Structure based discovery of small molecules to regulate the activity of human insulin degrading enzyme

Background

Insulin-degrading enzyme (IDE) is an allosteric Zn⁺² metalloprotease involved in the degradation of many peptides including amyloid-β, and insulin that play key roles in Alzheimer''s disease (AD) and type 2 diabetes mellitus (T2DM), respectively. Therefore, the use of therapeutic agents that regulate the activity of IDE would be a viable approach towards generating pharmaceutical treatments for these diseases. Crystal structure of IDE revealed that N-terminal has an exosite which is ∼30 Å away from the catalytic region and serves as a regulation site by orientation of the substrates of IDE to the catalytic site. It is possible to find small molecules that bind to the exosite of IDE and enhance its proteolytic activity towards different substrates.

Methodology/Principal Findings

In this study, we applied structure based drug design method combined with experimental methods to discover four novel molecules that enhance the activity of human IDE. The novel compounds, designated as D3, D4, D6, and D10 enhanced IDE mediated proteolysis of substrate V, insulin and amyloid-β, while enhanced degradation profiles were obtained towards substrate V and insulin in the presence of D10 only.

Conclusion/Significance

This paper describes the first examples of a computer-aided discovery of IDE regulators, showing that in vitro and in vivo activation of this important enzyme with small molecules is possible.     

Soil and cultivar type shape the bacterial community in the potato rhizosphere

The rhizospheres of five different potato cultivars (including a genetically modified cultivar) obtained from a loamy sand soil and two from a sandy peat soil, next to corresponding bulk soils, were studied with respect to their community structures and potential function. For the former analyses, we performed bacterial 16S ribosomal RNA gene-based PCR denaturing gradient gel electrophoresis (PCR-DGGE) on the basis of soil DNA; for the latter, we extracted microbial communities and subjected these to analyses in phenotype arrays (PM1, PM2, and PM4, Biolog), with a focus on the use of different carbon, sulfur and phosphorus sources. In addition, we performed bacterial PCR-DGGE on selected wells to assess the structures of these substrate-responsive communities. Effects of soil type, the rhizosphere, and cultivar on the microbial community structures were clearly observed. Soil type was the most determinative parameter shaping the functional communities, whereas the rhizosphere and cultivar type also exerted an influence. However, no genetically modified plant effect was observed. The effects were imminent based on general community analysis and also single-compound analysis. Utilization of some of the carbon and sulfur sources was specific per cultivar, and different microbial communities were found as defined by cultivar. Thus, both soil and cultivar type shaped the potato root-associated bacterial communities that were responsive to some of the substrates in phenotype arrays.     

Enhanced Topical Delivery of Terbinafine Hydrochloride with Chitosan Hydrogels

Chitosan-based carriers have important potential applications for the administration of drugs. In the present study, topical gel formulations of terbinafine hydrochloride (T-HCl) were prepared using different types of chitosan at different molecular weight, and the antifungal inhibitory activity was evaluated to suggest an effective formulation for the treatment of fungal infections. The characteristics of gel formulations were determined with viscosity measurements and texture profile analysis. Stability studies were performed at different temperatures during 3 months. The ex vivo permeation properties were studied through rat skin by using Franz diffusion cells. The antifungal inhibitory activity of formulations on Candida species and filamentous fungi was also examined with agar-cup method. The microbiological assay was found suitable for determination of in vitro antifungal activity of T-HCl. A marketed product was used to compare the results. The antifungal activity of T-HCl significantly increased when it was introduced into the chitosan gels. A higher drug release and the highest zone of inhibition were obtained from gels prepared with the lowest molecular weight chitosan (Protasan UP CL 213) compared to that of other chitosan gels and marketed product. These results indicated the advantages of the suggested formulations for topical antifungal therapy against Candida species and filamentous fungi.     

Microwave assisted synthesis of novel tetrazole/sulfonamide derivatives based on octahydroacridine,xanthene and chromene skeletons as inhibitors of the carbonic anhydrases isoforms I,II, IV and VII

The synthesis of novel tetrazole/sulfonamide derivatives based on octahydroacridine, xanthene and chromene scaffold by using microwave (MW) assisted techniques is reported in this study. These synthesized hybrid compounds were assayed for the inhibition of carbonic anhydrase (CA, EC 4.2.1.1). The inhibitory activities were determined against three cytosolic human isoforms (hCA I, II and VII) and one membrane-associated (hCA IV) isoform. Some of the newly synthesized sulfonamides showed micromolar to nanomolar inhibitory activity against these enzymes.     

A one-year aeropalynological study at Ankara, Turkey

Atmospheric pollen was collected with a Burkard sporetrap in Ankara, Turkey from January 1993 to January1994. The sum of the annual totals of the dailyconcentration of pollen, belonging to 44 taxa, was57,735. A relatively high pollen concentration wasrecorded in June which could be caused by higher windspeed and lower rainfall. Pinaceae,Cupressaceae/Taxaceae, Gramineae, Platanus, Populus,Moraceae, Chenopodiaceae/Amaranthaceae, Acer, Quercus,Betula, Salix, Rumex and Plantago are found tobe the dominant pollen types in the atmosphere inAnkara.     

Determination of the interaction between the receptor binding domain of 2019-nCoV spike protein,TMPRSS2, cathepsin B and cathepsin L,and glycosidic and aglycon forms of some flavonols

The novel coronavirus (COVID-19, SARS-CoV-2) is a rapidly spreading disease with a high mortality. In this research, the interactions between specific flavonols and the 2019-nCoV receptor binding domain (RBD), transmembrane protease, serine 2 (TMPRSS2), and cathepsins (CatB and CatL) were analyzed. According to the relative binding capacity index (RBCI) calculated based on the free energy of binding and calculated inhibition constants, it was determined that robinin (ROB) and gossypetin (GOS) were the most effective flavonols on all targets. While the binding free energy of ROB with the spike glycoprotein RBD, TMPRSS2, CatB, and CatL were –5.02, –7.57, –10.10, and –6.11 kcal/mol, the values for GOS were –4.67, –5.24, –8.31, and –6.76, respectively. Furthermore, both compounds maintained their stability for at least 170 ns on respective targets in molecular dynamics simulations. The molecular mechanics Poisson–Boltzmann surface area (MM/PBSA) calculations also corroborated these data. Considering Lipinski’s rule of five, ROB and GOS exhibited 3 (MW>500, N or O>10, NH or OH>5), and 1 (NH or OH>5) violations, respectively. Neither ROB nor GOS showed AMES toxicity or hepatotoxicity. The LD50 of these compounds in rats were 2.482 and 2.527 mol/kg, respectively. Therefore, we conclude that these compounds could be considered as alternative therapeutic agents in the treatment of COVID-19. However, the possible inhibitory effects of these compounds on cytochromes (CYPs) should be verified by in vitro or in vivo tests and their adverse effects on cellular energy metabolism should be minimized by performing molecular modifications if necessary.     

The susceptibility to stress-induced gastric injury of rats exposed to cadmium

In this experimental study, the effect of cadmium on cold and restraint stress-induced gastric lesions has been studied. Rats received 15 μg/mL cadmium-containing water for 30 d, and at the end of this period, they were subjected to cold and restraint stress. Cadmium accumulation in gastric mucosa was associated with increased mucosal lesions, as well as decreased mucin and PGE₂ levels in rats exposed to cadmium. Stress-induced mucosal injury was more pronounced, and the hemoglobin leakage into gastric lumen owing to breakdown in the barrier was 17.30±3.45 μg/mL in control and 35.71±6.18 μg/mL in treated rats. Our data suggest that high cadmium intake facilitates the occurence of stress-induced mucosal lesions by diminishing the mucin content and PGE₂ generation in gastric mucosa.     

Genotoxicity of Thermopsis turcica on Allium cepa L. roots revealed by alkaline comet and random amplified polymorphic DNA assays

This study was undertaken to evaluate genotoxic potential of Thermopsis turcica aqueous extracts on the roots of onion bulb (Allium cepa L.) by comet assay and random amplified polymorphic DNA technique. The Allium root growth inhibition test indicated that the EC₅₀ and 2×EC₅₀ values were 8 and 16 mg/ml concentrations of T. turcica aqueous extracts, respectively. The negative control (distilled water), positive control (methyl methane sulfonate, 10 mg/l) and 8 and 16 mg/ml concentrations of T. turcica extracts were introduced to the roots of onion bulbs for 24 and 96 h. The root growth, DNA damage in root cells and randomly amplified polymorphic DNA (RAPD) profiles of root tissue were used as endpoints of the genotoxicity. The comet assay clearly indicated that dose-dependent single strand DNA breaks in the root nuclei of onions were determined for the treatment concentrations of T. turcica extracts. In comparison to RAPD profile of negative control group, RAPD polymorphisms became evident as disappearance and/or appearance of RAPD bands in treated roots. The diagnostic and phenetic numerical analyses of RAPD profiles obviously indicated dose-dependent genotoxicity induced by Thermopsis extracts. In conclusion, the results clearly indicated that water extract of T. turcica has genotoxic potential on the roots of onion bulbs as shown by comet assay and RAPD technique.     

Biological control potential of Turkish entomopathogenic nematodes against the Colorado potato beetle,Leptinotarsa decemlineata

Three Turkish isolates of the entomopathogenic nematodes Steinernema carpocapsae, Steinernema feltiae and Heterorhabditis bacteriophora were evaluated under laboratory conditions (in different temperatures and doses) for their biocontrol efficiency against last instar Leptinotarsa decemlineata. Herein, the effects of infective juveniles in aqueous suspension against L. decemlineata were evaluated. S. feltiae appeared to be the most pathogenic nematode among the tested species.     

Phylogenetics support an ancient common origin of two scientific icons: Devils Hole and Devils Hole pupfish

The Devils Hole pupfish (Cyprinodon diabolis; DHP) is an icon of conservation biology. Isolated in a 50 m² pool (Devils Hole), DHP is one of the rarest vertebrate species known and an evolutionary anomaly, having survived in complete isolation for thousands of years. However, recent findings suggest DHP might be younger than commonly thought, potentially introduced to Devils Hole by humans in the past thousand years. As a result, the significance of DHP from an evolutionary and conservation perspective has been questioned. Here we present a high‐resolution genomic analysis of DHP and two closely related species, with the goal of thoroughly examining the temporal divergence of DHP. To this end, we inferred the evolutionary history of DHP from multiple random genomic subsets and evaluated four historical scenarios using the multispecies coalescent. Our results provide substantial information regarding the evolutionary history of DHP. Genomic patterns of secondary contact present strong evidence that DHP were isolated in Devils Hole prior to 20–10 ka and the model best supported by geological history and known mutation rates predicts DHP diverged around 60 ka, approximately the same time Devils Hole opened to the surface. We make the novel prediction that DHP colonized and have survived in Devils Hole since the cavern opened, and the two events (colonization and collapse of the cavern's roof) were caused by a common geologic event. Our results emphasize the power of evolutionary theory as a predictive framework and reaffirm DHP as an important evolutionary novelty, worthy of continued conservation and exploration.