Structure-Based Discovery of Dual-Target Hits for Acetylcholinesterase and the α7 Nicotinic Acetylcholine Receptors: In Silico Studies and In Vitro Confirmation.

Despite extensive efforts in the development of drugs for complex neurodegenerative diseases, treatment often remains challenging or ineffective, and hence new treatment strategies are necessary. One approach is the design of multi-target drugs, which can potentially address the complex nature of disorders such as Alzheimer's disease. We report a method for high throughput virtual screening aimed at identifying new dual target hit molecules. One of the identified hits, N,N-dimethyl-1-(4-(3-methyl-[1,2,4]triazolo[4,3-a]pyrimidin-6-yl)phenyl)ethan-1-amine (Ý;mir-2), has dual-activity as an acetylcholinesterase (AChE) inhibitor and as an α7 nicotinic acetylcholine receptor (α7 nAChR) agonist. Using computational chemistry methods, parallel and independent screening of a virtual compound library consisting of 3,848,234 drug-like and commercially available molecules from the ZINC15 database, resulted in an intersecting set of 57 compounds, that potentially possess activity at both of the two protein targets. Based on ligand efficiency as well as scaffold and molecular diversity, 16 of these compounds were purchased for in vitro validation by Ellman's method and two-electrode voltage-clamp electrophysiology. Ý;mir-2 was shown to exhibit the desired activity profile (AChE IC50 = 2.58 ± 0.96 µM; α7 nAChR activation = 7.0 ± 0.9% at 200 µM) making it the first reported compound with this particular profile and providing further evidence of the feasibility of in silico methods for the identification of novel multi-target hit molecules.

Novel Approach for the Search for Chemical Scaffolds with Dual Activity with Acetylcholinesterase and the α7 Nicotinic Acetylcholine Receptor-A Perspective for the Treatment of Neurodegenerative Disorders.

Neurodegenerative disorders, including Alzheimer's disease, belong to the group of the most difficult and challenging conditions with very limited treatment options. Attempts to find new drugs in most cases fail at the clinical stage. New tactics to develop better drug candidates to manage these diseases are urgently needed. It is evident that better understanding of the neurodegeneration process is required and targeting multiple receptors may be essential. Herein, we present a novel approach, searching for dual active compounds interacting with acetylcholinesterase (AChE) and the α7 nicotinic acetylcholine receptor (nAChR) using computational chemistry methods including homology modelling and high throughput virtual screening. Activities of identified hits were evaluated at the two targets using the colorimetric method of Ellman and two-electrode voltage-clamp electrophysiology, respectively. Out of 87,250 compounds from a ZINC database of natural products and their derivatives, we identified two compounds, 8 and 9, with dual activity and balanced IC50 values of 10 and 5 µM at AChE, and 34 and 14 µM at α7 nAChR, respectively. This is the first report presenting successful use of virtual screening in finding compounds with dual mode of action inhibiting both the AChE enzyme and the α7 nAChR and shows that computational methods can be a valuable tool in the early lead discovery process.

Galantamine is not a positive allosteric modulator of human α4ß2 or α7 nicotinic acetylcholine receptors.

BACKGROUND AND PURPOSE: The alkaloid galantamine was originally isolated from the green snowdrop Galanthus woronowii and is currently marketed as a drug for treatment of mild to moderate dementia in patients with Alzheimer's disease. In addition to a well-documented proficiency to inhibit acetylcholinesterase, galantamine has been reported to increase neuronal nicotinic ACh (nACh) receptor function by acting as a positive allosteric modulator. Yet there remains controversy regarding these findings in the literature. To resolve this conundrum, we evaluated galantamine actions at α4ß2 and α7, which represent the nACh receptors most commonly associated with mammalian cognitive domains. EXPERIMENTAL APPROACH: α4ß2 [in (α4)3 (ß2)2 and (α4)2 (ß2)3 stoichiometries] and α7 nACh receptors were expressed in Xenopus laevis oocytes and subjected to two-electrode voltage-clamp electrophysiological experiments. Galantamine (10 nM to 100 µM) was evaluated for direct agonist effects and for positive modulation by co-application with sub-maximally efficacious concentrations of ACh. In addition, similar experiments were performed with α7 nACh receptors stably expressed in HEK293 cells using patch-clamp electrophysiology. KEY RESULTS: In concentrations ranging from 10 nM to 1 µM, galantamine did not display direct agonism nor positive modulatory effects at any receptor combination tested. At concentrations from 10 µM and above, galantamine inhibited the activity with a mechanism of action consistent with open-channel pore blockade at all receptor types. CONCLUSION AND IMPLICATIONS: Based on our data, we conclude that galantamine is not a positive allosteric modulator of α7 or α4ß2 receptors, which represent the majority of nACh receptors in mammalian brain.

DNA barcoding and LC-MS metabolite profiling of the lichen-forming genus Melanelia: Specimen identification and discrimination focusing on Icelandic taxa.

Taxa in the genus Melanelia (Parmeliaceae, Ascomycota) belong to a group of saxicolous lichens with brown to black foliose thalli, which have recently undergone extensive changes in circumscription. Taxa belonging to Parmeliaceae are prolific producers of bioactive compounds, which have also been traditionally used for chemotaxonomic purposes. However, the chemical diversity of the genus Melanelia and the use of chemical data for species discrimination in this genus are largely unexplored. In addition, identification based on morphological characters is challenging due to few taxonomically informative characters. Molecular identification methods, such as DNA barcoding, have rarely been applied to this genus. This study aimed to identify the Melanelia species from Iceland using DNA barcoding approach, and to explore their chemical diversity using chemical profiling. Chemometric tools were used to see if lichen metabolite profiles determined by LC-MS could be used for the identification of Icelandic Melanelia species. Barcoding using the fungal nuclear ribosomal internal transcribed spacer region (nrITS) successfully identified three Melalenlia species occurring in Iceland, together with Montanelia disjuncta (Basionym: Melanelia disjuncta). All species formed monophyletic clades in the neighbor-joining nrITS gene tree. However, high intraspecific genetic distance of M. stygia suggests the potential of unrecognized species lineages. Principal component analysis (PCA) of metabolite data gave a holistic overview showing that M. hepatizon and M. disjuncta were distinct from the rest, without the power to separate M. agnata and M. stygia due to their chemical similarity. Orthogonal partial least-squares to latent structures-discriminate analysis (OPLS-DA), however, successfully distinguished M. agnata and M. stygia by identifying statistically significant metabolites, which lead to class differentiation. This work has demonstrated the potential of DNA barcoding, chemical profiling and chemometrics in identification of Melanelia species.

Exopolysaccharides from Cyanobacterium aponinum from the Blue Lagoon in Iceland increase IL-10 secretion by human dendritic cells and their ability to reduce the IL-17+RORγt+/IL-10+FoxP3+ ratio in CD4+ T cells.

Regular bathing in the Blue Lagoon in Iceland has beneficial effects on psoriasis. Cyanobacterium aponinum is a dominating member of the Blue Lagoon's microbial ecosystem. The aim of the study was to determine whether exopolysaccharides (EPSs) secreted by C. aponinum (EPS-Ca) had immunomodulatory effects in vitro. Human monocyte-derived dendritic cells (DCs) were matured in the absence or presence of EPS-Ca and the effects were determined by measuring the secretion of cytokines by ELISA and the expression of surface molecules by flow cytometry. DCs matured with EPS-Ca at 100 µg/ml secreted higher levels of IL-10 than untreated DCs. Subsequently, DCs matured in the presence or absence of EPS-Ca were co-cultured with allogeneic CD4(+) T cells and their effects on T cell activation analysed by measuring expression of intracellular and surface molecules and cytokine secretion. Supernatant from allogeneic T cells co-cultured with EPS-Ca-exposed DCs had raised levels of IL-10 compared with control. A reduced frequency of IL-17(+)RORγt(+) T cells was observed when co-cultured with EPS-Ca-exposed DCs and a tendency towards increased frequency of FoxP3(+)IL-10(+) T cells, resulting in a lower IL-17(+)RORγt(+)/FoxP3(+)IL-10(+) ratio. The study shows that EPSs secreted by C. aponinum stimulate DCs to produce vast amounts of the immunosuppressive cytokine IL-10. These DCs induce differentiation of allogeneic CD4(+) T cells with an increased Treg but decreased Th17 phenotype. These data suggest that EPSs from C. aponinum may play a role in the beneficial clinical effect on psoriasis following bathing in the Blue Lagoon.

Synergistic cytotoxic effect of the microtubule inhibitor marchantin A from Marchantia polymorpha and the Aurora kinase inhibitor MLN8237 on breast cancer cells in vitro.

Macrocyclic bisbibenzyls are a class of characteristic compounds, exclusively produced by liverworts. They are attracting increasing attention due to their wide range of biological activities, including antibacterial, antifungal, and antioxidative properties as well as cytotoxicity. Marchantin A is a cyclic bisbibenzyl that has previously been isolated from Marchantia polymorpha and other liverwort species and has been shown to exert cytotoxic effects. In the present study we found that the Icelandic M. polymorpha species produces marchantin A and through an in vitro cell growth inhibition assay, marchantin A was shown to induce a reduction in cell viability of breast cancer cell lines A256 (IC50 = 5.5 µM), MCF7 (IC50 = 11.5 µM), and T47D (IC50 = 15.3 µM). The effect was considerably increased in all cell lines in a synergistic manner when the Aurora-A kinase inhibitor MLN8237 was added simultaneously. Fluorescence microscopy confirmed the antimicrotubular effect of marchantin A, and cell cycle analysis indicated enhanced cell division failure when combining this mitotic-spindle inhibitor with the checkpoint modulator.

Ethanol extract from birch bark (Betula pubescens) suppresses human dendritic cell mediated Th1 responses and directs it towards a Th17 regulatory response in vitro.

Extracts and fractions from birch bark have been used to treat various diseases, such as skin disorders and rheumatism, and for analgesic effects. Results from studies in vitro and in vivo have shown that birch bark extracts can have immunoregulatory effects. These effects have mainly been attributed to the various triterpenes found in birch bark. The effects of birch bark from Betula pubescens on immune responses have not been reported. Ethanol extract was prepared from dry birch bark (DBBEE) and five fractions made using various ratios of dichloromethane and methanol (fractions I-V). Human monocyte-derived dendritic cells (DCs) were matured with or without DBBEE or fractions I-V at several concentrations. The effects of the extract and fractions on DC maturation were determined by measuring cytokine secretion by ELISA and expression of surface molecules by flow cytometry. DBBEE and fractions III and IV reduced DC secretion of IL-6, IL-10 and IL-12p40 and expression of CD83, CD86, CCR7 and DC-SIGN compared with control DCs. Proliferation of allogeneic CD4(+) T cells co-cultured with DCs matured with fraction IV, as measured by (3)H-thymidine incorporation, was similar to proliferation of allogeneic CD4(+) T cells co-cultured with control DCs. However, IFN-γ secretion was reduced and IL-10 and IL-17 secretion was increased, a cytokine profile consistent with a Th17 regulatory phenotype. These results indicate that bark from Betula pubescens contains compound(s) that can modulate DCs so that their interaction with T cells leads to an immunoregulatory response.

Structural characterisation of novel lichen heteroglycans by NMR spectroscopy and methylation analysis.

Two galactofuranomannans, Ths-4 and Ths-5, were isolated from the lichen, Thamnolia vermicularis var. subuliformis, using ethanol fractionation and anion-exchange and size-exclusion chromatography. The average molecular weights of Ths-4 and Ths-5 were estimated to be 19 and 200 kDa, respectively. Structural characterisation of Ths-4, Ths-5 and their partially hydrolysed derivatives was performed by methanolysis and methylation analysis. The intact and partially hydrolysed Ths-4 was further analysed using NMR spectroscopy (1D, COSY, NOESY, TOCSY, HSQC and HMBC). According to the data obtained, the heteroglycans Ths-4 and Ths-5 have similar structures, but have large differences in molecular weight. The structure is composed of 3-O-linked and 5-O-linked galactofuranosyl chains linked to a mannan core. The mannan core consists of a main chain of alpha-(1-->6)-linked mannopyranosyl residues, substituted at O-2 with either a single alpha-mannopyranosyl unit or an alpha-Manp-(1-->2)-alpha-Manp-(1-->2)-alpha-Manp group in the ratio of approximately 1:3, respectively. The polysaccharides have idealised repeating blocks as is shown.

Selagoline, a new alkaloid from Huperzia selago.

A new natural product, named selagoline, and two known alkaloids, huperzine A and serratidine, were isolated from Huperzia selago (Lycopodiaceae) collected in Iceland. Their structures were determined using 600 and 800MHz one- and two-dimensional NMR methods supported by Fourier-transform mass spectrometry. Possible role of selagoline as a precursor of 5,15-oxidolycopodane, a component of the classical alkaloids L28 and L31, is discussed.

Cyanogenesis in glucosinolate-producing plants: Carica papaya and Carica quercifolia.

(R)-2-(beta-D-Glucopyranosyloxy)-2-phenylacetonitrile (prunasin) was isolated from Carica papaya L. and C. quercifolia (A. St.-Hil.) Hieron. (syn. C. hastata Brign.). Earlier reported presence of cyclopentanoid cyanohydrin glycosides in C. papaya could not be confirmed, and no cyclopentanoid amino acids could be detected in extracts of C. papaya and C. quercifolia. Conversion of [2,3,4,5,6-3H]phenylalanine into tritiated prunasin was demonstrated in both species. On the other hand, when the plants were administered [2-14C]-2-(2'cyclopentenyl)glycine, extracted, and the extracts hydrolyzed with beta-glucosidase (Helix pomatia), formation of labelled cyanide was not observed. The absence of cyclopentanoids, which are typical for the Passifloraceae, and the inability of Carica species to utilize 2-(2'-cyclopentenyl)glycine as a precursor of cyanogenic glycosides are in agreement with the relative phylogenetic position of the Caricaceae and the Passifloraceae. Carica species are thus rare examples of taxa in which glucosinolates and cyanogenic glycosides co-occur, both types of natural products being derived from the same amino acid, phenylalanine.

Cyanohydrin glycosides of Passiflora: distribution pattern, a saturated cyclopentane derivative from P. guatemalensis, and formation of pseudocyanogenic alpha-hydroxyamides as isolation artefacts.

Nineteen species of Passiflora (Passifloraceae) were examined for the presence of cyanogenic glycosides. Passibiflorin, a bisglycoside containing the 6-deoxy-beta-D-gulopyranosyl residue, was isolated from P. apetala, P. biflora, P. cuneata, P. indecora, P. murucuja and P. perfoliata. In some cases this glycoside co-occurs with simple beta-D-glucopyranosides: tetraphyllin A, deidaclin, tetraphyllin B, volkenin, epivolkenin and taraktophyllin. P. citrina contains passicapsin, a rare glycoside with the 2,6-dideoxy-beta-D-xylo-hexopyranosyl moiety, while P. herbertiana contains tetraphyllin A, deidaclin, epivolkenin and taraktophyllin, P. discophora tetraphyllin B and volkenin, and P. x violacea tetraphyllin B sulfate. The remaining species were noncyanogenic. The glycosides were identified by 1H and 13C NMR spectroscopy following isolation by reversed-phase preparative HPLC. From P. guatemalensis, a new glucoside named passiguatemalin was isolated and identified as a 1-(beta-D-glucopyranosyloxy)-2,3-dihydroxycyclopentane-1-carbonitrile. An isomeric glycoside was prepared by catalytic hydrogenation of gynocardin. alpha-Hydroxyamides corresponding to the cyanogenic glycosides were isolated from several Passiflora species. These alpha-hydroxyamides, presumably formed during processing of the plant material, behave as cyanogenic compounds when treated with commercial Helix pomatia crude enzyme preparation. Thus, the enzyme preparation appears to contain an amide dehydratase, which converts alpha-hydroxyamides to cyanohydrins that liberate cyanide; this finding is of interest in connection with analysis of plant tissues and extracts using Helix pomatia enzymes.