Development of a novel apigenin prodrug programmed for alkaline-phosphatase instructed self-inhibition to combat cancer.

Elevated levels of alkaline phosphatase (ALP) in the tumor microenvironment (TME) are a hallmark of cancer progression and thus inhibition of ALP could serve as an effective approach against cancer. Herein, we developed a novel prodrug approach to tackle cancer that bears self-inhibiting alkaline phosphatase-responsiveness properties that can enhance at the same time the solubility of the parent compound. To probe this novel concept, we selected apigenin as the cytotoxic agent since we first unveiled, that it directly interacts and inhibits ALP activity. Consequently, we rationally designed and synthesized, using a self-immolative linker, an ALP responsive apigenin-based phosphate prodrug, phospho-apigenin. Phospho-apigenin markedly increased the stability of the parent compound apigenin. Furthermore, the prodrug exhibited enhanced antiproliferative effect in malignant cells with elevated ALP levels, compared to apigenin. This recorded potency of the developed prodrug was further confirmed in vivo where phospho-apigenin significantly suppressed by 52.8% the growth of PC-3 xenograft tumors.Communicated by Ramaswamy H. Sarma.

ANTIAGE-DB: A Database and Server for the Prediction of Anti-Aging Compounds Targeting Elastase, Hyaluronidase, and Tyrosinase.

Natural products bear a multivariate biochemical profile with antioxidant, anti-inflammatory, antibacterial, and antitumoral properties. Along with their natural sources, they have been widely used both as anti-aging and anti-melanogenic agents due to their effective contribution in the elimination of reactive oxygen species (ROS) caused by oxidative stress. Their anti-aging activity is mainly related to their capacity of inhibiting enzymes like Human Neutrophil Elastase (HNE), Hyaluronidase (Hyal) and Tyrosinase (Tyr). Herein, we accumulated literature information (covering the period 1965-2020) on the inhibitory activity of natural products and their natural sources towards these enzymes. To navigate this information, we developed a database and server termed ANTIAGE-DB that allows the prediction of the anti-aging potential of target compounds. The server operates in two axes. First a comparison of compounds by shape similarity can be performed against our curated database of natural products whose inhibitory potential has been established in the literature. In addition, inverse virtual screening can be performed for a chosen molecule against the three targeted enzymes. The server is open access, and a detailed report with the prediction results is emailed to the user. ANTIAGE-DB could enable researchers to explore the chemical space of natural based products, but is not limited to, as anti-aging compounds and can predict their anti-aging potential. ANTIAGE-DB is accessed online.

Exploration of Betalains and Determination of the Antioxidant and Cytotoxicity Profile of Orange and Purple Opuntia spp. Cultivars in Greece.

Replacing synthetic dyes with natural pigments has gained great attention over the past years in the food industry, due to the increased alertness of consumers for nontoxic and natural additives. Betalains are water-soluble nitrogenous natural pigments that are used as natural colorants in food industries, due to their applicability and their rich pharmacological profile including antioxidant, antimicrobial, and anticancer properties. Therefore, there is a need for a detailed exploration of betalains to fully exploit their properties. Opuntia spp. plants are one of the primary sources of betalains. The objective of this study was to identify betalain phytochemical content in prickly pear cactus of two different Opuntia species from Greece (an Opuntia ficus-indica (L.) Mill (OFI) orange prickly pear cultivar and an Opuntia spp. purple prickly pear cultivar) using modern analytical techniques as also to evaluate their antioxidant and cytotoxicity profile. To achieve this we used an array of analytical techniques, including ultra-violet-vis (UV-Vis) spectroscopy, nuclear magnetic resonance (NMR) spectroscopy, and liquid chromatography-high resolution mass spectrometry (LC-HRMS) as also cell based in vitro assays. These enabled us to establish a rapid approach that can distinguish the different Opuntia spp. cultivars based on their phytochemical constituents through untargeted metabolomics analysis using ultra-high performance liquid chromatography-mass spectrometry - quadrupole time-of-flight (UPLC/MS Q-TOF). These findings could allow a further exploitation of Opuntia species and especially their enriched betalain phytochemical profile as viable source of natural food colorants.

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.

Anti-Ageing Potential of S. euboea Heldr. Phenolics.

In recent years, the use of Sideritis species as bioactive agents is increasing exponentially. The present study aimed to investigate the chemical constituents, as well as the anti-ageing potential of the cultivated Sideritis euboea Heldr. The chemical fingerprinting of the ethyl acetate residue of this plant was studied using 1D and 2D-NMR spectra. Isomeric compounds belonging to acylated flavone derivatives and phenylethanoid glycosides were detected in the early stage of the experimental process through 2D-NMR techniques. Overall, thirty-three known compounds were isolated and identified. Some of them are reported for the first time not only in S. euboea, but also in genus Sideritis L. The anti-ageing effect of the ethyl acetate residue and the isolated specialized products was assessed as anti-hyaluronidase activity. In silico docking simulation revealed the interactions of the isolated compounds with hyaluronidase. Furthermore, the in vitro study on the inhibition of hyaluronidase unveiled the potent inhibitory properties of ethyl acetate residue and apigenin 7-O-ß-d-glucopyranoside. Though, the isomers of apigenin 7-O-p-coumaroyl-glucosides and also the 4'-methyl-hypolaetin 7-O-[6'''-O-acetyl-ß-d-allopyranosyl]-(1→2)-ß-d-glucopyranoside exerted moderate hyaluronidase inhibition. This research represents the first study to report on the anti-hyaluronidase activity of Sideritis species, confirming its anti-inflammatory, cytotoxic and anti-ageing effects and its importance as an agent for cosmetic formulations as also anticancer potential.

Interplay of cholesterol, membrane bilayers and the AT1R: A cholesterol consensus motif on AT1R is revealed.

Hypertension, mediated by the Angiotensin II receptor type 1 (AT1R), is still the major cause of premature death despite the discovery of novel therapeutics, highlighting the importance of an in depth understanding of the drug-AT1R recognition mechanisms coupled with the impact of the membrane environment on the interaction of drugs with AT1R. Herein, we examine the interplay of cholesterol-lipid-candesartan and the AT1R using Molecular Dynamics simulations of a model membrane consisting of 60:40 mol%. DPPC:cholesterol, candesartan and the AT1R, mimicking the physiological cholesterol concentration in sarcolemma membranes. The simulations of the model membrane of 60:40 mol%. DPPC:cholesterol were further validated using DOSY NMR experiments. Interestingly, our results suggest a significant role of cholesterol in the AT1R function imposed through a Cholesterol Consensus Motif (CCM) in the receptor, which could be crucial in the drug binding process. Candesartan diffusion towards AT1R through incorporation into lipid bilayers, appears to be retarded by the presence of cholesterol. However, its direct approach towards AT1R may be facilitated through the mobility induced on the N-terminus by the cholesterol binding on the CCM these novel insights could pave the way towards the development of more potent pharmaceutical agents to combat hypertension more effectively.

Valorisation of stachysetin from cultivated Stachys iva Griseb. as anti-diabetic agent: a multi-spectroscopic and molecular docking approach.

Stachys species are considered as important medicinal plants with numerous health benefit effects. In continuation of our research on the Greek Stachys species, the chemical profile of the aerial parts of cultivated S. iva Griseb. has been explored. The NMR profiles of the plant extract/infusion were used to guide the isolation process, leading to the targeted isolation of seventeen known compounds. The rare acylated flavonoid, stachysetin, was isolated for the third time from plant species in the international literature. Identification of the characteristic signals of stachysetin in the 1D 1H-NMR spectrum of the crude extract was presented. In order to evaluate the potential of the identified chemical space in Stachys to bear possible bioactivity against diabetes, we performed an in silico screening against 17 proteins implicated in diabetes, as also ligand based similarity metrics against established anti-diabetic drugs. The results capitalized the anti-diabetic potency of stachysetin. Its binding profile to the major drug carrier plasma protein serum albumin was also explored along with its photophysical properties suggesting that stachysetin could be recognized and delivered in plasma through serum albumin and also could be tracked through near-infrared imaging. Communicated by Ramaswamy H. Sarma.

Charting the structural and thermodynamic determinants in phenolic acid natural product - cyclodextrin encapsulations.

Cyclodextrins are pliable platforms that have served to optimize the pharmaceutic profile of numerous compounds and to enhance the stability of natural food additives. Caffeic and rosmarinic acid are natural products with proven health benefits, though their full therapeutic potential has not been exploited. To enhance their pharmaceutic profile, we developed cyclodextrin-based formulates and unveiled their thermodynamic and structural principles. The complexes' stoichiometry was determined by ESI-MS. Solid-state and liquid NMR spectroscopy revealed the interactions and the topographical location of the caffeic and rosmarinic acid inside the cyclodextrin cavity. The theoretically analyzed HP-ß-CD's degree of substitution (DS) of caffeic and rosmarinic acids can explain the intensities obtained by 2D NOESY experiments. The thermodynamics and the affinity of the complexes were evaluated through isothermal titration calorimetry. In addition, the rosmarinic and caffeic acids as, also, their complexes showed considerable antimicrobial activity against common food spoilage and pathogenic bacteria. The generated data could provide the basis to understand the structural and thermodynamic determinants implicated in natural products - CD recognition and to develop platforms for the optimization of their pharmaceutical and stability profiles in order to be utilized as safe and stable natural antimicrobial food additives.Communicated by Ramaswamy H. Sarma.

Bioinspired tailoring of fluorogenic thiol responsive antioxidant precursors to protect cells against H2O2-induced DNA damage.

Natural antioxidants, like phenolic acids, possess a unique chemical space that can protect cellular components from oxidative stress. However, their polar carboxylic acid chemotype reduces full intracellular antioxidant potential due to limited diffusion through biological membranes. Here, we have designed and developed a new generation of hydrophobic turn-on fluorescent antioxidant precursors that upon penetration of the cell membrane, reveal a more polar and more potent antioxidant core and simultaneously become fluorescent allowing their intracellular tracking. Their activation is stimulated by polarity alteration by sensing intracellular signals and specifically biothiols. In our design, the carboxylic group of phenolic acids that originally restricts cell entrance is derivatized and conjugated through Copper (I)-catalyzed azide-alkyne cycloaddition (CuAAC) to a coumarin derivative that its fluorescence properties are quenched with a biothiol activatable element. This more hydrophobic precursor readily penetrates cell membrane and once inside the cell the antioxidant core is revealed upon sensing glutathione, its fluorescence is restored in a turn-on manner and the generation of a more polar character traps the molecule inside the cell. This turn-on fluorescent antioxidant precursor that can be applied to phenolic acids, was developed for rosmarinic acid and the conjugate was named as RCG. The selectivity and responsiveness of RCG towards the most abundant biothiols was monitored through a variety of biophysical techniques including UV-Vis, fluorescence and NMR spectroscopy. The electrochemical behavior and free radical scavenging capacity of the precursor RCG and the active compound (RC) was evaluated and compared with the parent compound (rosmarinic acid) through cyclic voltammetry and EPR spectroscopy, respectively. The stability of the newly synthesized bioactive conjugate RC was found significantly higher than the parent rosmarinic acid when exposed to oxygen. Cell uptake experiments were conducted and revealed the internalization of RCG. The degree of intracellular DNA protection offered by RCG and its active drug (RC) on exposure to H2O2 was also evaluated in Jurkat cells.

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.

Exploring the role of the membrane bilayer in the recognition of candesartan by its GPCR AT1 receptor.

Cardiovascular diseases and hypertension in particular are major health risks worldwide and the improvement on their treatment will be beneficial for the human health. AT1R antagonists belong to the sartans family that targets the renin-angiotensin aldosterone system (RAAS) through blocking the hormone angiotensin II to exert its detrimental effects in pathological states. As a consequence, they are beneficial to treat hypertension, diabetes related kidney failure and hyperaemic episodes. Long unbiased Molecular Dynamics (MD) simulations are performed in order to explore candesartan's possible 2D and 3D diffusion mechanisms towards AT1R receptor. 3D diffusion mechanism is referred to the direct binding of the AT1 antagonist candesartan to the AT1R 3D structure (PDB ID: 4YAY). 2D diffusion mechanism involves first, the incorporation of candesartan in the bilayer core and then its localization on the AT1R binding cavity, through a diffusion mechanism. The obtained results indicate that membranes interact significantly with the neutral form of candesartan, which is indeed approaching the receptors' active site through diffusion via the lipids. On the other hand, the deprotonated form of the drug is interacting with AT1R's extracellular loop and fails to enter the membrane, pointing out the importance of the pH microenvironment around the receptor. To validate the calculated diffusion coefficients of the drug in the lipid bilayers 2D DOSY NMR experiments were recorded and they were in good agreement. Information on the impact that has the interaction of candesartan with the membrane is very important for the rationally design and development of potent ARBs. Thus, its conformational features as well as its localization in the membrane core have to be thoroughly explored.

NMR-Based Μetabolomics of the Lipid Fraction of Organic and Conventional Bovine Milk.

Origin and quality identification in dairy products is an important issue and also an extremely challenging and complex experimental procedure. The objective of the present work was to compare the metabolite profile of the lipid fraction of organic and conventional bovine milk using NMR metabolomics analysis. ¹H-NMR and 1D TOCSY NMR methods of analysis were performed on extracted lipid fraction of lyophilized milk. For this purpose, 14 organic and 16 conventional retail milk samples were collected monthly, and 64 bulk-tank (58 conventional and 6 organics) milk samples were collected over a 14-month longitudinal study in Cyprus. Data were treated with multivariate methods (PCA, PLS-DA). Minor components were identified and quantified, and modification of the currently used equations is proposed. A significantly increased % content of conjugated (9-cis, 11-trans)18:2 linoleic acid (CLA), α-linolenic acid, linoleic acid, allylic protons and total unsaturated fatty acids (UFA) and decreased % content for caproleic acid were observed in the organic samples compared to the conventional ones. The present work confirms that lipid profile is affected by contrasting management system (organic vs. conventional), and supports the potential of NMR-based metabolomics for the rapid analysis and authentication of the milk from its lipid profile.

Selective One-Dimensional Total Correlation Spectroscopy Nuclear Magnetic Resonance Experiments for a Rapid Identification of Minor Components in the Lipid Fraction of Milk and Dairy Products: Toward Spin Chromatography?

We report a rapid, direct, and unequivocal spin-chromatographic separation and identification of minor components in the lipid fraction of milk and common dairy products with the use of selective one-dimensional (1D) total correlation spectroscopy (TOCSY) nuclear magnetic resonance (NMR) experiments. The method allows for the complete backbone spin-coupling network to be elucidated even in strongly overlapped regions and in the presence of major components from 4 × 10(2) to 3 × 10(3) stronger NMR signal intensities. The proposed spin-chromatography method does not require any derivatization steps for the lipid fraction, is selective with excellent resolution, is sensitive with quantitation capability, and compares favorably to two-dimensional (2D) TOCSY and gas chromatography-mass spectrometry (GC-MS) methods of analysis. The results of the present study demonstrated that the 1D TOCSY NMR spin-chromatography method can become a procedure of primary interest in food analysis and generally in complex mixture analysis.