Synthesis and biological evaluation of novel quinazoline-triazole hybrid compounds with potential use in Alzheimer’s disease

A library of twelve quinazoline-triazole hybrid compounds were designed, synthesized and evaluated as a novel class of acetylcholinesterase inhibitors to treat Alzheimer’s disease (AD). The biological assay results demonstrated the ability of several hybrid compounds to inhibit AChE enzyme (IC₅₀ range = 0.2–83.9 µM). To understand the high potential activity of these compounds, molecular docking simulations were performed to get better insights into the mechanism of binding of quinazoline-triazole hybrid compounds. As expected, compounds 8a and 9a-b bind to both catalytic anionic site (CAS) and peripheral anionic site (PAS) in the active site of AChE enzyme, which implicates that these compounds could act as dual binding site inhibitors. These compounds were not cytotoxic and they also displayed appropriated physicochemical as well as pharmacokinetic profile to be developed as novel anti-AD drug candidates.     

Structural basis for glutamate racemase inhibition

d-Glutamic acid is a required biosynthetic building block for peptidoglycan, and the enzyme glutamate racemase (GluR) catalyzes the inter-conversion of D and L-glutamate enantiomers. Therefore, GluR is considered as an attractive target for the design of new antibacterial drugs. Here, we report the crystal structures of GluR from Streptococcus pyogenes in both inhibitor-free and inhibitor-bound forms. The inhibitor free GluR crystallized in two different forms, which diffracted to 2.25 Å and 2.5 Å resolution, while the inhibitor-bound crystal diffracted to 2.5 Å resolution. GluR is composed of two domains of α/β protein that are related by pseudo-2-fold symmetry and the active site is located at the domain interface. The inhibitor, γ-2-naphthylmethyl-d-glutamate, which was reported earlier as a novel potent competitive inhibitor, makes several hydrogen bonds with protein atoms, and the naphthyl moiety is located in the hydrophobic pocket. The inhibitor binding induces a disorder in one of the loops near the active site. In both crystal forms, GluR exists as a dimer and the interactions seen at the dimer interface are almost identical. This agrees well with the results from gel filtration and dynamic light-scattering studies.     

Early human occupations at the westernmost tip of Eurasia: The lithic industries from Menez–Dregan I (Plouhinec,Finistère,France)

The stratigraphical complex preserved at the site of Menez–Dregan I (Brittany, France) displays an alternating sequence of 17 occupation levels and of 4 marine deposits, between ca. 450 and 150,000 years (stratigraphical units 9a and 3b). The lithic industry retrieved at the site is extremely abundant, with more than 153,000 artefacts over 3 cm in length, as well as millions of knapping debris, and corresponds to a regional facies of the Acheulean, with heavy-duty tools essentially represented by choppers, as well as a few handaxes and cleavers. This paper presents a first synthesis of the data produced for almost 30 years on the site of Menez–Dregan I. The typological study of the entire lithic collection is now complete and allows us to assess the different human occupations that occurred at this site during the Lower Palaeolithic.     

Vertical Redistribution of Soil Organic Carbon Pools After Twenty Years of Nitrogen Addition in Two Temperate Coniferous Forests

Nitrogen (N) inputs from atmospheric deposition can increase soil organic carbon (SOC) storage in temperate and boreal forests, thereby mitigating the adverse effects of anthropogenic CO₂ emissions on global climate. However, direct evidence of N-induced SOC sequestration from low-dose, long-term N addition experiments (that is, addition of < 50 kg N ha⁻¹ y⁻¹ for > 10 years) is scarce worldwide and virtually absent for European temperate forests. Here, we examine how tree growth, fine roots, physicochemical soil properties as well as pools of SOC and soil total N responded to 20 years of regular, low-dose N addition in two European coniferous forests in Switzerland and Denmark. At the Swiss site, the addition of 22 kg N ha⁻¹ y⁻¹ (or 1.3 times throughfall deposition) stimulated tree growth, but decreased soil pH and exchangeable calcium. At the Danish site, the addition of 35 kg N ha⁻¹ y⁻¹ (1.5 times throughfall deposition) impaired tree growth, increased fine root biomass and led to an accumulation of N in several belowground pools. At both sites, elevated N inputs increased SOC pools in the moderately decomposed organic horizons, but decreased them in the mineral topsoil. Hence, long-term N addition led to a vertical redistribution of SOC pools, whereas overall SOC storage within 30 cm depth was unaffected. Our results imply that an N-induced shift of SOC from older, mineral-associated pools to younger, unprotected pools might foster the vulnerability of SOC in temperate coniferous forest soils.

     

Effects of hydroxysafflor yellow A on ALDH1: Inhibition kinetics and molecular dynamics simulation

Hydroxysafflor yellow A (HSYA) is a potent natural antioxidant that displays important neuroprotective activity. Inhibition of acetaldehyde dehydrogenase 1 (ALDH1) has attracted the attention of researchers due to its overexpression in several types of cancers. We studied the effects of HSYA on ALDH1 by evaluating the inhibitory kinetics based on its antioxidant properties and performing computational simulation integrating methods. HSYA reversibly inhibited human recombinant ALDH1 via non-competitive inhibition (K_i = 0.267 ± 0.024 mM). We also investigated the tertiary structural changes via measuring intrinsic and ANS-binding fluorescence. The results indicated that the inactivation induced by HSYA was associated with structural changes. To obtain further information, we simulated the 3D structure of ALDH1 and conducted computational docking simulations as well as molecular dynamics simulations. The results indicated that 4 rings of HSYA interact with several residues near the ALDH1 active site. Our study provides insight into the inhibition of ALDH1 accompanied by structural changes. Based on its ALDH1-inhibiting effect and its potential as a natural antioxidant, HSYA is a potential agent for treating ALDH1-associated cancers.     

Roc-La-Tour I, le site des Esprits : L’art du Magdalénien VI à Monthermé (Ardennes)

For 100 or 200 years during the late Bölling this site in the Ardenne, with its fine outlook, was the summer camp for a small group (10-15 persons) from the Paris Basin. No bone has been preserved (acid soil). Each of the four levels corresponds to at least five to 10 visits. The visitors were attracted to the site by the viewpoint, the dry ground and the presence of ochre and schist. Thousands of plaques and plaquettes were used and broken up before and after engraving. There are more engraved pieces than burins and borers: the visitors must have engraved every day. The engravings include five palimpsests, 32 tangled groups of incisions (« tangles ») and 75 fragmentary images of animals and human beings, with the same menagerie as in the South-West of France; a large majority of the figurative subjects are shown only partially, or merely roughly sketched; there are several human beings and one vulva. The 410 abstract signs can be classified into 14 categories, mostly different from those found in the South-West, and there are 215 groups of parallel lines. These engraved, smoothed or shaped plaquettes, together with 123 deposits of ochre, all contrast with the lack of similar finds on sites in the Paris Basin which are, however, 10 times richer as far as tools are concerned. There was an evolutionary course in the engravings (figures and signs): inception, development, acme and the start of a decline. This site complements those in the Paris Basin and the Ardenne left by the same persons; it is an integral, inseparable part of the remains of the same group of human beings.     

Carbohydrate-binding site of a novel mannose-specific lectin from fugu (Takifugu rubripes) skin mucus

Pufflectin-s, identified in the skin mucus of the fugu Takifugu rubripes, is a novel mannose-specific lectin with similar structure to monocotyledonous plant lectins. In the present study, mutational analysis was used to reveal the mannose-binding sites of pufflectin-s. Putative binding sites were mutated as follows: binding site 1; rPL-D32E (Asp³² → Glu³²), rPL-N34S (Asn³⁴ → Ser³⁴) and rPL-V36A (Val³⁶ → Ala³⁶) whereas binding site 2; rPL-D61E (Asp⁶¹ → Glu⁶¹), rPL-N63S (Asn⁶³ → Ser⁶³) and rPL-V65A (Val⁶⁵ → Ala⁶⁵). All recombinant proteins were expressed in Escherichia coli, purified with two chromatographic steps, and then subjected to mannose-binding assay by affinity chromatography. Recombinant wild-type pufflectin-s (rPL-wt) as well as three mutants with changes in binding site 2 could bind to mannose, in contrast to the three mutants with changes in binding site 1 in which mannose-binding activity was completely lost. These results clearly demonstrate that, at the least, binding site 1 is critical to mannose-binding activity in pufflectin-s.     

Regeneration of Carnegiea gigantea (Cactaceae) since 1850 in three populations in the northern Sonoran Desert

Saguaro cacti (Carnegiea gigantea) are long-lived and exhibit great variability in growth that makes age estimation problematic. A few single-site studies have focused on those locales where long-term data (e.g. 85 years) are available. Using a newly developed technique, 733 saguaros were sampled in three locales (Silverbell, Harcuvar, Kofa) across Arizona and their age structure reconstructed for the last 150 years based on a mathematical model of the heights of individuals. This is the first study to compare regeneration at multiple locations across the species' range. Regression analysis for each site (years and frequency of individuals established during that year) was run and residuals extracted to determine peaks and troughs in regeneration over time. Correlation was run on the residuals between sites, and chi-square analysis was employed to compare frequency of good and bad regeneration years between Kofa and Silverbell. Peaks and troughs represent regeneration as well as survivorship and mortality. Several large cohorts established at Kofa and Harcuvar since 1850, while at Silverbell well over 80% of sampled saguaros established after the late 1930s. This more recent recruitment at Silverbell may be related to the major freezing event of 1937 whose impact was likely greater at the cooler Silverbell site. Despite the widely different population structures at Silverbell and Kofa, recruitment trends in both populations were statistically linked as both locales often benefited from the same favorable periods for regeneration (P < 0.001). The Harcuvar population shares some common peaks and troughs in regeneration over time with Kofa and Silverbell, but its relationship to them is not statistically significant. Some trends overlap in some locales, such as the favorable regeneration period in the late 1800s and early 1900s, particularly at Silverbell and Kofa as well as at other known sites. However, each population has its own signature. Silverbell is a youthful population likely shaped by severe freezing events, while Kofa has many individuals representing regeneration at several different periods. Regeneration, mortality and subsequent population structure is shaped by both regional-scale influences as well as more localized conditions over the long and short terms.     

Long-distance travellers stopover for longer: a case study with spoonbills staying in North Iberia

Long-distance migration is widespread among birds, connecting breeding and wintering areas through a set of stopover localities where individuals refuel and/or rest. The extent of the stopover is critical in determining the migratory strategy of a bird. Here, we examined the relationship between minimum length of stay of PVC-ringed birds in a major stopover site and the remaining flight distance to the overwintering area in the Eurasian spoonbill (Platalea l. leucorodia) during four consecutive autumn migrations. We also analysed the potential effect of timing (arrival date), as well as the role of experience in explaining stopover duration of spoonbills. Overall, birds wintering in Africa, and facing long-distance travel from the stopover site (ca. 3,000 km) stay for longer (2.7 ± 0.4 days) than Iberian winterers (1.5 ± 0.2 days) that perform a much shorter migration (ca. 800 km). These differences were consistent between years. Stopover duration was not significantly affected by the age of the bird. However, there was a significant reduction as migration advanced. Our results suggest that spoonbills develop different stopover strategies depending on the expected distance to the wintering grounds. Adults, especially long-distance migratory ones, could reduce the potential negative effects of density-dependence processes by avoiding stopover at the end of the migration period. These findings are of significant relevance for understanding differences in migratory behaviour within single populations, especially for declining waterbirds, as well as stress the relevance of preserving stopover localities for the conservation of intraspecific diversity in migratory species.     

Isolation of cross-reacting antigen candidates by mRNA-display using a mixed cDNA library

The identification of cross-reacting antigens is an important process in the characterization of antibodies. We describe here the application of mRNA-display technology with a cDNA library for the isolation of cross-reacting antigens using a monoclonal antibody against human tumor protein p53 (hTP53) as a model. A mixed cDNA library constructed from mRNAs prepared from several human tissues and cell-lines was used for the mRNA-display. After several rounds of panning, five annotated polypeptides, topoisomeraseII-binding protein 1 (TOBP1), RAS protein activator like 2 isoform 1 (RASAL2), endosome-associated FYVE-domain protein (ZFYVE16), and utrophin (UTRN) as well as hTP53, were identified as cross-reactive antigen candidates. All of them had a consensus motif, -S-D-L-( )-K-L-, which was included in the known epitope of the antibody. They were synthesized in vitro, and their binding was compared by conducting a pull-down assay. In cross-activity to the antibody, they ranked as follows: ZYVE16 ≅ hTP53 ≅ TOBP1 > UTRN > RASAL2.     

Synthesis and biological evaluation of 1,2,4-trisubstituted imidazoles as inhibitors of transforming growth factor-β type I receptor (ALK5)

A series of 2-(6-methylpyridin-2-yl)-1H-imidazoles were synthesized and evaluated for ALK5 inhibitory activity in cell-based luciferase reporter assays. The compound 4-(((1-(benzo[d][1,3]dioxol-5-yl)-2-(6-methylpyridin-2-yl)-1H-imidazol-4-yl)methyl)amino)benzenesulfonamide (27a) exhibited slightly higher inhibition (IC₅₀ = 0.24 μM) than SB431542 (IC₅₀ = 0.35 μM), a well known potent ALK5 inhibitor. The binding mode of 27a generated by flexible docking study shows that it fits well into the site cavity of ALK5 by forming several tight interactions.     

Towards alpha-glucosidase folding induced by trifluoroethanol: Kinetics and computational prediction

Alpha-glucosidase (EC 3.2.1.20) is an enzyme, which is related with diabetes mellitus type 2 clinically, and is also generally used to convert starch to fermentable sugars in the industry. Therefore, study on this enzyme structures and functions is important. In this study, we investigated structural changes in the alpha-glucosidase during trifluoroethanol (TFE)-induced unfolding. The activity of alpha-glucosidase was significantly inactivated by TFE in a dose-dependent manner. The inactivation was composed of two-phases. TFE inhibited alpha-glucosidase in a parabolic mixed-type reaction (K_i = 0.72 ± 0.08 M). TFE directly induced the unfolding and hydrophobic exposure of alpha-glucosidase. We also simulated the docking between alpha-glucosidase and TFE, as well as molecular dynamics. The computational simulations suggested that several residues, such as ASP68, TYR71, VAL108, HIS111, PHE177, ASP214, THR215, GLU276, HIS348, ASP349, and ARG439, interact with TFE. The molecular dynamics simulation confirmed the binding mechanisms, between the alpha-glucosidase and TFE, and suggested that TFE inhibits the glucose binding site. Our study provides insights into the alpha-glucosidase folding behaviors, and cosolvent binding under a 3D structural simulation.     

Habitat selection by a despotic passerine,the Bell Miner (Manorina melanophrys): When restoring habitat through Lantana (Lantana camara) removal is not enough

The Bell Miner (Manorina melanophrys) occurs in logged eucalypt forest in northern NSW with a dense understorey of the invasive Neotropical shrub Lantana (Lantana camara) that is used for nesting. The link between Bell Miners and Lantana is important as the birds aggressively exclude all smaller and similar‐sized birds from their colonies, reducing avian diversity in forest occupied by the species. We monitored the impact of Lantana removal on Bell Miner persistence in several plots in two logged forest sites, along with untreated control plots at one of the sites. Lantana control was successful over 7 years at both sites, with regeneration of native understorey, midstorey and canopy species compensating for the loss of live Lantana cover in the understorey. Bell Miner individuals vacated the treated plots in one site (Creek's Bend) but persisted in the control and treated plots at the second site (Toonumbar National Park). Bell Miner response was correlated with forest structure: birds vacated forest with a sparse understorey (<5 m) but dense midstorey (5–15 m) and canopy (>15 m) at Creek's Bend, but remained at the site with a dense understorey but sparse midstorey and canopy at Toonumbar. We therefore predict that forest restoration that simultaneously reduces Lantana understorey and increases midstorey density will be most successful in reducing the abundance of the despotic Bell Miner and increasing avian diversity in rehabilitated sites.     

Insulin receptor-inspired soluble insulin binder

The insulin receptor (IR) is a 320 kDa membrane receptor tyrosine kinase mediating the pleiotropic actions of insulin, leading to phosphorylation of several intracellular substrates including serine/threonine-protein kinase (AKT1), and IR autophosphorylation. Structural details of the IR have been recently revealed. A high-binding insulin site, L1 (K_d =2 nM), consists of two distant domains in the primary sequence of the IR. Our design simplified the L1 binding site and transformed it into a soluble insulin binder (sIB). The sIB, a 17 kDa protein, binds insulin with 38 nM affinity. The sIB competes with IR for insulin and reduces by more than 50% phosphorylation of AKT1 in HEK 293 T cells, with similar effects on IR autophosphorylation. The sIB represents a new tool for research of insulin binding and signaling properties.     

A sensitive voltammetric sensor for determination of synthetic corticosteroid triamcinolone, abused for doping

Edge plane pyrolytic graphite electrode (EPPGE) modified with single-wall carbon nanotubes (SWNTs) has been used as a sensor to determine triamcinolone, abused by athletes for doping. A comparison of the voltammetric behavior between SWNTs modified EPPGE and fullerene – C₆₀-modified EPPGE indicated that SWNTs modified EPPGE is more sensitive. The electrode exhibited an effective catalytic response with good reproducibility and stability. The effect of several parameters such as pH, square wave frequency and steroid concentration were studied. The square wave voltammetric response of the electrode to triamcinolone is linear in the range 0.1–25 nM with a detection limit and sensitivity of 8.9 × 10⁻¹⁰ M and 2.06 μA nM⁻¹, respectively. The method was applied for the determination of triamcinolone in several commercially available pharmaceuticals and real urine samples obtained from patients undergoing pharmacological treatment with triamcinolone. A comparison of the observed results with HPLC analysis indicated a good agreement. The product obtained after reduction of triamcinolone was also characterized using ¹H NMR and GC–MS and the site of reduction is found to be carbonyl group at position 20. The method described is rapid, simple and accurate and can be easily applied for detecting cases of doping.     

The utility of geometrical and chemical restraint information extracted from predicted ligand-binding sites in protein structure refinement

Exhaustive exploration of molecular interactions at the level of complete proteomes requires efficient and reliable computational approaches to protein function inference. Ligand docking and ranking techniques show considerable promise in their ability to quantify the interactions between proteins and small molecules. Despite the advances in the development of docking approaches and scoring functions, the genome-wide application of many ligand docking/screening algorithms is limited by the quality of the binding sites in theoretical receptor models constructed by protein structure prediction. In this study, we describe a new template-based method for the local refinement of ligand-binding regions in protein models using remotely related templates identified by threading. We designed a Support Vector Regression (SVR) model that selects correct binding site geometries in a large ensemble of multiple receptor conformations. The SVR model employs several scoring functions that impose geometrical restraints on the Cα positions, account for the specific chemical environment within a binding site and optimize the interactions with putative ligands. The SVR score is well correlated with the RMSD from the native structure; in 47% (70%) of the cases, the Pearson’s correlation coefficient is >0.5 (>0.3). When applied to weakly homologous models, the average heavy atom, local RMSD from the native structure of the top-ranked (best of top five) binding site geometries is 3.1 Å (2.9 Å) for roughly half of the targets; this represents a 0.1 (0.3) Å average improvement over the original predicted structure. Focusing on the subset of strongly conserved residues, the average heavy atom RMSD is 2.6 Å (2.3 Å). Furthermore, we estimate the upper bound of template-based binding site refinement using only weakly related proteins to be ～2.6 Å RMSD. This value also corresponds to the plasticity of the ligand-binding regions in distant homologues. The Binding Site Refinement (BSR) approach is available to the scientific community as a web server that can be accessed at http://cssb.biology.gatech.edu/bsr/.     

Crystal structures of the pro-inflammatory cytokine interleukin-23 and its complex with a high-affinity neutralizing antibody

Interleukin (IL)-23 is a pro-inflammatory cytokine playing a key role in the pathogenesis of several autoimmune and inflammatory diseases. We have determined the crystal structures of the heterodimeric p19-p40 IL-23 and its complex with the Fab (antigen-binding fragment) of a neutralizing antibody at 2.9 and 1.9 Å, respectively. The IL-23 structure closely resembles that of IL-12. They share the common p40 subunit, and IL-23 p19 overlaps well with IL-12 p35. Along the hydrophilic heterodimeric interface, fewer charged residues are involved for IL-23 compared with IL-12. The binding site of the Fab is located exclusively on the p19 subunit, and comparison with published cytokine-receptor structures suggests that it overlaps with the IL-23 receptor binding site.     

Below‐ground biomass and productivity of a grazed site and a neighbouring ungrazed exclosure in a grassland in central Argentina

Abstract We estimated the below‐ground net plant productivity (BNPP) of different biomass components in an intensively and continuously 45‐ha grazed site and in a neighbouring exclosure ungrazed for 16 years for a natural mountain grassland in central Argentina. We measured approximately twice as much dead below‐ground biomass in the grazed site as in the ungrazed site, with a strong concentration of total below‐ground biomass towards the upper 10 cm of the soil layer in both sites. The main contribution to total live biomass was accounted for by very fine (<0.5 mm) and fine roots (0.5–1.0 mm) both at the grazed (79%) and at the ungrazed (81%) sites. We measured more dead biomass for almost all root components, more live biomass of rhizomes, tap roots and bulbs, and less live biomass of thicker roots (>1 mm) in the grazed site. The seasonal variation of total live below‐ground biomass mainly reflected climate, with the growing season being limited to the warmer and wetter portion of the year, but such variation was higher in the grazed site. Using different methods of estimation of BNPP, we estimated maximum values of 1241 and 723 g m^?2 year^?1 for the grazed and ungrazed sites, respectively. We estimated that very fine root productivity was almost twice as high at the grazed site as at the ungrazed one, despite the fact that both sites had similar total live biomass, and root turnover rate was twofold at the grazed site.     

Secondary structural modeling of the second internal transcribed spacer (ITS2) from Pfiesteria-like dinoflagellates (Dinophyceae)

Pfiesteria piscicida is a harmful bloom-forming alga that has received a great deal of attention due to its potential association with large fish kills and neurological problems in humans. Since the discovery of Pfiesteria, several other Pfiesteria-like dinoflagellates (PLDs) have also been identified. Genetic identification and phylogenetic relationships among the PLDs commonly utilize sequence data from the genes and spacers of the ribosomal DNA (rDNA) operon. Of these, the internal transcribed spacers (ITSs) have been previously shown to fold into secondary structures that are critical for proper ribosomal processing. In this study, we modeled the secondary structure of the second internal transcribed spacer (ITS2) from 16 PLDs (as well as an outgroup taxon) using phylogenetic comparative methods and minimum free energy. The secondary structural models predicted for these dinoflagellates consisted of four paired helices separated by five unpaired regions, consistent with those reported from many eukaryotes. All of the structures were highly stable (ΔG = ?66.1 to ?122.3 kcal·mol at 37 °C) and several structural characters were found to be conserved either across the PLDs or were specific to monophyletic subgroups, strengthening previously inferred phylogenetic relationships among taxa. Additionally, an 18 bp motif was identified in the PLDs whose position corresponds to a ribosomal processing site described from other eukaryotes. Potential applications of these ITS2 secondary structures include utility in strain and species identification, phylogenetic inference and serving as a tool for identifying and excluding rDNA pseudogenes when assessing biodiversity within the PLDs.