Additionally, the enumeration of research on CYP2J2 identifies the future needs for the molecular characterization of CYP2J2 to enable a new axis of therapeutic design.

Endocannabinoid Virodhamine Is an Endogenous Inhibitor of Human Cardiovascular CYP2J2 Epoxygenase.

The human body contains endogenous cannabinoids (endocannabinoids) that elicit effects similar to those of Δ9-tetrahydrocanabinol, the principal bioactive component of cannabis. The endocannabinoid virodhamine (O-AEA) is the constitutional isomer of the well-characterized cardioprotective and anti-inflammatory endocannabinoid anandamide (AEA). The chemical structures of O-AEA and AEA contain arachidonic acid (AA) and ethanolamine; however, AA in O-AEA is connected to ethanolamine via an ester linkage, whereas AA in AEA is connected through an amide linkage. O-AEA is involved in regulating blood pressure and cardiovascular function. We show that O-AEA is found at levels 9.6-fold higher than that of AEA in porcine left ventricle. On a separate note, the cytochrome P450 (CYP) epoxygenase CYP2J2 is the most abundant CYP in the heart where it catalyzes the metabolism of AA and AA-derived eCBs to bioactive epoxides that are involved in diverse cardiovascular functions. Herein, using competitive binding studies, kinetic metabolism measurements, molecular dynamics, and wound healing assays, we have shown that O-AEA is an endogenous inhibitor of CYP2J2 epoxygenase. As a result, the role of O-AEA as an endogenous eCB inhibitor of CYP2J2 may provide a new mode of regulation to control the activity of cardiovascular CYP2J2 in vivo and suggests a potential cross-talk between the cardiovascular endocannabinoids and the cytochrome P450 system.

Cross-talk of cannabinoid and endocannabinoid metabolism is mediated via human cardiac CYP2J2.

Phytocannabinoids have well-known cardiovascular implications. For instance, Δ9-tetrahydrocannabinol (Δ9-THC), the principal component of cannabis, induces tachycardia in humans. In order to understand the impact of phytocannabinoids on human cardiovascular health, there is a need to study the metabolism of phytocannabinoids by cardiac cytochromes p450 (CYPs). CYP2J2, the primary CYP of cardiomyocytes, is responsible for the metabolism of the endocannabinoid, anandamide (AEA), into cardioprotective epoxides (EET-EAs). Herein, we have investigated the kinetics of the direct metabolism of six phytocannabinoids (Δ9-THC, Δ8-tetrahydrocannabinol, cannabinol, cannabidiol, cannabigerol, and cannabichromene) by CYP2J2. CYP2J2 mainly produces 1'/1″-OH metabolites of these phytocannabinoids. These phytocannabinoids are metabolized with greater catalytic efficiency compared to the metabolism of AEA by CYP2J2. We have also determined that the phytocannabinoids are potent inhibitors of CYP2J2-mediated AEA metabolism, with Δ9-THC being the strongest inhibitor. Most of the inhibition of CYP2J2 by the phytocannabinoids follow a noncompetitive inhibition model, and therefore dramatically reduce the formation of EET-EAs by CYP2J2. Taken together, these data demonstrate that phytocannabinoids are directly metabolized by CYP2J2 and inhibit human cardiac CYP2J2, leading to a reduction in the formation of cardioprotective EET-EAs.

Arachidonic Acid Metabolism by Human Cardiovascular CYP2J2 Is Modulated by Doxorubicin.

Doxorubicin (DOX) is a chemotherapeutic that is used in the treatment of a wide variety of cancers. However, it causes cardiotoxicity partly because of the formation of reactive oxygen species. CYP2J2 is a human cytochrome P450 that is strongly expressed in cardiomyocytes. It converts arachidonic acid (AA) into four different regioisomers of epoxyeicosatrienoic acids (EETs). Using kinetic analyses, we show that AA metabolism by CYP2J2 is modulated by DOX. We show that cytochrome P450 reductase, the redox partner of CYP2J2, metabolizes DOX to 7-deoxydoxorubicin aglycone (7-de-aDOX). This metabolite then binds to CYP2J2 and inhibits and alters the preferred site of metabolism of AA, leading to a change in the ratio of the EET regioisomers. Furthermore, molecular dynamics simulations indicate that 7-de-aDOX and AA can concurrently bind to the CYP2J2 active site to produce these changes in the site of AA metabolism. To determine if these observations are unique to DOX/7-de-aDOX, we use noncardiotoxic DOX analogues, zorubicin (ZRN) and 5-iminodaunorubicin (5-IDN). ZRN and 5-IDN inhibit CYP2J2-mediated AA metabolism but do not change the ratio of EET regioisomers. Altogether, we demonstrate that DOX and 7-de-aDOX inhibit CYP2J2-mediated AA metabolism and 7-de-aDOX binds close to the active site to alter the ratio of cardioprotective EETs. These mechanistic studies of CYP2J2 can aid in the design of new alternative DOX derivatives.

Heterologous expression and characterization of plant Taxadiene-5α-Hydroxylase (CYP725A4) in Escherichia coli.

Taxadiene-5α-Hydroxylase (CYP725A4) is a membrane-bound plant cytochrome P450 that catalyzes the oxidation of taxadiene to taxadiene-5α-ol. This oxidation is a key step in the production of the valuable cancer therapeutic and natural plant product, taxol. In this work, we report the bacterial expression and purification of six different constructs of CYP725A4. All six of these constructs are N-terminally modified and three of them are fused to cytochrome P450 reductase to form a chimera construct. The construct with the highest yield of CYP725A4 protein was then selected for substrate binding and kinetic analysis. Taxadiene binding followed type-1 substrate patterns with an observed KD of 2.1 ± 0.4 µM. CYP725A4 was further incorporated into nanoscale lipid bilayers (nanodiscs) and taxadiene metabolism was measured. Taxadiene metabolism followed Michaelis-Menten kinetics with an observed Vmax of 30 ± 8 pmol/min/nmolCYP725A4 and a KM of 123 ± 52 µM. Additionally, molecular operating environment (MOE) modeling was performed in order to gain insight into the interactions of taxadiene with CYP725A4 active site. Taken together, we demonstrate the successful expression and purification of the functional membrane-bound plant CYP, CYP725A4, in E. coli.

Asymmetric Binding and Metabolism of Polyunsaturated Fatty Acids (PUFAs) by CYP2J2 Epoxygenase.

Cytochrome P450 (CYP) 2J2 is the primary epoxygenase in the heart and is responsible for the epoxidation of arachidonic acid (AA), an ω-6 polyunsaturated fatty acid (PUFA), into anti-inflammatory epoxide metabolites. It also epoxidizes other PUFAs such as docosahexaenoic acid (DHA), linoleic acid (LA), and eicosapentaenoic acid (EPA). Herein, we have performed detailed thermodynamic and kinetic analyses to determine how DHA, LA, and EPA modulate the metabolism of AA by CYP2J2. We use the Nanodisc system to stabilize CYP2J2 and its redox partner, CYP reductase (CPR). We observe that DHA strongly inhibits CYP2J2-mediated AA metabolism, LA only moderately inhibits AA metabolism, and EPA exhibits insignificant inhibition. We also characterized the binding of these molecules using ebastine competitive binding assays and show that DHA binds significantly tighter to CYP2J2 than AA, EPA, or LA. Furthermore, we utilize a combined approach of molecular dynamics (MD) simulations and docking to predict key residues mediating the tight binding of DHA. We show that although all the tested fatty acids form similar contacts to the active site residues, the affinity of DHA for CYP2J2 is tighter because of the interaction of DHA with residues Arg-321, Thr-318, and Ser-493. To demonstrate the importance of these residues in binding, we mutated these residues to make two mutant variants, CYP2J2-T318A and CYP2J2-T318V/S493A. Both mutant variants showed weaker binding than the wild type (WT) to DHA and AA; DHA inhibition of AA was also mitigated in the mutants compared to the WT. Therefore, using a combined experimental and MD simulation approach, we establish that CYP2J2 inhibition of AA metabolism by DHA, EPA, and LA is asymmetric because of tighter binding of DHA to select residues in the active site.

Substrate binding to cytochrome P450-2J2 in Nanodiscs detected by nanoplasmonic Lycurgus cup arrays.

Cytochrome P450s are the primary enzymes involved in phase I drug metabolism. They are an important target for early drug discovery research. However, high-throughput drug screening of P450s is limited by poor protein stability and lack of consistent measurement of binding events. Here we present the detection of substrate binding to cytochrome P450-2J2 (CYP2J2), the predominant P450 in the human heart, using a combination of Nanodisc technology and a nanohole plasmonic sensor called nanoplasmonic Lycurgus cup array (nanoLCA). The Nanodisc, a nanoscale membrane bilayer disc, is used to stabilize the protein on the metallic plasmonic surface. Absorption spectroscopy of seven different substrates binding to CYP2J2 in solution showed that they are all type I, resulting in shifting of the protein bands to lower wavelengths (blue shift). Detection on the nanoLCA sensor also showed spectral blue shifts of CYP2J2 following substrate binding. Finite Difference Time Domain (FDTD) electromagnetic simulation suggested that the blue shift on the nanoLCA is because of the hybridization of plasmon polariton Bloch wave and the electronic resonance of the heme group of CYP2J2. We found the plasmonic properties of the nanoLCA sensor to be highly reproducible, which allowed comparisons among the different substrates at different concentrations. Further, due to the unique spectral properties of the nanoLCA sensor, including the transmission of a single color, we were able to perform colorimetric detection of the binding events. These results indicate that a resonance plasmonic sensing mechanism can be used to distinguish between different substrates of the same binding type at different concentrations binding to P450s and that the nanoLCA sensor has the potential to provide consistent high-throughput measurements of this system.

Endocannabinoid metabolism by cytochrome P450 monooxygenases.

The endogenous cannabinoid system was first uncovered following studies of the recreational drug Cannabis sativa. It is now recognized as a vital network of signaling pathways that regulate several physiological processes. Following the initial discovery of the cannabinoid receptors 1 (CB1) and 2 (CB2), activated by Cannabis-derived analogs, many endogenous fatty acids termed "endocannabinoids" are now known to be partial agonists of the CB receptors. At present, the most thoroughly studied endocannabinoid signaling molecules are anandamide (AEA) and 2-arachidonylglycerol (2-AG), which are both derived from arachidonic acid. Both AEA and 2-AG are also substrates for the eicosanoid-synthesizing pathways, namely, certain cyclooxygenase (COX), lipoxygenase (LOX), and cytochrome P450 (CYP) enzymes. In the past, research in the endocannabinoid field focused on the interaction of AEA and 2-AG with the COX and LOX enzymes, but accumulating evidence also points to the involvement of CYPs in modulating endocannabinoid signaling. The focus of this review is to explore the current understanding of CYP-mediated metabolism of endocannabinoids.

{2,2'-[N,N'-Bis(pyridin-2-ylmeth-yl)propane-1,3-diyldi(nitrilo)]di-acetato}-cobalt(III) hexa-fluoridophosphate aceto-nitrile 0.064-solvate.

In the title compound, [Co(C19H22N4O4)]PF6·0.064CH3CN, commonly known as [Co(bppd)]PF6·0.064CH3CN, where bppd represents the historical ligand name N,N'-bis(2-pyridylmethyl)-1,3-diaminopropane-N,N'-diacetate, the Co(III) atom is coordinated in a distorted octa-hedral geometry with an N4O2 donor atom set. The acetate O atoms, which exhibit monodentate coordination, are oriented in a trans configuration with respect to each other, whereas the pyridyl N atoms are coordinated in a cis configuration. The compound crystallizes with two crystallographically unique cations and two anions per asymmetric unit along with a disordered, partially occupied (occupancy = 0.128) aceto-nitrile solvent mol-ecule. Crystals of the title complex were found to be twinned by pseudomerohedry with a 180° rotation around [10-1] and a refined contribution of 90.5 (3)% of the major twin component.