Highly Potent and Selective Ectonucleoside Triphosphate Diphosphohydrolase (ENTPDase1, 2, 3 and 8) Inhibitors Having 2-substituted-7- trifluoromethyl-thiadiazolopyrimidones Scaffold

Background: The ecto-nucleoside triphosphate diphosphohydrolases (NTPDases) terminate nucleotide signaling via the hydrolysis of extracellular nucleoside-5'-triphosphate and nucleoside- 5'-diphosphate, to nucleoside-5'-monophosphate and composed of eight Ca2+/Mg2+ dependent ectonucleotidases (NTPDase1-8). Extracellular nucleotides are involved in a variety of physiological mechanisms. However, they are rapidly inactivated by ectonucleotidases that are involved in the sequential removal of phosphate group from nucleotides with the release of inorganic phosphate and their respective nucleoside. Ectonucleoside triphosphate diphosphohydrolases (NTPDases) represent the key enzymes responsible for nucleotides hydrolysis and their overexpression has been related to certain pathological conditions. Therefore, the inhibitors of NTPDases are of particular importance in order to investigate their potential to treat various diseases e.g., cancer, ischemia and other disorders of the cardiovascular and immune system. Methods: Keeping in view the importance of NTPDase inhibitors, a series of thiadiazolopyrimidones were evaluated for their potential inhibitory activity towards NTPDases by the malachite green assay. Results: The results suggested that some of the compounds were found as non-selective inhibitors of isozyme of NTPDases, however, most of the compounds act as potent and selective inhibitors. In case of substituted amino derivatives (4c-m), the compounds 4m (IC50 = 1.13 ± 0.09 μM) and 4g (IC50 = 1.72 ± 0.08 μM) were found to be the most potent inhibitors of h-NTPDase1 and 2, respectively. Whereas, compound 4d showed the best inhibitory potential for both h-NTPDase3 (IC50 = 1.25 ± 0.06 μM) and h-NTPDase8 (0.21 ± 0.02 μM). Among 5a-t derivatives, compounds 5e (IC50 = 2.52 ± 0.15 μM), 5p (IC50 = 3.17 ± 0.05 μM), 5n (IC50 = 1.22 ± 0.06 μM) and 5b (IC50 = 0.35 ± 0.001 μM) were found to be the most potent inhibitors of h-NTPDase1, 2, 3 and 8, respectively. Interestingly, the inhibitory concentration values of above-mentioned inhibitors were several folds greater than suramin, a reference control. In order to determine the binding interactions, molecular docking studies of the most potent inhibitors were conducted into the homology models of NTPDases and the putative binding analysis further confirmed that selective and potent compounds bind deep inside the active pocket of the respective enzymes. Conclusions: The docking analysis proposed that the inhibitory activity correlates with the hydrogen bonds inside the binding pocket. Thus, these derivatives are of interest and may further be investigated for their importance in medicinal chemistry.

Rational Identification of Hsp90 Inhibitors as Anticancer Lead Molecules by Structure Based Drug Designing Approach

Background: Heat shock protein 90 (Hsp90) is an encouraging anticancer target for the development of clinically significant molecules. Schiff bases play a crucial role in anticancer research because of their ease of synthesis and excellent antiproliferative effect against multiple cancer cell lines. Therefore, we started our research work with the discovery of resorcinol/4-chloro resorcinol derived Schiff bases as Hsp90 inhibitors, which resulted in the discovery of a viable anticancer lead molecule. Objective: The objective of the study is to discover more promising lead molecules using our previously established drug discovery program, wherein the rational drug design is achieved by molecular docking studies. Methods: The docking studies were carried out by using Surflex Geom X programme of Sybyl X-1.2 version software. The molecules with good docking scores were synthesized and their structures were confirmed by IR, 1H NMR and mass spectral analysis. Subsequently, the molecules were evaluated for their potential to attenuate Hsp90 ATPase activity by Malachite green assay. The anticancer effect of the molecules was examined on PC3 prostate cancer cell lines by utilizing 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay methodology. Results: Schiff bases 11, 12, 20, 23 and 27 exhibiting IC50 value below 1μM and 15μM, in malachite green assay and MTT assay, respectively, emerged as viable lead molecules for future optimization. Conclusion: The research work will pave the way for the rational development of cost-effective Schiff bases as Hsp90 inhibitors as the method employed for the synthesis of the molecules is simple, economic and facile.

The In Vitro Anti-Cancer Activities of 17βH-Neriifolin Isolated from Cerbera odollam and its Binding Activity on Na+, K+-ATPase

Background: 17βH-neriifolin, a cardiac glycoside compound had been successfully isolated from Cerbera odollam leaves based on the bioassay guided-isolation procedure. The aim of these studies were to determine the in vitro anti-cancer and binding effects of 17βH-neriifolin on Na+, K+-ATPase. Methods: The in vitro anti-cancer effects were evaluated using Sulphorhodamine B and Hoescht 33342 assays. The Na+, K+-ATPase assay was carried out using Malachite Green assay. In silico molecular docking studies and in vitro malachite green assay were used to predict the binding activities of 17βH-neriifolin on Na+, K+-ATPase and ouabain was also included as for comparison studies. Results: The compound was tested against breast (MCF-7, T47D), colorectal (HT-29), ovarian (A2780, SKOV-3) and skin (A375) cancer cell lines that gave IC50 values ranged from 0.022 ± 0.0015 to 0.030 ± 0.0018 μM. The mechanism of cell death of 17βH-neriifolin was further evaluated using Hoescht 33342 assay and it was found that the compound killed the cancer cells via apoptosis. 17βHneriifolin and ouabain both bound at α-subunit in Na+, K+-ATPase and their binding energy were - 8.16 ± 0.74 kcal/mol and -8.18 ± 0.48 kcal/mol respectively. Conclusion: The results had confirmed the anti-proliferative effects exerted by 17βH-neriifolin in the breast, colorectal, ovarian and skin cancer cell lines. 17βH-neriifolin had shown to cause apoptotic cell death in the respective cancer cell lines.17βH-neriifolin and ouabain both bound at α-subunit in Na+, K+-ATPase and their binding energy were -8.16 ± 0.74 kcal/mol and -8.18 ± 0.48 kcal/mol respectively. This is the first report to reveal that 17βH-neriifolin managed to bind to the pocket of α-subunit of Na+.K+-ATPase.

Surface Functionalization by Hydrophobin-EPSPS Fusion Protein Allows for the Fast and Simple Detection of Glyphosate

Glyphosate, the most widely used pesticide worldwide, is under debate due to its potentially cancerogenic effects and harmful influence on biodiversity and environment. Therefore, the detection of glyphosate in water, food or environmental probes is of high interest. Currently detection of glyphosate usually requires specialized, costly instruments, is labor intensive and time consuming. Here we present a fast and simple method to detect glyphosate in the nanomolar range based on the surface immobilization of glyphosate’s target enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) via fusion to the hydrophobin Ccg2 and determination of enzyme activity with a malachite green assay, which is a common photometric technique to measure inorganic phosphate (Pi). The assay demonstrates a new approach for a fast and simple detection of pesticides.

A novel isotope pool dilution approach to quantify gross rates of key abiotic and biological processes in the soil phosphorus cycle

Efforts to understand and model the current and future behavior of the global phosphorus (P) cycle are limited by the availability of global data on rates of soil P processes, as well as their environmental controls. Here, we present a novel isotope pool dilution approach using 33P labeling of live and sterile soils, which allows for high-quality data on gross fluxes of soil inorganic P (Pi) sorption and desorption, as well as of gross fluxes of organic P mineralization and microbial Pi uptake to be obtained. At the same time, net immobilization of 33Pi by soil microbes and abiotic sorption can be easily derived and partitioned. Compared with other approaches, we used short incubation times (up to 48 h), avoiding tracer remineralization, which was confirmed by the separation of organic P and Pi using isobutanol fractionation. This approach is also suitable for strongly weathered and P-impoverished soils, as the sensitivity is increased by the extraction of exchangeable bioavailable Pi (Olsen Pi; 0.5 M NaHCO3) followed by Pi measurement using the malachite green assay. Biotic processes were corrected for desorption/sorption processes using adequate sterile abiotic controls that exhibited negligible microbial and extracellular phosphatase activities. Gross rates were calculated using analytical solutions of tracer kinetics, which also allowed for the study of gross soil P dynamics under non-steady-state conditions. Finally, we present major environmental controls of gross P-cycle processes that were measured for three P-poor tropical forest and three P-rich temperate grassland soils.

A novel isotope pool dilution approach to quantify gross rates of key abiotic and biological processes in the soil phosphorus cycle

Efforts to understand and model the current and future behavior of the global phosphorus (P) cycle are limited by the availability of global data on gross rates of soil P processes, as well as its environmental controls. We here present a novel isotope pool dilution approach using 33P labelling of live and sterile soils, which allows to obtain high quality data on gross fluxes of soil inorganic P (Pi) sorption and desorption, as well as of gross fluxes of organic P mineralization and microbial Pi uptake. At the same time, net immobilization of 33Pi by soil microbes and abiotic sorption can be easily derived and partitioned. Compared to other approaches, we used short incubation times (up to 48 h), avoiding tracer re-mineralization, which was confirmed by separation of organic P and Pi using isobutanol fractionation. This approach is also suitable for strongly weathered and P impoverished soils, as sensitivity is increased by extraction of exchangeable bio-available Pi (Olsen Pi; 0.5 M NaHCO3) followed by Pi measurement using the malachite green assay. Biotic processes were corrected for desorption/sorption processes by using adequate sterile abiotic controls that exhibited negligible microbial and extracellular phosphatase activities. Gross rates are calculated using analytical solutions of tracer kinetics, which also allows to study gross soil P dynamics under non-steady-state conditions. Finally, we present major environmental controls of gross and net P cycle processes that were measured for three P-poor tropical forest and three P-rich temperate grassland soils.