Microchip-based high-throughput screening analysis of combinatorial libraries

In recent years, there have been significant advances in biochemical assay miniturization and integration of microchip-based technologies with combinatorial library screening for high-throughput and large-scale applications. Small-molecule microarrays, protein arrays and cell-based arrays and conventional DNA arrays as well as microfluidic approaches in HTS are discussed in this review.     

A convenient, high-throughput assay for measuring the relative cell permeability of synthetic compounds

We describe here a convenient procedure for assessing the relative cell permeability of chemical compounds. The assay can be used in a high-throughput mode and is particularly applicable for the evaluation of the relative permeability of compounds in a combinatorial library. The compound of interest is conjugated to a dexamethasone derivative. The entry of the conjugate into living mammalian cells triggers the nuclear transport of a Gal4 DNA binding domain-glucocorticoid receptor ligand binding domain-VP16 activation domain fusion protein and, consequently, the activation of a Gal4-responsive luciferase reporter gene. The relative cell permeability is thus described quantitatively by the level of luciferase expression. The experiments take only a few days once chemical synthesis and conjugation are finished.     

A high-throughput pH indicator assay for screening glycosyltransferase saturation mutagenesis libraries

Protein engineering using directed evolution or saturation mutagenesis at hot spots is often used to improve enzyme properties such as their substrate selectivity or stability. This requires access to robust high-throughput assays to facilitate the analysis of enzyme libraries. However, relatively few studies on directed evolution or saturation mutagenesis of glycosyltransferases have been reported in part due to a lack of suitable screening methods. In the present study we report a general screening assay for glycosyltransferases that has been developed using the blood group α-(1→3)-galactosyltransferase (GTB) as a model. GTB utilizes UDP-Gal as a donor substrate and α-L-Fucp-(1→2)-β-D-Galp-O-R (H antigen) as an acceptor substrate and synthesizes the blood group B antigen α-D-Galp-(1→3)-[α-L-Fucp-(1→2)]-β-D-Galp-O-R. A closely related α-(1→3)-N-acetylgalactosaminyltransferase (GTA) uses UDP-GalNAc as a donor with the same H acceptor, yielding the A antigen α-D-Galp-NAc-(1→3)-[α-L-Fuc(1→2)]-β-D-Gal-O-R. GTA and GTB are highly homologous enzymes differing in only 4 of 354 amino acids, Arg/Gly-176, Gly/Ser-235, Leu/Met-266, and Gly/Ala-268. The screening assay is based on the color change of the pH indicator bromothymol blue when a proton is released during the transfer of Gal/GalNAc from UDP-Gal/UDP-GalNAc to the acceptor substrate. Saturation mutagenesis of GTB enzyme at M214, a hot spot adjacent to the ²¹¹DVD²¹³ metal binding motif, was performed and the resulting library was screened for increases in UDP-GalNAc transfer activity. Two novel mutants, M214G and M214S, identified by pH indicator screening, were purified and kinetically characterized. M214S and M214G both exhibited two-fold higher k_cat and specific activity than wild-type GTB for UDP-GalNAc. The results confirm the importance of residue M214 for donor enzyme specificity.     

Circular polymerase extension cloning for high-throughput cloning of complex and combinatorial DNA libraries

High-throughput genomics, proteomics and synthetic biology studies require ever more efficient and economical strategies to clone complex DNA libraries or variants of biological modules. In this paper, we provide a protocol for a sequence-independent approach for cloning complex individual or combinatorial DNA libraries, and routine or high-throughput cloning of single or multiple DNA fragments. The strategy, called circular polymerase extension cloning (CPEC), is based on polymerase overlap extension and is therefore free of restriction digestion, ligation or single-stranded homologous recombination. CPEC is highly efficient, accurate and user friendly. Once the inserts and the linear vector have been prepared, the CPEC reaction can be completed in 10 min to 3 h, depending on the complexity of the gene libraries.     

The one-bead two-compound assay for solid phase screening of combinatorial libraries

Fluorescent quenched substrate libraries are a very powerful tool for investigation of protease activity and specificity. Particularly, libraries where the fluorescent resonance energy transfer (FRET) pair is 3-nitrotyrosine and 2-amino-benzamide are easy to prepare by split and combine synthesis to yield a one-bead one-compound library format. The solid support is critical for the successful hydrolysis of the resin-bound substrates. For this purpose, a range of highly porous poly(ethylene glycol) (PEG)-based resins have been developed. Active substrates yield highly fluorescent beads and these are selected under a fluorescence microscope or isolated on a bead sorter. Edman sequence analysis yields the substrate sequence, the cleavage point, and the degree of conversion. The method gives a complete map of the substrate specificity, and substrates with high affinity for the active site can be selected. These may in turn be used as inhibition indicators in a second solid phase library assay for enzyme inhibition where each single bead is transformed into an assay container. The substrate is attached to temporarily shielded functional groups after completion of inhibitor library synthesis. By using a photolabile linker and ladder synthesis, the active inhibitors may be rapidly identified by mass spectrometry. In each bead, the putative inhibitor competes with the substrate attached for binding to the enzyme, and when the inhibitor binds strongly, the substrate remains intact and quenched. Thus dark beads indicate inhibitors, and these may be isolated using a bead-sorter and the structure determined by mass spectrometry. A selection of the best substrates and inhibitors should always be resynthesized for solution kinetics and confirmation of the results obtained on solid support. The inhibitor assay is almost free from false positives, which is a consequence of combining the binding of the protease to the inhibitor with observation of activity toward a FRET substrate. The K(i) of the identified inhibitors are typically in the nM range.     

A quantitative high-throughput endothelial cell migration assay

By combining the use of BD Biosciences FluoroBlok membrane-based Boyden chambers with the Cellomics HCS ArrayScan, a more sensitive method for measuring cell migration has been developed. This assay is based on counting nuclei of migrated cells on the bottom of the filter rather than conventional approaches, which use measurement of total well fluorescence. This cell migration assay provides approximately 10-fold increased signal/background compared to conventional approaches and can be used to assess the effects of growth factors on endothelial cell migration and to screen chemical compounds for inhibitory effects on growth factor-mediated endothelial cell migration.     

A nonradioactive high-throughput assay for screening and characterization of adenylation domains for nonribosomal peptide combinatorial biosynthesis

Adenylation domains are critical enzymes that dictate the identity of the amino acid building blocks to be incorporated during nonribosomal peptide (NRP) biosynthesis. NRPs display a wide range of biological activities and are some of the most important drugs currently used in clinics. Traditionally, activity of adenylation domains has been measured by radioactive ATP-[³²P]pyrophosphate (PP_i) exchange assays. To identify adenylation domains for future combinatorial production of novel NRPs as potential drugs, we report a convenient high-throughput nonradioactive method to measure activity of these enzymes. In our assay, malachite green is used to measure orthophosphate (P_i) concentrations after degradation by inorganic pyrophosphatase of the PP_i released during aminoacyl-AMP formation by action of the adenylation domains. The assay is quantitative, accurate, and robust, and it can be performed in 96- and 384-well plate formats. The performance of our assay was tested by using NcpB-A₄, one of the seven adenylation domains involved in nostocyclopeptide biosynthesis. The kinetics of pyrophosphate release monitored by this method are much slower than those measured by a traditional ATP-[³²P]PP_i exchange assay. This observation indicates that the formation of the adenylated amino acid and its release are the rate-limiting steps during the catalytic turnover.     

A novel multiplex cell viability assay for high-throughput RNAi screening

Cell-based high-throughput RNAi screening has become a powerful research tool in addressing a variety of biological questions. In RNAi screening, one of the most commonly applied assay system is measuring the fitness of cells that is usually quantified using fluorescence, luminescence and absorption-based readouts. These methods, typically implemented and scaled to large-scale screening format, however often only yield limited information on the cell fitness phenotype due to evaluation of a single and indirect physiological indicator. To address this problem, we have established a cell fitness multiplexing assay which combines a biochemical approach and two fluorescence-based assaying methods. We applied this assay in a large-scale RNAi screening experiment with siRNA pools targeting the human kinome in different modified HEK293 cell lines. Subsequent analysis of ranked fitness phenotypes assessed by the different assaying methods revealed average phenotype intersections of 50.7±2.3%-58.7±14.4% when two indicators were combined and 40-48% when a third indicator was taken into account. From these observations we conclude that combination of multiple fitness measures may decrease false-positive rates and increases confidence for hit selection. Our robust experimental and analytical method improves the classical approach in terms of time, data comprehensiveness and cost.     

A cell death assay for assessing the mitochondrial targeting of proteins

The mitochondrial proteome comprises 1000 to 1500 proteins, in addition to proteins for which the mitochondrial localization is uncertain. About 800 diseases have been linked with mutations in mitochondrial proteins. We devised a cell survival assay for assessing the mitochondrial localization in a high-throughput format. This protocol allows us to assess the mitochondrial localization of proteins and their mutants, and to identify drugs and nutrients that modulate the mitochondrial targeting of proteins. The assay works equally well for proteins directed to the outer mitochondrial membrane, inner mitochondrial membrane mitochondrial and mitochondrial matrix, as demonstrated by assessing the mitochondrial targeting of the following proteins: carnitine palmitoyl transferase 1 (consensus sequence and R123C mutant), acetyl-CoA carboxylase 2, uncoupling protein 1 and holocarboxylase synthetase. Our screen may be useful for linking the mitochondrial proteome with rare diseases and for devising drug- and nutrition-based strategies for altering the mitochondrial targeting of proteins.     

A high-throughput screening assay for small molecules that disrupt yeast cell integrity

Lead compounds for antifungal drug development are urgently needed because invasive fungal infections are an important cause of morbidity and mortality in immunocompromised patients. Here, a high-throughput screening assay for small molecules that cause yeast cell lysis is described. The assay is based on the detection of the intracellular enzyme adenylate kinase in the culture medium as a reporter of yeast cell lysis. Features of the assay protocol include 1) the ability to detect cell lysis at drug concentrations that cause no apparent growth defect, 2) specificity for fungicidal molecules, 3) a simple 1-plate, add-and-read protocol using a commercially available adenylate kinase assay kit, 4) short, 5-h incubation time, and 5) low cost. The assay is applicable to the model yeast Saccharomyces cerevisiae and to Candida albicans, the most common human fungal pathogen. The adenylate kinase assay is validated in a pilot screen of 4505 compounds. Consistent with its specificity for fungicidal molecules, the largest class of molecules identified in 2 libraries of known bioactive molecules targeted the plasma membrane. Fungistatic compounds are not detected by the assay. Adenylate kinase-based screening appears to be a useful approach to the direct identification of small molecules that kill yeast cells. ( Journal of Biomolecular Screening 2008:657-664).     

A comparative evaluation of corneal epithelial cell cultures for assessing ocular permeability

The purpose of this study was to evaluate the potential value of different epithelial cell culture systems as in vitro models for studying corneal permeability. Transformed human corneal epithelial (HCE-T) cells and Statens Serum Institut rabbit corneal (SIRC) cells were cultured on permeable filters. SkinEthic human corneal epithelium (S-HCE) and Clonetics human corneal epithelium (C-HCE) were received as ready-to-use systems. Excised rabbit corneas (ERCs) and human corneas (EHCs) were mounted in Ussing chambers, and used as references. Barrier properties were assessed by measuring transepithelial electrical resistance, and by determining the apparent permeability of markers with different physico-chemical properties, namely, fluorescein, sodium salt; propranolol hydrochloride; moxaverine hydrochloride; timolol hydrogenmaleate; and rhodamine 123. SIRC cells and the S-HCE failed to develop epithelial barrier properties, and hence were unable to distinguish between the permeation markers. Barrier function and the power to differentiate compound permeabilities were evident with HCE-T cells, and were even more pronounced in the case of C-HCE, corresponding very well with data from ERCs and EHCs. A net secretion of rhodamine 123 was not observed with any of the models, suggesting that P-glycoprotein or similar efflux systems have no significant effects on corneal permeability. Currently available corneal epithelial cell culture systems show differences in epithelial barrier function. Systems lacking functional cell-cell contacts are of limited value for assessing corneal permeability, and should be critically evaluated for other purposes.     

A chromatographic tool for preparing combinatorial quinone-thiol conjugate libraries

Quinones are well established as key players in the production of reactive oxygen species within cellular environments. Many factors govern their cytotoxicity but most studies have been restricted to a few, core, derivatives. A new strategy for the in situ production of quinone derivatives has been developed such that libraries of diverse functionality can be rapidly created without recourse to extensive synthetic procedures. The approach relies upon nucleophilic addition by reduced thiol derivatives to the quinone core within a pre-culture assay mixture and provides a generic strategy that exploits the large reservoir of commercial thiols currently available. A readily accessible chromatographic method has been developed that allows the derivatisation process to be easily monitored and the purity of the resulting one pot preparation to be assessed. The viability of the combinatorial approach has been fully validated through comparison with a range of quinone-S-conjugates prepared using conventional bench synthesis. The latter have been fully characterised.     

Bicyclic carbohydrate-derived scaffolds for combinatorial libraries

A bicyclic scaffold derived from the natural monosaccharide d-glucose, and possessing several diversity sites, was linked to various resins through the primary (C-6) hydroxyl and decorated on the solid phase: the hydroxyl group at C-4 was functionalized as ester, ether, and carbamate, the amino group in the second cycle (C-3' position) was functionalized as amide, sulfonamide, and ureido- and thioureido-derivatives. The compounds synthesized on the solid phase were tested for their antiproliferative activity on tumor cell lines.     

Computational design strategies for combinatorial libraries

Medicinal chemistry principles are being increasingly applied to the design of smaller, high purity, information-rich libraries. Recent computational advances in statistical methodology, the design of libraries to reduce ADMET problems, targeting protein families and revisiting natural products as sources of inspiration for scaffolds and reagents are all areas of progressive research.     

Optical detection methods for combinatorial libraries

The main interests in the development of new combinatorial assays are the reduction of time for screening and an increase in the number of samples measured in parallel. The variety of detection methods is increasing, but the optimal one has not yet been determined. In the past two years, the first parallel detection methods for non-labelled compounds have been developed.     

A fluorescence-based high-throughput assay for antimicrotubule drugs

With the advent of combinatorial chemistry and the extensive libraries of potential drugs produced from it, there is a growing need for rapid sensitive, high-throughput screening for drug potency. Microtubules are important targets for anticancer agents, and new antimicrotubule compounds are of continued interest in drug development. The in vitro potency of antimicrotubule drugs may be evaluated by measuring the extent of tubulin assembly. The extent of polymerization is proportional to the turbidity of the solution, which usually has been measured as apparent absorption. The turbidity method has inherent problems that hinder its adaptation to a high-throughput format, such as a requirement for high protein concentrations and a high coefficient of variation. We present here a high-throughput assay for antimicrotubule activity in which fluorescence is used to monitor microtubule assembly. Both assembly-inhibiting and assembly-promoting compounds can be evaluated. The assay is rapid and easy to perform, and the data are reliable, with good accuracy and reproducibility.     

A fluorescence-based glycosyltransferase assay for high-throughput screening

Glycosyltransferases catalyze the transfer of a monosaccharide unit from a nucleotide or lipid sugar donor to polysaccharides, lipids, and proteins in a stereospecific manner. Considerable effort has been invested in engineering glycosyltransferases to diversify sugar-containing drugs. An important requirement for glycosyltransferase engineering is the availability of a glycosyltransferase assay system for high-throughput screening of glycosyltransferase mutants. In this study, a general glycosyltransferase assay system was developed based on an ATP sensor. This system showed submicromolar sensitivity and compatibility with both purified enzymes and crude cell extracts. The assay system will be useful for glycosyltransferase engineering based on high-throughput screening, as well as for general glycosyltransferase assays and kinetics.     

A high-throughput capillary assay for bacterial chemotaxis

We present a high-throughput capillary assay in order to characterize the chemotactic response of the E. coli bacterium. We measure the number of organisms attracted into an array of 96 capillary tubes containing the attractant L-aspartate. The effect of bacterial concentration on the chemotactic response is reported. Such high-throughput assay can be used to characterize bacterial chemotaxis function of a wide range of biochemical parameters.     

A high-throughput 3-parameter flow cytometry-based cell death assay.

Apoptosis plays a role in many disease states, and the evaluation of novel therapeutics that alters the apoptotic cascade is an area of intense investigation. However, many generally available methods to evaluate cell death are either time consuming, imprecise, or both. We report a system that permits simultaneous evaluation of three apoptotic markers (cell membrane integrity, mitochondrial membrane potential, and cell cycle progression) with minimal technical manipulation. This system is particularly well-suited for toxicologic evaluation of novel compounds and profiling of new apoptosis-inducing agents.