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.

Exploiting the beneficial effects of Salvia officinalis L. extracts in human health and assessing their activity as potent functional regulators of food microbiota.

Salvia officinalis L. has attracted scientific and industrial interest due to its pharmacological properties. However, its detailed phytochemical profile and its correlation with beneficial effects in the human microbiome and oxidative stress remained elusive. To unveil this, S. officinalis was collected from the region of Epirus and its molecular identity was verified with DNA barcoding. Phytochemical profile for both aqueous and ethanol-based extracts was determined by high-pressure liquid chromatography-tandem mass spectrometry and 103 phytochemicals were determined. The effect of S. officinalis extracts as functional regulators of food microbiota by stimulating the growth of Lacticaseibacillus rhamnosus strains and by suppressing evolution of pathogenic bacteria was verified. Furthermore, we recorded that both extracts exhibited a significant cellular protection against H2O2-induced DNA damage. Finally, both extracts exhibited strong inhibitory effect towards LDL oxidation. This study provides a comprehensive characterization of S. officinalis on its phytochemical components as also its potential impact in human microbiome and oxidative stress.

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.

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.

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.

Advancing the Therapeutic Efficacy of Bioactive Molecules by Delivery Vehicle Platforms.

Drugs have to overcome numerous barriers to reach their desired therapeutic targets. In several cases, drugs, especially the highly lipophilic molecules, suffer from low solubility and bioavailability and therefore their desired targeting is hampered. In addition, undesired metabolic products might be produced or off-targets could be recognized. Along these lines, nanopharmacology has provided new technological platforms, to overcome these boundaries. Specifically, numerous vehicle platforms such as cyclodextrins and calixarenes have been widely utilized to host lipophilic drugs such as antagonists of the angiotensin II AT1 receptor (AT1R), as well as quercetin and silibinin. The encapsulation of these drugs in supramolecules or other systems refines their solubility and metabolic stability, increases their selectivity and therefore decreases their effective dose and improves their therapeutic index. In this mini review we report on the formulations of silibinin and AT1R antagonist candesartan in a 2-HP-ß-cyclodextrin host molecule, which displayed enhanced cytotoxicity and increased silibinin's and candesartan's stability, respectively. Moreover, we describe the encapsulation of quercetin in gold nanoparticles bearing a calixarene supramolecular host. Also, the encapsulation of temozolomide in a calixarene nanocapsule has been described. Finally, we report on the activity enhancement that has been achieved upon using these formulations as well as the analytical and computational methods we used to characterize these formulations and explore the molecular interactions between the host and quest molecules.

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.

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.

Encapsulation of Small Drugs in a Supramolecule Enhances Solubility, Stability, and Therapeutic Efficacy Against Glioblastoma Multiforme.

Cancer occupies a high rank in the global morbidity and mortality scale with glioblastoma multiforme (GBM) accounting for almost 80% of all primary tumors of the brain. Despite the increasing availability of targeted and immunotherapeutic agents, chemotherapy still plays an important role in the treatment of neoplastic diseases. Limitations to the effective use of chemotherapy such as low aqueous solubility and high toxicity have directed the scientific community's interest to the development of new therapeutic agents with enhanced efficacy and limited toxicity. Supramolecular chemistry has offered an alternative way on the design and development of new therapeutic agents as a result of their unique properties. Supramolecules can be used as drug carriers since their cavities can host a wide range of small drugs and surpass in this way the drawbacks of current therapeutic agents. Herein, we present the principles that should be followed for the encapsulation of small drugs in supramolecules with enhanced physicochemical properties and increased efficacy against glioblastoma multiforme.

Enhancement of glioblastoma multiforme therapy through a novel Quercetin-Losartan hybrid.

Glioblastoma multiforme (GBM) is the most common and aggressive primary malignant brain tumor. Maximal surgical resection followed by radiotherapy and concomitant chemotherapy with temozolomide remains the first-line therapy, prolonging the survival of patients by an average of only 2.5 months. There is therefore an urgent need for novel therapeutic strategies to improve clinical outcomes. Reactive oxygen species (ROS) are an important contributor to GBM development. Here, we describe the rational design and synthesis of a stable hybrid molecule tethering two ROS regulating moieties, with the aim of constructing a chemopreventive and anticancer chemical entity that retains the properties of the parent compounds. We utilized the selective AT1R antagonist losartan, leading to the inhibition of ROS levels, and the antioxidant flavonoid quercetin. In GBM cells, we show that this hybrid retains the binding potential of losartan to the AT1R through competition-binding experiments and simultaneously exhibits ROS inhibition and antioxidant capacity similar to native quercetin. In addition, we demonstrate that the hybrid is able to alter the cell cycle distribution of GBM cells, leading to cell cycle arrest and to the induction of cytotoxic effects. Last, the hybrid significantly and selectively reduces cancer cell proliferation and angiogenesis in primary GBM cultures with respect to the isolated parent components or their simple combination, further emphasizing the potential utility of the current hybridization approach in GBM.

The NMR tube bioreactor.

Enzymes are pliable systems and core cellular components allowing the performance of several processes. They can also be utilized as "green" synthetic factories to generate bioactive therapeutic, diagnostic or theranostic compounds. Methods to enable the mapping of enzyme substrates as well as the understanding of the interactions of the formed products with target proteins could be of importance. This chapter describes the utilization of the "NMR tube bioreactor" method. This method, carried out inside an NMR tube, can allow for the prediction of compounds that are able to serve as potential enzyme substrates, and also exploit the regioselectivity of the enzymatic reactions. Furthermore, it enables the real time monitoring of multiple-biotransformation products in the NMR tube without the need of fractionation or isolation of each individual component. Finally, it allows for the screening of the resulting biotransformation products as ligands for protein targets.

Encapsulation of Temozolomide in a Calixarene Nanocapsule Improves Its Stability and Enhances Its Therapeutic Efficacy against Glioblastoma.

The alkylating agent temozolomide (TMZ) is the first-line chemotherapeutic for glioblastoma (GBM), a common and aggressive primary brain tumor in adults. However, its poor stability and unfavorable pharmacokinetic profile limit its clinical efficacy. There is an unmet need to tailor the therapeutic window of TMZ, either through complex derivatization or by utilizing pharmaceutical excipients. To enhance stability and aqueous solubility, we encapsulated TMZ in a p-sulphonatocalix[4]arene (Calix) nanocapsule and used 1H-NMR, LC-MS, and UV-Vis spectroscopy to chart the stability of this novel TMZ@Calix complex according to FDA and European Medicines Agency guidelines. LC-MS/MS plasma stability assays were conducted in mice to further explore the stability profile of TMZ@Calix in vivo The therapeutic efficacy of TMZ@Calix was compared with that of unbound TMZ in GBM cell lines and patient-derived primary cells with known O6-methylguanine-DNA methyltransferase (MGMT) expression status and in vivo in an intracranial U87 xenograft mouse model. Encapsulation significantly enhanced the stability of TMZ in all conditions tested. TMZ@Calix was more potent than native TMZ at inhibiting the growth of established GBM cell lines and patient-derived primary lines expressing MGMT and highly resistant to TMZ. In vivo, native TMZ was rapidly degraded in mouse plasma, whereas the stability of TMZ@Calix was enhanced threefold with increased therapeutic efficacy in an orthotopic model. In the absence of new effective therapies, this novel formulation is of clinical importance, serving as an inexpensive and highly efficient treatment that could be made readily available to patients with GBM and warrants further preclinical and clinical evaluation.

Rational design and structure-activity relationship studies of quercetin-amino acid hybrids targeting the anti-apoptotic protein Bcl-xL.

Anti-apoptotic proteins, like the Bcl-2 family proteins, present an important therapeutic cancer drug target. Their activity is orchestrated through neutralization upon interaction of pro-apoptotic protein counterparts that leads to immortality of cancer cells. Therefore, generating compounds targeting these proteins is of immense therapeutic importance. Herein, Induced Fit Docking (IFD) and Molecular Dynamics (MD) simulations were performed to rationally design quercetin analogues that bind in the BH3 site of the Bcl-xL protein. IFD calculations determined their binding cavity while Molecular Mechanics Poisson Boltzmann Surface Area (MM-PBSA) and Molecular Mechanics Generalised Born Surface Area (MM-GBSA) calculations provided an insight into the binding enthalpies of the analogues. The quercetin analogues were synthesized and their binding to Bcl-xL was verified with fluorescence spectroscopy. The binding affinity and the thermodynamic parameters between Bcl-xL and quercetin-glutamic acid were estimated through Isothermal Titration Calorimetry. 2D 1H-15N HSQC NMR chemical shift perturbation mapping was used to chart the binding site of the quercetin analogues in the Bcl-xL that overlapped with the predicted poses generated by both IFD and MD calculations. Furthermore, evaluation of the four conjugates against the prostate DU-145 and PC-3 cancer cell lines, revealed quercetin-glutamic acid and quercetin-alanine as the most potent conjugates bearing the higher cytostatic activity. This pinpoints that the chemical space of natural products can be tailored to exploit new hits for difficult tractable targets such as protein-protein interactions.