Combinatorial Chemistry & High Throughput Screening - Volume 14, Issue 7, 2011

A detailed knowledge of hydrogen bond geometry and its directional preferences is vital for in silico investigations of the ligand-receptor short-range non-covalent interactions. The spatial arrangement of the carbonyl and hydroxyl groups seems to determine the capability of β-ketoenol derivatives to recognize the surrounding environment by forming inter- and intra-molecular hydrogen bonds (IHB). In the current study we examined the application of the MoStBioDat platform for a massive database screening of the IHB motifs in β-ketoenol subunits (O=C-C=C-OH). Then, the virtual 3D structural data derived from ZINC and PubChem repository were compared to the experimentally determined CSD data. Differences specific for each database were discovered, which indicated inaccuracies in the simulated data.

An efficient solution-phase parallel procedure to perform the structural diversification of some formyl-nitrogen heterocycles (A) using the reusable TBD supported base is described. The library synthesis is based in a consecutive Alkylation-Knoevenagel functionalisation that uses alkyl halides (B), Michael acceptors (C) and activated methylene compounds (D) as diversity elements.

Research involving non-adherent cell lines, primary cells and blood cells is definitely important, but its application in image-based assays, especially in high-content systems, is highly limited. Accordingly, efficient highcontent methods to study non-adherent cells are needed not only to improve diagnostics but also for early screening of targeted drugs. A plate-based assay using adhesion reagents for multiparametric measurement with single non-adherent and non-anchored cells in a large cell population in high-content cytometry was developed and optimized. The cells preserved their identity even during extensive biomanipulations. The proposed method is highly robust for better imaging and can be used in various assays in different cellular backgrounds. Furthermore, as exemplary experiments, novel optimized assay protocols were used to study extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) activity after cell inhibition with imatinib in chronic myelocytic leukemia K562 cells, revealing the phosphorylation kinetics of ERK MAPK. The results showed that the proposed assay detects kinase phosphorylation with good sensitivity and may be used in rapid drug screening.

PDZ (PSD-95/Discs-large/ZO-1 homology) domains represent putative targets in several diseases including cancer, stroke, addiction and neuropathic pain. Here we describe the application of a simple and fast screening assay based on fluorescence polarization (FP) to identify inhibitors of the PDZ domain in PICK1 (protein interacting with C kinase 1). We screened 43,380 compounds for their ability to inhibit binding of an Oregon Green labeled C-terminal dopamine transporter peptide (OrG-DAT C13) to purified PICK1 in solution. The assay was highly reliable with excellent screening assay parameters (Z'≈ 0.7 and Z≈ 0.6). Out of ∼ 200 compounds that reduced FP to less than 80% of the control wells, six compounds were further characterized. The apparent affinities of the compounds were determined in FP competition binding experiments and ranged from ∼ 5.0 μ M to ∼ 193 μ M. Binding to the PICK1 PDZ domain was confirmed for five of the compounds (CSC-03, CSC-04, CSC-43, FSC-231 and FSC-240) in a non-fluorescence based assay by their ability to inhibit pull-down of PICK1 by a C-terminal DAT GST fusion protein. CSC-03 displayed the highest apparent affinity (5.0 μ M) in the FP assay, and was according to fluorescence resonance energy transfer (FRET) experiments capable of inhibiting the interaction between the C-terminus of the GluR2 subunit of the AMPA-type glutamate receptor and PICK1 in live cells. Additional experiments suggested that CSC-03 most likely is an irreversible inhibitor but with specificity for PICK1 since it did not bind three different PDZ domains of PSD-95. Summarized, our data suggest that FP based screening assays might be a widely applicable tool in the search for small molecule inhibitors of PDZ domain interactions.

A linear quantitative structure-visible light absorption wavelength (λ) relationship model for one hundred and forty-two photosensitizers was proposed using heuristic method and multiple linear regression analysis. The statistical parameters of the model were R2 = 0.916; F = 372.16; and RMSE = 5.0873. A fivefold cross-validation algorithm was applied, and the results indicated that the model has a satisfactory statistical stability and validity. The proposed model was evaluated for predictive ability with an external validation set, and the statistical parameters obtained were R2 EXT = 0.908; Q2 EXT = 0.897; F = 118.14; and RMSEEXT = 5.6338 for the external test set. The results obtained demonstrated that the simple linear quantitative structure-wavelength relationship model was robust and satisfactory. It could be a feasible and effective tool for predicting λ of photosensitizers, which is an important parameter for their effect on photodynamic therapy for cancer, and could be a potential way for instructing synthesis of this kind of new photosensitizers.

All medicines pose a potential health risk, be they Eastern or Western medicines. Newly developed Western drugs must undergo rigorous testing to ensure their efficacy and safety, while with Eastern drugs, safety has generally been established because of their long histories of safe usage as traditional medicines. The regulation of Western medicines is much stronger than that of Eastern medicines, partly as pure chemicals are used and their effects and side effects are more likely to be acute. Eastern medicines consist of multiple components, generally extracted from a single or several plants or other natural sources, and their effects are not so acute, with delayed onset of side effects. However, the chronic usage of many Eastern medicines may result in the gradual accumulation of toxic compounds in the body. For example, Agaricus blazei extracts have been used as alternative medicines for cancer, but contain the known carcinogen agaritine (this carcinogen is also present in Agaricus bisporus). To ensure the safety of this alternative medicine, agaritine should be removed or its content reduced if the extract is to be taken chronically. Clearly, the safety of not only pure medicines, but also alternative medicines and daily foods, should be carefully controlled.

Several different approaches have been taken to development of homogeneous fluorescent aptamer assays including end-labeled beacons and signaling aptamers which are intrinsically quenched by nucleotides. Two new strategies dubbed “intrachain” and “competitive” FRET-aptamer assays are summarized in this review. Intrachain and competitive FRET-aptamers can be engineered on the molecular level through a series of exploratory experiments involving prior knowledge of aptamer secondary or tertiary structures and hypotheses about aptamer conformational changes. However, there is an intrinsic risk of altering aptamer affinity or specificity associated with chemical modifications of an aptamer. Natural selection methods for FRET-aptamers have also been devised to potentially obviate the chemical modification problem. The naturally selected aptamers are subjected to fluorophore (F)- and or quencher (Q)-conjugated nucleotide triphosphate (NTP) incorporation by polymerase chain reaction (PCR) with permissive polymerases such as Deep Vent exo-, but still demonstrate sensitive and specific assay performance despite modified bases, because they are ultimately selected after decoration with F and Q. This paper summarizes work in this area and presents some new examples of engineered and naturally selected FRET-aptamers for detection of vitamin D.

When targeting G-protein coupled receptors (GPCRs) in early stage drug discovery, or for novel targets, the type of ligand most likely to produce the desired therapeutic effect may be unknown. Therefore, it can be desirable to identify potential lead compounds from multiple categories: agonists, antagonists, and allosteric modulators. In this study, we developed a triple addition calcium flux assay using FLIPR Tetra to identify multiple ligand classes for the metabotropic glutamate receptor 3 (mGlu3), using a cell line stably co-expressing the human G-protein-coupled mGlu3 receptor, a promiscuous G-protein (Gα16), and rat Glast, a glutamate transporter. Compounds were added to the cells followed by stimulation with EC10 and then EC80 concentration of glutamate, the physiological agonist for mGlu receptors. This format produced a robust assay, facilitating the identification of agonists, positive allosteric modulators and antagonists/negative allosteric modulators. Follow up experiments were conducted to exclude false positives. Using this approach, we screened a library of approximately 800,000 compounds using FLIPR Tetra and identified viable leads for all three ligand classes. Further characterization revealed the selectivity of individual ligands.

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