eNOS protects from atherosclerosis despite relevant superoxide production by the enzyme in apoE mice

Background

All three nitric oxide synthase (NOS) isoforms are expressed in atherosclerotic plaques. NOS enzymes in general catalyse NO production. However, under conditions of substrate and cofactor deficiency, the enzyme directly catalyse superoxide formation. Considering this alternative chemistry, the effects of NOS on key events in spontaneous hyperlipidemia driven atherosclerosis have not been investigated yet. Here, we evaluate how endothelial nitric oxide synthase (eNOS) modulates leukocyte/endothelial- (L/E) and platelet/endothelial- (P/E) interactions in atherosclerosis and the production of nitric oxide (NO) and superoxide by the enzyme.

Principal Findings

Intravital microscopy (IVM) of carotid arteries revealed significantly increased L/E-interactions in apolipoproteinE/eNOS double knockout mice (apoE^−/−/eNOS^−/−), while P/E-interactions did not differ, compared to apoE^−/−. eNOS deficiency increased macrophage infiltration in carotid arteries and vascular cell adhesion molecule-1 (VCAM-1) expression, both in endothelial and smooth muscle cells. Despite the expression of other NOS isoforms (inducible NOS, iNOS and neuronal NOS, nNOS) in plaques, Electron Spin Resonance (ESR) measurements of NO showed significant contribution of eNOS to total circulating and vascular wall NO production. Pharmacological inhibition and genetic deletion of eNOS reduced vascular superoxide production, indicating uncoupling of the enzyme in apoE^−/− vessels.

Conclusion

Overt plaque formation, increased vascular inflammation and L/E- interactions are associated with significant reduction of superoxide production in apoE^−/−/eNOS^−/− vessels. Therefore, lack of eNOS does not cause an automatic increase in oxidative stress. Uncoupling of eNOS occurs in apoE^−/− atherosclerosis but does not negate the enzyme''s strong protective effects.     

Different vasoactive effects of chronic endothelial and neuronal NO-synthase inhibition in young Wistar rats

While the unequivocal pattern of endothelial nitric oxide synthase (eNOS) inhibition in cardiovascular control is recognized, the role of NO produced by neuronal NOS (nNOS) remains unclear. The aim of this study was to compare the effects of chronic treatment with 7-nitroindazole (7-NI, nNOS inhibitor) and N^G-nitro-l-arginine methylester (l-NAME, general and predominantly eNOS inhibitor) on cardiovascular system of young normotensive rats. Wistar rats (4 weeks old) were used: controls and rats administered either 7-NI (10 mg/kg bw/day) or l-NAME (50 mg/kg bw/day) in drinking water for 6 weeks. The systolic blood pressure (sBP) was measured by plethysmographic method, and the vasoactivity of isolated arteries was recorded. 7-NI-treatment did not affect sBP; however, the sBP was increased after l-NAME-treatment. l-NAME inhibited acetylcholine-induced relaxation of thoracic aorta (TA), whereas it remained unchanged after 7-NI-treatment. The response of TA to sodium nitroprusside was increased in both experimental groups. The expression of eNOS and nNOS in TA was unchanged in both experimental groups, whereas the activity of NOS was decreased in l-NAME-treated group. Noradrenaline- and angiotensin II-induced contractions of TA were reduced in l-NAME-treated group; however, the contractions remained unchanged in 7-NI-treated group. In all groups, the endogenous angiotensin II participated in adrenergic contraction of TA; this contribution was significantly increased in l-NAME-treated group. Neurogenic contractions in mesenteric artery (MA) remained unchanged after 7-NI-treatment, but increased after l-NAME-treatment. Results show that NO deficiency induced by administration of 7-NI and l-NAME had different cardiovascular effects: eNOS and nNOS triggered distinct signaling pathways in young normotensive rats.     

Nickel/zinc-catalyzed decarbonylative addition of anhydrides to alkynes: A DFT study

Density functional theory (DFT) was used to investigate the nickel- or nickel(0)/zinc- catalyzed decarbonylative addition of phthalic anhydrides to alkynes. All intermediates and transition states were optimized completely at the B3LYP/6-31+G(d,p) level. Calculated results indicated that the decarbonylative addition of phthalic anhydrides to alkynes was exergonic, and the total free energy released was ?87.6 kJ mol^?1. In the five-coordinated complexes M4a and M4b, the insertion reaction of alkynes into the Ni-C bond occurred prior to that into the Ni-O bond. The nickel(0)/zinc-catalyzed decarbonylative addition was much more dominant than the nickel-catalyzed one in whole catalytic decarbonylative addition. The reaction channel CA→M1'→T1'→M2'→T2'→M3a'→M4a'→T3a1'→M5a1' →T4a1'→M6a'→P was the most favorable among all reaction pathways of the nickel- or nickel(0)/zinc- catalyzed decarbonylative addition of phthalic anhydrides to alkynes. And the alkyne insertion reaction was the rate-determining step for this channel. The additive ZnCl₂ had a significant effect, and it might change greatly the electron and geometry structures of those intermediates and transition states. On the whole, the solvent effect decreased the free energy barriers.

Density functional studies on photophysical properties and chemical reactivities of the triarylboranes: effect of the constraint of planarity

The geometric and electronic structures, absorption spectra, transporting properties, chemical reactivity indices and electrostatic potentials of the planar three-coordinate organoboron compounds 1-2 and twisted reference compound Mes ₃ B, have been investigated by employing density functional theory (DFT) and conceptual DFT methods to shed light on the planarity effects on the photophysical properties and the chemical reactivity. The results show that the planar compounds 1-2 exhibit significantly lower HOMO level than Mes ₃ B, owing to the stronger electronic induction effect of boron centers. This feature conspicuously induces a blue shifted absorption for 1, although 1 seemingly possesses more extended conjugation framework than Mes ₃ B. Importantly, the reactivity strength of the boron atoms in 1-2 is much lower than that in Mes ₃ B, despite the fact that the tri-coordinate boron centers of 1-2 are completely naked. The interesting and abnormal phenomenon is caused by the strong p-π electronic interactions, that is, the empty p-orbital of boron center is partly filled by π-electron of the neighbor carbon atoms in 1-2, which are confirmed by the analysis of Laplacian of the electron density and natural bond orbitals. Furthermore, the negative electrostatic potentials of the boron centers in 1-2 also interpret that they are not the most preferred sites for incoming nucleophiles. Moreover, it is also found that the planar compounds 1-2 can act as promising electron transporting materials since the internal reorganization energies for electron are really small.

Cytotoxic effect and molecular docking of 4-ethoxycarbonylmethyl-1-(piperidin-4-ylcarbonyl)-thiosemicarbazide—a novel topoisomerase II inhibitor

The preliminary cytotoxic effect of 4-ethoxycarbonylmethyl-1-(piperidin-4-ylcarbonyl)-thiosemicarbazide hydrochloride (1)—a potent topoisomerase II inhibitor—was measured using a MTT assay. It was found that the compound decreased the number of viable cells in both estrogen receptor-positive MCF-7 and estrogen receptor-negative MDA-MB-231breast cancer cells, with IC₅₀ values of 146?±?2 and 132?±?2 μM, respectively. To clarify the molecular basis of the inhibitory action of 1, molecular docking studies were carried out. The results suggest that 1 targets the ATP binding pocket.

Theoretical investigation into optical and electronic properties of 1,8-naphthalimide derivatives

A series of 1,8-naphthalimide derivatives has been designed to explore their optical, electronic, and charge transport properties as charge transport and/or luminescent materials for organic light-emitting diodes (OLEDs). The frontier molecular orbitals (FMOs) analysis have shown that the vertical electronic transitions of absorption and emission are characterized as intramolecular charge transfer (ICT) for electron-donating and aromatic groups substituted derivatives. However, the ICT character of the electron-withdrawing substituted derivatives is not significant. The calculated results show that their optical and electronic properties are affected by the substituent groups in 4-position of 1,8-naphthalimide. Our results suggest that 1,8-naphthalimide derivatives with electron-donating ?OCH₃ and ?N(CH₃)₂ (1 and 2), electron-withdrawing ?CN and?COCH₃ (3 and 4), 2-(thiophen-2-yl)thiophene (5), 2,3-dihydrothieno[3,4-b][1, 4]dioxine (6), 2-phenyl-1,3,4-oxadiazole (7), and benzo[c][1,2,5]thiadiazole (8) fragments are expected to be promising candidates for luminescent materials for OLEDs, particularly for 5 and 7. In addition, 3 and 7 can be used as promising hole transport materials for OLEDs. This study should be helpful in further theoretical investigations on such kind of systems and also to the experimental study for charge transport and/or luminescent materials for OLEDs.

Computationally designed prodrugs of statins based on Kirby’s enzyme model

DFT calculations at B3LYP/6-31G(d,p) for intramolecular proton transfer in Kirby’s enzyme models 1–7 demonstrated that the reaction rate is dependent on the distance between the two reacting centers, r_GM, and the hydrogen bonding angle, α, and the rate of the reaction is linearly correlated with r_GM and α. Based on these calculation results three simvastatin prodrugs were designed with the potential to provide simvastatin with higher bioavailability. For example, based on the calculated log EM for the three proposed prodrugs, the interconversion of simvastatin prodrug ProD 3 to simvastatin is predicted to be about 10 times faster than that of either simvastatin prodrug ProD 1 or simvastatin ProD 2. Hence, the rate by which the prodrug releases the statin drug can be determined according to the structural features of the promoiety (Kirby’s enzyme model).

Modeling the effect of H-bonding interactions and molecular packing on the molecular structure of [Ag(ethylnicotinate)2]NO3 complex

The gas phase molecular structure of a single isolated molecule of [Ag(Etnic)₂NO₃];1 where Etnic = Ethylnicotinate was calculated using B3LYP method. The H-bonding interaction between 1 with one (complex 2) and two (complex 3) water molecules together with the dimeric formula [Ag(Etnic)₂NO₃]₂;4 and the tetrameric formula [Ag(Etnic)₂NO₃]₄;5 were calculated using the same level of theory to model the effect of intermolecular interactions and molecular packing on the molecular structure of the titled complex. The H-bond dissociation energies of complexes 2 and 3 were calculated to be in the range of 12.220–14.253 and 30.106–31.055 kcal?mol^?1, respectively, indicating the formation of relatively strong H-bonds between 1 and water molecules. The calculations predict bidentate nitrate ligand in the case of 1 and 2, leading to distorted tetrahedral geometry around the silver ion with longer Ag–O distances in case of 2 compared to 1, while 3 has a unidentate nitrate ligand leading to a distorted trigonal planar geometry. The packing of two [Ag(Etnic)₂NO₃] complex units; 4 does not affect the molecular geometry around Ag(I) ion compared to 1. In the case of 5, the two asymmetric units of the formula [Ag(Etnic)₂NO₃] differ in the bonding mode of the nitrate group, where the geometry around the silver ion is distorted tetrahedral in one unit and trigonal planar in the other. The calculations predicted almost no change in the charge densities at the different atomic sites except at the sites involved in the C–H?O interactions as well as at the coordinated nitrogen of the pyridine ring.

Prodrugs of fumarate esters for the treatment of psoriasis and multiple sclerosis—a computational approach

Density functional theory (DFT) calculations at B3LYP/6-31 G (d,p) and B3LYP/6-311?+?G(d,p) levels for the substituted pyridine-catalyzed isomerization of monomethyl maleate revealed that isomerization proceeds via four steps, with the rate-limiting step being proton transfer from the substituted pyridinium ion to the C=C double bond in INT1. In addition, it was found that the isomerization rate (maleate to fumarate) is solvent dependent. Polar solvents, such as water, tend to accelerate the isomerization rate, whereas apolar solvents, such as chloroform, act to slow down the reaction. A linear correlation was obtained between the isomerization activation energy and the dielectric constant of the solvent. Furthermore, linearity was achieved when the activation energy was plotted against the pK _a value of the catalyst. Substituted-pyridine derivatives with high pK _a values were able to catalyze isomerization more efficiently than those with low pK _a values. The calculated relative rates for prodrugs 1–6 were: 1 (406.7), 2 (7.6?×?10⁶), 3 (1.0), 4 (20.7), 5 (13.5) and 6 (2.2?×?10³). This result indicates that isomerizations of prodrugs 1 and 3–5 are expected to be slow and that of prodrugs 2 and 6 are expected to be relatively fast. Hence, prodrugs 2 and 3–5 have the potential to be utilized as prodrugs for the slow release of monomethylfumarate in the treatment of psoriasis and multiple sclerosis.

Discovery of novel low-molecular-weight HIV-1 inhibitors interacting with cyclophilin A using in silico screening and biological evaluations

Cyclophilin A has attracted attention recently as a new target of anti-human immunodeficiency virus type 1 (HIV-1) drugs. However, so far no drug against HIV-1 infection exhibiting this mechanism of action has been approved. To identify new potent candidates for inhibitors, we performed in silico screening of a commercial database of more than 1,300 drug-like compounds by using receptor-based docking studies. The candidates selected from docking studies were subsequently tested using biological assays to assess anti-HIV activities. As a result, two compounds were identified as the most active. Specifically, both exhibited anti-HIV activity against viral replication at a low concentration and relatively low cytotoxicity at the effective concentration inhibiting viral growth by 50 %. Further modification of these molecules may lead to the elucidation of potent inhibitors of HIV-1.

Prodrugs for masking bitter taste of antibacterial drugs—a computational approach

DFT calculations for the acid-catalyzed hydrolysis of several maleamic acid amide derivatives revealed that the reaction rate-limiting step is determined on the nature of the amine leaving group. Further, it was established that when the amine leaving group was a secondary amine, acyclovir or cefuroxime moiety the tetrahedral intermediate formation was the rate-limiting step such as in the cases of acyclovir ProD 1- ProD 4 and cefuroxime ProD 1- ProD 4. In addition, the linear correlation between the calculated and experimental rates provided a credible basis for designing prodrugs for masking bitter taste of the corresponding parental drugs which have the potential to release the parent drug in a sustained release fashion. For example, based on the DFT calculated rates the predicted t_1/2 (a time needed for 50 % of the reactant to be hydrolyzed to products) for cefuroxime prodrugs, cefuroxime ProD 1- ProD 4, were 12 min, 18 min, 200 min and 123 min, respectively.

In search of potent and selective inhibitors of neuronal nitric oxide synthase with more simple structures

In certain neurodegenerative diseases damaging levels of nitric oxide (NO) are produced by neuronal nitric oxide synthase (nNOS). It, therefore, is important to develop inhibitors selective for nNOS that do not interfere with other NOS isoforms, especially endothelial NOS (eNOS), which is critical for proper functioning of the cardiovascular system. While we have been successful in developing potent and isoform-selective inhibitors, such as lead compounds 1 and 2, the ease of synthesis and bioavailability have been problematic. Here we describe a new series of compounds including crystal structures of NOS-inhibitor complexes that integrate the advantages of easy synthesis and good biological properties compared to the lead compounds. These results provide the basis for additional structure–activity relationship (SAR) studies to guide further improvement of isozyme selective inhibitors.     

Spin-flip reactions of Zr + C2H6 researched by relativistic density functional theory

Density functional theory (DFT) with relativistic corrections of zero-order regular approximation (ZORA) has been applied to explore the reaction mechanisms of ethane dehydrogenation by Zr atom with triplet and singlet spin-states. Among the complicated minimum energy reaction path, the available states involves three transition states (TS), and four stationary states (1) to (4) and one intersystem crossing with spin-flip (marked by ?): ³ Zr + C ₂ H ₆ → ³ Zr-CH ₃ -CH ₃ ( ³ 1) → ³ TS _1/2 → ³ ZrH-CH ₂ -CH ₃ ( ³ 2) → ³ TS _2/3 ? ¹ ZrH₂-CH₂ = CH₂ ( ¹ 3) → ¹ TS _3/4 → ¹ ZrH ₃ -CH = CH ₂ ( ¹ 4). The minimum energy crossing point is determined with the help of the DFT fractional-occupation-number (FON) approach. The spin inversion leads the reaction pathway transferring from the triplet potential energy surface (PES) to the singlet’s accompanying with the activation of the second C-H bond. The overall reaction is calculated to be exothermic by about 231 kJ mol^?1. Frequency and NBO analysis are also applied to confirm with the experimental observed data.

Electron correlation effects and density analysis of the first-order hyperpolarizability of neutral guanine tautomers

Dipole moments (μ), charge distributions, and static electronic first-order hyperpolarizabilities (β _μ ) of the two lowest-energy keto tautomers of guanine (7H and 9H) were determined in the gas phase using Hartree–Fock, Møller–Plesset perturbation theory (MP2 and MP4), and DFT (PBE1PBE, B97-1, B3LYP, CAM-B3LYP) methods with Dunning’s correlation-consistent aug-cc-pVDZ and d-aug-cc-pVDZ basis sets. The most stable isomer 7H exhibits a μ value smaller than that of the 9H form by a factor of ca. 3.5. The β _μ value of the 9H tautomer is strongly dependent on the computational method employed, as it dramatically influences the β _μ (9H)/β _μ (7H) ratio, which at the highest correlated MP4/aug-cc-pVDZ level is predicted to be ca. 5. The Coulomb-attenuating hybrid exchange-correlation CAM-B3LYP method is superior to the conventional PBE1PBE, B3LYP, and B97-1 functionals in predicting the β _μ values. Differences between the largest diagonal hyperpolarizability components were clarified through hyperpolarizability density analyses. Dipole moment and first-order hyperpolarizability are molecular properties that are potentially useful for distinguishing the 7H from the 9H tautomer.

Theoretical description of halogen bonding – an insight based on the natural orbitals for chemical valence combined with the extended-transition-state method (ETS-NOCV)

In the present study we have characterized the halogen bonding in selected molecules H₃N–ICF₃ (1-NH ₃ ), (PH₃)₂C–ICF₃ (1-CPH ₃ ), C₃H₇Br–(IN₂H₂C₃)₂C₆H₄ (2-Br), H₂–(IN₂H₂C₃)₂C₆H₄ (2-H ₂ ) and Cl–(IC₆F₅)₂C₇H₁₀N₂O₅ (3-Cl), containing from one halogen bond (1-NH ₃ , 1-CPH ₃ ) up to four connections in 3-Cl (the two Cl–HN and two Cl–I), based on recently proposed ETS-NOCV analysis. It was found based on the NOCV-deformation density components that the halogen bonding C–X ^… B (X-halogen atom, B-Lewis base), contains a large degree of covalent contribution (the charge transfer to X ^… B inter-atomic region) supported further by the electron donation from base atom B to the empty σ*(C–X) orbital. Such charge transfers can be of similar importance compared to the electrostatic stabilization. Further, the covalent part of halogen bonding is due to the presence of σ-hole at outer part of halogen atom (X). ETS-NOCV approach allowed to visualize formation of the σ-hole at iodine atom of CF₃I molecule. It has also been demonstrated that strongly electrophilic halogen bond donor, [C₆H₄(C₃H₂N₂I)₂][OTf]₂, can activate chemically inert isopropyl bromide (2-Br) moiety via formation of Br–I bonding and bind the hydrogen molecule (2-H ₂ ). Finally, ETS-NOCV analysis performed for 3-Cl leads to the conclusion that, in terms of the orbital-interaction component, the strength of halogen (Cl–I) bond is roughly three times more important than the hydrogen bonding (Cl–HN).

Why tazobactam and sulbactam have different intermediates population with SHV-1 β-lactamase: a molecular dynamics study

The imine intermediates of tazobactam and sulbactam bound to SHV-1 β-lactamase were investigated by molecular dynamics (MD) simulation respectively. Hydrogen bond networks around active site were found different between tazobactam and sulbactam acyl-enzymes. In tazobactam imine intermediate, it was observed that the triazolyl ring formed stable hydrogen bonds with Asn170 and Thr167. The results suggest that conformation of imine determined the population of intermediates. In imine intermediate of tazobactam, the triazolyl ring is trapped in Thr_Asn pocket, and it restricts the rotation of C5-C6 bond so that tazobactam can only generate trans enamine intermediate. Further, conformational cluster analyses are performed to substantiate the results. These findings provide an explanation for the corresponding experimental results, and will be potentially useful in the development of new inhibitors.

Structural basis for dipeptide amide isoform-selective inhibition of neuronal nitric oxide synthase

Three nitric oxide synthase (NOS) isoforms, eNOS, nNOS and iNOS, generate nitric oxide (NO) crucial to the cardiovascular, nervous and host defense systems, respectively. Development of isoform-selective NOS inhibitors is of considerable therapeutic importance. Crystal structures of nNOS-selective dipeptide inhibitors in complex with both nNOS and eNOS were solved and the inhibitors were found to adopt a curled conformation in nNOS but an extended conformation in eNOS. We hypothesized that a single-residue difference in the active site, Asp597 (nNOS) versus Asn368 (eNOS), is responsible for the favored binding in nNOS. In the D597N nNOS mutant crystal structure, a bound inhibitor switches to the extended conformation and its inhibition of nNOS decreases >200-fold. Therefore, a single-residue difference is responsible for more than two orders of magnitude selectivity in inhibition of nNOS over eNOS by L-N(omega)-nitroarginine-containing dipeptide inhibitors.