Impact of high-throughput screening in biomedical research.

High-throughput screening (HTS) has been postulated in several quarters to be a contributory factor to the decline in productivity in the pharmaceutical industry. Moreover, it has been blamed for stifling the creativity that drug discovery demands. In this article, we aim to dispel these myths and present the case for the use of HTS as part of a proven scientific tool kit, the wider use of which is essential for the discovery of new chemotypes.

A generic fast solid-phase extraction high-performance liquid chromatography/mass spectrometry method for high-throughput drug discovery.

High-performance liquid chromatography/mass spectrometry (HPLC/MS) is increasingly perceived to be an essential tool in drug discovery at many key steps, like drug screening, lead identification, ADME profiling, and drug metabolism and pharmacology studies. High-throughput screenings in the early phase for metabolic stability, protein binding, permeability (ADME) and bioavailability are widely used to weed out compounds that do not exhibit the necessary characteristics. For such high-throughput LC/MS studies, a generic LC/MS method that can be used for a variety of compounds is desired. In this study, we used a small set of compounds with a wide range of properties to guide method development, and achieved a sample throughput of 1.7 min/sample. Here, we present a generic fast method that achieves good peak separation and peak shape on conventional HPLC systems using a column-switching mechanism for on-line solid-phase extraction (SPE)-HPLC/MS analysis. The method has a linear response range from 1 to 500 nM for the tested compounds. When a larger set of 658 randomly picked small molecules were analyzed using this method, 612 were observed with good signal intensity and HPLC peak shapes. This generic fast SPE-LC/MS method has been used to screen more than 1.5 million compounds repetitively against over 200 protein targets for hit confirmation and semi-quantitation of binding constants from biological assays. Over 7000 different compounds for a variety of protein-binding assays have been studied using this method for quantitative analysis as well.

An ultraefficient affinity-based high-throughout screening process: application to bacterial cell wall biosynthesis enzyme MurF.

The authors describe the discovery of a new class of inhibitors to an essential Streptococcus pneumoniae cell wall biosyn-thesis enzyme, MurF, by a novel affinity screening method. The strategy involved screening very large mixtures of diverse small organic molecules against the protein target on the basis of equilibrium binding, followed by iterative ultrafiltration steps and ligand identification by mass spectrometry. Hits from any affinity-based screening method often can be relatively nonselective ligands, sometimes referred to as "nuisance" or "promiscuous" compounds. Ligands selective in their binding affinity for the MurF target were readily identified through electronic subtraction of an empirically determined subset of promiscuous compounds in the library without subsequent selectivity panels. The complete strategy for discovery and identification of novel specific ligands can be applied to all soluble protein targets and a wide variety of ligand libraries.

Kinase drug discovery by affinity selection/mass spectrometry (ASMS): application to DNA damage checkpoint kinase Chk1.

Kinase enzymes are involved in a vast array of biological processes associated with human disease; therefore, selective kinase inhibition by small molecules and therapeutic antibodies is an area of intense study. The authors show that drug candidates with immediate value for biological preclinical evaluation can be identified directly through ultra-efficient affinity screening of kinase enzymes and random compound mixtures. The screening process comprises sampling and trapping equilibrium binding between candidate ligands and protein in solution, followed by removal of unbound ligands via 3 rounds of ultrafiltration and direct identification of bound ligands by mass spectrometry. Evaluation of significant peaks is facilitated by automated integration and collation of the mass spectral data and import into custom software for analysis. One Chk1-selective ligand found by using this process is presented in detail. The compound is potent in both enzymatic and Chk1-dependent cellular assays, and specific contacts in the Chk1 active site are shown by X-ray crystallography.

An offline-addition format for identifying GPCR modulators by screening 384-well mixed compounds in the FLIPR.

Although fluorescence imaging plate reader (FLIPR)-based assays have been widely used in high-throughput screening, improved efficiencies in throughput and fidelity continue to be investigated. This study presents an offline compound addition protocol coupled with a testing strategy using mixtures of compounds in a 384-well format to identify antagonists of the neurokinin-1 receptor expressed in the human astrocytoma cell line (U373 MG). Substance P evoked a concentration-dependent increase in intracellular cellular Ca(2+) with an EC(50) value of 0.30 +/- 0.17 nM, which was inhibited by neurokinin-1 (NK1) antagonists L-733,060 and L-703,606. Test compounds, as mixtures of 10 compounds/well, were added to the cells offline using an automated dispensing unit and incubated prior to performing the assay in the FLIPR. Using the offline protocol, a higher through put of ~200,000 compounds was achieved in an 8-h working day, and several novel structural classes of compounds were identified as antagonists for the NK1 receptor. These studies demonstrate that the offline compound addition format using a mixture of compounds in a 384-well FLIPR assay provides an efficient platform for screening and identifying modulators for G-protein-coupled receptors.

A cell-based microarrayed compound screening format for identifying agonists of G-protein-coupled receptors.

The identification of agonist and antagonist leads for G-protein-coupled receptors (GPCRs) is of critical importance to the pharmaceutical and biotechnology industries. We report on the utilization of a novel, high-density, well-less screening platform known as microarrayed compound screening microARCS) that tests 8640 compounds in the footprint of a standard microtiter plate for the identification of novel agonists for a specific G-protein-coupled receptor. Although receptors coupled to the G alpha(q) protein can readily be assessed by fluorescence-based Ca(2+) release measurements, many GPCRs that are coupled to G alpha(s) or G alpha(i/o) proteins are not amenable to functional evaluation in such a high-throughput manner. In this study, the human dopamine D(4.4) receptor, which normally couples through the G alpha(i/o) protein to inhibit adenylate cyclase and to reduce levels of intracellular cAMP, was coupled to intracellular Ca(2+) release by stably coexpressing this receptor with a chimeric G(alpha qo5) protein in HEK-293 cells. In microARCS format, the cells expressing D(4.4) receptor and G alpha(qo5) protein were preloaded with fluo-4, cast into a 1% agarose gel, placed above the compound sheets, and imaged successively using a ViewLux charge-coupled device imaging system. Dopamine and other agonists evoked an increase in fluorescence response that appeared as bright spots in a time- and concentration-dependent manner. Utilizing this technology, a library of 260,000 compounds was rapidly screened and led to the identification of several novel agonists. These agonists were further characterized using a fluorometric imaging plate reader assay. Excellent confirmation rates coupled with enhanced efficiency and throughput enable microARCS to serve as an alternative platform for the screening and identification of novel GPCR agonists.

Identification and characterization of novel antagonists of the CCR3 receptor.

Eotaxin, an inducer of eosinophil migration and activation, exerts its activity by binding to CCR3, the C-C chemokine receptor 3. An inhibitor of the eotaxin-CCR3 binding interaction may have potential as an anti-inflammatory drug for treatment of asthma, parasitic infections, and allergic disorders. A radioligand binding assay was developed using HEK cells transfected with CCR3, with (125)I eotaxin as the ligand. Whole cells grown on polylysine-coated plates were used as the receptor source for the screen. Screening of more than 200,000 compounds with this assay yielded a number of screening hits, and of these, 2 active novel antagonists were identified. These compounds showed inhibitory effects on eosinophil chemotaxis in both in vitro and in vivo assays.

Integration of NMR and high-throughput screening.

NMR-based screening has become a powerful method for the identification and analysis of low-molecular weight organic compounds that bind to protein targets and can be utilized in drug discovery programs. In particular, heteronuclear NMR-based screening can yield information about both the affinity and binding location of potential lead compounds. In addition, heteronuclear NMR-based screening has wide applications in complementing and facilitating conventional high-throughout screening programs. This article will describe several strategies for the integration of NMR-based screening and high-throughput screening. The marriage of these two techniques promises to be of tremendous benefit in the triage of hits that come from HTS, and can aid the medicinal chemist in the identification of quality leads that have high potential for further optimization.

Application of Micro Arrayed Compound Screening (microARCS) to identify inhibitors of caspase-3.

Micro Arrayed Compound Screening (microARCS) is a miniaturized ultra-high-throughput screening platform developed at Abbott Laboratories. In this format, 8,640 discrete compounds are spotted and dried onto a polystyrene sheet, which has the same footprint as a 96-well plate. A homogeneous time-resolved fluorescence assay format (LANCE) was applied to identify the inhibitors of caspase-3 using a peptide substrate labeled with a fluorescent europium chelate and a dabcyl quencher. The caspase-3 enzyme was cast into a thin agarose gel, which was placed on a sheet containing test compounds. A second gel containing caspase substrate was then laid above the enzyme gel to initiate the reaction. Caspase-3 cleaves the substrate and separates the europium from the quencher, giving rise to a time-resolved fluorescent signal, which was detected using a ViewLux charge-coupled device imaging system. Potential inhibitors of caspase-3 appeared as dark spots on a bright fluorescent background. Results from the microARCS assay format were compared to those from a conventional 96-well plate-screening format.