Medium-throughput Assay Research Articles

A Cell-Based Assay for Measuring Endogenous BcrAbl Kinase Activity and Inhibitor Resistance.

Kinase enzymes are an important class of drug targets, particularly in cancer. Cell-based kinase assays are needed to understand how potential kinase inhibitors act on their targets in a physiologically relevant context. Current cell-based kinase assays rely on antibody-based detection of endogenous substrates, inaccurate disease models, or indirect measurements of drug action. Here we expand on previous work from our lab to introduce a 96-well plate compatible approach for measuring cell-based kinase activity in disease-relevant human chronic myeloid leukemia cell lines using an exogenously added, multi-functional peptide substrate. Our cellular models natively express the BcrAbl oncogene and are either sensitive or have acquired resistance to well-characterized BcrAbl tyrosine kinase inhibitors. This approach measures IC50 values comparable to established methods of assessing drug potency, and its robustness indicates that it can be employed in drug discovery applications. This medium-throughput assay could bridge the gap between single target focused, high-throughput in vitro assays and lower-throughput cell-based follow-up experiments.

Uncoupling neuronal death and dysfunction in Drosophila models of neurodegenerative disease.

Common neurodegenerative proteinopathies, such as Alzheimer’s disease (AD) and Parkinson’s disease (PD), are characterized by the misfolding and aggregation of toxic protein species, including the amyloid beta (Aß) peptide, microtubule-associated protein Tau (Tau), and alpha-synuclein (αSyn) protein. These factors also show toxicity in Drosophila; however, potential limitations of prior studies include poor discrimination between effects on the adult versus developing nervous system and neuronal versus glial cell types. In addition, variable expression paradigms and outcomes hinder systematic comparison of toxicity profiles. Using standardized conditions and medium-throughput assays, we express human Tau, Aß or αSyn selectively in neurons of the adult Drosophila retina and monitor age-dependent changes in both structure and function, based on tissue histology and recordings of the electroretinogram (ERG), respectively. We find that each protein causes a unique profile of neurodegenerative pathology, demonstrating distinct and separable impacts on neuronal death and dysfunction. Strikingly, expression of Tau leads to progressive loss of ERG responses whereas retinal architecture and neuronal numbers are largely preserved. By contrast, Aß induces modest, age-dependent neuronal loss without degrading the retinal ERG. αSyn expression, using a codon-optimized transgene, is characterized by marked retinal vacuolar change, progressive photoreceptor cell death, and delayed-onset but modest ERG changes. Lastly, to address potential mechanisms, we perform transmission electron microscopy (TEM) to reveal potential degenerative changes at the ultrastructural level. Surprisingly, Tau and αSyn each cause prominent but distinct synaptotoxic profiles, including disorganization or enlargement of photoreceptor terminals, respectively. Our findings highlight variable and dynamic properties of neurodegeneration triggered by these disease-relevant proteins in vivo, and suggest that Drosophila may be useful for revealing determinants of neuronal dysfunction that precede cell loss, including synaptic changes, in the adult nervous system.Electronic supplementary materialThe online version of this article (doi:10.1186/s40478-016-0333-4) contains supplementary material, which is available to authorized users.

Abstract C162: A medium-throughput single cell CRISPR-Cas9 assay to assess gene essentiality

Abstract Target selection for oncology and other indications is a critical step in the successful development of therapeutics, however it remains one of the most challenging areas of drug discovery. In fact, up to two-thirds of oncology relevant targets reported in literature have not been confirmed on follow-up studies, indicating that target validation in oncology is especially challenging. Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 gene editing of specific loci offers an alternative method to RNA interference and complements small molecule inhibitors for determining whether or not a cell line is dependent on a specific gene product for proliferation and/or survival. Importantly, CRISPR-Cas9 may be advantageous for some studies as it offers efficient and specific gene knock-out leading to complete loss of protein function. This may be especially useful for some target classes, including epigenetic targets, which appear to require near complete loss of protein function to observe phenotypes. In our initial studies using CRISPR-Cas9 to verify the essential nature of EZH2 (Enhancer of Zest 2) expression for the proliferation of SMARCB1/SNF5/INI1 mutant malignant rhabdoid tumor cell lines, we observed that the initial reduction in proliferation was lost over time. We hypothesized that in the few cells that retain proliferative capacity at least one allele of EZH2 remains functional, and this hypothesis suggests that carrying out CRISPR-Cas9 studies for individual target genes without analyzing single cell clones could produce misleading results. To verify this, we developed a medium throughput assay to analyze 10s-100s of single cell clones for target gene disruption using CRISPR-Cas9 gene knockout, followed by a restriction digest and fluorescent undigested fragment length analysis to successfully assess EZH2 allele status. Significantly, these data support our hypothesis that retention of one functional copy of EZH2 is required for the proliferation of EZH2-dependent cell lines and that this can be rapidly assessed by our assay. Thus, the assessment of zygosity of the gene of interest can be evaluated in a medium-throughput manner in single cell clones. The assay is thereby able to unambiguously indicate whether or not a specific gene is essential for survival and/or proliferation in a given cell line, and offers a unique approach for target validation using gene editing. Importantly, this approach should be applicable to any target of interest that is expected to affect cell proliferation or survival. Such data can aid in the development of more robust cancer therapeutics by increasing confidence in target selection. Citation Format: Alexandra R. Grassian, Tim Scales, Sarah K. Knutson, Nicola J. McCarthy, Chris E. Lowe, Jon D. Moore, Robert A. Copeland, Heike Keilhack, Jesse J. Smith, Julie A. Wickendon, Scott Ribich. A medium-throughput single cell CRISPR-Cas9 assay to assess gene essentiality. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C162.

Development and validation of a luminescence-based, medium-throughput assay for drug screening in Schistosoma mansoni.

BackgroundSchistosomiasis, one of the world’s greatest neglected tropical diseases, is responsible for over 280,000 human deaths per annum. Praziquantel, developed in the 1970s, has high efficacy, excellent tolerability, few and transient side effects, simple administration procedures and competitive cost and it is currently the only recommended drug for treatment of human schistosomiasis. The use of a single drug to treat a population of over 200 million infected people appears particularly alarming when considering the threat of drug resistance. Quantitative, objective and validated methods for the screening of compound collections are needed for the discovery of novel anti-schistosomal drugs.Methodology/Principal FindingsThe present work describes the development and validation of a luminescence-based, medium-throughput assay for the detection of schistosomula viability through quantitation of ATP, a good indicator of metabolically active cells in culture. This validated method is demonstrated to be fast, highly reliable, sensitive and automation-friendly. The optimized assay was used for the screening of a small compound library on S. mansoni schistosomula, showing that the proposed method is suitable for a medium-throughput semi-automated screening. Interestingly, the pilot screening identified hits previously reported to have some anti-parasitic activity, further supporting the validity of this assay for anthelminthic drug discovery.ConclusionsThe developed and validated schistosomula viability luminescence-based assay was shown to be successful and suitable for the identification of novel compounds potentially exploitable in future schistosomiasis therapies.

Sensitivity and Specificity of the In Vitro Guinea Pig Papillary Muscle Action Potential Duration for the Assessment of Drug-Induced Torsades De Pointes Liability in Humans.

The ICH S7B document, which provides guidance for the preclinical cardiovascular evaluation of pharmaceutical new chemical entities (NCE), is essentially focused on drug-induced QT lengthening, a biomarker for the proarrhythmic adverse drug reaction, torsades de pointes (TdP). In 2005, this guidance recommended the IKr assay and the in vivo QT telemetry study as mandatory assays for detecting potential torsades de pointes liability and relegated the cardiac action potential (AP) assay as a follow-up study. The IKr assay has become a mandatory screening tool in the early development and safety assessment process. Using only the IKr assay as a go/no go decision arbiter is regrettable since, due to the low specificity of the model (positives that are false for proarrhythmia liability, e.g. verapamil), promising, safe NCEs may be inadvertently discarded. Inclusion of additional medium throughput assays should be performed early to confirm or balance the putatively unfavourable IKr result with positive discovery model output (Pugsley et al., J Pharmacol Toxicol Methods 60:24-27, 2009). In the present chapter, the predictive value of in vitro guinea pig papillary muscle action potential assay will be discussed in terms of sensitivity and specificity and compared to currently available preclinical models such as IKr/hERG assay, dog Purkinje fibre action potential and in vivo QT measurements in dog and cynomolgus monkey.

Using a non-image-based medium-throughput assay for screening compounds targeting N-myristoylation in intracellular Leishmania amastigotes.

We have refined a medium-throughput assay to screen hit compounds for activity against N-myristoylation in intracellular amastigotes of Leishmania donovani. Using clinically-relevant stages of wild type parasites and an Alamar blue-based detection method, parasite survival following drug treatment of infected macrophages is monitored after macrophage lysis and transformation of freed amastigotes into replicative extracellular promastigotes. The latter transformation step is essential to amplify the signal for determination of parasite burden, a factor dependent on equivalent proliferation rate between samples. Validation of the assay has been achieved using the anti-leishmanial gold standard drugs, amphotericin B and miltefosine, with EC50 values correlating well with published values. This assay has been used, in parallel with enzyme activity data and direct assay on isolated extracellular amastigotes, to test lead-like and hit-like inhibitors of Leishmania N-myristoyl transferase (NMT). These were derived both from validated in vivo inhibitors of Trypanosoma brucei NMT and a recent high-throughput screen against L. donovani NMT. Despite being a potent inhibitor of L. donovani NMT, the activity of the lead T. brucei NMT inhibitor (DDD85646) against L. donovani amastigotes is relatively poor. Encouragingly, analogues of DDD85646 show improved translation of enzyme to cellular activity. In testing the high-throughput L. donovani hits, we observed macrophage cytotoxicity with compounds from two of the four NMT-selective series identified, while all four series displayed low enzyme to cellular translation, also seen here with the T. brucei NMT inhibitors. Improvements in potency and physicochemical properties will be required to deliver attractive lead-like Leishmania NMT inhibitors.

Detecting, visualizing and quantitating the generation of reactive oxygen species in an amoeba model system.

Reactive oxygen species (ROS) comprise a range of reactive and short-lived, oxygen-containing molecules, which are dynamically interconverted or eliminated either catalytically or spontaneously. Due to the short life spans of most ROS and the diversity of their sources and subcellular localizations, a complete picture can be obtained only by careful measurements using a combination of protocols. Here, we present a set of three different protocols using OxyBurst Green (OBG)-coated beads, or dihydroethidium (DHE) and Amplex UltraRed (AUR), to monitor qualitatively and quantitatively various ROS in professional phagocytes such as Dictyostelium. We optimised the beads coating procedures and used OBG-coated beads and live microscopy to dynamically visualize intraphagosomal ROS generation at the single cell level. We identified lipopolysaccharide (LPS) from E. coli as a potent stimulator for ROS generation in Dictyostelium. In addition, we developed real time, medium-throughput assays using DHE and AUR to quantitatively measure intracellular superoxide and extracellular H2O2 production, respectively.

Multiple Myeloma-Derived Bone-Marrow Mesenchymal Stem Cells: Microrna-, Gene Expression-Profiling and Functional Characterization

Abstract 1837 Introduction:Current research has demonstrated that cancer cells are not only influenced by their microenvironment, but are also able to drastically alter their surroundings to further accelerate cancer progression. Hematological malignancies create a forward feedback system with local mesenchymal stromal cells within the bone marrow, but the exact mechanisms and cellular changes within the stroma are largely unknown. Our work has aimed at understanding the bidirectional interaction between multiple myeloma (MM) cells and human bone marrow-derived mesenchymal stromal cells (hMSCs) by characterizing hMSCs derived from either MM patients or healthy individuals, using medium-throughput assays and 2D and 3D in vitro bone marrow models. Methods:First, primary human myeloma patient MSCs (MM-MSCs) and normal donor MSCs (ND-MSCs) were characterized in terms of proliferation when cultured with and without MM.1S cancer cells in direct and indirect co-culture. Next, primary ND- and MM-MSCs were profiled for their microRNA (miRNA) (n=3 for normal, n=7 for MM) and mRNA (n=5 for normal, n=5 for MM) expression using Nanostring technologies. We analyzed 800 human miRNAs from miRBase v.18 and 230 human cancer-related genes using the nCounter® Human Cancer Reference Kit, which allowed for much greater specificity and reliability than microarray technologies. Lastly, a more representative culture system was designed to better mimic myeloma growth within the bone marrow. We developed a 3D in vitro model using hMSCS and fluorescent, luciferase-labeled MM cell lines seeded into porous, autofluorescent silk scaffolds. Scaffolds were made using silk fibroin protein isolated from silkworm cocoons and were formed into biocompatible cylinders with pores of 500–600 microns in diameter. Both ND- and MM-MSCs were labeled with fluorescent cell-tracker dyes and cultured on scaffolds with or without MM1S-GFP+/Luc+ cells. These were non-destructively assessed using confocal microscopy and bioluminescent imaging (BLI) for their ability to promote cancer cell growth and protect cancer cells from chemotherapeutics. Comparison with a conventional 2D in vitro model was performed. Scaffolds were also assessed for their ability to support in vitro culture of primary MM cells using confocal microscopy. Results:MM-MSCs differed from ND-MSCs at miRNA, mRNA, and functional levels. MM-MSCs proliferated slower than ND-MSCs and direct, but not indirect, co-culture of ND-MSCs with MM.1S was able to recapitulate this slowed proliferation in vitro. Indeed, we found that 22 microRNAs were significantly dis-regulated (14 up-regulated and 8 down-regulated in MM patients versus healthy individuals). These included up-regulation of miRNA-222, -181, -146, and -382; together with down-regulation of miRNA-15a, -143, and -199a, in MM-derived hMSCs, compared to ND-MSCs (p<0.05). Moreover, gene expression profiling showed higher expression of CDKN1A, CDKN2A, STAT1, FOSL2, and BCL6 mRNAs, in MM- versus ND- MSCs (fold changes of 3.94, 2.3207, 2.3207, 1.735 and 1.933 respectively, all p<0.05 value).MM1S-GFP+/Luc+ cells were seeded onto scaffolds in presence of either normal- or MM-derived MSCs, in the presence or absence of bortezomib (5nM), and cultured for up to 1 month. Importantly, the 3D model demonstrated stromal-induced protection of MM1S, thus allowing for a more realistic culture system. MM1S cells were protected from bortezomib-induced death by stroma in both 2D and 3D culture over a short time period (48 hours), but, after a 2 week period, this protection was only found in 3D, compared to the 2D model, where no MM1S remained (with or without stroma). Stroma-seeded scaffolds also showed evidence of enhancing culture longevity of primary patient MM cells. Conclusion:MiRNA, mRNA, and functional differences between ND- and MM- MSCs demonstrate changes induced in local stroma in response to MM cells that may encourage tumor growth and require further investigation. The different growth patterns and drug responses of MM.1S to therapies in 2D vs 3D culture suggests that a 3D co-culture environment may represent a more realistic model of drug resistance and minimal residual disease than 2D or monoculture models. Both MM and ND- MSCs were able to induce protection of MM.1S cells in the 3D environment; the mechanisms behind this protection need to be characterized to increase efficacy of current therapeutics within the bone marrow. Disclosures:Ghobrial:Millennium: Advisory Board Other; Novartis: Advisory Board, Advisory Board Other.

Development and Validation of a Medium-Throughput Electrophysiological Assay for KCNQ2/3 Channel Openers Using QPatch HT

The KCNQ2/3 channel has emerged as a drug target for a number of neurological disorders including pain and epilepsy. Known KCNQ2/3 openers have effects on two distinct biophysical properties of the channel: (1) a hyperpolarizing shift in the voltage dependence of channel activation (V(1/2)), and (2) an increase in channel open probability or peak whole-cell current. The current high-throughput screening assays for KCNQ2/3 openers measure changes of channel activity at sub-peak conductances and the output measure is a combination of effects on V(1/2) shift and peak current. Here, we describe a medium-throughput electrophysiological assay for screening KCNQ2/3 openers using the QPatch HT platform. We employed a double-pulse protocol that measures the shift in V(1/2) and the change in current amplitude at peak conductance voltage. Retigabine along with novel KCNQ2/3 openers were evaluated in this assay. Three classes of KCNQ2/3 openers were identified based on the hyperpolarizing shift in V(1/2) and the change in peak current. All three classes of compounds caused a hyperpolarizing shift in V(1/2), but they were differentiated by their respective effects on peak current amplitude (increase, decrease, or only modestly affecting peak current amplitude). KCNQ2/3 blockers were also identified with this assay. These compounds blocked currents without affecting voltage-dependent activation. In summary, we have developed a medium-throughput assay that can reliably detect changes in the biophysical properties of the KCNQ2/3 channel, V(1/2), and peak current amplitude, and therefore may serve as a reliable assay to evaluate KCNQ2/3 openers and blockers.

Catechins protect neurons against mitochondrial toxins and HIV proteins via activation of the BDNF pathway.

Currently, there is no effective treatment for neurological complications of infection with the human immunodeficiency virus that persists despite the use of combination antiretroviral therapy. A medium throughput assay was developed for screening neuroprotective compounds using primary mixed neuronal cells and mitochondrial toxin 3-nitropropionic acid. Using this assay, a library of 2,000 compounds was screened. Out of 256 compounds that showed variable degrees of neuroprotection, nine were related to epicatechin, a monomeric flavonoid found in cocoa and green tea leaves that readily crosses the blood-brain barrier. Hence, catechin, epicatechin, and the related compound, epigallocatechin gallate (EGCG) were further screened for their neuroprotective properties against HIV proteins Tat and gp120, and compared to those of resveratrol. Epicatechin and EGCG targets the brain-derived neurotrophic factor (BDNF) and its precursor proBDNF signaling pathways, normalizing both Tat-mediated increases in proapoptotic proBDNF and concomitant Tat-mediated decreases in the mature BDNF protein in hippocampal neurons. Epicatechin and epigallocatechin gallate were more potent than catechin or resveratrol as neuroprotectants. Due to its simpler structure and more efficient blood-brain barrier penetration properties, epicatechin might be the best therapeutic candidate for neurodegenerative diseases including HIV-associated neurocognitive disorders where oxidative stress is an important pathophysiological mechanism.

RNA interference screen for RGS protein specificity at muscarinic and protease-activated receptors reveals bidirectional modulation of signaling.

Regulator of G protein signaling (RGS) proteins are considered key modulators of G protein-coupled receptor (GPCR)-mediated signal transduction. These proteins act directly on Galpha subunits in vitro to increase their intrinsic rate of GTP hydrolysis; this activity is central to the prevailing view of RGS proteins as negative regulators of agonist-initiated GPCR signaling. However, the specificities of action of particular RGS proteins toward specific GPCRs in an integrated cellular context remain unclear. Here, we developed a medium-throughput assay to address this question in a wholly endogenous context using RNA interference. We performed medium-throughput calcium mobilization assays of agonist-stimulated muscarinic acetylcholine and protease-activated receptors in human embryonic kidney 293 (HEK293) cells transfected with individual members of a "pooled duplex" short interfering RNA library targeting all conventional human RGS transcripts. Only knockdown of RGS11 increased both carbachol-mediated calcium mobilization and inositol phosphate accumulation. Surprisingly, we found that knockdown of RGS8 and RGS9, but not other conventional RGS proteins, significantly decreased carbachol-mediated calcium mobilization, whereas only RGS8 knockdown decreased protease-activated receptor-1 (PAR-1)-mediated calcium mobilization. Loss of responsiveness toward carbachol and PAR-1 agonist peptide upon RGS8 knockdown appears due, at least in part, to a loss in respective receptor cell surface expression, although this is not the case for RGS9 knockdown. Our data suggest a cellular role for RGS8 in the stable surface expression of M3 muscarinic acetylcholine receptor and PAR-1, as well as a specific and opposing set of functions for RGS9 and RGS11 in modulating carbachol responsiveness similar to that seen in Caenorhabditis elegans.

A Discrete Time Model for the Analysis of Medium-Throughput C. elegans Growth Data

BackgroundAs part of a program to predict the toxicity of environmental agents on human health using alternative methods, several in vivo high- and medium-throughput assays are being developed that use C. elegans as a model organism. C. elegans-based toxicological assays utilize the COPAS Biosort flow sorting system that can rapidly measure size, extinction (EXT) and time-of-flight (TOF), of individual nematodes. The use of this technology requires the development of mathematical and statistical tools to properly analyze the large volumes of biological data.Methodology/Principal FindingsFindings A Markov model was developed that predicts the growth of populations of C. elegans. The model was developed using observations from a 60 h growth study in which five cohorts of 300 nematodes each were aspirated and measured every 12 h. Frequency distributions of log(EXT) measurements that were made when loading C. elegans L1 larvae into 96 well plates (t = 0 h) were used by the model to predict the frequency distributions of the same set of nematodes when measured at 12 h intervals. The model prediction coincided well with the biological observations confirming the validity of the model. The model was also applied to log(TOF) measurements following an adaptation. The adaptation accounted for variability in TOF measurements associated with potential curling or shortening of the nematodes as they passed through the flow cell of the Biosort. By providing accurate estimates of frequencies of EXT or TOF measurements following varying growth periods, the model was able to estimate growth rates. Best model fits showed that C. elegans did not grow at a constant exponential rate. Growth was best described with three different rates. Microscopic observations indicated that the points where the growth rates changed corresponded to specific developmental events: the L1/L2 molt and the start of oogenesis in young adult C. elegans.ConclusionsQuantitative analysis of COPAS Biosort measurements of C. elegans growth has been hampered by the lack of a mathematical model. In addition, extraneous matter and the inability to assign specific measurements to specific nematodes made it difficult to estimate growth rates. The present model addresses these problems through a population-based Markov model.

New anti-tuberculosis agents amongst known drugs

Mycobacterium tuberculosis has an on-going impact on global public health and new therapeutics to treat tuberculosis are urgently required. The emergence of drug resistant tuberculosis poses a serious threat to the control of this pathogen, and the development of drugs that are active against the resistant strains is vital. A medium-throughput assay using the Alamar Blue reagent was set-up to identify novel inhibitors of M. tuberculosis from a library of known drugs, for which there has already been extensive research investigating their suitability and safety as human therapeutics. Of the 1514 compounds screened, 53 were demonstrated to possess inhibitory properties against M. tuberculosis at a concentration of 5microM or below. Of these, 17 were novel inhibitors while 36 were known tuberculosis drugs or had been previously described as possessing anti-tuberculosis activity. Five compounds were selected as those which represent the most promising starting points for new anti-tuberculosis agents. It was demonstrated that all five were active against intracellular M. tuberculosis in a macrophage model of infection. The anti-tuberculosis agents identified in this screen represent promising new scaffolds on which future drug development efforts can be focused.

Development and Evaluation of a FACS-Based Medium-Throughput Assay for HCV Entry Inhibitors

The interaction between the hepatitis C virus (HCV) envelope glycoprotein E2 and the human tetraspanin protein CD81 is one of the key events involved in HCV cell entry. Therefore, compounds that interfere with this interaction may be useful tools for basic research and potential drugs for the treatment of HCV infection. The authors describe a medium-throughput assay for ligands of the E2 binding site on the CD81 receptor. In the assay, human hepatoma cells are incubated with the test compounds and stained with a fluorescently labeled anti-CD81 antibody (JS81). Flow cytometry is used to detect the level of bound antibody, reflecting the inhibitory potencies of the compounds. Eighty percent of compounds active in the assay show efficacy in an infection assay using luciferase reporter genome in cell culture. Thus, the assay can be used as a fast screening system for inhibitors of interaction of viral E2 to host cell CD81-LELs.

Melanin Concentrating Hormone Receptor Antagonists as Antiobesity Agents: From M2 to MCHR-1

Melanin concentrating hormone (MCH) is a cyclic, nonadecapeptide expressed in the CNS of all vertebrates that regulates feeding behavior and energy homeostasis. The MCH-1 receptor (MCH-R1) has been identified as a key target in MCH regulation, as small molecule antagonists of MCH-R1 have demonstrated activity in vivo. Herein, we chronicle our efforts to optimize a hit identified via high throughput screening of our proprietary compound library. Several challenges such as selectivity over other receptors, toxicity of a potential metabolite and determining receptor occupancy via a medium throughput assay will be reviewed.

Development of a Screening Assay for Surrogate Markers of Chk1 Inhibitor-Induced Cell Cycle Release

Chk1 is a key regulator of the S and G2/M checkpoints and is activated following DNA damage by agents such as the topoisomerase I inhibitor camptothecin (CPT). It has been proposed that Chk1 inhibitors used in combination with such a DNA damaging agent to treat tumors would potentiate cytotoxicity and increase the therapeutic index, particularly in tumors lacking functional p53. The aim of this study was to determine whether gene expression analysis could be used to inform lead optimization of a novel series of Chk1 inhibitors. The candidate small-molecule Chk1 inhibitors were used in combination with CPT to identify potential markers of functional Chk1 inhibition, as well as resulting cell cycle progression, using cDNA-based microarrays. Differential expression of several of these putative marker genes was further validated by RT-PCR for use as a medium-throughput assay. In the presence of DNA damage, Chk1 inhibitors altered CPT-dependent effects on the expression of cell cycle and DNA repair genes in a manner consistent with a Chk1-specific mechanism of action. Furthermore, differential expression of selected marker genes, cyclin E2, EGR1, and DDIT3, was dose dependent for Chk1 inhibition. RT-PCR results for these genes following treatment with a panel of Chk1 inhibitors showed a strong correlation between marker gene response and the ability of each compound to abrogate cell cycle arrest in situ following CPT-induced DNA damage. These results demonstrate the utility of global expression analysis to identify surrogate markers, providing an alternative method for rapid compound characterization to support advancement decisions in early drug discovery.

Quantifying the association and dissociation rates of unlabelled antagonists at the muscarinic M3receptor

Slow receptor dissociation kinetics has been implicated in the long clinical duration of action of the muscarinic receptor antagonist tiotropium. However, despite the potential benefits of new drugs with slow dissociation kinetics, the rate parameters of new compounds are seldom measured due to technical difficulties and financial implications associated with radiolabeling multiple ligands. Here we describe the development and optimisation of a medium throughput assay which is capable of measuring the kinetic parameters of novel, unlabelled compounds. Radioligand binding studies were performed with membranes derived from CHO cells recombinantly expressing the human M(3) muscarinic receptor.Initial characterisation of the radioligand [(3)H]-NMS yielded on and off rates of 4.1+/-0.2 x 10(8) M(-1) min(-1) and 0.015+/-0.0005 min(-1), respectively. The specific binding of [(3)H]-NMS was measured over time in the presence and absence of several concentrations of unlabelled competitor compounds. These data were analysed using a competition kinetic model to provide on and off rates for the unlabelled competitor. Comparison of the kinetically derived Kd (k(off)/k(on)) with K(i) values generated at equilibrium showed an excellent correlation (r(2)=0.99), providing good validation of the method. The on and off rates were also used in theoretical computer simulations to successfully predict the effect of incubation time on apparent IC(50) values. This study demonstrates that a medium-throughput competition kinetic binding assay can be used to determine accurate on and off rates of unlabelled compounds, providing the opportunity to optimise for kinetic parameters early in the drug discovery process.

The development of a medium throughput assay for lanosterol synthase from Leptosphaeria nodorum: Comparison of the enzyme from L. nodorum, Saccharomyces cerevisiae, and two species of Fusarium

Lanosterol synthase (LS) was isolated from microsomes from the plant pathogenic fungus Leptosphaeria nodorum ( L. nodorum) and characterized with respect to its physical and kinetic properties. A medium throughput radiometric assay was developed for LS in order to screen potential inhibitors and to determine its kinetic properties. Quantification of LS activity relies on the separation of (±)2,3-oxidosqualene from lanosterol by reverse-phase high-pressure liquid chromatography (RP-HPLC) followed by analysis in a flow scintillation counter. LS from L. nodorum has an apparent K m (oxidosqualene) of 43 μM and an apparent native molecular weight of 150 kDa as determined by size exclusion chromatography. L. nodorum LS was compared to LS from Fusarium culmorum ( F. culmorum), Fusarium graminearum ( F. graminearum), and Saccharomyces cerevisiae ( S. cerevisiae) for kinetic properties and inhibitory activity of a known LS inhibitor (4-bromophenyl)[2-fluoro-4-[[6-(methyl-2-propenylamino)hexyl]oxy]phenyly]-methanone (compound 1) and a derivative of this inhibitor (compound 2). While the K m (oxidosqualene) was similar for LS from all four organisms, there were differences in the activity of the two inhibitors against LS from these species. The characteristics of the assay and its use for screening molecules to find fungal inhibitors are described. To compare the biological activity of the inhibitors with their activity against LS, they were tested to determine the extent to which they suppressed the growth of L. nodorum and F. culmorum. The results suggest that the fungicidal activity of these inhibitors is related to their inhibitory effect on LS.

Sensitive Assay for Laboratory Evolution of Hydroxylases toward Aromatic and Heterocyclic Compounds

Powerful directed evolution methods have been developed for tailoring proteins to our needs in industrial applications. Here, the authors report a medium-throughput assay system designed for screening mutant libraries of oxygenases capable of inserting a hydroxyl group into a C-H bond of aromatic or O-heterocyclic compounds and for exploring the substrate profile of oxygenases. The assay system is based on 4-aminoantipyrine (4-AAP), a colorimetric phenol detection reagent. By using 2 detection wavelengths (509 nm and 600 nm), the authors achieved a linear response from 50 to 800 microM phenol and standard deviations below 11% in 96-well plate assays. The monooxygenase P450 BM-3 and its F87A mutant were used as a model system for medium-throughput assay development, identification of novel substrates (e.g., phenoxytoluene, phenylallyether, and coumarone), and discovery of P450 BM-3 F87A mutants with 8-fold improvement in 3-phenoxytoluene hydroxylation activity. This activity increase was achieved by screening a saturation mutagenesis library of amino acid position Y51 using the 4-AAP protocol in the 96-well format.

The carboxyterminal processing protease of D1 protein: expression, purification and enzymology of the recombinant and native spinach proteins

The carboxyterminal processing protease of D1 protein (CtpA) is predicted to be an excellent target for a general broad-spectrum herbicide. The gene for spinach CtpA has been expressed in Escherichia coli. The expressed protein that was found mainly in inclusion bodies has been purified and refolded on a nickel-chelate column. Active recombinant CtpA was recovered. Two assays for CtpA activity were developed, a medium-throughput HPLC assay using a fluorescent substrate and a high-throughput assay based on fluorescence polarization capable of application in a high-throughput 96-well plate format. This high-throughput assay was developed to screen chemistry for CtpA inhibitors. Native spinach CtpA was partially purified and the native and recombinant enzymes were compared kinetically for their K(m) and V(max) values using different peptide substrates. Native CtpA partially purified from spinach was shown to have similar kinetic properties to recombinant CtpA. Antibodies developed against the recombinant protein were used to estimate the in planta abundance of the native enzyme in spinach. Since only a small proportion of the recombinant protein is refolded during isolation and it appears that only a small proportion of this enzyme is active, size-exclusion chromatography and light scattering experiments were performed on rCtpA in order to gain insight into its structure and the reasons why most of the protein is not active. The use of rCtpA to screen for herbicidal compounds and the more general question of how good a herbicide target the enzyme is are discussed.