Combinatorial Chemistry & High Throughput Screening - Volume 16, Issue 6, 2013

The Notch pathway plays a crucial role in cell fate decisions through controlling various cellular processes. Overactive Notch signal contributes to cancer development from leukemias to solid tumors. γ-Secretase is an intramembrane protease responsible for the final proteolytic step of Notch that releases the membrane-tethered Notch fragment for signaling. Therefore,γ-secretase is an attractive drug target in treating Notch-mediated cancers. However, the absence of high throughput γ-secretase assay using Notch substrate has limited the identification and development of γ- secretase inhibitors that specifically target the Notch signaling pathway. Here, we report on the development of a 1536- well γ-secretase assay using a biotinylated recombinant Notch1 substrate. We effectively assimilated and miniaturized this newly developed Notch1 substrate with the AlphaLISA detection technology and demonstrated its robustness with a calculated Z’ score of 0.66. We further validated this optimized assay by performing a pilot screening against a chemical library consisting of ∼5,600 chemicals and identified known γ-secretase inhibitors e.g. DAPT, and Calpeptin; as well as a novel γ-secretase inhibitor referred to as KD-I-085. This assay is the first reported 1536-well AlphaLISA format and represents a novel high throughput Notch1-γ-secretase assay, which provides an unprecedented opportunity to discover Notch-selective γ -secretase inhibitors that can be potentially used for the treatment of cancer and other human disorders.

Present work deals with generation of virtual samples as mathematical modeling of empirical data on the basis of empirical data. The generated samples were used for development of QSAR model. The method deals with extrapolation of sample vector in such a manner that there is conservation of the empirical data distribution. The data distribution has been judged with statistical parameters. The method was implemented with anticancer activity of Gossypol acetic acid against BCL2 target for colorectal cancer. Considering the virtual samples only for model development, model training showed a regression coefficient for leave one out cross validation as 0.996 with 66 virtual samples, and a regression coefficient with external test set data (51 samples) as 0.993. External test set data which were never used in the virtual sample generation showed predicted regression coefficient value of >0.61. On the basis of QSAR model, nine compounds were suggested as anti-BCL2 active compounds. The suggested compounds were further validated by docking study with Gossypol acetic acid and ‘Tetrahydroisoquinoline amide substituted phenyl pyrazole’ cocrystallized with chimeric BCL2-XL (PDBID: 2W3L) protein.

An efficient synthesis of new spiro[chroman-3,6‘-furo[2,3-d]pyrimidine]- tetraones by an organocatalyzed three-component condensation reaction of aldehydes, barbituric acids and 3-bromo-4-hydroxy-2H-chromen-2-one in refluxing acetic acid in the presence of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) is reported. We are hopeful that the products will lead to a wider range pharmacological and physiological activity.

One-bead-one-compound (OBOC) combinatorial library screening has been broadly utilized for the last two decades to identify small molecules, peptides or peptidomimetics targeting variable screening probes such as cell surface receptors, bacteria, protein kinases, phosphatases, proteases etc. In previous screening methods, library beads were suspended in solution and screened against one single probe. Only the positive beads were tracked and isolated for additional screens and finally selected for chemical decoding. During this process, the remaining negative beads were not tracked and discarded. Here we report a novel bead immobilization method such that a bead library array can be conveniently prepared and screened in its entirety, sequentially many times with a series of distinct probes. This method not only allows us to increase the screening efficiency but also permits us to determine the binding profile of each and every library bead against a large number of target receptors. As proof of concept, we serially screened a random OBOC disulfide containing cyclic heptapeptide library with three water soluble dyes as model probes: malachite green, bromocresol purple and indigo carmine. This multiplicative screening approach resulted in a rapid determination of the binding profile of each and every bead respective to each of the three dyes. Beads that interacted with malachite green only, bromocresol purple only, or both indigo carmine and bromocresol purple were isolated, and their peptide sequences were determined with microsequencer. Ultimately, the novel OBOC multiplicative screening approach could play a key role in the enhancement of existing on-bead assays such as whole cell binding, bacteria binding, protein binding, posttranslational modifications etc. with increased efficiency, capacity, and specificity.

Screening and identifying multi-target ligands becomes a daunting task when there are very few matching pharmacophoric features among the proteins. Herein, we describe a novel screening strategy to identify multi-target ligands for proteins having varying pharmacophoric features with their ligands. This strategy was adopted to identify multi-target ligands for death-associated protein kinase (DAPk) family. The role of the kinase activity of DAPk in eukaryotic cell apoptosis and the ability of bioavailable DAPk inhibitors to rescue neuronal death after brain injury have made it a drug-discovery target for neurodegenerative disorders. In this work, we employed a novel strategy using the existing computational approaches to design multi-target inhibitors, which can potentially inhibit one or any combination of the three DAPk family members. The strategy employs a combination of merged pharmacophore matching, database screening and molecular docking to reliably identify potential multi-target inhibitors targeted against DAPk protein family.

Silica nanoparticles were synthesized from rice husk ash at room temperature by using high energy planetary ball mill. The milling time and mill rotational speed were varied in four levels. The morphology of the synthesized powders was investigated by the FE-SEM and TEM image as well as XRD patterns. The results have revealed that the nano-sized amorphous silica particles are formed after about 6 h ball milling and they are spherical in shape. The average particle size of the silica powders is found to be around 70 nm which decreases with increasing ball milling time or mill rotational speed. The as-synthesized silica nanoparticles were subsequently employed as drug carrier to investigate in vitro release behavior of Penicillin-G in simulated body fluid. UV-Vis spectroscopy was used to determine the amount of Penicillin-G released from the carrier. Penicillin-G release profile from silica nanoparticles exhibited a delayed release effect.

The evaluation of the relationships between the hormones involved in the urogenital tract cancer, including bladder, kidney, prostate, and testis, could prove important from diagnostic point of view. The determination of the steroid hormone profiles may likely provide a biomarker for discrimination of hormone-related diseases, as well as for differentiation of healthy volunteers from patients with cancer. The aim of the study was to demonstrate the changes in the steroid hormone profile (comprising corticosteroids, androgens and progesterone) in the urine of patients with the urogenital tract cancer versus urine from healthy subjects. A reliable analytical method based on liquid chromatography coupled with mass spectrometry was successfully applied to determine the urinary profiles of 6 endogenous steroids: cortisol, cortisone, corticosterone, testosterone, epitestosterone and progesterone for 92 urogenital tract cancer patients and 100 healthy controls. The obtained data was further evaluated by in-depth chemometric analysis, including the applied standardized Kennard- Stone's algorithm to pre-process the data. Mann-Whitney U test revealed statistically significant (p < 0.05) differences in concentration of androgens and progesterone in the case of bladder cancer for male and female population, for male also cortisol and cortisone levels were significantly increased. PCA analysis proved a reasonable trend for differentiating healthy and cancer patients, and finally, applying PLS-DA model we were able to correctly classify 80.56%of cancer patients. Our results indicate that steroid hormone profile determination could be a promising approach for early diagnosis of urogenital tract cancer. However our preliminary results require an extension both in patient number and steroid profile.

Tetranychus urticae Koch is an important pest affecting citrus, for which biological control has not yet been achieved; therefore, acaricides are commonly used instead. The goal of the work reported in this paper was to measure the efficacy of different new compounds —uracil derivatives— on this mite and conduct a quantitative structure-activity relationship (QSAR) study based on the results obtained, in order to set up a model capable of predicting the acaricidal activity of further new compounds. Some of the tested new products proved highly effective against T. urticae. Besides, topological indices were used as structural descriptors. The result was a topological model consisting of two discriminant functions for distinguishing between active and inactive compounds, and a predictive equation for the adult mortality percentage on the sixth day. This model was then sequentially applied to a large database of compounds with unknown activity against the Tetranychus urticae plague. Finally, a preliminary toxicity study of the most effective novel compounds supports their non-toxicity, performing even better than commercial referents.

Heterogeneous copper-in-charcoal-catalyzed click synthesis in 96-well polypropylene filter plates is an efficient method for the rapid generation of sufficient pure 2-alkoxyl-2-(1,2,3-triazole-1-yl) acetamide derivatives library by simple filtration, which directly assay the products for larvicidal activity against mosquitoes. In this procedure, copper nanoparticles on charcoal were arrayed into each well on a 96-well plate, reagents were delivered using a pipette gun, and a constant temperature shaker bath was used to complete the click reaction in 24-72 hours under temperature-controlled conditions. The results of bioassays indicated that the target compounds possessed excellent larvacidal activities against mosquitoes. In particular, the larvacidal activities against mosquitoes of compounds 8[2,3] and 8[7,1] at 2µg.mL-1 were 100 % and 73 % respectively.

A simple and fast homogeneous fluorescent polarization immunoassay (FPIA) was developed for the determination of furaltadone and its metabolite 3-amino-5-methylmorpholino-2-oxazolidinone (AMOZ). Monoclonal antibody with high cross-reactivity to furaltadone and the nitrophenyl derivative of AMOZ (NPAMOZ) were produced against a novel immunogen and the effects of several synthesized tracers on FPIA sensitivity studied. The proposed FPIA, using an optimum antibody and tracer pair, had an IC50 of 4.3µg L–1 and limit of detection at 0.6µg L–1 for furaltadone, and 2.7 µgL–1and0.3µg L–1 for NPAMOZ. Recoveries of furaltadone from animal feeds by FPIA ranged from 79.6 to 87.7%, while recoveries of AMOZ from animal tissues ranged from 72.9 to 83.1&percent. Good correlation (R>0.99) between the results of this FPIA and a standard analytical method was obtained. The FPIA does not require separation or washing steps and the total time required for equilibrium of the antibody-tracer interaction is only 10 min. These results indicated that the proposed FPIA offers great potential and utility for the high throughput screening of furaltadone residues in animal feed and its metabolite AMOZ residues in animal tissues.