Combinatorial Chemistry & High Throughput Screening - Volume 11, Issue 4, 2008

This study shows how chemistry knowledge and reasoning are taken into account for building a new methodology that aims at automatically grouping data having a chronological structure. We consider combinatorial catalytic experiments where the evolution of a reaction (e.g., conversion) over time is expected to be analyzed. The mathematical tool has been developed to compare and group curves taking into account their shape. The strategy, which consists on combining a hierarchical clustering with the k-means algorithm, is described and compared with both algorithms used separately. The hybridization is shown to be of great interest. Then, a second application mode of the proposed methodology is presented. Once meaningful clusters according to chemist's preferences and goals are successfully achieved, the induced model may be used in order to automatically classify new experimental results. The grouping of the new catalysts tested for the Heck coupling reaction between styrene and iodobenzene verified the set of criteria “defined” during the initial clustering step, and facilitated a quick identification of the catalytic behaviors following user's preferences.

Symptoms associated with menopause can greatly affect the quality of life for women. Botanical dietary supplements have been viewed by the public as safe and effective despite a lack of evidence indicating a urgent necessity to standardize these supplements chemically and biologically. Seventeen plants were evaluated for estrogenic biological activity using standard assays: competitive estrogen receptor (ER) binding assay for both alpha and beta subtypes, transient transfection of the estrogen response element luciferase plasmid into MCF-7 cells expressing either ER alpha or ER beta, and the Ishikawa alkaline phosphatase induction assay for both estrogenic and antiestrogenic activities. Based on the combination of data pooled from these assays, the following was determined: a) a high rate of false positive activity for the competitive binding assays, b) some extracts had estrogenic activity despite a lack of ability to bind the ER, c) one extract exhibited selective estrogen receptor modulator (SERM) activity, and d) several extracts show additive/synergistic activity. Taken together, these data indicate a need to reprioritize the order in which the bioassays are performed for maximal efficiency of programs involving bioassay-guided fractionation. In addition, possible explanations for the conflicts in the literature over the estrogenicity of Cimicifuga racemosa (black cohosh) are suggested.

Many natural privileged scaffolds contain a basic nitrogen atom, which often is a key element of pharmacophore and a chemically reactive centre as well. In our ongoing research program devoted to the design of targeted libraries based on acidic templates, we developed methods to convert privileged basic compounds -like natural alkaloids or drugs into acidic compounds. This conversion led to a profound alteration of the pharmacophore, without changing the overall shape and lipophilicity of the molecule. We expect such modifications to generate unexpected biological activities. Recently, we focused on derivatives of squaric acid, a vinylogous carboxylic acid. Two series were studied. First we describe a new, selective parallel synthesis of squaramic acids from a dissymmetric diester (3-tert-butoxy-4-ethoxycyclobut- 3-en-1,2-dione). This efficient procedure avoids the synthesis of the undesired squaramides. Secondly we describe a microplate parallel synthesis (15 μmol-scale) of squaric acid hydroxamate amides from a squaric hydroxamate ester.

Generation of in vitro cellular assays using fluorescence measurements at heterologously expressed NMDA receptors would speed up the process of ligand characterization and enable high-throughput screening. The major drawback to the development of such assays is the cytotoxicity caused by Ca2+-flux into the cell via NMDA receptors upon prolonged activation by agonists present in the culture medium. In the present study, we established four cell lines with stable expression of NMDA receptor subtypes NR1/NR2A, NR1/NR2B, NR1/NR2C, or NR1/NR2D in BHK-21 cells. To assess the usefulness of the stable cell lines in conjunction with intracellular calcium ([Ca2+]i) measurements for evaluation of NMDA receptor pharmacology, several ligands were characterized using this method. The results were compared to parallel data obtained by electrophysiological recordings at NMDA receptors expressed in Xenopus oocytes. This comparison showed that agonist potencies determined by [Ca2+]i measurements and electrophysiological recordings correlated well, meaning that the stable cell lines in conjunction with [Ca2+]i measurements provide a useful tool for characterization of NMDA receptor ligands. The agonist series of conformationally constrained glutamate analogues (2S,3R,4S)-α- (carboxycyclopropyl)glycine (CCG), 1-aminocyclobutane-r-1,cis-3-dicarboxylic acid (trans-ACBD), and (±)-1- aminocyclopentane-r-1,cis-3-dicarboxylic acid (cis-ACPD), as well as the highly potent agonist tetrazolylglycine were among the characterized ligands that were assessed with respect to subtype selectivity at NMDA receptors. However, none of the characterized agonists displays more than 2-3 fold selectivity towards a specific NMDA receptor subtype. Thus, the present study provides a broad pharmacological characterization of structurally diverse ligands at recombinant NMDA receptor subtypes.

Continuous identification and validation of novel drug targets require the development of rapid, reliable, and sensitive cell-based high-throughput screening (HTS) methods for proposed targets. Recently, the 5-HT6 receptor (5- HT6R), a member of the class of recently discovered 5-HT receptors, has received considerable attention for its possible implications in depression, cognition, and anxiety. However, the cellular signaling mechanisms of 5-HT6R are poorly understood due to the lack of selective 5-HT6R ligands. In the present study, we examined functional coupling of the human 5-HT6R, 5-HT7AR, or 5-HT7BR with various Gα-proteins (Gα15, Gαqs5, or GαqG66Ds5) to develop a reliable cell-based HTS method for 5-HT receptors. Among variable couplings between 5-HT receptors and G-proteins, we found that functional coupling of human 5-HT6R with GαqG66Ds5 produced the highest levels of Ca2+ signaling in HEK293 cells as measured by the fluorescence-based HTS plate reader, FDSS6000. After validation of this new 5-HT6R HTS system (Z´-factor = 0.56) in 96-well plates and characterization of the pharmacological profile of the 5-HT6R, we screened ∼500 synthetic chemical compounds including butanamide and benzenesulfonamide derivatives. Based on this preliminary screening, we found that the butanamide derivative LSG11104 produced an IC50 value of 6.3 μM. This compound will serve as a lead structure for further chemical modification to develop novel 5-HT6R ligands. Furthermore, we demonstrated that this HTS method can be utilized to identify proteins that modulate 5-HT6R function and present Fyn tyrosine kinase as an example, which is already known as a 5-HT6R interacting protein. Taken together, these results suggest that the 5-HT6R/GαqG66Ds5 FDSS6000 system can be utilized to screen for selective 5-HT6R ligands and to examine any functional relationships between 5-HT6R and its binding proteins.

In the demanding field of proteomics, there is an urgent need for affinity-catcher molecules to implement effective and high throughput methods for analysing the human proteome or parts of it. Antibodies have an essential role in this endeavour, and selection, isolation and characterisation of specific antibodies represent a key issue to meet success. Alternatively, it is expected that new, well-characterised affinity reagents generated in rapid and cost-effective manners will also be used to facilitate the deciphering of the function, location and interactions of the high number of encoded protein products. Combinatorial approaches combined with high throughput screening (HTS) technologies have become essential for the generation and identification of robust affinity reagents from biological combinatorial libraries and the lead discovery of active/mimic molecules in large chemical libraries. Phage and yeast display provide the means for engineering a multitude of antibody-like molecules against any desired antigen. The construction of peptide libraries is commonly used for the identification and characterisation of ligand-receptor specific interactions, and the search for novel ligands for protein purification. Further improvement of chemical and biological resistance of affinity ligands encouraged the “intelligent” design and synthesis of chemical libraries of low-molecular-weight bio-inspired mimic compounds. No matter what the ligand source, selection and characterisation of leads is a most relevant task. Immunological assays, in microtiter plates, biosensors or microarrays, are a biological tool of inestimable value for the iterative screening of combinatorial ligand libraries for tailored specificities, and improved affinities. Particularly, enzyme-linked immunosorbent assays are frequently the method of choice in a large number of screening strategies, for both biological and chemical libraries.