Exploring the Potential of Sulfonamide-Dihydropyridine Hybrids as Multitargeted Ligands for Alzheimer's Disease Treatment.

Alzheimer's disease (AD) is a multifactorial neurodegenerative disease that has a heavy social and economic impact on all societies and for which there is still no cure. Multitarget-directed ligands (MTDLs) seem to be a promising therapeutic strategy for finding an effective treatment for this disease. For this purpose, new MTDLs were designed and synthesized in three steps by simple and cost-efficient procedures targeting calcium channel blockade, cholinesterase inhibition, and antioxidant activity. The biological and physicochemical results collected in this study allowed us the identification two sulfonamide-dihydropyridine hybrids showing simultaneous cholinesterase inhibition, calcium channel blockade, antioxidant capacity and Nrf2-ARE activating effect, that deserve to be further investigated for AD therapy.

Evidence against involvement of kynurenate branch of kynurenine pathway in pathophysiology of Fuchs' dystrophy and keratoconus.

Kynurenine aminotransferases (KAT) are enzymes catalyzing formation of kynurenic acid (KYNA) from kynurenine. KYNA is a Janus-faced molecule of high biological activity. On the one hand KYNA was identified as a UV filter and neuroprotectant with free radical scavenging properties, but on the other hand it may contribute to photodamage of lens proteins resulting in cataract formation. Fuchs endothelial corneal dystrophy (FECD) and keratoconus (KC) are common, vision threatening corneal dystrophies whose etiology is not fully understood. In our previous works, we confirmed the presence of KATs in the human cornea together with GPR35, a receptor for KYNA. This prompted us to investigate the potential changes in the expression of three isoforms: KAT I, KAT II, and KAT III in normal and FECD- and KC-affected corneas. Immunohistochemistry accompanied by gene expression data mining revealed that the levels of neither KAT I, KAT II, nor KAT III are affected in FECD and KC. This constitutes evidence against the involvement of KATs in the pathophysiology of FECD and KC.

Effects of Linkers and Substitutions on Multitarget Directed Ligands for Alzheimer's Diseases: Emerging Paradigms and Strategies.

Alzheimer's disease (AD) is multifactorial, progressive and the most predominant cause of cognitive impairment and dementia worldwide. The current "one-drug, one-target" approach provides only symptomatic relief to the condition but is unable to cure the disease completely. The conventional single-target therapeutic approach might not always induce the desired effect due to the multifactorial nature of AD. Hence, multitarget strategies have been proposed to simultaneously knock out multiple targets involved in the development of AD. Herein, we provide an overview of the various strategies, followed by the multitarget-directed ligand (MTDL) development, rationale designs and efficient examples. Furthermore, the effects of the linkers and substitutional functional groups on MTDLs against various targets of AD and their modes of action are also discussed.

Biginelli Reaction Synthesis of Novel Multitarget-Directed Ligands with Ca2+ Channel Blocking Ability, Cholinesterase Inhibition, Antioxidant Capacity, and Nrf2 Activation.

Novel multitarget-directed ligands BIGI 4a-d and BIGI 5a-d were designed and synthesized with a simple and cost-efficient procedure via a one-pot three-component Biginelli reaction targeting acetyl-/butyrylcholinesterases inhibition, calcium channel antagonism, and antioxidant ability. Among these multitarget-directed ligands, BIGI 4b, BIGI 4d, and BIGI 5b were identified as promising new hit compounds showing in vitro balanced activities toward the recognized AD targets. In addition, these compounds showed suitable physicochemical properties and a good druglikeness score predicted by Data Warrior software.

Comparative Antiseizure Analysis of Diverse Natural Coumarin Derivatives in Zebrafish.

Coumarins are a well-known group of plant secondary metabolites with various pharmacological activities, including antiseizure activity. In the search for new antiseizure drugs (ASDs) to treat epilepsy, it is yet unclear which types of coumarins are particularly interesting as a systematic analysis has not been reported. The current study performed behavioral antiseizure activity screening of 18 different coumarin derivatives in the larval zebrafish pentylenetetrazole (PTZ) model using locomotor measurements. Activity was confirmed for seven compounds, which lowered seizure-like behavior as follows: oxypeucedanin 38%, oxypeucedanin hydrate 74%, notopterol 54%, nodakenetin 29%, hyuganin C 35%, daphnoretin 65%, and pimpinellin 60%. These coumarins, together with nodakenin, underwent further antiepileptiform analysis by local field potential recordings from the zebrafish opticum tectum (midbrain). All of them, except for nodakenetin, showed pronounced antiepileptiform activity, decreasing PTZ-induced elevation in power spectral density (PSD) by 83-89% for oxypeucedanin, oxypeucedanin hydrate, and notopterol, 77% for nodakenin, 26% for nodakenetin, 65% for hyuganin C, 88% for daphnoretin, and 81% for pimpinellin. These data demonstrate the potential of diverse coumarin scaffolds for ASD discovery. Finally, the structural differences between active and inactive coumarins were investigated in silico for oxypeucedanin hydrate and byacangelicin for their interaction with GABA-transaminase, a hypothetical target.

Structural Insights into Ligand-Receptor Interactions Involved in Biased Agonism of G-Protein Coupled Receptors.

G protein-coupled receptors (GPCRs) are versatile signaling proteins that mediate complex cellular responses to hormones and neurotransmitters. Ligand directed signaling is observed when agonists, upon binding to the same receptor, trigger significantly different configuration of intracellular events. The current work reviews the structurally defined ligand - receptor interactions that can be related to specific molecular mechanisms of ligand directed signaling across different receptors belonging to class A of GPCRs. Recent advances in GPCR structural biology allow for mapping receptors' binding sites with residues particularly important in recognition of ligands' structural features that are responsible for biased signaling. Various studies show particular role of specific residues lining the extended ligand binding domains, biased agonists may alternatively affect their interhelical interactions and flexibility what can be translated into intracellular loop rearrangements. Studies on opioid and angiotensin receptors indicate importance of residues located deeper within the binding cavity and direct interactions with receptor residues linking the ortosteric ligand binding site with the intracellular transducer binding domain. Collection of results across different receptors may suggest elements of common molecular mechanisms which are responsible for passing alternative signals from biased agonists.

Synthesis of Hantzsch Adducts as Cholinesterases and Calcium Flux inhibitors, Antioxidants and Neuroprotectives.

We report herein the design, synthesis, biological evaluation, and molecular modelling of new inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE), able to block Ca+2 channels also showing antioxidant and neuroprotective activities. The new MTDL, dialkyl 2,6-dimethyl-4-(4-((5-aminoalkyl)oxy)phenyl)-1,4-dihydropyridine-3,5-dicarboxylate 3a-p, have been obtained via Hantzsch reaction from appropriate and commercially available precursors. Pertinent biological analysis has prompted us to identify MTDL 3h [dimethyl-4-(4-((5-(4-benzylpiperidin-1-yl)pentyl)oxy)phenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate] as an attractive inhibitor of AChE (1.8 µM) and BuChE (2 µM), Ca+2 channel antagonist (47.72% at 10 µM), and antioxidant (2.54 TE) agent, showing significant neuroprotection 28.68% and 38.29% against H2O2, and O/R, respectively, at 0.3 µM, thus being considered a hit-compound for further investigation in our search for anti-Alzheimer's disease agents.

Design, Synthesis and Biological Evaluation of New Antioxidant and Neuroprotective Multitarget Directed Ligands Able to Block Calcium Channels.

We report herein the design, synthesis and biological evaluation of new antioxidant and neuroprotective multitarget directed ligands (MTDLs) able to block Ca2+ channels. New dialkyl 2,6-dimethyl-4-(4-(prop-2-yn-1-yloxy)phenyl)-1,4-dihydropyridine-3,5-dicarboxylate MTDLs 3a-t, resulting from the juxtaposition of nimodipine, a Ca2+ channel antagonist, and rasagiline, a known MAO inhibitor, have been obtained from appropriate and commercially available precursors using a Hantzsch reaction. Pertinent biological analysis has prompted us to identify the MTDL 3,5-dimethyl-2,6-dimethyl-4-[4-(prop-2-yn-1-yloxy)phenyl]-1,4-dihydro- pyridine- 3,5-dicarboxylate (3a), as an attractive antioxidant (1.75 TE), Ca2+ channel antagonist (46.95% at 10 µM), showing significant neuroprotection (38%) against H2O2 at 10 µM, being considered thus a hit-compound for further investigation in our search for anti-Alzheimer's disease agents.

Rutamarin: Efficient Liquid-Liquid Chromatographic Isolation from Ruta graveolens L. and Evaluation of Its In Vitro and In Silico MAO-B Inhibitory Activity.

Naturally occurring coumarins are a group of compounds with many documented central nervous system (CNS) activities. However, dihydrofuranocoumarins have been infrequently investigated for their bioactivities at CNS level. Within the frame of this study, an efficient liquid-liquid chromatography method was developed to rapidly isolate rutamarin from Ruta graveolens L. (Rutaceae) dichloromethane extract (DCM). The crude DCM (9.78 mg/mL) and rutamarin (6.17 M) were found to be effective inhibitors of human monoamine oxidase B (hMAO-B) with inhibition percentages of 89.98% and 95.26%, respectively. The inhibitory activity against human monoamine oxidase A (hMAO-A) for the DCM extract was almost the same (88.22%). However, for rutamarin, it significantly dropped to 25.15%. To examine the molecular interaction of rutamarin with hMAO- B, an in silico evaluation was implemented. A docking study was performed for the two enantiomers (R)-rutamarin and (S)-rutamarin. The (S)-rutamarin was found to bind stronger to the hMAO-B binging cavity.

The presence and distribution of G protein-coupled receptor 35 (GPR35) in the human cornea - Evidences from in silico gene expression analysis and immunodetection.

We provide the evidence for G protein-coupled receptor 35 (GPR35) presence and distribution in the human cornea. The initial data on GPR35 gene expression were retrieved from microarray repositories and were further confirmed by western blotting and immunohistochemical analysis. Immunoblotting suggested that GPR35 exists predominantly as a dimer in corneal tissue. Moreover, corneal tissues were significantly richer in GPR35 compared to the adjacent sclera. Immunoreactivity for GPR35 was detected in normal corneas, keratoconus and Fuchs' dystrophy, mainly in the corneal epithelium and endothelium. In corneas with Fuchs' dystrophy, less intensive immunoreactivity for GPR35 in endothelium was revealed. The physiological relevance of this phenomenon requires further investigation.

Multi-target 1,4-dihydropyridines showing calcium channel blockade and antioxidant capacity for Alzheimer's disease therapy.

In this work we describe the synthesis, Ca+2 channel blockade capacity and antioxidant power of N3,N5-bis(2-(5-methoxy-1H-indol-3-yl)ethyl)-2,6-dimethyl-4-aryl-1,4-dihydropyridine-3,5-dicarboxamides 1-9, a number of multi-target small 1,4-dihydropyridines (DHP), designed by juxtaposition of melatonin and nimodipine. As a result, we have identified antioxidant DHP 7 (Ca2+ channel blockade: 55%, and 8.78 Trolox/Equivalents), the most balanced DHP analyzed here, for potential Alzheimer's disease therapy.

Determination of affinity and efficacy of acetylcholinesterase inhibitors using isothermal titration calorimetry.

BACKGROUND: Acetylcholinesterase (AChE), an enzyme rapidly terminating nerve signals at synapses of cholinergic neurons is an important drug target in treatment of Alzheimer's disease and related memory loss conditions. Here we present comprehensive use of isothermal titration calorimetry (ITC) for investigation of AChE kinetics and AChE-inhibitor interactions. METHODS: Acetylcholinesterase (AChE, EC 3.1.1.7) from Electrophorus electricus was assayed for interactions with five well known AChE inhibitors, galanthamine, tacrine, donepezil, edrophonium and ambenonium. In ITC experiments the inhibitors were injected to the enzyme solution solely (for thermodynamic characterization of binding) or in presence of the substrate, acetylcholine (for determination of inhibitors potency). RESULTS: Detailed description of various experimental protocols is presented, allowing evaluation of inhibitors potency (in terms of IC50 and Ki) and thermodynamic parameters of the binding. The potency of tested inhibitors was in nano to micromolar range which corresponded to activities determined in conventional method. Binding of all inhibitors showed to be enthalpy driven and obtained Ka values demonstrated good correlation with the data from standard Ellman's assay. CONCLUSIONS: Obtained results confirmed the usability of the ITC technique for comprehensive characterization of AChE-inhibitor interactions and AChE kinetics. The method reduced the complexity of reaction mixture and interference problems with the advantage of using natural substrates. GENERAL SIGNIFICANCE: The work reports complete thermodynamic characteristics of the AChE - inhibitor complexes. Due to the universal character of ITC measurements, described protocols can be easily adapted to other enzymatic systems.

GPR55 receptor antagonist decreases glycolytic activity in PANC-1 pancreatic cancer cell line and tumor xenografts.

The Warburg effect is a predominant metabolic pathway in cancer cells characterized by enhanced glucose uptake and its conversion to l-lactate and is associated with upregulated expression of HIF-1α and activation of the EGFR-MEK-ERK, Wnt-ß-catenin, and PI3K-AKT signaling pathways. (R,R')-4'-methoxy-1-naphthylfenoterol ((R,R')-MNF) significantly reduces proliferation, survival, and motility of PANC-1 pancreatic cancer cells through inhibition of the GPR55 receptor. We examined (R,R')-MNF's effect on glycolysis in PANC-1 cells and tumors. Global NMR metabolomics was used to elucidate differences in the metabolome between untreated and (R,R')-MNF-treated cells. LC/MS analysis was used to quantify intracellular concentrations of ß-hydroxybutyrate, carnitine, and l-lactate. Changes in target protein expression were determined by Western blot analysis. Data was also obtained from mouse PANC-1 tumor xenografts after administration of (R,R')-MNF. Metabolomics data indicate that (R,R')-MNF altered fatty acid metabolism, energy metabolism, and amino acid metabolism and increased intracellular concentrations of ß-hydroxybutyrate and carnitine while reducing l-lactate content. The cellular content of phosphoinositide-dependent kinase-1 and hexokinase 2 was reduced consistent with diminished PI3K-AKT signaling and glucose metabolism. The presence of the GLUT8 transporter was established and found to be attenuated by (R,R')-MNF. Mice treated with (R,R')-MNF had significant accumulation of l-lactate in tumor tissue relative to vehicle-treated mice, together with reduced levels of the selective l-lactate transporter MCT4. Lower intratumoral levels of EGFR, pyruvate kinase M2, ß-catenin, hexokinase 2, and p-glycoprotein were also observed. The data suggest that (R,R')-MNF reduces glycolysis in PANC-1 cells and tumors through reduced expression and function at multiple controlling sites in the glycolytic pathway.

The presence of kynurenine aminotransferases in the human cornea: Evidence from bioinformatics analysis of gene expression and immunohistochemical staining.

PURPOSE: Kynurenine aminotransferases (KATs) catalyze the synthesis of kynurenic acid (KYNA), a compound of significant biological activity. The aim of this study is to investigate the presence and distribution of KAT immunoreactivity in the healthy human cornea. METHODS: Data on gene expression in human eye structures were extracted from public microarray experiments using Genevestigator software. Immunohistochemistry was conducted using polyclonal antibodies against KAT I, II, and III on sections of eight enucleated eyes from patients with choroidal melanoma. RESULTS: Bioinformatics analysis showed that all four KAT isoforms were actively transcribed in the cornea and the conjunctiva. Immunohistochemical analysis revealed the presence of KAT I, II, and III in all examined corneal sections. The corneal endothelium showed the strongest reactivity for all three KAT isoforms. There was a slight positive staining of the corneal stroma for KAT I and II. KAT III immunoreactivity was found only in the stroma of the limbal region. In the corneal epithelium, the expression of all three KAT isoforms showed a specific pattern of the stain with fine squatter granules throughout the cytoplasm. This reactivity was more pronounced in the basal cell layers. The intermediate cell layers showed only faint immunoreactivity, and occasionally, there was no staining. KAT I, II, and III were also present in the adjacent limbal conjunctiva. CONCLUSIONS: The results indicate that kynurenine can be metabolized to KYNA in the corneal epithelium, stroma, and endothelium.

New approach in evaluation of ceramic-polymer composite bioactivity and biocompatibility.

Regeneration of bone defects was promoted by a novel ß-glucan/carbonate hydroxyapatite composite and characterized by Raman spectroscopy, microCT and electron microscopy. The elastic biomaterial with an apatite-forming ability was developed for bone tissue engineering and implanted into the critical-size defects of rabbits' tibiae. The bone repair process was analyzed on non-decalcified bone/implant sections during a 6-month regeneration period. Using spectroscopic methods, we were able to determine the presence of amides, lipids and assign the areas of newly formed bone tissue. Raman spectroscopy was also used to assess the chemical changes in the composite before and after the implantation process. SEM analyses showed the mineralization degree in the defect area and that the gap size decreased significantly. Microscopic images revealed that the implant debris were interconnected to the poorly mineralized inner side of a new bone tissue. Our study demonstrated that the composite may serve as a biocompatible background for collagen ingrowth and exhibits the advantages of applying Raman spectroscopy, SEM and microCT in studying these samples.

Molecular mechanism of action and safety of 5-(3-chlorophenyl)-4-hexyl-2,4-dihydro-3H-1,2,4-triazole-3-thione - a novel anticonvulsant drug candidate.

Previously, it was found that 5-(3-chlorophenyl)-4-hexyl-2,4-dihydro-3H-1,2,4-triazole-3-thione (TP-315) effectively protects mice from maximal electroshock-induced seizures. The aim of this study was to determine possible interactions between TP-315 and different molecular targets, i.e. GABAA receptors, voltage-gated sodium channels, and human neuronal α7 and α4ß2 nicotinic acetylcholine receptors. The influence of TP-315 on the viability of human hepatic HepG2 cells was also established using PrestoBlue and ToxiLight assays. It was found that the anticonvulsant activity of TP-315 results (at least partially) from its influence on voltage-gated sodium channels (VGSCs). Moreover, the title compound slightly affected the viability of human hepatic cells.

Pharmacological and molecular studies on the interaction of varenicline with different nicotinic acetylcholine receptor subtypes. Potential mechanism underlying partial agonism at human α4ß2 and α3ß4 subtypes.

To determine the structural components underlying differences in affinity, potency, and selectivity of varenicline for several human (h) nicotinic acetylcholine receptors (nAChRs), functional and structural experiments were performed. The Ca2+ influx results established that: (a) varenicline activates (µM range) nAChR subtypes with the following rank sequence: hα7>hα4ß4>hα4ß2>hα3ß4>>>hα1ß1γδ; (b) varenicline binds to nAChR subtypes with the following affinity order (nM range): hα4ß2~hα4ß4>hα3ß4>hα7>>>Torpedo α1ß1γδ. The molecular docking results indicating that more hydrogen bond interactions are apparent for α4-containing nAChRs in comparison to other nAChRs may explain the observed higher affinity; and that (c) varenicline is a full agonist at hα7 (101%) and hα4ß4 (93%), and a partial agonist at hα4ß2 (20%) and hα3ß4 (45%), relative to (±)-epibatidine. The allosteric sites found at the extracellular domain (EXD) of hα3ß4 and hα4ß2 nAChRs could explain the partial agonistic activity of varenicline on these nAChR subtypes. Molecular dynamics simulations show that the interaction of varenicline to each allosteric site decreases the capping of Loop C at the hα4ß2 nAChR, suggesting that these allosteric interactions limit the initial step in the gating process. In conclusion, we propose that in addition to hα4ß2 nAChRs, hα4ß4 nAChRs can be considered as potential targets for the clinical activity of varenicline, and that the allosteric interactions at the hα3ß4- and hα4ß2-EXDs are alternative mechanisms underlying partial agonism at these nAChRs.

The impact of multiple sclerosis relapse treatment on migration of effector T cells--Preliminary study.

UNLABELLED: Migration of inflammatory cells from the blood to the central nervous system (CNS) is crucial for development of multiple sclerosis (MS). Inhibition of this process would allow to control disease activity. The first step confirming this approach would be the analysis of the impact of effective MS relapse therapy on migration of effector T cells. The aim of the study was to analyze the influence of methylprednisolone (MP) on the migratory activity of effector CD4+ T cells from MS patients. Moreover, to study the potential mechanism of this process we studied expression of chemokine receptors on migrating cells. MATERIAL AND METHODS: Peripheral blood samples were obtained from relapsing-remitting MS (RR-MS) patients during relapse (n=23) and from control group (n=23). After isolation CD4+ T cells were incubated with various concentrations of MP. Then they were stimulated in chemotaxis assay with chemokines CCL3 or CXCL10 or were used to CCR1 and CXCR3 expression analysis. RESULTS: CXCL10- and CCL3-stimulated migration of CD4+ T cells was significantly increased in MS. MP was able to reduce in vitro migration of effector T cells induced by CXCL10, but not by CCL3. Inhibition by MP was dose-dependent. Expression of analyzed chemokine receptors was unaltered after MP incubation. CONCLUSIONS: MP reduced CD4+ T cells migration induced by CXCL10 without affecting CXCR3 expression. These observations demonstrate one of the potential mechanisms of MP action in MS, distinct from inducing cell apoptosis, and suggests the new targets for development of more effective MS treatments.

Tyrosine 308 is necessary for ligand-directed Gs protein-biased signaling of ß2-adrenoceptor.

Interaction of a given G protein-coupled receptor to multiple different G proteins is a widespread phenomenon. For instance, ß2-adrenoceptor (ß2-AR) couples dually to Gs and Gi proteins. Previous studies have shown that cAMP-dependent protein kinase (PKA)-mediated phosphorylation of ß2-AR causes a switch in receptor coupling from Gs to Gi. More recent studies have demonstrated that phosphorylation of ß2-AR by G protein-coupled receptor kinases, particularly GRK2, markedly enhances the Gi coupling. We have previously shown that although most ß2-AR agonists cause both Gs and Gi activation, (R,R')-fenoterol preferentially activates ß2-AR-Gs signaling. However, the structural basis for this functional selectivity remains elusive. Here, using docking simulation and site-directed mutagenesis, we defined Tyr-308 as the key amino acid residue on ß2-AR essential for Gs-biased signaling. Following stimulation with a ß2-AR-Gs-biased agonist (R,R')-4'-aminofenoterol, the Gi disruptor pertussis toxin produced no effects on the receptor-mediated ERK phosphorylation in HEK293 cells nor on the contractile response in cardiomyocytes expressing the wild-type ß2-AR. Interestingly, Y308F substitution on ß2-AR enabled (R,R')-4'-aminofenoterol to activate Gi and to produce these responses in a pertussis toxin-sensitive manner without altering ß2-AR phosphorylation by PKA or G protein-coupled receptor kinases. These results indicate that, in addition to the phosphorylation status, the intrinsic structural feature of ß2-AR plays a crucial role in the receptor coupling selectivity to G proteins. We conclude that specific interactions between the ligand and the Tyr-308 residue of ß2-AR stabilize receptor conformations favoring the receptor-Gs protein coupling and subsequently result in Gs-biased agonism.

Agonist binding by the ß2-adrenergic receptor: an effect of receptor conformation on ligand association-dissociation characteristics.

The ß2-adrenergic receptor (ß2-AR), a G protein-coupled receptor (GPCR), is a physiologically important transmembrane protein that is a target for drugs used for treatment of asthma and cardiovascular diseases. Study of the first steps of ligand recognition and the molecular basis of ligand binding to the orthosteric site is essential for understanding the pharmacological properties of the receptor. In this work we investigated the characteristic features of the agonist association-dissociation process to and from the different conformational forms of ß2-AR by use of advanced molecular modeling techniques. The investigation was focused on estimating the free energy profiles (FEPs) corresponding to the process of a full agonist ((R,R)-fenoterol) and an inverse agonist (carazolol) binding and unbinding to and from ß2-AR. The two different conformational forms of ß2-AR, i.e. active ß2-AR-PDB: 3P0G and inactive ß2-AR-PDB: 2RH1 were included in this stage of the study. We revealed several significant qualitative differences between FEPs characteristic of both conformational forms. Both FEPs suggest the existence of three transient binding sites in the extracellular domain of ß2-AR. Comparison of the residues surrounding these transient binding sites in both ß2-AR states revealed the importance of the aromatic residues F194, H93(2.64), H296(6.58), and H178 (extracellular part of ß2-AR) in the early stages of the binding process. In addition, slightly different exit and entry paths are preferred by the ligand molecule in the extracellular part of ß2-AR, depending on the conformation of the receptor.