Bioluminescent kinase strips: A novel approach to targeted and flexible kinase inhibitor profiling

In addition to target efficacy, drug safety is a major requirement during the drug discovery process and is influenced by target specificity. Therefore, it is imperative that every new drug candidate be profiled against various liability panels that include protein kinases. Here, an effective methodology to streamline kinase inhibitor profiling is described. An accessible standardized profiling system for 112 protein kinases covering all branches of the kinome was developed. This approach consists of creating different sets of kinases and their corresponding substrates in multi-tube strips. The kinase stocks are pre-standardized for optimal kinase activity and used for inhibitor profiling using a bioluminescent ADP detection assay. We show that these strips can routinely generate inhibitor selectivity profiles for small or broad kinase family panels. Lipid kinases were also assembled in strip format and profiled together with protein kinases. We identified two specific PI3K inhibitors that have off-target effects on CK2 that were not reported before and would have been missed if compounds were not profiled against lipid and protein kinases simultaneously. To validate the accuracy of the data generated by this method, we confirmed that the inhibition potencies observed are consistent with published values produced by more complex technologies such as radioactivity assays.     

Three mechanistically distinct kinase assays compared: Measurement of intrinsic ATPase activity identified the most comprehensive set of ITK inhibitors

Numerous assay methods have been developed to identify small-molecule effectors of protein kinases, but no single method can be applied to all isolated kinases. The authors developed a set of 3 high-throughput screening (HTS)-compatible biochemical assays that can measure 3 mechanistically distinct properties of a kinase active site, with the goal that at least 1 of the 3 would be applicable to any kinase selected as a target for drug discovery efforts. Two assays measure catalytically active enzyme: A dissociation-enhanced lanthanide fluoroimmuno assay (DELFIA) uses an antibody to quantitate the generation of phosphorylated substrate; a second assay uses luciferase to measure the consumption of adenosine triphosphate (ATP) during either phosphoryl-transfer to a peptide substrate or to water (intrinsic ATPase activity). A third assay, which is not dependent on a catalytically active enzyme, measures the competition for binding to kinase between an inhibitor and a fluorescent ATP binding site probe. To evaluate the suitability of these assays for drug discovery, the authors compared their ability to identify inhibitors of a nonreceptor protein tyrosine kinase from the Tec family, interleukin-2-inducible T cell kinase (ITK). The 3 assays agreed on 57% of the combined confirmed hit set identified from screening a 10,208-compound library enriched with known kinase inhibitors and molecules that were structurally similar. Among the 3 assays, the one measuring intrinsic ATPase activity produced the largest number of unique hits, the fewest unique misses, and the most comprehensive hit set, missing only 2.7% of the confirmed inhibitors identified by the other 2 assays combined. Based on these data, all 3 assay formats are viable for screening and together provide greater options for assay design depending on the targeted kinase.     

Using aptamers as capture reagents in bead-based assay systems for diagnostics and hit identification

Most applications of xMAP (Luminex) bead-based assay technology in diagnostics and drug discovery use immobilized antigens or antibodies. Here the authors describe the development of novel assay systems in which synthetic oligonucleotides that specifically bind and inhibit other biomolecules--so-called aptamers--are directly immobilized on beads. The robustness, specificity, and sensitivity of aptamer-based assays were demonstrated in a test system that detected human alpha-thrombin in serum samples. xMAP technology was also adapted to competitive screening formats where an aptamer/protein complex was disrupted by a functionally analogous competitor. The results indicate that such assays are excellently suited for diagnostic applications or drug screening, where aptamers serve as competitive binding probes for the identification of small-molecule hits. These methods should be transferable to a large number of applications because specific aptamers can be rapidly generated for almost any protein target.     

High-Throughput 1,536-Well Fluorescence Polarization Assays for α1-Acid Glycoprotein and Human Serum Albumin Binding

Two major plasma proteins in humans are primarily responsible for drug binding, the α₁-acid-glycoprotein (AGP) and human serum albumin (HSA). The availability of at least a semiquantitative high-throughput assay for assessment of protein binding is expected to aid in bridging the current gap between high-throughput screening and early lead discovery, where cell-based and biochemical assays are deployed routinely to test up to several million compounds rapidly, as opposed to the late-stage candidate drug profiling methods which test at most dozens of compounds at a time. Here, we describe the miniaturization of a pair of assays based on the binding- and displacement-induced changes in fluorescence polarization (FP) of fluorescent small molecule probes known to specifically target the drug-binding sites of these two proteins. A robust and reproducible assay performance was achieved in ≤4 µL assay volume in 1,536-well format. The assays were tested against a validation set of 10 known protein binders, and the results compared favorably with data obtained using protein-coated beads with high-performance liquid chromatography analysis. The miniaturized assays were taken to a high-throughput level in a screen of the LOPAC¹²⁸⁰ collection of 1,280 pharmacologically active compounds. The adaptation of the AGP and HSA FP assays to a 1,536-well format should allow their use in early-stage profiling of large-size compound sets.     

Multiplexed G-protein-coupled receptor Ca2+ flux assays for high-throughput screening

An early drug discovery approach focusing on gene families can benefit from strategies that exploit common signaling mechanisms to more effectively identify and characterize novel chemical lead structures. Multiplexing, defined as the screening of multiple targets within the same experiment, is an example of this strategy. Here, the authors describe a technique that allows multiplexing of a common assay type used to study G-protein-coupled receptors: changes in intracellular Ca2+ levels as measured by Molecular Device's fluorometric imaging plate reader (FLIPR). The multiplexed FLIPR assays showed the expected pharmacological properties of single assays, with good reproducibility and Z* factors. The authors used them to screen large compound libraries in 2 multiplexed assay designs. The 1st used a single-cell line expressing 2 different receptors and the 2nd a mixture of 2 cell lines of the same type each expressing distinct receptors. Screening using these multiplexed assays produced significant savings in reagents, time, and human resources and allowed the authors to quickly identify specific and selective hits.     

A standard operating procedure for assessing liquid handler performance in high-throughput screening

The thrust of early drug discovery in recent years has been toward the configuration of homogeneous miniaturized assays. This has allowed organizations to contain costs in the face of exponential increases in the number of screening assays that need to be run to remain competitive. Miniaturization brings with it an increasing dependence on instrumentation, which over the past several years has seen the development of nanodispensing capability and sophisticated detection strategies. To maintain confidence in the data generated from miniaturized assays, it is critical to ensure that both compounds and reagents have been delivered as expected to the target wells. The authors have developed a standard operating procedure for liquid-handling quality control that has enabled them to evaluate performance on 2 levels. The first level provides for routine daily testing on existing instrumentation, and the second allows for more rigorous testing of new dispensing technologies. The procedure has shown itself to be useful in identifying both method programming and instrumentation performance shortcomings and has provided a means to harmonizing instrumentation usage by assay development and screening groups. The goal is that this type of procedure be used for facilitating the exchange of liquid handler performance data across the industry.     

A new strategy for improved secondary screening and lead optimization using high-resolution SPR characterization of compound-target interactions

Biophysical label-free assays such as those based on SPR are essential tools in generating high-quality data on affinity, kinetic, mechanistic and thermodynamic aspects of interactions between target proteins and potential drug candidates. Here we show examples of the integration of SPR with bioinformatic approaches and mutation studies in the early drug discovery process. We call this combination 'structure-based biophysical analysis'. Binding sites are identified on target proteins using information that is either extracted from three-dimensional structural analysis (X-ray crystallography or NMR), or derived from a pharmacore model based on known binders. The binding site information is used for in silico screening of a large substance library (e.g. available chemical directory), providing virtual hits. The three-dimensional structure is also used for the design of mutants where the binding site has been impaired. The wild-type target and the impaired mutant are then immobilized on different spots of the sensor chip and the interactions of compounds with the wild-type and mutant are compared in order to identify selective binders for the binding site of the target protein. This method can be used as a cost-effective alternative to high-throughput screening methods in cases when detailed binding site information is available. Here, we present three examples of how this technique can be applied to provide invaluable data during different phases of the drug discovery process.     

A high-throughput absorbance-based assay for methionine produced by methionine aminopeptidase using S-adenosyl-L-methionine synthetase

Methionine aminopeptidase (MAP) (E.C. 3.4.11.18) is a metallopeptidase that cleaves the N-terminal methionine (Met) residue from some proteins. MAP is essential for growth of several bacterial pathogens, making it a target for antibacterial drug discovery. MAP enzymes are also present in eukaryotic cells, and one is a target for antiangiogenic cancer therapy. To screen large compound libraries for MAP inhibitors as the starting point for drug discovery, a high-throughput-compatible assay is valuable. Here the authors describe a novel assay, which detects the Met product of MAP-catalyzed peptide cleavage by coupling it to adenosine triphosphate (ATP)-dependent production of S-adenosyl-L-methionine (SAM) and inorganic phosphate (P(i)) by SAM synthetase (MetK) combined with inorganic pyrophosphatase. The three P(i) ions produced for each Met consumed are detected using Malachite Green/molybdate reagent. This assay can use any unmodified peptide MAP substrate with an N-terminal Met. The assay was used to measure kinetic constants for Escherichia coli MAP using Mn(2+) as the activator and the peptide Met-Gly-Met-Met as the substrate, as well as to measure the potency of a MAP inhibitor. A Mn(2+) buffer is described that can be used to prevent free Mn(2+) depletion by chelating compounds from interfering in screens for MAP inhibitors.     

Optimization of a higher throughput microsomal stability screening assay for profiling drug discovery candidates

Metabolic stability plays an important role in the success of drug candidates. First-pass metabolism is one of the major causes of poor oral bioavailability and short half-life. Traditionally, metabolic stability was evaluated at a later stage of drug discovery and required laborious manual manipulations. With the advance of high-throughput screening, combinatorial chemistry, and early profiling of drug-like properties, automated and rapid stability assays are needed to meet the increasing demand of throughput, speed, and reproducibility at earlier stages of drug discovery. The authors describe optimization of a simple, robust, high-throughput microsomal stability assay developed in a 96-well format. The assay consists of 2 automated components: robotic sample preparation for incubation and cleanup and rapid liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS) analysis to determine percent remaining of the parent compound. The reagent solutions and procedural steps were optimized for automation. Variables affecting assay results were investigated. The variability introduced by microsome preparations from different sources (various vendors and batches) was studied and indicates the need for careful control. Quality control and normalization of the stability results are critical when applying the screening data, generated at different times or research sites, to discovery projects.     

Thymidine incorporation is highly predictive of colony formation and can be used for high-throughput screening

The primary goal of anticancer chemotherapy is to kill cancer cells. Therefore, it is of critical importance that any assay that is used to determine the toxicity of a potential anticancer drug accurately measures viability. While colony formation is widely regarded as the most accurate measure of viability following drug treatment, it is laborious, time consuming, and difficult to carry out with non-adherent cells. For these reasons, it is not suitable for moderate- to high-throughput screening applications. We sought to identify a convenient and reliable assay that would accurately reproduce colony formation results and be amenable to high-throughput applications. Here, we describe a modification of the 3H-thymidine incorporation assay that meets these criteria. The assay can be carried out in 96-well plates with minimal handling of reagents and media. It can be performed with non-adherent and adherent cell lines. Most importantly, LC50 values obtained with this assay show excellent agreement with colony formation results. Taken together, these advantages make the modified 3H-thymidine incorporation assay well suited for high-throughput viability assays in anticancer drug discovery and development.     

Development of a high-throughput screening method for LIM kinase 1 using a luciferase-based assay of ATP consumption

Kinases are attractive drug targets because of the central roles they play in signal transduction pathways and human diseases. Their well-formed adenosine triphosphate (ATP)-binding pockets make ideal targets for small-molecule inhibitors. For drug discovery purposes, many peptide-based kinase assays have been developed that measure substrate phosphorylation using fluorescence-based readouts. However, for some kinases these assays may not be appropriate. In the case of the LIM kinases (LIMK), an inability to phosphorylate peptide substrates resulted in previous high-throughput screens (HTS) using radioactive labeling of recombinant cofilin protein as the readout. We describe the development of an HTS-compatible assay that measures relative ATP levels using luciferase-generated luminescence as a function of LIMK activity. The assay was inexpensive to perform, and proof-of-principle screening of kinase inhibitors demonstrated that compound potency against LIMK could be determined; ultimately, the assay was used for successful prosecution of automated HTS. Following HTS, the secondary assay format was changed to obtain more accurate measures of potency and mechanism of action using more complex (and expensive) assays. The luciferase assay nonetheless provides an inexpensive and reliable primary assay for HTS that allowed for the identification of LIMK inhibitors to initiate discovery programs for the eventual treatment of human diseases.     

BioAssay ontology annotations facilitate cross-analysis of diverse high-throughput screening data sets

High-throughput screening data repositories, such as PubChem, represent valuable resources for the development of small-molecule chemical probes and can serve as entry points for drug discovery programs. Although the loose data format offered by PubChem allows for great flexibility, important annotations, such as the assay format and technologies employed, are not explicitly indexed. The authors have previously developed a BioAssay Ontology (BAO) and curated more than 350 assays with standardized BAO terms. Here they describe the use of BAO annotations to analyze a large set of assays that employ luciferase- and β-lactamase-based technologies. They identified promiscuous chemotypes pertaining to different subcategories of assays and specific mechanisms by which these chemotypes interfere in reporter gene assays. Results show that the data in PubChem can be used to identify promiscuous compounds that interfere nonspecifically with particular technologies. Furthermore, they show that BAO is a valuable toolset for the identification of related assays and for the systematic generation of insights that are beyond the scope of individual assays or screening campaigns.     

Sensitive cell viability assay for use in drug screens and for studying the mechanism of action of drugs in Dictyostelium discoideum

In order to increase the effectiveness of Dictyostelium discoideum as a lead genetic model for drug discovery, a luminescence-based assay has been adapted and standardized for sensitive and rapid cell viability measurements. The applicability of the assay was demonstrated by measuring the cytotoxicity of several drugs in wild-type and mutant cells. The robustness and ease of the assay demonstrate that it can be used in high-throughput applications such as drug or mutant screens. Conclusions from these studies are applicable to evaluating cell viability assays in other systems as well.     

Analysis of multiple compound–protein interactions reveals novel bioactive molecules

The discovery of novel bioactive molecules advances our systems‐level understanding of biological processes and is crucial for innovation in drug development. For this purpose, the emerging field of chemical genomics is currently focused on accumulating large assay data sets describing compound–protein interactions (CPIs). Although new target proteins for known drugs have recently been identified through mining of CPI databases, using these resources to identify novel ligands remains unexplored. Herein, we demonstrate that machine learning of multiple CPIs can not only assess drug polypharmacology but can also efficiently identify novel bioactive scaffold‐hopping compounds. Through a machine‐learning technique that uses multiple CPIs, we have successfully identified novel lead compounds for two pharmaceutically important protein families, G‐protein‐coupled receptors and protein kinases. These novel compounds were not identified by existing computational ligand‐screening methods in comparative studies. The results of this study indicate that data derived from chemical genomics can be highly useful for exploring chemical space, and this systems biology perspective could accelerate drug discovery processes.     

Evaluation of a high-throughput fluorescence assay method for HERG potassium channel inhibition

The number of projects in drug development that fail in late phases because of cardiac side effects such as QT prolongation can impede drug discovery and development of projects. The molecular target responsible for QT prolongation by a wide range of pharmaceutical agents is the myocardial hERG potassium channel. It is therefore desirable to screen for compound interactions with the hERG channel at an early stage of drug development. Here, the authors report a cell-based fluorescence assay using membrane potential-sensitive fluorescent dyes and stably transfected hERG channels from CHO cells. The assay allows semiautomated screening of compounds for hERG activity on 384-well plates and is sufficiently rapid for testing a large number of compounds. The assay is robust as indicated by a Z' factor larger than 0.6. The throughput is in the range of 10,000 data points per day, which is significantly higher than any other method presently available for hERG. The data obtained with the fluorescence assay were in qualitative agreement with those from patch-clamp electrophysiological analysis. There were no false-positive hits, and the rate of false-negative compounds is currently 12% but might be further reduced by testing compounds at higher concentration. Quantitative differences between fluorescence and electrophysiological methods may be due to the use- or voltage-dependent activity of the antagonists.     

Assessment of Pseudomonas aeruginosa N5,N10-methylenetetrahydrofolate dehydrogenase-cyclohydrolase as a potential antibacterial drug target

The bifunctional enzyme methylenetetrahydrofolate dehydrogenase – cyclohydrolase (FolD) is identified as a potential drug target in Gram-negative bacteria, in particular the troublesome Pseudomonas aeruginosa. In order to provide a comprehensive and realistic assessment of the potential of this target for drug discovery we generated a highly efficient recombinant protein production system and purification protocol, characterized the enzyme, carried out screening of two commercial compound libraries by differential scanning fluorimetry, developed a high-throughput enzyme assay and prosecuted a screening campaign against almost 80,000 compounds. The crystal structure of P. aeruginosa FolD was determined at 2.2 Å resolution and provided a template for an assessment of druggability and for modelling of ligand complexes as well as for comparisons with the human enzyme. New FolD inhibitors were identified and characterized but the weak levels of enzyme inhibition suggest that these compounds are not optimal starting points for future development. Furthermore, the close similarity of the bacterial and human enzyme structures suggest that selective inhibition might be difficult to attain. In conclusion, although the preliminary biological data indicates that FolD represents a valuable target for the development of new antibacterial drugs, indeed spurred us to investigate it, our screening results and structural data suggest that this would be a difficult enzyme to target with respect to developing the appropriate lead molecules required to underpin a serious drug discovery effort.     

Evaluation of cellular dielectric spectroscopy, a whole-cell, label-free technology for drug discovery on Gi-coupled GPCRs

Cellular dielectric spectroscopy (CDS) is an emerging technology capable of detecting a range of whole-cell responses in a label-free manner. A new CDS-based instrument, CellKey, has been developed that is optimized for G-protein coupled receptor (GPCR) detection and has automated liquid handling in microplate format, thereby making CDS accessible to lead generation/optimization drug discovery. In addition to having sufficient throughput, new assay technologies must pass rigorous standards for assay development, signal window, dynamic range, and reproducibility to effectively support drug discovery SAR studies. Here, the authors evaluated CellKey with 3 different G(i)-coupled GPCRs for suitability in supporting SAR studies. Optimized assay conditions compatible with the precision, reproducibility, and throughput required for routine screening were quickly achieved for each target. Across a 1000-fold range in compound potencies, CellKey results correlated with agonist and antagonist data obtained using classical methods ([(35)S]GTPgammaS binding and cAMP production). For partial agonists, relative efficacy measurements also correlated with GTPgammaS data. CellKey detection of positive allosteric modulators appeared superior to GTPgammaS methodology. Agonist and antagonist activity could be accurately quantified under conditions of low receptor expression. CellKey is a new technology platform that uses label-free detection in a homogeneous assay that is unaffected by color quenching and is easily integrated into existing microtiter-based compound testing and data analysis procedures for drug discovery.     

Statistical analysis of systematic errors in high-throughput screening

High-throughput screening (HTS) is an efficient technology for drug discovery. It allows for screening of more than 100,000 compounds a day per screen and requires effective procedures for quality control. The authors have developed a method for evaluating a background surface of an HTS assay; it can be used to correct raw HTS data. This correction is necessary to take into account systematic errors that may affect the procedure of hit selection. The described method allows one to analyze experimental HTS data and determine trends and local fluctuations of the corresponding background surfaces. For an assay with a large number of plates, the deviations of the background surface from a plane are caused by systematic errors. Their influence can be minimized by the subtraction of the systematic background from the raw data. Two experimental HTS assays from the ChemBank database are examined in this article. The systematic error present in these data was estimated and removed from them. It enabled the authors to correct the hit selection procedure for both assays.     

A novel cell-based, high-content assay for phosphorylation of Lats2 by Aurora A

Aurora A kinase is a key regulator of mitosis, which is upregulated in several human cancers, making it a potential target for anticancer therapeutics. Consequently, robust medium- to high-throughput cell-based assays to measure Aurora A kinase activity are critical for the development of small-molecule inhibitors. Here the authors compare measurement of the phosphorylation of two Aurora A substrates previously used in high-content screening Aurora A assays, Aurora A itself and TACC3, with a novel substrate Lats2. Using antibodies directed against phosphorylated forms of Aurora A (pThr288), P-TACC3 (pSer558), and P-Lats2 (pSer83), the authors investigate their suitability in parallel for development of a cell-based assay using several reference Aurora inhibitors: MLN8054, VX680, and AZD1152-HQPA. They validate a combined assay of target-specific phosphorylation of Lats2 at the centrosome and an increase in mitotic index as a measure of Aurora A activity. The assay is both sensitive and robust and has acceptable assay performance for high-throughput screening or potency estimation from concentration-response assays. It has the advantage that it can be carried out using a commercially available monoclonal antibody against phospho-Lats2 and the widely available Cellomics ArrayScan HCS reader and thus represents a significant addition to the tools available for the identification of Aurora A specific inhibitors.