Inhibition of the angiotensin II type 2 receptor AT2R is a novel therapeutic strategy for glioblastoma.

Glioblastoma (GBM) is an aggressive malignant primary brain tumor with limited therapeutic options. We show that the angiotensin II (AngII) type 2 receptor (AT2R) is a therapeutic target for GBM and that AngII, endogenously produced in GBM cells, promotes proliferation through AT2R. We repurposed EMA401, an AT2R antagonist originally developed as a peripherally restricted analgesic, for GBM and showed that it inhibits the proliferation of AT2R-expressing GBM spheroids and blocks their invasiveness and angiogenic capacity. The crystal structure of AT2R bound to EMA401 was determined and revealed the receptor to be in an active-like conformation with helix-VIII blocking G-protein or ß-arrestin recruitment. The architecture and interactions of EMA401 in AT2R differ drastically from complexes of AT2R with other relevant compounds. To enhance central nervous system (CNS) penetration of EMA401, we exploited the crystal structure to design an angiopep-2-tethered EMA401 derivative, A3E. A3E exhibited enhanced CNS penetration, leading to reduced tumor volume, inhibition of proliferation, and increased levels of apoptosis in an orthotopic xenograft model of GBM.

A Novel Fluorescent Gemcitabine Prodrug That Follows a Nucleoside Transporter-Independent Internalization and Bears Enhanced Therapeutic Efficacy With Respect to Gemcitabine.

The multiplexity of cancer has rendered it the second leading cause of mortality worldwide and theragnostic prodrugs have gained popularity in recent years as a means of treatment. Theragnostic prodrugs enable the simultaneous diagnosis and therapy of tumors via high-precision real-time drug release monitoring. Herein, we report the development of the small theragnostic prodrug GF, based on the nucleoside anticancer agent gemcitabine and the fluorescent dye 5(6)-carboxyfluorescein. We have successfully demonstrated its efficient internalization in tumor cells, showing localization throughout both the early and late endocytic pathways. Its mechanism of cell internalization was evaluated, confirming its independence from nucleoside transporters. Its cellular localization via confocal microscopy revealed a clathrin-mediated endocytosis mechanism, distinguishing it from analogous compounds studied previously. Furthermore, GF exhibited stability across various pH values and in human blood plasma. Subsequently, its in vitro cytotoxicity was assessed in three human cancer cell lines (A549, U87 and T98). Additionally, its pharmacokinetic profile in mice was investigated and the consequent drug release was monitored. Finally, its in vivo visualization was accomplished in zebrafish xenotransplantation models and its in vivo efficacy was evaluated in A549 xenografts. The results unveiled an intriguing efficacy profile, positioning GF as a compelling candidate warranting further investigation.

Development of FluoAHRL: A Novel Synthetic Fluorescent Compound That Activates AHR and Potentiates Anti-Inflammatory T Regulatory Cells.

Aryl Hydrocarbon Receptor (AHR) ligands, upon binding, induce distinct gene expression profiles orchestrated by the AHR, leading to a spectrum of pro- or anti-inflammatory effects. In this study, we designed, synthesized and evaluated three indole-containing potential AHR ligands (FluoAHRL: AGT-4, AGT-5 and AGT-6). All synthesized compounds were shown to emit fluorescence in the near-infrared. Their AHR agonist activity was first predicted using in silico docking studies, and then confirmed using AHR luciferase reporter cell lines. FluoAHRLs were tested in vitro using mouse peritoneal macrophages and T lymphocytes to assess their immunomodulatory properties. We then focused on AGT-5, as it illustrated the predominant anti-inflammatory effects. Notably, AGT-5 demonstrated the ability to foster anti-inflammatory regulatory T cells (Treg) while suppressing pro-inflammatory T helper (Th)17 cells in vitro. AGT-5 actively induced Treg differentiation from naïve CD4+ cells, and promoted Treg proliferation, cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) expression and interleukin-10 (IL-10) production. The increase in IL-10 correlated with an upregulation of Signal Transducer and Activator of Transcription 3 (STAT3) expression. Importantly, the Treg-inducing effect of AGT-5 was also observed in human tonsil cells in vitro. AGT-5 showed no toxicity when applied to zebrafish embryos and was therefore considered safe for animal studies. Following oral administration to C57BL/6 mice, AGT-5 significantly upregulated Treg while downregulating pro-inflammatory Th1 cells in the mesenteric lymph nodes. Due to its fluorescent properties, AGT-5 could be visualized both in vitro (during uptake by macrophages) and ex vivo (within the lamina propria of the small intestine). These findings make AGT-5 a promising candidate for further exploration in the treatment of inflammatory and autoimmune diseases.

Development of a Novel Apigenin Dosage form as a Substitute for the Modern Triple Antithrombotic Regimen.

The simultaneous administration of three antiplatelet agents has been proposed as an efficient strategy for the secondary prevention of atherothrombotic events and is included in the European guidelines. However, this strategy presented an increased risk of bleeding; therefore, the identification of new antiplatelet agents, with improved efficacy and diminished side effects, is of great importance. In silico studies, UPLC/MS Q-TOF plasma stability, in vitro platelet aggregation experiments, and pharmacokinetic studies were exploited. In the present study, it has been predicted that the flavonoid apigenin could target different platelet activation pathways, including P2Y12, protease-activated receptor-1 (PAR-1), and cyclooxygenase 1 (COX-1). To enhance apigenin's potency, hybridization with docosahexaenoic acid (DHA) was performed, as fatty acids have illustrated potent efficacy against cardiovascular diseases (CVDs). The new molecular hybrid, termed 4'-DHA-apigenin, demonstrated enhanced inhibitory activity against platelet aggregation induced by thrombin receptor activator peptide-6 (TRAP-6), adenosine diphosphate (ADP), and arachidonic acid (AA), with respect to the parent apigenin. The 4'-DHA-apigenin hybrid illustrated an almost 2-fold enhanced inhibitory activity, with respect to apigenin, and an almost 3-fold enhanced inhibitory activity, with respect to DHA, for the ADP-induced platelet aggregation. Additionally, the hybrid presented a more than 12-fold enhanced inhibitory activity with respect to DHA for the TRAP-6 induced platelet aggregation. Furthermore, a 2-fold enhanced inhibitory activity was recorded for the 4'-DHA-apigenin hybrid for the AA-induced platelet aggregation with respect to apigenin. To surmount the reduced LC-MS based plasma stability, a novel dosage form in olive oil has been developed. The 4'-DHA-apigenin olive oil-based formulation presented an enhanced antiplatelet inhibitory effect in three activation pathways. To further explore the pharmacokinetic profile of 4'-DHA-apigenin in olive oil formulations, a UPLC/MS Q-TOF protocol has been established to quantify the serum levels of apigenin after oral administration to C57BL/6J wild type mice. The olive oil-based formulation of 4'-DHA-apigenin demonstrated an increase in apigenin bioavailability of 262 %. This study may offer a new therapeutic strategy tailored to improve the treatment of CVDs.

Comparative Interaction Studies of Quercetin with 2-Hydroxyl-propyl-ß-cyclodextrin and 2,6-Methylated-ß-cyclodextrin.

Quercetin (QUE) is a well-known natural product that can exert beneficial properties on human health. However, due to its low solubility its bioavailability is limited. In the present study, we examine whether its formulation with two cyclodextrins (CDs) may enhance its pharmacological profile. Comparative interaction studies of quercetin with 2-hydroxyl-propyl-ß-cyclodextrin (2HP-ß-CD) and 2,6-methylated cyclodextrin (2,6Me-ß-CD) were performed using NMR spectroscopy, DFT calculations, and in silico molecular dynamics (MD) simulations. Using T1 relaxation experiments and 2D DOSY it was illustrated that both cyclodextrin vehicles can host quercetin. Quantum mechanical calculations showed the formation of hydrogen bonds between QUE with 2HP-ß-CD and 2,6Μe-ß-CD. Six hydrogen bonds are formed ranging between 2 to 2.8 Å with 2HP-ß-CD and four hydrogen bonds within 2.8 Å with 2,6Μe-ß-CD. Calculations of absolute binding free energies show that quercetin binds favorably to both 2,6Me-ß-CD and 2HP-ß-CD. MM/GBSA results show equally favorable binding of quercetin in the two CDs. Fluorescence spectroscopy shows moderate binding of quercetin in 2HP-ß-CD (520 M-1) and 2,6Me-ß-CD (770 M-1). Thus, we propose that both formulations (2HP-ß-CD:quercetin, 2,6Me-ß-CD:quercetin) could be further explored and exploited as small molecule carriers in biological studies.

Phenethyl Ester of Gallic Acid Ameliorates Experimental Autoimmune Encephalomyelitis.

Gallic acid is a phenolic acid present in various plants, nuts, and fruits. It is well known for its anti-oxidative and anti-inflammatory properties. The phenethyl ester of gallic acid (PEGA) was synthesized with the aim of increasing the bioavailability of gallic acid, and thus its pharmacological potential. Here, the effects of PEGA on encephalitogenic cells were examined, and PEGA was found to modulate the inflammatory activities of T cells and macrophages/microglia. Specifically, PEGA reduced the release of interleukin (IL)-17 and interferon (IFN)-γ from T cells, as well as NO, and IL-6 from macrophages/microglia. Importantly, PEGA ameliorated experimental autoimmune encephalomyelitis, an animal model of chronic inflammatory disease of the central nervous system (CNS)-multiple sclerosis. Thus, PEGA is a potent anti-inflammatory compound with a perspective to be further explored in the context of CNS autoimmunity and other chronic inflammatory disorders.

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.

Using conformational constraints at position 6 of Angiotensin II to generate compounds with enhanced AT2R selectivity and proteolytic stability.

The Renin-Angiotensin System (RAS) plays a crucial role in numerous pathological conditions. Two of the critical RAS players, the angiotensin receptors AT1R and AT2R, possess differential functional profiles, although they share high sequence similarity. Although the main focus has been placed on AT1R, several epidemiological studies have evidenced that activation of AT2R could operate as a multimodal therapeutic target for different diseases. Thus, the development of selective AT2R ligands could have a high clinical potential for different therapeutic directions. Furthermore, they could serve as a powerful tool to interrogate the molecular mechanisms that are mediated by AT2R. Based on our recently established high affinity and AT2R selective compound [Y]6-AII we developed several analogues through modifying aminoacids located at positions 6 and 7 with various conformationally constrained analogues to enhance both the selectivity and stability. We report the development of high-affinity AT2R binders, which displayed high selectivity for AT2R versus AT1R. Furthermore, all analogues presented enhanced stability in human plasma with respect to the parent hormone Angiotensin II as also [Y]6-AII.

Characterization of Protocatechuate 4,5-Dioxygenase from Pseudarthrobacter phenanthrenivorans Sphe3 and In Situ Reaction Monitoring in the NMR Tube.

The current study aims at the functional and kinetic characterization of protocatechuate (PCA) 4,5-dioxygenase (PcaA) from Pseudarthrobacter phenanthrenivorans Sphe3. This is the first single subunit Type II dioxygenase characterized in Actinobacteria. RT-PCR analysis demonstrated that pcaA and the adjacent putative genes implicated in the PCA meta-cleavage pathway comprise a single transcriptional unit. The recombinant PcaA is highly specific for PCA and exhibits Michaelis-Menten kinetics with Km and Vmax values of 21 ± 1.6 µM and 44.8 ± 4.0 U × mg-1, respectively, in pH 9.5 and at 20 °C. PcaA also converted gallate from a broad range of substrates tested. The enzymatic reaction products were identified and characterized, for the first time, through in situ biotransformation monitoring inside an NMR tube. The PCA reaction product demonstrated a keto-enol tautomerization, whereas the gallate reaction product was present only in the keto form. Moreover, the transcriptional levels of pcaA and pcaR (gene encoding a LysR-type regulator of the pathway) were also determined, showing an induction when cells were grown on PCA and phenanthrene. Studying key enzymes in biodegradation pathways is significant for bioremediation and for efficient biocatalysts development.

Rational Design and Synthesis of AT1R Antagonists.

Hypertension is one of the most common diseases nowadays and is still the major cause of premature death despite of the continuous discovery of novel therapeutics. The discovery of the Renin Angiotensin System (RAS) unveiled a path to develop efficient drugs to fruitfully combat hypertension. Several compounds that prevent the Angiotensin II hormone from binding and activating the AT1R, named sartans, have been developed. Herein, we report a comprehensive review of the synthetic paths followed for the development of different sartans since the discovery of the first sartan, Losartan.

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.

Chemical Profiling, Bioactivity Evaluation and the Discovery of a Novel Biopigment Produced by Penicillium purpurogenum CBS 113139.

Biobased pigments are environmentally friendly alternatives to synthetic variants with an increased market demand. Production of pigments via fermentation is a promising process, yet optimization of the production yield and rate is crucial. Herein, we evaluated the potential of Penicillium purpurogenum to produce biobased pigments. Optimum sugar concentration was 30 g/L and optimum C:N ratio was 36:1 resulting in the production of 4.1-4.5 AU (namely Pigment Complex A). Supplementation with ammonium nitrate resulted in the production of 4.1-4.9 AU (namely Pigment Complex B). Pigments showed excellent pH stability. The major biopigments in Pigment Complex A were N-threonyl-rubropunctamin or the acid form of PP-R (red pigment), N-GABA-PP-V (violet pigment), PP-O (orange pigment) and monascorubrin. In Pigment Complex B, a novel biopigment annotated as N-GLA-PP-V was identified. Its basic structure contains a polyketide azaphilone with the same carboxyl-monascorubramine base structure as PP-V (violet pigment) and γ-carboxyglutamic acid (GLA). The pigments were not cytotoxic up to 250 µg/mL.

Biophysical Evaluation and In Vitro Controlled Release of Two Isomeric Adamantane Phenylalkylamines with Antiproliferative/Anticancer and Analgesic Activity.

The aqueous dissolution profile of the isomeric synthetic adamantane phenylalkylamine hydrochlorides I and II was probed. These adducts have shown significant antiproliferative/anticancer activity associated with an analgesic profile against neuropathic pain. They are both devoid of toxic effects and show appreciable enzymatic human plasma stability. The structures of these two compounds have been elucidated using 2D NMR experiments, which were used to study their predominant conformations. Compound II's scaffold appeared more flexible, as shown by the NOE spatial interactions between the alkyl bridge chain, the aromatic rings, and the adamantane nucleus. Conversely, compound I appeared very rigid, as it did not share significant NOEs between the aforementioned structural segments. MD simulations confirmed the NOE results. The aqueous dissolution profile of both molecules fits well with their minimum energy conformers' features, which stem from the NOE data; this was nicely demonstrated, especially in the case of compound II.

Single Peptide Backbone Surrogate Mutations to Regulate Angiotensin GPCR Subtype Selectivity.

Mutating the side-chains of amino acids in a peptide ligand, with unnatural amino acids, aiming to mitigate its short half-life is an established approach. However, it is hypothesized that mutating specific backbone peptide bonds with bioisosters can be exploited not only to enhance the proteolytic stability of parent peptides, but also to tune its receptor subtype selectivity. Towards this end, four [Y]6 -Angiotensin II analogues are synthesized where amide bonds have been replaced by 1,4-disubstituted 1,2,3-triazole isosteres in four different backbone locations. All the analogues possessed enhanced stability in human plasma in comparison with the parent peptide, whereas only two of them achieved enhanced AT2 R/AT1 R subtype selectivity. This diversification has been studied through 2D NMR spectroscopy and unveiled a putative more structured microenvironment for the two selective ligands accompanied with increased number of NOE cross-peaks. The most potent analogue, compound 2, has been explored regarding its neurotrophic potential and resulted in an enhanced neurite growth with respect to the established agent C21.

Discovery of a stable tripeptide targeting the N-domain of CRF1 receptor.

The corticotropin-releasing factor (CRF) and its CRF1 receptor (CRF1R) play a central role in the maintenance of homeostasis. Malfunctioning of the CRF/CRF1R unit is associated with several disorders, such as anxiety and depression. Non-peptide CRF1R-selective antagonists have been shown to exert anxiolytic and antidepressant effects on experimental animals. However, none of them is in clinical use today because of several side effects, thus demonstrating the need for the development of other more suitable CRF1R antagonists. In an effort to develop novel CRF1R antagonists we designed, synthesized and chemically characterized two tripeptide analogues of CRF, namely (R)-LMI and (S)-LMI, having their Leu either in R (or D) or in S (or L) configuration, respectively. Their design was based on the crystal structure of the N-extracellular domain (N-domain) of CRF1R/CRF complex, using a relevant array of computational methods. Experimental evaluation of the stability of synthetic peptides in human plasma has revealed that (R)-LMI is proteolytically more stable than (S)-LMI. Based on this finding, (R)-LMI was selected for pharmacological characterization. We have found that (R)-LMI is a CRF antagonist, inhibiting (1) the CRF-stimulated accumulation of cAMP in HEK 293 cells expressing the CRF1R, (2) the production of interleukins by adipocytes and (3) the proliferation rate of RAW 264.7 cells. (R)-LMI likely blocked agonist actions by interacting with the N-domain of CRF1R as suggested by data using a constitutively active chimera of CRF1R. We propose that (R)-LMI can be used as an optimal lead compound in the rational design of novel CRF antagonists.

Preparation and Biophysical Characterization of Quercetin Inclusion Complexes with ß-Cyclodextrin Derivatives to be Formulated as Possible Nose-to-Brain Quercetin Delivery Systems.

Quercetin (Que) is a flavonoid associated with high oxygen radical scavenging activity and potential neuroprotective activity against Alzheimer's disease. Que's oral bioavailability is limited by its low water solubility and extended peripheral metabolism; thus, nasal administration may be a promising alternative to achieve effective Que concentrations in the brain. The formation of Que-2-hydroxypropylated-ß-cyclodextrin (Que/HP-ß-CD) complexes was previously found to increase the molecule's solubility and stability in aqueous media. Que-methyl-ß-cyclodextrin (Que/Me-ß-CD) inclusion complexes were prepared, characterized, and compared with the Que/HP-ß-CD complex using biophysical and computational methods (phase solubility, fluorescence and NMR spectroscopy, differential scanning calorimetry (DSC), and molecular dynamics simulations (MDS)) as candidates for the preparation of nose-to-brain Que's delivery systems. DSC thermograms, NMR, fluorescence spectroscopy, and MDS confirmed the inclusion complex formation of Que with both CDs. Differences between the two preparations were observed regarding their thermodynamic stability and inclusion mode governing the details of molecular interactions. Que's solubility in aqueous media at pH 1.2 and 4.5 was similar and linearly increased with both CD concentrations. At pH 6.8, Que's solubility was higher and positively deviated from linearity in the presence of HP-ß-CD more than with Me-ß-CD, possibly revealing the presence of more than one HP-ß-CD molecule involved in the complex. Overall, water solubility of lyophilized Que/Me-ß-CD and Que/HP-ß-CD products was approximately 7-40 times and 14-50 times as high as for pure Que at pH 1.2-6.8. In addition, the proof of concept experiment on ex vivo permeation across rabbit nasal mucosa revealed measurable and similar Que permeability profiles with both CDs and negligible permeation of pure Que. These results are quite encouraging for further ex vivo and in vivo evaluation toward nasal administration and nose-to-brain delivery of Que.

DIA-DB: A Database and Web Server for the Prediction of Diabetes Drugs.

The DIA-DB is a web server for the prediction of diabetes drugs that uses two different and complementary approaches: (a) comparison by shape similarity against a curated database of approved antidiabetic drugs and experimental small molecules and (b) inverse virtual screening of the input molecules chosen by the users against a set of therapeutic protein targets identified as key elements in diabetes. As a proof of concept DIA-DB was successfully applied in an integral workflow for the identification of the antidiabetic chemical profile in a complex crude plant extract. To this end, we conducted the extraction and LC-MS based chemical profile analysis of Sclerocarya birrea and subsequently utilized this data as input for our server. The server is open to all users, registration is not necessary, and a detailed report with the results of the prediction is sent to the user by email once calculations are completed. This is a novel public domain database and web server specific for diabetes drugs and can be accessed online through http://bio-hpc.eu/software/dia-db/.

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.

On the Rational Drug Design for Hypertension through NMR Spectroscopy.

Antagonists of the AT1receptor (AT1R) are beneficial molecules that can prevent the peptide hormone angiotensin II from binding and activating the specific receptor causing hypertension in pathological states. This review article summarizes the multifaced applications of solid and liquid state high resolution nuclear magnetic resonance (NMR) spectroscopy in antihypertensive commercial drugs that act as AT1R antagonists. The 3D architecture of these compounds is explored through 2D NOESY spectroscopy and their interactions with micelles and lipid bilayers are described using solid state 13CP/MAS, 31P and 2H static solid state NMR spectroscopy. Due to their hydrophobic character, AT1R antagonists do not exert their optimum profile on the AT1R. Therefore, various vehicles are explored so as to effectively deliver these molecules to the site of action and to enhance their pharmaceutical efficacy. Cyclodextrins and polymers comprise successful examples of effective drug delivery vehicles, widely used for the delivery of hydrophobic drugs to the active site of the receptor. High resolution NMR spectroscopy provides valuable information on the physical-chemical forces that govern these drug:vehicle interactions, knowledge required to get a deeper understanding on the stability of the formed complexes and therefore the appropriateness and usefulness of the drug delivery system. In addition, it provides valuable information on the rational design towards the synthesis of more stable and efficient drug formulations.

NMR-Based Chemical Profiling, Isolation and Evaluation of the Cytotoxic Potential of the Diterpenoid Siderol from Cultivated Sideritis euboea Heldr.

Diterpenes are characteristic compounds from the genus Sideritis L., possessing an array of biological activities. Siderol is the main constituent of the ent-kaurene diterpenes in Sideritis species. In order to isolate the specific compound and evaluate for the first time its cytotoxic activity, we explored the dichloromethane extract of cultivated Sideritis euboea Heldr. To track the specific natural bioactive agent, we applied NMR spectroscopy to the crude plant extract, since NMR can serve as a powerful and rapid tool both to navigate the targeted isolation process of bioactive constituents, and to also reveal the identity of bioactive components. Along these lines, from the rapid 1D 1H NMR spectrum of the total crude plant extract, we were able to determine the characteristic proton NMR signals of siderol. Furthermore, with the same NMR spectrum, we were able to categorize several secondary metabolites into chemical groups as a control of the isolation process. Therefore, this non-polar extract was explored, for the first time, revealing eleven compounds-one fatty acid ester; 2-(p-hydroxyphenyl)ethylstearate (1), three phytosterols; ß-sitosterol (2), stigmasterol (3), and campesterol (4); one triterpenoid; ursolic acid (5), four diterpenoids; siderol (6), eubol (7), eubotriol (8), 7-epicandicandiol (9) and two flavonoids; xanthomicrol (10) and penduletin (11). The main isolated constituent was siderol. The antiproliferative potential of siderol was evaluated, using the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assay, on three human cancer cell lines DLD1, HeLa, and A549, where the IC50 values were estimated at 26.4 ± 3.7, 44.7 ± 7.2, and 46.0 ± 4.9 µΜ, respectively. The most potent activity was recorded in the human colon cancer cell line DLD1, where siderol exhibited the lowest IC50. Our study unveiled the beneficial potential of siderol as a remarkable cytotoxic agent and the significant contribution of NMR spectroscopy towards the isolation and identification of this potent anticancer agent.