False Positive Hits Research Articles

Abstract 2919: Discovery of small-molecule compounds targeting c-MYC using a novel cell-based screen

Abstract Elevated expression of the c-MYC oncogene (either due to gene amplification, translocation, abnormality in upstream signaling pathways and/or protein stabilization) is one of the most common abnormalities in human cancers. Efforts to identify direct pharmacological inhibitors of c-MYC function have not yet yielded drug-like molecules. Therefore, we sought to pursue alternative screening strategies for this classically “undruggable” transcription factor. We developed a novel antibody-based high-throughput HTRF screening assay that specifically detects endogenous c-MYC protein levels in a MYC amplified cancer cell line. Taking advantage of the short half-life of c-MYC, both at the protein and mRNA level, we conducted a cell-based screen of the GSK screening collection to identify compounds that can rapidly decrease c-MYC protein levels. Elimination of false positive hits using stringent triage assays successfully identified two valid hit series exemplified by GSK970 and GSK417. Molecular mode of action studies revealed these molecules inhibit MYC transcription by binding to the minor groove of DNA with AT sequence specificity. Unfortunately, this mechanism of c-MYC inhibition demonstrated poor in vivo translatability as tissue DNA acts as a molecular sink, effectively sequestering compound, and limiting its pharmacodnyamic response. Citation Format: Biju Mangatt, Anthony D. Pietra, Anna Waszkiewicz, Jon-Paul Jaworski, Sonja Ghidelli-Disse, Thomas J. Berrodin, Christian S. Sherk, Derrick W. Meinhold, Anna Rutkowska-Klute, Shanker K. Sundaram, Gopinath Ganji, Wendy S. Halsey, George P. Livi, William Li, James Mack, Stuart P. Romeril, Elisabeth A. Minthorn, Rakesh Kumar, Gerard C. Drewes, Dirk A. Heerding, Lorena A. Kallal, Carolyn A. Buser, Jesus R. Medina. Discovery of small-molecule compounds targeting c-MYC using a novel cell-based screen [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2919.

Abstract LB-068: Development of a screening cascade to identify selective small molecule inhibitors of DNMT1

Abstract DNA Methyltransferases (or DNMTs) play an important role in genomic integrity. The DNMT family of enzymes catalyse the transfer of methyl groups to specific CpG structures on DNA, with DNMT1 acting as a maintenance methyltransferase and DNMT3a and 3b acting as de novo methyltransferases. Upon cell division, epigenetic profiles within DNA strands are copied, with DNMT1 ensuring that methyl marks are transferred correctly to the newly synthesised DNA strand, thereby conferring heritability. Aberrant DNA hypermethylation within DNA promoter regions can lead to gene silencing, which is a hallmark of human cancer. Treatment with hypomethylating agents such as azacytidine and decitabine reverse methyl marks and show clinical benefit for the treatment of Acute Myeloid Leukemia (AML) and Chronic Myelomonocytic Leukemia (CMML) and are also the standard of care for Myelodysplastic Syndrome (MDS). However, these agents also show dose limiting toxicity due to their irreversible and non-selective mechanism of action and generally poor pharmacokinetic properties. Over the past decades both the Pharma and Biotech industries have deployed significant resource in a bid to identify and develop potent, selective DNMT1 inhibitors. These attempts have delivered little, if any, success until now. Collaboration between the CRUK Manchester Institute and GSK has resulted in the successful development of a screening cascade to identify compounds that selectively inhibit DNMT1. A collection of over 1 million compounds was screened at GSK using a radioactive scintillation proximity assay (SPA). Preliminary hits were identified and triaged through our screening cascade to determine genuine hits from false positive results. Firstly, ‘hits' from the SPA screen were tested in rapid-fire mass spectrometry and also in fluorescence intensity coupled ‘break-light' assays. Though these orthogonal screens apparently confirmed activity, IC50 screening of many of these compounds revealed high Hill slopes and high maximal inhibition profiles which are indicative of non-specific inhibition mechanisms. Detailed mechanism of inhibition studies alongside differential scanning fluorimetry was employed to rule out those compounds that were binding non-specifically to the oligonucleotide substrate. One series of compounds displayed IC50 profiles consistent with specific inhibition of DNMT1, with a 1:1 stoichiometry and no evidence for non-specific binding to the oligonucleotide substrate. Selectivity screening against DNMT3a and 3b also showed that within the DNMT family this series of compounds was selective for DNMT1 only. All compounds that displayed reasonable biochemical potency, (sub 1µΜ), were tested further in cellular assays to confirm target engagement. Through our extensive range of assays and thorough profiling of hit compounds, we were able to quickly de-validate the significant number of false positive hits that have plagued previous drug discovery screens in this area and identify a series of genuine, selective, small molecule inhibitors of DNMT1. Citation Format: Alexandra Stowell, Graeme Thomson, Mark Cockerill, Charlotte Burt, Emma Fairweather, Ian Waddell, Ali Raoof, Allan Jordan, Donald 0gilvie, Melissa Pappalardi, Juan Luengo, Mehul Patel, Ryan Kruger, Chris Carpenter. Development of a screening cascade to identify selective small molecule inhibitors of DNMT1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-068.

Abstract 1944: High-throughput fluorescence-based assay for screening of Arginase I inhibitors for cancer immunotherapy

Abstract Arginase I (ArgI) is an important small molecule drug target in cancer immunotherapy. Arg1 converts L-arginine into L-ornithine and urea. Recruitment of ArgI-expressing myeloid-derived suppressor cells (MSDCs) at a tumor site results in the depletion of L-arginine, which causes reduced proliferation of T-cells and natural killer cells and inhibition of the antitumor immune response. In patient material, MSDC-induced T-cell suppression can be reverted by arginase inhibitors. ArgI inhibitors work synergistically with checkpoint inhibitor therapy in syngeneic mouse models. In order to find novel inhibitors for ArgI, we developed a set of tools to enable screening and hit validation. The first is an activity assay that enables the high-throughput screening (HTS) of compound libraries. Previously reported arginase assays are poorly compatible with HTS due to the requirement of multiple reactions steps, harsh assay conditions, or the use of low-turnover substrates other than L-arginine. We developed a novel ArgI activity assay, which has a homogenous format and requires only two addition steps before readout. The assay makes use of a fluorescence readout and has a high robustness (Z'-factor > 0.7). Progression of the assay can be followed in real time, allowing for kinetic experiments. To investigate the false positive hit rate, the assay was screened at the Pivot Park Screening Centre with a library consisting of 233 compounds with known interference in other assay formats. Using a simple background signal control, the number of false positives in this library was minimized to below 0.2%. The assay was subsequently used to accurately determine the dissociation constants and binding kinetics of the reference inhibitors ABH, NOHA and CB1158. In order to validate the binding of screening hits to ArgI, we developed a thermal shift assay. We observed a shift in the ArgI melting temperature of up to 3.8°C after addition of the most potent inhibitors. In addition, we developed a Surface Plasmon Resonance binding assay. This showed that ABH and CB1158 have long residence times on ArgI. To allow the development of novel inhibitors through rational drug design, we successfully crystallized human ArgI in a novel space group with higher symmetry (P63) compared to those previously reported (space group P3), and without the presence of hemihedral twinning. We determined a series of high resolution (< 1.7 Å) crystal structures at various pH values and with several ligands. These demonstrate that a series of peptide flips lies at the basis of the pH-dependent symmetry of ArgI and its unusually high pH optimum of 9.0 to 9.5. Finally, to assess the cellular activity of ArgI inhibitors, we examined 102 cancer cell lines for ArgI activity and correlated the results to public gene expression profiles. The novel assay portfolio will help to deliver a new generation of ArgI inhibitors. Citation Format: Yvonne Grobben, Joost C. Uitdehaag, Nicole Willemsen-Seegers, Werner W. Tabak, Martine B. Prinsen, Suzanne J. van Gerwen, Jan van Groningen, Johan Friesen, Helma Rutjes, Jos de Man, Rogier C. Buijsman, Guido J. Zaman. High-throughput fluorescence-based assay for screening of Arginase I inhibitors for cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1944.

Novel cell-based high-throughput hybridoma screening method using the Celigo image cytometer for antibody discovery

Abstract Hybridoma screening is a highly important process that can identify potential clones from hybridoma cultures against the desired target antigen. Traditional screening methods using ELISA and flow cytometry presented technical issues such as limited accuracy, reduced high-throughput capability, increased time and cost. Here, we demonstrate a novel cell-based high-throughput screening method using the Celigo Image Cytometer, which avoids nonspecific signals by contrasting antibody binding signals directly on living cells, with and without recombinant antigen expression. The image cytometry screening method was optimized by detecting the binding of hybridoma supernatants to the recombinant antigen CD39 expressed on CHO cells. Both target CHO and CFSE-stained wild type were co-cultured to simultaneously detect target antibodies and nonspecific binding, which can eliminate the need to produce a separate control sample for each hybridoma supernatant. Next, the image cytometer was used to screen 672 hybridoma supernatants to develop the screening workflow. The Celigo was used to quickly image and analyze antibody binding using Hoechst, Alexa Fluor 594 and CFSE fluorescence to identify high, medium, and low binding hits. In addition, the Celigo was used to measure the binding affinities of the target antibodies to membrane-bound CD39. Furthermore, measuring direct antibody binding to living cells eliminated both false positive and false negative hits. The image cytometry method was highly sensitive and versatile, and could detect positive antibody in supernatants at concentrations as low as ~5 ng/mL. We propose that this method will greatly improve screening technologies and facilitate more efficient antibody discovery.

Transcriptome Profiling Reveals PHLDA1 as a Novel Molecular Marker for Ischemic Cardiomyopathy

Ischemic cardiomyopathy (ICM) represents a worldwide health issue owning to its high sudden death rate. Easy diagnosis and effective treatment of ICM are still lacking. Identification of novel molecular markers will help illustrate the pathophysiology of ICM and facilitate its diagnosis and targeted treatment. Transcription profiling could be an easy and efficient way for identifying new markers. However, the mega data in the available database may contain a large number of false-positive hits. To identify the true marker for ICM, we systematically compared available microarray datasets in the GEO database and identified 26 genes that are shared by all datasets. We further verified the expression pattern of these 26 genes in ICM rat model. Only 12 genes show significant differential expression in our animal model. Among them, we focused on PHLDA1, a well-documented pro-apoptotic factor. Expression of PHLDA1 was elevated in both ischemic cardiac cell lines and in rat model. Overexpression of PHLDA1 promotes apoptosis of cardiac muscle cell. Meanwhile, PHLDA1 not only inhibited AKT pathway, but also activated p53 pathway. We thus confirmed PHLDA1 as a true molecular marker for ICM.

Integration of virtual screening and computational simulation identifies photodynamic therapeutics against human Protoporphyrinogen Oxidase IX (hPPO)

Photodynamic therapy (PDT) is a rapidly evolving area of cancer management against solid tumors. PDT is either administrated by injecting photosensitizer (porphyrins) or by accumulation of intracellular protoporphyrin IX via the inhibition of human Protoporphyrinogen Oxidase IX (hPPO). In this study, novel inhibitors of hPPO have been investigated by integrating virtual screening, molecular docking, and molecular dynamics (MD) simulation. A ligand-based pharmacophore was generated from a training set of 22 inhibitors of hPPO. The selected pharmacophore had four chemical features including three hydrogen bond acceptors and one hydrophobic. The pharmacophore was characterized by highest correlation coefficient of 0.96, cost difference of 53.20, and lowest root mean square deviation of 0.73. The resultant pharmacophore was validated by Fischer’s Randomization and Test Set Validation methods. The validated pharmacophore was used as a 3D query to screen chemical databases including NCI, Asinex, Chembridge, and Maybridge. The screening of chemical databases and the subsequent application of Lipinski’s Rule of Five, and ADMET Assessment Test, retrieved 1176 drug-like compounds. The drug-like compounds were subjected to molecular docking studies in the active site of hPPO to eliminate false positive hits and to elucidate their true binding orientation. Top three candidate molecules with high docking scores and hydrogen bond interactions with catalytic active residues were selected as best candidate inhibitors against hPPO. The binding stability of selected candidate inhibitors was evaluated by MD simulation. The MD simulation of hits portrayed strong hydrogen bonds and key hydrophobic interactions with catalytic active residues of hPPO including R59, R97, G159, G332 and flavin moiety of FAD (coenzyme of hPPO). Our study predicts three hit compounds against hPPO, which could possibly accumulate high concentration of protoporphyrinogen-IX, and thereby acting as an intracellular photosensitizer against tumor cells through photodynamic therapy.

Systematic survey of non-retroviral virus-like elements in eukaryotic genomes

Endogenous viral elements (EVEs) are viral sequences that are endogenized in the host cell. Recently, several eukaryotic genomes have been shown to contain EVEs. To improve the understanding of EVEs in eukaryotes, we have developed a system for detecting EVE-like sequences in eukaryotes and conducted a large-scale nucleotide sequence similarity search using all available eukaryotic and viral genome assembly sequences (excluding those from retroviruses) stored in the National Center for Biotechnology Information genome database (as of August 14, 2017). We found that 3856 of 7007 viral genomes were similar to 4098 of 4102 eukaryotic genomes. For those EVE-like sequences, we constructed a database, Predicted Endogenous Viral Elements (pEVE, http://peve.med.u-tokai.ac.jp) which provides comprehensive search results summarized from an evolutionary viewpoint. A comparison of EVE-like sequences among closely related species may be useful to avoid false-positive hits. We believe that our search system and database will facilitate studies on EVEs.

Cellular and Biophysical Pipeline for the Screening of Peroxisome Proliferator-Activated Receptor Beta/Delta Agonists: Avoiding False Positives.

Peroxisome proliferator-activated receptor beta/delta (PPARß/δ) is considered a therapeutic target for metabolic disorders, cancer, and cardiovascular diseases. Here, we developed one pipeline for the screening of PPARß/δ agonists, which reduces the cost, time, and false-positive hits. The first step is an optimized 3-day long cellular transactivation assay based on reporter-gene technology, which is supported by automated liquid-handlers. This primary screening is followed by a confirmatory transactivation assay and by two biophysical validation methods (thermal shift assay (TSA) and (ANS) fluorescence quenching), which allow the calculation of the affinity constant, giving more information about the selected hits. All of the assays were validated using well-known commercial agonists providing trustworthy data. Furthermore, to validate and test this pipeline, we screened a natural extract library (560 extracts), and we found one plant extract that might be interesting for PPARß/δ modulation. In conclusion, our results suggested that we developed a cheaper and more robust pipeline that goes beyond the single activation screening, as it also evaluates PPARß/δ tertiary structure stabilization and the ligand affinity constant, selecting only molecules that directly bind to the receptor. Moreover, this approach might improve the effectiveness of the screening for agonists that target PPARß/δ for drug development.

A multifaceted approach to identify non-specific enzyme inhibition: Application to Mycobacterium tuberculosis shikimate kinase

Single dose high-throughput screening (HTS) followed by dose-response evaluations is a common strategy for the identification of initial hits for further development. Early identification and exclusion of false positives is a cost-saving and essential step in early drug discovery. One of the mechanisms of false positive compounds is the formation of aggregates in assays. This study evaluates the mechanism(s) of inhibition of a set of 14 compounds identified previously as actives in Mycobacterium tuberculosis (Mt) cell culture screening and in vitro actives in Mt shikimate kinase (MtSK) assay. Aggregation of hit compounds was characterized using multiple experimental methods, LC-MS, 1HNMR, dynamic light scattering (DLS), transmission electron microscopy (TEM), and visual inspection after centrifugation for orthogonal confirmation. Our results suggest that the investigated compounds containing oxadiazole-amide and aminobenzothiazole moieties are false positive hits and non-specific inhibitors of MtSK through aggregate formation.

Dual direction CRISPR transcriptional regulation screening uncovers gene networks driving drug resistance

Pooled CRISPR–Cas9 knock out screens provide a valuable addition to the methods available for novel drug target identification and validation. However, where gene editing is targeted to amplified loci, the resulting multiple DNA cleavage events can be a cause of false positive hit identification. The generation of nuclease deficient versions of Cas9 has enabled the development of two additional techniques – CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) – that enable the repression or overexpression, respectively, of target genes. Here we report the first direct combination of all three approaches (CRISPRko, CRISPRi and CRISPRa) in the context of genome-wide screens to identify components that influence resistance and sensitivity to the BRAF inhibitor, vemurafenib. The pairing of both loss- and gain-of-function datasets reveals complex gene networks which control drug response and illustrates how such data can add substantial confidence to target identification and validation analyses.

Hybrid splicing minigene and antisense oligonucleotides as efficient tools to determine functional protein/RNA interactions

Alternative splicing is a complex process that provides a high diversity of proteins from a limited number of protein-coding genes. It is governed by multiple regulatory factors, including RNA-binding proteins (RBPs), that bind to specific RNA sequences embedded in a specific structure. The ability to predict RNA-binding regions recognized by RBPs using whole-transcriptome approaches can deliver a multitude of data, including false-positive hits. Therefore, validation of the global results is indispensable. Here, we report the development of an efficient and rapid approach based on a modular hybrid minigene combined with antisense oligonucleotides to enable verification of functional RBP-binding sites within intronic and exonic sequences of regulated pre-mRNA. This approach also provides valuable information regarding the regulatory properties of pre-mRNA, including the RNA secondary structure context. We also show that the developed approach can be used to effectively identify or better characterize the inhibitory properties of potential therapeutic agents for myotonic dystrophy, which is caused by sequestration of specific RBPs, known as muscleblind-like proteins, by mutated RNA with expanded CUG repeats.

UC2 search: using unique connectivity of uncharged compounds for metabolite annotation by database searching in mass spectrometry-based metabolomics.

SummaryFor metabolite annotation in metabolomics, variations in the registered states of compounds (charged molecules and multiple components, such as salts) and their redundancy among compound databases could be the cause of misannotations and hamper immediate recognition of the uniqueness of metabolites while searching by mass values measured using mass spectrometry. We developed a search system named UC2 (Unique Connectivity of Uncharged Compounds), where compounds are tentatively neutralized into uncharged states and stored on the basis of their unique connectivity of atoms after removing their stereochemical information using the first block in the hash of the IUPAC International Chemical Identifier, by which false-positive hits are remarkably reduced, both charged and uncharged compounds are properly searched in a single query and records having a unique connectivity are compiled in a single search result.Availability and implementationThe UC2 search tool is available free of charge as a REST web service (http://webs2.kazusa.or.jp/mfsearcher) and a Java-based GUI tool.Supplementary information Supplementary data are available at Bioinformatics online.

Abstract B21: CRISPR screens provide a comprehensive assessment of cancer vulnerabilities but generate false-positive hits for highly amplified genomic regions

Abstract CRISPR/Cas9 has emerged as a transformative new tool to systematically probe gene function. In this study, we compare the performance of CRISPR to RNAi-based loss-of-function screens for the identification of cancer dependencies by performing parallel deep-coverage shRNA and CRISPR screens targeting 2722 genes across several cancer cell lines. CRISPR-based dropout screens identified more lethal genes compared to RNAi in all five cancer models, indicating that the identification of many cellular dependencies may require full gene inactivation, as induced by CRISPR but not RNAi. However, in two aneuploid cancer models we found that all genes within highly amplified regions, including non-expressed genes, scored as lethal by CRISPR, revealing an unanticipated class of false-positive hits in CRISPR-based screens. Using a CRISPR tiling array that encompassed all possible sgRNAs against the coding regions of 139 genes, sgRNAs targeting essential domains provide the most robust dropout phenotypes, suggesting that this approach might be used to define the protein domains that are required for cancer dependence. Collectively, these findings demonstrate the utility of CRISPR-based screens in the identification of cancer-dependent genes, but also reveal the need to carefully control for false-positive results especially in chromosomally unstable cancer lines. Note: This abstract was not presented at the conference. Citation Format: Diana Munoz, Frank P. Stegmeier, Michael Schlabach. CRISPR screens provide a comprehensive assessment of cancer vulnerabilities but generate false-positive hits for highly amplified genomic regions [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr B21.

Identification of SPPL2a Inhibitors by Multiparametric Analysis of a High-Content Ultra-High-Throughput Screen

The intramembrane protease signal peptide peptidase-like 2a (SPPL2a) is a potential drug target for the treatment of autoimmune diseases due to an essential role in B cells and dendritic cells. To screen a library of 1.4 million compounds for inhibitors of SPPL2a, we developed an imaging assay detecting nuclear translocation of the proteolytically released cytosolic substrate fragment. The state-of-the-art hit calling approach based on nuclear translocation resulted in numerous false-positive hits, mainly interrupting intracellular protein trafficking. To filter the false positives, we extracted 340 image-based readouts and developed a novel multiparametric analysis method that successfully triaged the primary hit list. The identified scaffolds were validated by demonstrating activity on endogenous SPPL2a and substrate CD74/p8 in B cells. The multiparametric analysis discovered diverse cellular phenotypes and provided profiles for the whole library. The principle of the presented imaging assay, the screening strategy, and multiparametric analysis are potentially applicable in future screening campaigns.

Combining De Novo Peptide Sequencing Algorithms, A Synergistic Approach to Boost Both Identifications and Confidence in Bottom-up Proteomics.

Complex mass spectrometry based proteomics data sets are mostly analyzed by protein database searches. While this approach performs considerably well for sequenced organisms, direct inference of peptide sequences from tandem mass spectra, i.e., de novo peptide sequencing, oftentimes is the only way to obtain information when protein databases are absent. However, available algorithms suffer from drawbacks such as lack of validation and often high rates of false positive hits (FP). Here we present a simple method of combining results from commonly available de novo peptide sequencing algorithms, which in conjunction with minor tweaks in data acquisition ensues lower empirical FDR compared to the analysis using single algorithms. Results were validated using state-of-the art database search algorithms as well specifically synthesized reference peptides. Thus, we could increase the number of PSMs meeting a stringent FDR of 5% more than 3-fold compared to the single best de novo sequencing algorithm alone, accounting for an average of 11 120 PSMs (combined) instead of 3476 PSMs (alone) in triplicate 2 h LC-MS runs of tryptic HeLa digestion.

QSAR, docking, ADMET, and system pharmacology studies on tormentic acid derivatives for anticancer activity

To explore the anticancer compounds from tormentic acid derivatives, a quantitative structure–activity relationship (QSAR) model was developed by the multiple linear regression methods. The developed QSAR model yielded a high activity–descriptors relationship accuracy of 94% referred by regression coefficient (r2 = .94) and a high activity prediction accuracy of 91%. The QSAR study indicates that chemical descriptors, chiV5, T_T_Cl_7, T_2_T_4, SsCH3count, and Epsilon3 are significantly correlated with anticancer activity. This validated model was further been used for virtual screening and thus identification of new potential breast cancer inhibitors. Lipinski’s rule of five, ADMET risk and synthetic accessibility are used to filter false positive hits. Filtered compounds were then docked to identify the possible target binding pocket, to obtain a set of aligned ligand poses and to prioritize the predicted active compounds. The scrutinized compounds, as well as their metabolites, were predicted and analyzed for different pharmacokinetics parameters such as absorption, distribution, metabolism, excretion, and toxicity. Finally, the top-ranked compound NB-12 was evaluated by system pharmacology approach. Later studied the metabolic networks, disease biomarker networks, pathway maps, drug-target networks and generate significant gene networks. The strategy applied in this research work may act as a framework for rational design of potential anticancer drugs.

Systematic Identification of MCU Modulators by Orthogonal Interspecies Chemical Screening

The mitochondrial calcium uniporter complex is essential for calcium (Ca2+) uptake into mitochondria of all mammalian tissues, where it regulates bioenergetics, cell death, and Ca2+ signal transduction. Despite its involvement in several human diseases, we currently lack pharmacological agents for targeting uniporter activity. Here we introduce a high-throughput assay that selects for human MCU-specificsmall-molecule modulators in primary drug screens. Using isolated yeast mitochondria, reconstituted with human MCU, its essential regulator EMRE, and aequorin, and exploiting a D-lactate- and mannitol/sucrose-based bioenergetic shunt that greatly minimizes false-positive hits, we identifymitoxantrone out of more than 600 clinically approved drugs as a direct selective inhibitor of human MCU. We validate mitoxantrone in orthogonal mammalian cell-based assays, demonstrating that our screening approach is an effective and robust tool for MCU-specific drug discovery and, more generally, for the identification of compounds that target mitochondrial functions.

NMR Methods for Identification of False Positives in Biochemical Screens.

Over the past few decades, NMR spectroscopy has become an established tool in drug discovery. This communication will highlight the potential of NMR spectroscopy as a method for identification of problematic compounds and as a valuable aid toward revealing some mechanisms of promiscuous behavior. NMR methods for detecting false positives will be analyzed on the basis of their performance, strengths, limitations, and potential pitfalls. Additionally, this communication aims to provide an insight into the limitations of NMR-based methodologies applied to ligand screening in the context of false-positive hits.

Biased selection of propagation-related TUPs from phage display peptide libraries.

Phage display is rapidly advancing as a screening strategy in drug discovery and drug delivery. Phage-encoded combinatorial peptide libraries can be screened through the affinity selection procedure of biopanning to find pharmaceutically relevant cell-specific ligands. However, the unwanted enrichment of target-unrelated peptides (TUPs) with no true affinity for the target presents an important barrier to the successful screening of phage display libraries. Propagation-related TUPs (Pr-TUPs) are an emerging but less-studied category of phage display-derived false-positive hits that are displayed on the surface of clones with faster propagation rates. Despite long regarded as an unbiased selection system, accumulating evidence suggests that biopanning may create biological bias toward selection of phage clones with certain displayed peptides. This bias can be dependent on or independent of the displayed sequence and may act as a major driving force for the isolation of fast-growing clones. Sequence-dependent bias is reflected by censorship or over-representation of some amino acids in the displayed peptide and sequence-independent bias is derived from either point mutations or rare recombination events occurring in the phage genome. It is of utmost interest to clean biopanning data by identifying and removing Pr-TUPs. Experimental and bioinformatic approaches can be exploited for Pr-TUP discovery. With no doubt, obtaining deeper insight into how Pr-TUPs emerge during biopanning and how they could be detected provides a basis for using cell-targeting peptides isolated from phage display screening in the development of disease-specific diagnostic and therapeutic platforms.

Novel high-throughput cell-based hybridoma screening methodology using the Celigo Image Cytometer

Hybridoma screening is a critical step for antibody discovery, which necessitates prompt identification of potential clones from hundreds to thousands of hybridoma cultures against the desired immunogen. Technical issues associated with ELISA- and flow cytometry-based screening limit accuracy and diminish high-throughput capability, increasing time and cost. Conventional ELISA screening with coated antigen is also impractical for difficult-to-express hydrophobic membrane antigens or multi-chain protein complexes. Here, we demonstrate novel high-throughput screening methodology employing the Celigo Image Cytometer, which avoids nonspecific signals by contrasting antibody binding signals directly on living cells, with and without recombinant antigen expression.The image cytometry-based high-throughput screening method was optimized by detecting the binding of hybridoma supernatants to the recombinant antigen CD39 expressed on Chinese hamster ovary (CHO) cells. Next, the sensitivity of the image cytometer was demonstrated by serial dilution of purified CD39 antibody. Celigo was used to measure antibody affinities of commercial and in-house antibodies to membrane-bound CD39. This cell-based screening procedure can be completely accomplished within one day, significantly improving throughput and efficiency of hybridoma screening. Furthermore, measuring direct antibody binding to living cells eliminated both false positive and false negative hits. The image cytometry method was highly sensitive and versatile, and could detect positive antibody in supernatants at concentrations as low as ~5ng/mL, with concurrent Kd binding affinity coefficient determination. We propose that this screening method will greatly facilitate antibody discovery and screening technologies.