Andrew J. Pope

High-throughput screening: new technology for the 21st century

New technologies in high-throughput screening have significantly increased throughput and reduced assay volumes. Key advances over the past few years include new fluorescence methods, detection platforms and liquid-handling technologies. Screening 100,000 samples per day in miniaturized assay volumes will soon become routine. Furthermore, new technologies are now being applied to information-rich cell-based assays, and this is beginning to remove one of the key bottlenecks downstream from primary screening.

The antimicrobial natural product chuangxinmycin and some synthetic analogues are potent and selective inhibitors of bacterial tryptophanyl tRNA synthetase.

The antimicrobial natural product chuangxinmycin has been found to be a potent and selective inhibitor of bacterial tryptophanyl tRNA synthetase (WRS). A number of analogues have been synthesised. The interaction with WRS appears to be highly constrained, as only sterically smaller analogues afforded significant inhibition. The only analogue to show inhibition comparable to chuangxinmycin also had antibacterial activity. WRS inhibition may contribute to the antibacterial action of chuangxinmycin.

Real Experiences of uHTS: A Prototypic 1536-Well Fluorescence Anisotropy-Based uHTS Screen and Application of Well-Level Quality Control Procedures

This paper describes, for the first time, a true ultra-high throughput screen (uHTS) based upon fluorescence anisotropy and performed entirely in 1536-well assay plates. The assay is based upon binding and displacement of a BODIPY-FL-labeled antibiotic to a specific binding site on 70S ribosomes from Escherichia coli (Kd 15 nM). The screen was performed at uHTS rates (i.e., >100,000 assay wells/24 h) using entirely commercially available equipment. In order to examine the reproducibility of detection of test compound effects, assays were performed in duplicate. Both overall assay statistics and reproducibility for individual compound results were excellent, at least equivalent to conventional HTS assays. Interference artifacts occurred mainly as a result of autofluorescence from test compounds. Well-level quality control procedures were developed to detect, eliminate, or even correct for such effects. Moreover, development of a brighter, longer wavelength probe (based upon Cy3B) markedly reduced such interferences. Overall, the data demonstrate that fluorescence anisotropy-based uHTS is now a practical reality.

Development and Validation of Reagents and Assays for EZH2 Peptide and Nucleosome High-Throughput Screens

Histone methyltransferases (HMT) catalyze the methylation of histone tail lysines, resulting in changes in gene transcription. Misregulation of these enzymes has been associated with various forms of cancer, making this target class a potential new area for the development of novel chemotherapeutics. EZH2 is the catalytic component of the polycomb group repressive complex (PRC2), which selectively methylates histone H3 lysine 27 (H3K27). EZH2 is overexpressed in prostate, breast, bladder, brain, and other tumor types and is recognized as a molecular marker for cancer progression and aggressiveness. Several new reagents and assays were developed to aid in the identification of EZH2 inhibitors, and these were used to execute two high-throughput screening campaigns. Activity assays using either an H3K27 peptide or nucleosomes as substrates for methylation are described. The strategy to screen EZH2 with either a surrogate peptide or a natural substrate led to the identification of the same tractable series. Compounds from this series are reversible, are [3H]-S-adenosyl-L-methionine competitive, and display biochemical inhibition of H3K27 methylation.

Developments in fluorescence lifetime-based analysis for ultra-HTS

Abstract Homogeneous fluorescence detection methods are proving key in the establishment of miniaturized ultra-HTS (uHTS). Fluorescence lifetime-based methods seem set to provide an additional dimension to these approaches and are likely to represent a significant further advance both in terms of increasing measurement robustness and in opening up completely new types of uHTS assay format.

Single-Molecule Detection Technologies in Miniaturized High-Throughput Screening: Fluorescence Intensity Distribution Analysis

Single-molecule detection technologies are becoming a powerful readout format to support ultra-high-throughput screening. These methods are based on the analysis of fluorescence intensity fluctuations detected from a small confocal volume element. The fluctuating signal contains information about the mass and brightness of the different species in a mixture. The authors demonstrate a number of applications of fluorescence intensity distribution analysis (FIDA), which discriminates molecules by their specific brightness. Examples for assays based on brightness changes induced by quenching/dequenching of fluorescence, fluorescence energy transfer, and multiple-binding stoichiometry are given for important drug targets such as kinases and proteases. FIDA also provides a powerful method to extract correct biological data in the presence of compound fluorescence. (Journal of Biomolecular Screening 2003:19-33)

Conformational restriction of methionyl tRNA synthetase inhibitors leading to analogues with potent inhibition and excellent gram-positive antibacterial activity.

Conformationally restricted analogues of the central linker unit of bacterial methionyl tRNA synthetase (MRS) inhibitors have been prepared. The (1S,2R)-cyclopentylmethyl moiety was identified as the preferred cyclic linker, with significant diastereo- and enantioselectivity of activity. Combination of this linker with an optimal substituted aryl right-hand side has resulted in a compound with exceptionally good antibacterial activity against staphylococci and enterococci, including antibiotic resistant strains.

Cell-Based Selection Expands the Utility of DNA-Encoded Small-Molecule Library Technology to Cell Surface Drug Targets: Identification of Novel Antagonists of the NK3 Tachykinin Receptor

DNA-encoded small-molecule library technology has recently emerged as a new paradigm for identifying ligands against drug targets. To date, this technology has been used with soluble protein targets that are produced and used in a purified state. Here, we describe a cell-based method for identifying small-molecule ligands from DNA-encoded libraries against integral membrane protein targets. We use this method to identify novel, potent, and specific inhibitors of NK3, a member of the tachykinin family of G-protein coupled receptors (GPCRs). The method is simple and broadly applicable to other GPCRs and integral membrane proteins. We have extended the application of DNA-encoded library technology to membrane-associated targets and demonstrate the feasibility of selecting DNA-tagged, small-molecule ligands from complex combinatorial libraries against targets in a heterogeneous milieu, such as the surface of a cell.

Potent synthetic inhibitors of tyrosyl tRNA synthetase derived from C-pyranosyl analogues of SB-219383.

Novel pyranosyl analogues of SB-219383 have been synthesised to elucidate the structure-activity relationships around the pyran ring. Analogues with highly potent stereoselective and bacterioselective inhibition of bacterial tyrosyl tRNA synthetase have been identified. A major reduction in the overall polarity of the molecule can be tolerated without loss of the nanomolar level of inhibition.

Perspectives on the Discovery of Small-Molecule Modulators for Epigenetic Processes

Epigenetic gene regulation is a critical process controlling differentiation and development, the malfunction of which may underpin a variety of diseases. In this article, we review the current landscape of small-molecule epigenetic modulators including drugs on the market, key compounds in clinical trials, and chemical probes being used in epigenetic mechanistic studies. Hit identification strategies for the discovery of small-molecule epigenetic modulators are summarized with respect to writers, erasers, and readers of histone marks. Perspectives are provided on opportunities for new hit discovery approaches, some of which may define the next generation of therapeutic intervention strategies for epigenetic processes.