Complementary Segmental Labeling of Large RNAs: Economic Preparation and Simplified NMR Spectra for Measurement of More RDCs

NMR structure determination of large RNAs is often restricted by limited RDC information caused by chemical shift degeneracy. We established a general, time- and cost-effective methodology for the preparation of 13C/15N complementary labeled RNAs from a single plasmid. Applying this method to the 25 kDa BC1-DTE RNA, we were able to resolve severe chemical shift degeneracy, thereby almost doubling the number of RDC restraints in comparison to the conventional 13C,15N uniform-labeled RNA

Rapid and Direct Low Micromolar NMR Method for the Simultaneous Detection of Hydrogen Peroxide and Phenolics in Plant Extracts

A rapid and direct low micromolar ¹H NMR method for the simultaneous identification and quantification of hydrogen peroxide and phenolic compounds in plant extracts was developed. The method is based on the highly deshielded ¹H NMR signal of H₂O₂ at ∼10.30 ppm in DMSO-<i>d</i>₆ and the combined use of picric acid and low temperature, near the freezing point of the solution, in order to achieve the minimum proton exchange rate. Line widths of H₂O₂ below 3.8 Hz were obtained for several Greek oregano extracts which resulted in a detection limit of 0.7 μmol L^–1. Application of an array of NMR experiments, including 2D ¹H–¹³C HMBC, spiking of the samples with H₂O₂, and variable temperature experiments, resulted in the unequivocal assignment of H₂O₂ precluding any confusion with interferences from intrinsic phenolics in the extract

Unveiling the interaction profile of rosmarinic acid and its bioactive substructures with serum albumin

Rosmarinic acid, a phytochemical compound, bears diverse pharmaceutical profile. It is composed by two building blocks: caffeic acid and a salvianic acid unit. The interaction profile, responsible for the delivery of rosmarinic acid and its two substructure components by serum albumin remains unexplored. To unveil this, we established a novel low-cost and efficient method to produce salvianic acid from the parent compound. To probe the interaction profile of rosmarinic acid and its two substructure constituents with the different serum albumin binding sites we utilised fluorescence spectroscopy and competitive saturation transfer difference NMR experiments. These studies were complemented with transfer NOESY NMR experiments. The thermodynamics of the binding profile of rosmarinic acid and its substructures were addressed using isothermal titration calorimetry. In silico docking studies, driven by the experimental data, have been used to deliver further atomic details on the binding mode of rosmarinic acid and its structural components.</p

Deconvoluting the Dual Antiplatelet Activity of a Plant Extract

A thorough evaluation of the antiplatelet activity profile of hexane olive leaf extract in human platelets indicated a potent activity accomplished through a two axis inhibition of platelet activation triggered both by ADP and thrombin. To delineate the extract components responsible for this dual activity, an NMR based method was established to determine and quantify the triterpenoid content leading to the characterization of uvaol, erythrodiol, and oleanolic acid. The antiplatelet profile of the total extract and of the 3 determined triterpenoids was evaluated against in vitro platelet aggregation induced by several platelet agonists as also on PAC-1 binding and P-selectin membrane expression both in healthy volunteers and in platelets from patients with an acute coronary syndrome receiving dual antiplatelet therapy with aspirin and ticagrelor. The extract was identified to inhibit ADP-induced platelet activation due to its erythrodiol content and TRAP-induced platelet activation due to the activity of uvaol and oleanolic acid

Development of a DHA-Losartan hybrid as a potent inhibitor of multiple pathway-induced platelet aggregation

Despite the scientific progression in the prevention and treatment of cardiovascular diseases (CVDs) they remain the leading cause of mortality and disability worldwide. The classic treatment involves the simultaneous dosing of two antiplatelet drugs, aspirin and clopidogrel/prasugrel. However, besides drug resistance, severe side effects have been also manifested including acute bleeding and toxicity. Thus, new therapeutic agents with enhanced efficacy and diminished side effects are of importance. Towards this end, omega-3 (ω-3) fatty acids have demonstrated potent efficacy against CVDs through inhibiting platelet aggregation that bears a pivotal role in atherothrombosis. Another factor that displays a critical role in the pathogenesis of cardiovascular diseases is the renin-angiotensin system (RAS), and especially the AT1R blocker losartan that has been reported to exert antiplatelet activity mediated by this receptor. Along these lines, we envisaged developing a molecular hybrid consisted of docosahexaenoic acid (ω-3 fatty acid) and losartan, that could exert a notable antiplatelet effect against CVDs. The design and synthesis of the new DHA-losartan hybrid, designated DHA-L, bestowed with the additive properties of the parent compounds, is reported. In silico studies were first exploited to validate the potential of DHA-L to retain losartan’s ability to bind AT1R. The antiplatelet activity of DHA-L was evaluated against in vitro platelet aggregation induced by several platelet agonists. Notably, the hybrid illustrated a pleiotropic antiplatelet profile inhibiting platelet aggregation through multiple platelet activation pathways including P2Y12, PAR-1 (Protease-Activated Receptor-1), PAF (Platelet Activating Factor), COX-1 (cyclooxygenase-1) and collagen receptors. The stability of DHA-L in human plasma and in a wide range of pH values was also evaluated over time using an HPLC protocol. The hybridization approach described herein could pave the way for the development of novel potent multitargeted therapeutics with enhanced antiplatelet profile. Communicated by Ramaswamy H. Sarma</p

Inclusion of Quercetin in Gold Nanoparticles Decorated with Supramolecular Hosts Amplifies Its Tumor Targeting Properties

Despite the anticancer potential of natural products (NPs), their limited bioavailability necessitates laborious derivatization or covalent conjugation to delivery vehicles. To unleash their potential, we developed a nanohybrid delivery platform with a noncovalently tunable surface. Initially, the active compound was encapsulated in a macrocycle, p-sulfonatocalix­[4]­arene, enabling a 62 000-fold aqueous solubility amplification as also a 2.9-fold enhancement in its cytotoxicity with respect to the parent compound in SW-620 colon cancer cells. A pH stimuli responsive behavior was recorded for this formulate, where a programmable release of quercetin from the macrocycle was monitored in an acidic environment. Then, a nanoparticle gold core was decorated with calixarene hosts to accommodate noncovalently NPs. The loaded nanocarrier with the NP quercetin dramatically enhanced the cytotoxicity (>50-fold) of the parent NP in colon cancer and altered its cell membrane transport mode. In vivo experiments in a mouse 4T1 tumor model showed a reduction of tumor volume in mice treated with quercetin-loaded nanoparticles without apparent toxic effects. Further analysis of the tumor-derived RNA highlighted that treatment with quercetin-loaded nanoparticles altered the expression of 27 genes related to apoptosis

Direct Binding of Bcl‑2 Family Proteins by Quercetin Triggers Its Pro-Apoptotic Activity

Bcl-2 family proteins are important regulators of apoptosis and its antiapoptotic members, which are overexpressed in many types of cancer, are of high prognostic significance, establishing them as attractive therapeutic targets. Quercetin, a natural flavonoid, has drawn much attention because it exerts anticancer effects, while sparing normal cells. A multidisciplinary approach has been employed herein, in an effort to reveal its mode of action including dose–response antiproliferative activity and induced apoptosis effect, biochemical and physicochemical assays, and computational calculations. It may be concluded that, quercetin binds directly to the BH3 domain of Bcl-2 and Bcl-xL proteins, thereby inhibiting their activity and promoting cancer cell apoptosis

Molecular investigation of artificial and natural sweeteners as potential anti-inflammatory agents

Repurposing existing drugs, as well as natural and artificial sweeteners for novel therapeutic indications could speed up the drug discovery process since numerous associated risks and costs for drug development can be surpassed. In this study, natural and artificial sweeteners have been evaluated by in silico and experimental studies for their potency to inhibit lipoxygenase enzyme, an enzyme participating in the inflammation pathway. A variety of different methods pinpointed that aspartame inhibits the lipoxygenase isoform 1 (LOX-1). In particular, “LOX-aspartame” complex, that was predicted by docking studies, was further evaluated by Molecular Dynamics (MD) simulations in order to assess the stability of the complex. The binding energy of the complex has been calculated after MD simulations using Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) method. Furthermore, Quantum Mechanics/Molecular Mechanics (QM/MM) calculations have been applied for geometry optimization of the “enzyme-ligand” complex. After having fully characterized the “LOX-aspartame” complex in silico, followed in vitro biological assays confirmed that aspartame inhibits LOX-1 (IC50=50 ± 3.0 μΜ) and blocks its biological response. The atomic details of aspartame’s interaction profile with LOX-1 were revealed through Saturation Transfer Difference (STD) NMR (Nuclear Magnetic Resonance). Finally, aspartame was also tested with Molecular Docking and Molecular Dynamics studies for its potent binding to a number of different LOX isoforms of many organisms, including human. The in silico methods indicated that aspartame could serve as a novel starting point for drug design against LOX enzyme. Communicated by Ramaswamy H. Sarma</p

Host–Guest Interactions between Candesartan and Its Prodrug Candesartan Cilexetil in Complex with 2‑Hydroxypropyl-β-cyclodextrin: On the Biological Potency for Angiotensin II Antagonism

Renin–angiotensin aldosterone system inhibitors are for a long time extensively used for the treatment of cardiovascular and renal diseases. AT1 receptor blockers (ARBs or sartans) act as antihypertensive drugs by blocking the octapeptide hormone Angiotensin II to stimulate AT1 receptors. The antihypertensive drug candesartan (CAN) is the active metabolite of candesartan cilexetil (Atacand, CC). Complexes of candesartan and candesartan cilexetil with 2-hydroxylpropyl-β-cyclodextrin (2-HP-β-CD) were characterized using high-resolution electrospray ionization mass spectrometry and solid state 13C cross-polarization/magic angle spinning nuclear magnetic resonance (CP/MAS NMR) spectroscopy. The 13C CP/MAS results showed broad peaks especially in the aromatic region, thus confirming the strong interactions between cyclodextrin and drugs. This experimental evidence was in accordance with molecular dynamics simulations and quantum mechanical calculations. The synthesized and characterized complexes were evaluated biologically in vitro. It was shown that as a result of CAN’s complexation, CAN exerts higher antagonistic activity than CC. Therefore, a formulation of CC with 2-HP-β-CD is not indicated, while the formulation with CAN is promising and needs further investigation. This intriguing result is justified by the binding free energy calculations, which predicted efficient CC binding to 2-HP-β-CD, and thus, the molecule’s availability for release and action on the target is diminished. In contrast, CAN binding was not favored, and this may allow easy release for the drug to exert its bioactivity