Combinatorial Chemistry & High Throughput Screening - Volume 13, Issue 1, 2010

After serving as Editor-in-Chief of Combinatorial Chemistry & High Throughput Screening since the beginning of its publication in 1998 and overseeing the first 12 volumes, I am stepping down from this position with the beginning of CCHTS volume 13. Since I will remain on the Editorial Board, I will still be involved with CCHTS. The new Editor-in-Chief of CCHTS will be Dr. Rathnam Chaguturu who directs the High Throughput Screening Laboratories at the University of Kansas. Since the publication of manuscripts takes several months and includes peer review, revision and proof reading under the oversight of the Editor-in-Chief followed by redacting, printing and publication by the production department, most of the articles in the next several issues of CCHTS will still have been prepared under my oversight. Gradually during the year, the transition to the new Editor-in-Chief will become complete. In a 2007 editorial [1], I reviewed the early years of CCHTS and listed the top ten most cited papers from the first 10 years of publication. It seems appropriate to update the list of our ten most cited papers since there have been some changes. At the top of the list this year is a paper from the laboratory of Prof. John Pezzuto concerning the screening of natural products for antioxidants. The honor of being first is appropriate since Prof. Pezzuto was involved in the founding of CCHTS. The second and tenth most cited papers in our top-ten list concern peptides and peptide therapeutic agents. In the first of these papers, Schulz-Knappe and colleagues coined the term “peptidomics” as a corollary to proteomics, recognizing that many peptides are biochemically, pharmacologically and therapeutically important. The other paper by Lohner and Blondelle is new to this list at position 10 and describes the rational design of peptide antibiotic agents targeting bacterial membranes. The popularity of these articles is a testament to the importance of proteomics and biological therapeutic agents in biomedical research today. The articles occupying the third and fifth positions of our top-ten list review the use of multicomponent reactions such as the Ugi reaction in combinatorial synthesis. It is no surprise that Prof. Ugi is an author of one of these review articles and his former student Prof. Domling is author of the other paper. The article by Xue and Bajorath rose from fifth to fourth place and is the only article on the top ten list addressing virtual screening, which has been a popular topic for many articles published in CCHTS. The paper by Naser and Jolley reviews the use of fluorescence polarization in high-throughput screening, which has been another popular topic. At position six for the second time in a row, the paper by Masimirembwa, et al., reviews how high throughput screening is being applied to drug development with a focus on drug metabolism. This is an important step in bringing new drugs to clinical trials that can be a bottleneck and a frequent cause of lead compound failure. The articles occupying positions eight and nine are new to the top ten list this year. The article by DeSimone, et al., at position nine concerns the discovery of scaffolds known as privileged structures which usually exhibit good drug-like properties and are useful for drug discovery. Finally, the paper by Yarrow and co-workers describes the use of fluorescent probes and cell imaging to screen combinatorial libraries for physiological changes in cells. The wide range of topics represented by these articles, which cover combinatorial synthesis, virtual screening, high-throughput screening, and drug development, is faithful to the scope and purpose of CCHTS which is to publish articles on all aspects of combinatorial chemistry and high-throughput screening. When my grandfather John van Breemen emigrated from the Netherlands to the Unites States of America more than 100 years ago, he became an apprentice in a Chicago print shop. He said that he learned English by reading what he printed. Later, my grandfather ran his own print shop in which my father learned the trade, too. Although my father became a university professor, he occasionally moonlighted in a print shop and sometimes let me help. Because of my heritage in printing, I have taken great pride in my job as editor of CCHTS. During the first several years of production, much of the correspondence for CCHTS was carried out by mail, telephone and Fax. Authors had to submit their articles in both print and electronic formats. Although the peer reviewed and accepted versions of all figures and text were submitted to our printing department in electronic form, it was still necessary to supply printed copies and photo ready art in case of problems with the electronic versions. I even had to mail a color slide of the cover art for each issue to our production department. After an issue went to press, only printed reprints were available in the early years, and electronic copies were not supplied. In fact, reprints of the first two volumes are CCHTS are still not available in electronic form. Gradually, the transition to electronic media was completed, and today, no printed material or slides need to be submitted by authors. The peer review process is also carried out electronically. Today, the accepted manuscripts, figures and even the cover art are transmitted electronically to our production department. Articles are available for download via the internet as soon as the printed versions are released. By the time my successor retires from this position, the print versions of CCHTS and perhaps most scientific journals might have been discontinued. I would like to thank all the members of the Editorial Board of CCHTS, most of whom have served since the formation of this journal, for providing their time and expertise in the peer review of articles under consideration for publication. I would also like to thank Regional Editors Dr. Andy Merritt, Prof. Peter Nielsen and Prof. Ikuo Fujii for handling the peer review of countless manuscripts of the years. Of course I am most grateful for the service of our publications department whose names do not appear on the inside cover of CCHTS and whose members include the printers of CCHTS.

Stimulation of CXC-type chemokine receptor 2 (CXCR2)-transfected cells by Gro-α or IL-8 induced (i) CXCR2 internalization, (ii) phosphorylation of ERK1/2 (pERK) and (iii) translocation of nuclear factor of activated T-cells (NFAT) into the nucleus. Employing high content screening (HCS; i.e. fluorimetric imaging combined with image analysis) these three ligand-induced events were quantified by using a CXCR2-specific antibody, an antibody recognizing phosphorylated ERK1/2 (pERK) and a red fluorescent protein (RFP) in fusion to transiently overexpressed NFAT, respectively. As an RFP, we applied a recently developed mutant of an Entacmaea quadricolor fluorescent protein with favorable properties for HCS, such as high fluorescence brightness, photostability, large Stokes shift, and stability with regard to formaldehyde. Receptor internalization was closely coupled with ERK signalling both when analyzed in regard of stimulation by physiological CXCR2 ligands and when observed in the presence of antagonistic test compounds. A means of increasing the throughput or of broadening the pharmacological characterization of test compounds is the use of multiplexed imaging. Indeed, CXCR2 internalization could be multiplexed with the NFAT nuclear translocation by fixation at ˜ 45 min after Gro-α stimulation. This multiplexing demonstrated that Gro-α-induced CXCR2 internalization was tightly correlated with Gro-α-induced NFAT translocation, also on the single cell level. The analysis of ERK phosphorylation, NFAT translocation and receptor internalization enabled the profiling of antagonistic test compounds with respect to G-protein signalling and possible receptor desensitization liabilities.

A dynamic, focused screening strategy that utilized a limited but diversified set of target-specific compounds was explored as an efficient means for the identification of inhibitors of the protein kinase PDK1. Approximately 21,500 compounds, including a 19,000 molecule kinase-focused compound collection (KFCC), were screened at two concentrations to identify initial leads. The KFCC included several empirically-derived, general kinase libraries and molecules chosen by PDK1-specific virtual screens. As was expected, this initial screen mostly identified potent leads with limited novelty. In order to overcome this limitation, the data from the screen were used to drive several rounds of a customized iterative focused screening (IFS) campaign. A machine-learning technique was used to build a predictive model to identify compounds to be screened in subsequent rounds. Molecules deemed not to be novel were removed from the training set for the next round, which allowed this campaign to progressively walk away from the chemical space covered by the KFCC. This resulted in the identification of PDK1 inhibitors which are uniquely different from publicly known chemotypes after just three rounds of screenings. A retrospective analysis of this IFS approach against an ultrahigh throughput screen (uHTS) indicated that while uHTS is still the most prolific paradigm for lead identification, this dynamic, focused screening approach was successful in discovering novel scaffolds for a medicinal chemistry effort. Finally, a theoretical optimization suggested the dynamic, focused screening approaches could provide either a complementary or alternative approach to uHTS for the efficient and rapid lead identification.

The malachite green assay is often used for measuring the presence of inorganic mono-phosphate concentrations. Some studies have adapted this assay for use in monitoring enzymatic reactions and have suggested its potential use in high throughput screening (HTS). With the increasing availability of laboratory automation, some studies are starting to explore the possibility of conducting limited, semi-automated versions of the assay. Here we report the optimization and complete adaptation of the malachite green assay to a fully automated, HTS platform that can be performed unattended with standard, commercially available, automated liquid-handling systems. The assay is universal for the majority of enzymes that release phosphate or pyrophosphate. Moreover, the assay is fully scalable from smaller drug screening efforts (˜ 20,000 wells per day) to ultra-high throughput environments (˜ 200,000 wells per day). The assay uses cost-effective, commercially available reagents, and can be used to perform automated IC50 value and kinetic parameter determination. Finally, we demonstrate the utility of the assay via the initial, primary screening of 100,080 compounds against two target enzymes from Bacillus anthracis, O-succinylbenzoyl-CoA synthetase and nicotinate mononucleotide adenylyltransferase.

A quantitative structure &ndash property relationship (QSPR) was used to predict the critical micelle concentration (cmc) of nonionic surfactants. The relation was developed for a diverse set of 23 nonionic surfactants (r = 0.99, F-test = 1042.5) employing the connectivity and valence connectivity indices only. The molecular connectivity indices were calculated for the whole molecule which was a simple and general approach.

With the increasingly available technology in automated synthesis and screening protocols, the combination of polymer-supported chemistry and biocatalysis, with their respective advantages over classical organic synthesis, has become more scientifically attractive, yet remains challenging. Since the development of solid-phase synthesis and its rapid expansion in combination with the advent of combinatorial techniques, a variety of alternative methodologies have been proposed and demonstrated to be viable for applications in high-throughput and multistep syntheses of the desired products. These alternative methodologies overcome the disadvantages of crosslinked polymer beads, which, as a consequence of their insolubility and their being necessarily heterogeneous in the reaction mixture, do have operational drawbacks. They often rely on a common strategy: tagging the target substrate of interest with other fragments (fluorous synthons, macromolecules, “precipitons”) in such a way that the tag-substrate covalent ensemble is then easily separated from the reaction mixture by physical methods (liquid-liquid extraction, precipitation, etc.). The efficiency of enzymes in transforming substrates is often enhanced when the stability limitations of the biocatalyst in unnatural conditions (i.e. organic solvents, high temperatures) are avoided by the use of immobilization-stabilization techniques. We comment here, with recent examples, on the use of linear macromolecules as recyclable tags capable of acting as covalent supports in combination with a biocatalyzed reaction.

Nature, especially the plant kingdom, is a rich source for novel bioactive compounds that can be used as lead compounds for drug development. In order to exploit this resource, the two neural network-based virtual screening techniques novelty detection with self-organizing maps (SOMs) and counterpropagation neural network were evaluated as tools for efficient lead structure discovery. As application scenario, significant descriptors for acetylcholinesterase (AChE) inhibitors were determined and used for model building, theoretical model validation, and virtual screening. Topranked virtual hits from both approaches were docked into the AChE binding site to approve the initial hits. Finally, in vitro testing of selected compounds led to the identification of forsythoside A and (+)-sesamolin as novel AChE inhibitors.

Nanoparticles of 35 individual metals as well as their binary combinations were synthesized using High Throughput pulsed laser ablation (PLA), and collected on Al2O3, CeO2, SiO2, TiO2, and ZrO2 pellets. These materials were then screened for their catalytic activities and selectivities for the partial oxidation of propylene, in particular for propylene oxide (PO), using array channel microreactors. Reaction conditions were the following: 1 atm pressure, gas hourly space velocity (GHSV) of 20,000 h-1, temperature 300 C, 333 C, and 367 C, and feed gas composition 20 vol% O2, 20 vol% C3H6 and balance He. Initial screening experiments resulted in the discovery of SiO2 supported Cr, Mn, Cu, Ru, Pd, Ag, Sn, and Ir as the most promising leads for PO synthesis. Subsequent experiments pointed to bimetallic Cu-on- Mn/SiO2, for which the PO yields increased several fold over single metal catalysts. For multimetallic materials, the sequence of deposition of the active metals was shown to have a significant effect on the resulting catalytic activity and selectivity.

Phage-display selection of combinatorial libraries is a powerful technique for identifying binding ligands against desired targets. Evaluation of target binding capacity of multiple clones recovered from phage display selection to a specific target is laborious, time-consuming, and a rate-limiting step. We constructed phage-display combinatorial peptide libraries in fusion with a β-lactamase enzyme, which acts as a reporter. Linear dodecapeptide and cysteineconstrained decapeptide libraries were created at the amino-terminus of the Enterobacter cloacae P99 cephalosporinase molecule (P99 β-lactamase). The overall and positional diversity of amino acids in both libraries was similar to other phage-display systems. The libraries were selected against the extracellular domain of ErbB2 receptor (ErbB2ECD). The target-selected clones were already conjugated to an enzyme reporter, therefore, did not require subcloning or any other post-panning modifications. We used β-lactamase enzyme activity-based assays for sample normalizations and clone binding evaluation. Clones were identified that bound to purified ErbB2ECD and ErbB2-overexpressing cell-lines. The peptide sequences of the selected binding clones shared significant motifs with several rationally designed peptide mimetics and phage-display derived peptides that have been reported to bind ErbB2ECD. β-Lactamase fusion to peptides saved time and resources otherwise required by the phage-ELISA of a typical phage display screening protocol. The β- lactamase enzyme assay protocols is a one-step process that does not require secondary proteins, several steps of lengthy incubations, or washings and can be finished in a few minutes instead of hours. The clone screening protocol can be adopted for a high throughput platform. Target-specific ??-lactamase-linked affinity reagents selected by this procedure can be produced in bulk, purified, and used, without any modification, for a variety of downstream applications, including targeted prodrug therapy.