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.     

Identification of potential Leptospira phosphoheptose isomerase inhibitors through virtual high-throughput screening

The hfe-threatening infections caused by Leptospira serovars demand the need for designing anti-leptospirosis drugs.The present study encompasses exploring inhibitors against phosphoheptose isomerase(GmhA)of Leptospira,which is vital for lipopolysaccharide(LPS)biosynthesis and is identified as a common drug target through the subtractive genomic approach.GmhA model was built in Modeller 9v7.Structural refinement and energy minimization of the predicted model was carried out using Maestro 9.0.The refined model reliability was assessed through Procheck,ProSA,ProQ and Profile 3D.The substrate-based virtual high-throughput screening(VHTS)in Ligand.Info Meta-Database tool generated an in-house library of 354 substrate structural analogs.Furthermore,structure-based VHTS from the in-house library with different conformations of each ligand provided 14 novel competitive inhibitors.The model together with insight gained from the VHTS would be a promising starting point for developing anti-leptospirosis competitive inhibitors targeting LPS biosynthesis pathway.     

Discovery of platelet-type 12-human lipoxygenase selective inhibitors by high-throughput screening of structurally diverse libraries

Human lipoxygenases (hLO) have been implicated in a variety of diseases and cancers and each hLO isozyme appears to have distinct roles in cellular biology. This fact emphasizes the need for discovering selective hLO inhibitors for both understanding the role of specific lipoxygenases in the cell and developing pharmaceutical therapeutics. To this end, we have modified a known lipoxygenase assay for high-throughput (HTP) screening of both the National Cancer Institute (NCI) and the UC Santa Cruz marine extract library (UCSC-MEL) in search of platelet-type 12-hLO (12-hLO) selective inhibitors. The HTP screen led to the characterization of five novel 12-hLO inhibitors from the NCI repository. One is the potent but non-selective michellamine B, a natural product, anti-viral agent. The other four compounds were selective inhibitors against 12-hLO, with three being synthetic compounds and one being alpha-mangostin, a natural product, caspase-3 pathway inhibitor. In addition, a selective inhibitor was isolated from the UCSC-MEL (neodysidenin), which has a unique chemical scaffold for a hLO inhibitor. Due to the unique structure of neodysidenin, steady-state inhibition kinetics were performed and its mode of inhibition against 12-hLO was determined to be competitive (K(i)=17microM) and selective over reticulocyte 15-hLO-1 (K(i) 15-hLO-1/12-hLO>30).     

Reporting data from high-throughput screening of small-molecule libraries

Publications reporting results of small-molecule screens are becoming more common as academic researchers increasingly make use of high-throughput screening (HTS) facilities. However, no standards have been formally established for reporting small-molecule screening data, and often key information important for the evaluation and interpretation of results is omitted in published HTS protocols. Here, we propose concise guidelines for reporting small-molecule HTS data.     

Identification and design of novel small molecule inhibitors against MERS-CoV papain-like protease via high-throughput screening and molecular modeling

The development of new therapeutic agents against the coronavirus causing Middle East Respiratory Syndrome (MERS) is a continuing imperative. The initial MERS-CoV epidemic was contained entirely through public health measures, but episodic cases continue, as there are currently no therapeutic agents effective in the treatment of MERS-CoV, although multiple strategies have been proposed. In this study, we screened 30,000 compounds from three different compound libraries against one of the essential proteases, the papain-like protease (PL^pro), using a fluorescence-based enzymatic assay followed by surface plasmon resonance (SPR) direct binding analysis for hit confirmation. Mode of inhibition assays and competition SPR studies revealed two compounds to be competitive inhibitors. To improve upon the inhibitory activity of the best hit compounds, a small fragment library consisting of 352 fragments was screened in the presence of each hit compound, resulting in one fragment that enhanced the IC₅₀ value of the best hit compound by 3-fold. Molecular docking and MM/PBSA binding energy calculations were used to predict potential binding sites, providing insight for design and synthesis of next-generation compounds.     

Comparative high-throughput RNAi screening methodologies in C. elegans and mammalian cells

The discovery of RNAi in Caenorhabditis elegans has generated a paradigm shift in how research is performed. Targeted gene knockdown using high throughput screening approaches is becoming a routine feature of the scientific landscape, and researchers can now evaluate the function of each gene in the genome in a relatively short period of time. This review compares and contrasts high throughput screening methodologies in C. elegans and mammalian cells and highlights the breadth of applications of this technology.     

Identification of inhibitors of N5-carboxyaminoimidazole ribonucleotide synthetase by high-throughput screening

The increasing risk of drug-resistant bacterial infections indicates that there is a growing need for new and effective antimicrobial agents. One promising, but unexplored area in antimicrobial drug design is de novo purine biosynthesis. Recent research has shown that de novo purine biosynthesis in microbes is different from that in humans. The differences in the pathways are centered around the synthesis of 4-carboxyaminoimidazole ribonucleotide (CAIR) which requires the enzyme N⁵-carboxyaminoimidazole ribonucleotide (N⁵-CAIR) synthetase. Humans do not require and have no homologs of this enzyme. Unfortunately, no studies aimed at identifying small-molecule inhibitors of N⁵-CAIR synthetase have been published. To remedy this problem, we have conducted high-throughput screening (HTS) against Escherichia coli N⁵-CAIR synthetase using a highly reproducible phosphate assay. HTS of 48,000 compounds identified 14 compounds that inhibited the enzyme. The hits identified could be classified into three classes based on chemical structure. Class I contains compounds with an indenedione core. Class II contains an indolinedione group, and Class III contains compounds that are structurally unrelated to other inhibitors in the group. We determined the Michaelis–Menten kinetics for five compounds representing each of the classes. Examination of compounds belonging to Class I indicates that these compounds do not follow normal Michaelis–Menten kinetics. Instead, these compounds inhibit N⁵-CAIR synthetase by reacting with the substrate AIR. Kinetic analysis indicates that the Class II family of compounds are non-competitive with both AIR and ATP. One compound in Class III is competitive with AIR but uncompetitive with ATP, whereas the other is non-competitive with both substrates. Finally, these compounds display no inhibition of human AIR carboxylase:SAICAR synthetase indicating that these agents are selective inhibitors of N⁵-CAIR synthetase.     

Structure-based tailoring of compound libraries for high-throughput screening: discovery of novel EphB4 kinase inhibitors

High-throughput docking is a computational tool frequently used to discover small-molecule inhibitors of enzymes or receptors of known three-dimensional structure. Because of the large number of molecules in chemical libraries, automatic procedures to prune multimillion compound collections are useful for high-throughput docking and necessary for in vitro screening. Here, we propose an anchor-based library tailoring approach (termed ALTA) to focus a chemical library by docking and prioritizing molecular fragments according to their binding energy which includes continuum electrostatics solvation. In principle, ALTA does not require prior knowledge of known inhibitors, but receptor-based pharmacophore information (hydrogen bonds with the hinge region) is additionally used here to identify molecules with optimal anchor fragments for the ATP-binding site of the EphB4 receptor tyrosine kinase. The 21,418 molecules of the focused library (from an initial collection of about 730,000) are docked into EphB4 and ranked by force-field-based energy including electrostatic solvation. Among the 43 compounds tested in vitro, eight molecules originating from two different anchors show low-micromolar activity in a fluorescence-based enzymatic assay. Four of them are active in a cell-based assay and are potential anti-angiogenic compounds.     

Identification by high-throughput screening of inhibitors of Schistosoma mansoni NAD(+) catabolizing enzyme

Schistosomiasis is a major tropical parasitic disease. For its treatment, praziquantel remains the only effective drug available and the dependence on this sole chemotherapy emphasizes the urgent need for new drugs to control this neglected disease. In this context, the newly characterized Schistosoma mansoni NAD(+) catabolizing enzyme (SmNACE) represents a potentially attractive drug target. This potent NAD(+)glycohydrolase, which is localized to the outer surface (tegument) of the adult parasite, is presumably involved in the parasite survival by manipulating the host's immune regulatory pathways. In an effort to identify SmNACE inhibitors, we have developed a sensitive and robust fluorometric high-throughput screening assay. The implementation of this assay to the screening of a highly diverse academic chemical library of 14,300 molecules yielded, after secondary assays and generation of dose-response curves, the identification of two natural product inhibitors, cyanidin and delphinidin. These confirmed hits inhibit SmNACE with IC(50) values in the low micromolar range. To rationalize the structure-activity relationship, several related flavonoids were tested, thereby leading to the identification of 15 additional natural product inhibitors. A selection of representative flavonoid inhibitors indicated that although they also inhibit the homologous human CD38, a selectivity in favor of SmNACE could be reached. Docking studies indicated that these inhibitors mimic the binding mode of the enzyme substrate NAD(+) and suggested the pharmacophoric features required for SmNACE active site recognition.     

Cardiac ryanodine receptor N-terminal region biosensors identify novel inhibitors via FRET-based high-throughput screening

The N-terminal region (NTR) of ryanodine receptor (RyR) channels is critical for the regulation of Ca²⁺ release during excitation–contraction (EC) coupling in muscle. The NTR hosts numerous mutations linked to skeletal (RyR1) and cardiac (RyR2) myopathies, highlighting its potential as a therapeutic target. Here, we constructed two biosensors by labeling the mouse RyR2 NTR at domains A, B, and C with FRET pairs. Using fluorescence lifetime (FLT) detection of intramolecular FRET signal, we developed high-throughput screening (HTS) assays with these biosensors to identify small-molecule RyR modulators. We then screened a small validation library and identified several hits. Hits with saturable FRET dose–response profiles and previously unreported effects on RyR were further tested using [³H]ryanodine binding to isolated sarcoplasmic reticulum vesicles to determine effects on intact RyR opening in its natural membrane. We identified three novel inhibitors of both RyR1 and RyR2 and two RyR1-selective inhibitors effective at nanomolar Ca²⁺. Two of these hits activated RyR1 only at micromolar Ca²⁺, highlighting them as potential enhancers of excitation–contraction coupling. To determine whether such hits can inhibit RyR leak in muscle, we further focused on one, an FDA-approved natural antibiotic, fusidic acid (FA). In skinned skeletal myofibers and permeabilized cardiomyocytes, FA inhibited RyR leak with no detrimental effect on skeletal myofiber excitation–contraction coupling. However, in intact cardiomyocytes, FA induced arrhythmogenic Ca²⁺ transients, a cautionary observation for a compound with an otherwise solid safety record. These results indicate that HTS campaigns using the NTR biosensor can identify compounds with therapeutic potential.     

Fluorescent high-throughput screening of chemical inducers of neuronal differentiation in skeletal muscle cells

This protocol describes detailed procedures for the fluorescent high-throughput screening of small molecules that induce neurogenesis in cultures of skeletal muscle cells. The detection of neurogenesis relies on a fluorescent dye, FM 1-43, which is used to study the neuronal property of depolarization-induced synaptic vesicle recycling. Thus, small molecules with neurogenesis-inducing activity in skeletal muscle cells can be rapidly identified by measuring the fluorescence intensity of the treated cells using a fluorescent microplate reader. This protocol uses murine myoblast C2C12 cells for screening, which are readily available and relatively easy to culture. Neurogenesis of PC12 cells induced by nerve growth factor is employed as a positive control for this screening. The screening time for this protocol is 8 d, which also includes the procedure to detect depolarization-induced synaptic vesicle recycling using FM 1-43.     

Identification of inhibitors of vacuolar proton-translocating ATPase pumps in yeast by high-throughput screening flow cytometry

Fluorescence intensity of the pH-sensitive carboxyfluorescein derivative 2,7-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) was monitored by high-throughput flow cytometry in living yeast cells. We measured fluorescence intensity of BCECF trapped in yeast vacuoles, acidic compartments equivalent to lysosomes where vacuolar proton-translocating ATPases (V-ATPases) are abundant. Because V-ATPases maintain a low pH in the vacuolar lumen, V-ATPase inhibition by concanamycin A alkalinized the vacuole and increased BCECF fluorescence. Likewise, V-ATPase-deficient mutant cells had greater fluorescence intensity than wild-type cells. Thus, we detected an increase of fluorescence intensity after short- and long-term inhibition of V-ATPase function. We used yeast cells loaded with BCECF to screen a small chemical library of structurally diverse compounds to identify V-ATPase inhibitors. One compound, disulfiram, enhanced BCECF fluorescence intensity (although to a degree beyond that anticipated for pH changes alone in the mutant cells). Once confirmed by dose-response assays (EC₅₀ = 26 μM), we verified V-ATPase inhibition by disulfiram in secondary assays that measured ATP hydrolysis in vacuolar membranes. The inhibitory action of disulfiram against V-ATPase pumps revealed a novel effect previously unknown for this compound. Because V-ATPases are highly conserved, new inhibitors identified could be used as research and therapeutic tools in cancer, viral infections, and other diseases where V-ATPases are involved.     

Identification of novel telomeric G-quadruplex-targeting chemical scaffolds through screening of three NCI libraries

Thirteen compounds with diverse chemical structures have been identified as selective telomeric G-quadruplex-binding ligands through screening the NCI Diversity Set II, the NCI Natural Products Set II and the NCI Mechanistic Diversity Set libraries containing a total of 2307 members against a human telomeric G-quadruplex using a FRET-based DNA melting assay. These compounds show significant selectivity towards a telomeric G-quadruplex compared to duplex DNA, fall within a molecular weight range of 327-533, and are generally consistent with the Lipinski Rule of Five for drug-likeness. Thus they provide new chemical scaffolds for the development of novel classes of G-quadruplex-targeting agents.     

The developing zebrafish (Danio rerio): a vertebrate model for high-throughput screening of chemical libraries

The zebrafish, Danio rerio, a small, tropical freshwater species native to Pakistan and India, has become a National Institutes of Health-sanctioned model organism and, due to its many advantages as an experimental vertebrate, it has garnered intense interest from the world's scientific community. Some have labeled the zebrafish, the "vertebrate Drosophila," due to its genetic tractability, small size, low cost, and rapid development. The transparency of the embryo, external development, and the many hundreds of mutant and transgenic lines available add to the allure. Now it appears, the zebrafish can be used for high-throughput screening (HTS) of drug libraries in the discovery process of promising new therapeutics. In this review, various types of screening methods are briefly outlined, as are a variety of screens for different disease models, to highlight the range of zebrafish HTS possibilities. High-content screening (HCS) has been available for cell-based screens for some time and, very recently, HCS is being adapted for the zebrafish. This will allow analysis, at high resolution, of drug effects on whole vertebrates; thus, whole body effects as well as those on specific organs and tissues may be determined.     

Optimized and automated protocols for high-throughput screening of amylosucrase libraries

This article describes the design and validation of a general procedure for the high-throughput isolation of amylosucrase variants displaying higher thermostability or increased resistance to organic solvents. This procedure consists of 2 successive steps: an in vivo selection that eliminates inactive variants followed by automated screening of active variants to isolate mutants displaying enhanced features. The authors chose an Escherichia coli expression vector, allowing a high production rate of the recombinant enzyme in miniaturized culture conditions. The screening assay was validated by minimizing variability for various parameters of the protocol, especially bacterial growth and protein production in cultures in 96-well microplates. Recombinant amylosucrase production was normalized by decreasing the coefficient of variance from 27% to 12.5%. Selective screening conditions were defined to select variants displaying higher thermostability or increased resistance to organic solvents. A first-generation amylosucrase variant library, constructed by random mutagenesis, was subjected to this procedure, yielding a mutant displaying a 25-fold increased stability at 50 degrees C compared to the parental wild-type enzyme.     

Identification of small molecule inhibitors of beta-amyloid cytotoxicity through a cell-based high-throughput screening platform

Calpain activation is hypothesized to be an early occurrence in the sequence of events resulting in neurodegeneration, as well as in the signaling pathways linking extracellular accumulation of beta-amyloid (Abeta) peptides and intracellular formation of neurofibrillary tangles. In an effort to identify small molecules that prevent neurodegeneration in Alzheimer's disease by early intervention in the cell death cascade, a cell-based assay in differentiated Sh-SY5Y cells was developed using calpain activity as a read-out for the early stages of death in cells exposed to extracellular Abeta. This assay was optimized for high-throughput screening, and a library of approximately 120,000 compounds was tested. It was expected that the compounds identified as calpain inhibitors would include those that act directly on the enzyme and those that prevented calpain activation by blocking an upstream step in the pathway. In fact, of the compounds that inhibited calpain activation by Abeta with IC(50) values of <10 microM and showed little or no toxicity at concentrations up to 30 microM, none inhibit the calpain enzyme directly.     

Genetic interventions in mammalian cells; applications and uses in high-throughput screening and drug discovery

Functional cellular assays are the bedrock of modern drug discovery. These utilise cellular systems that yield a measurable biochemical product or physiological response to drug stimulation. Often, these functional responses are studied by the introduction of the molecular target of choice into an inert cellular background to create a more discriminating system. There are as many techniques for delivery of the required target gene as there are techniques for studying their function. This article will consider the genetic modification of cell lines in vitro to develop cell-based assays for drug discovery and high-throughput screening.     

Discovery of furan carboxylate derivatives as novel inhibitors of ATP-citrate lyase via virtual high-throughput screening

The enzyme ATP citrate lyase (ACL) catalyzes the formation of cytosolic acetyl CoA, the starting material for de novo lipid and cholesterol biosynthesis. The dysfunction and upregulation of ACL in numerous cancers makes it an attractive target for developing anticancer therapies. ACL inhibition by shRNA knockdown limits cancer cell proliferation and reduces cancer stemness. We designed and implemented a dual docking protocol to select virtual ACL inhibitors that were scored among the top 10 percentiles by both the Autodock Vina and the Glamdock algorithms. Via this in silico screens of a focused furoic acid library, we discovered four subtypes of furans and benzofurans as novel ACL inhibitors. The hit rate of our in silico protocol was 45.8% with 11 of 24 virtual hits confirmed as active in an in vitro ACL enzymatic assay. The IC₅₀ of the most potent ACL inhibitor A1 is 4.1 μM. Our results demonstrated remarkable hit rate by the dual docking approach and provided novel chemical scaffolds for the development of ACL inhibitors for the treatment of cancer.     

Identification of important bases for the self-cleavage activity at two single-stranded regions of genomic HDV ribozyme.

In order to determine important bases at two single-stranded regions [SSrA (726-731 nt) and SSrB (762-766)] derived mainly from secondary structure models in genomic hepatitis delta virus (HDV) ribozyme possessing self-cleavage activity, we have constructed several point mutants at these two regions on the HDV88 molecule (683-770). Among the bases at SSrA and SSrB regions C763 was found to play an essential role during self-cleavage process since substitutions to any other bases viz. A or G or U completely abolished the activity.