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PubMed2002

Pharmacogenomics in cancer drug discovery and development: inhibitors of the Hsp90 molecular chaperone.

Drug discovery is being revolutionised by a number of technological developments. These include high throughput screening, combinatorial chemistry and genomics. The impact of the new technologies is to accelerate the pace of anticancer discovery. The completion of the Human Genome Project and the ongoing high throughput sequencing of cancer genomes will facilitate the identification of a range of new molecular targets for drug discovery. Over the next few years we will have a complete molecular understanding of the various combinations of genes and cognate pathways that drive the malignant phenotype and tumour progression. The vision for postgenomic cancer drug discovery must be to identify therapeutic agents that correct or exploit each of these molecular abnormalities. In this way, it will be possible to develop personalised drug combinations that are targeted to the molecular make up of individual tumours. It is anticipated that these therapies will be more effective and less toxic than current approaches, although combinations of novel agents with existing cytotoxic therapies are likely to continue for some time. Examples of postgenomic, mechanism-based drugs include Glivec, Herceptin and Iressa, with many more agents undergoing preclinical and clinical development. An interesting new approach involves the development of inhibitors of heat shock protein (Hsp90) molecular chaperone. Because Hsp90 is required for the correct folding, stability and function of a range of oncoproteins that are mutated or over expressed in cancer, Hsp90 inhibitors have the potential to provide a simultaneous, combinatorial attack on multiple oncogenic pathways. By depleting the levels of multiple oncoproteins in cancer cells and blocking a wide range of oncogenic pathways, Hsp90 inhibitors have the potential to inhibit all of the hallmark characteristics of cancer cells. Progress in the preclinical and clinical development of Hsp90 inhibitors will be described, including an update on clinical studies with the first-in-class agent 17AAG. The use of the postgenomic technology of gene expression microarrays in cancer pharmacology and drug development will be exemplified.

Animals↗
PubMed2005

Screening of peptide affinity tags using immobilised metal affinity chromatography in 96-well plate format.

A method for high throughput screening of Green Fluorescent Proteins carrying metal binding tags in bacteria was developed. A random four amino acids tag-peptide library was successfully generated in E. coli. A 96-microtiter plate assembled with metal-iminodiacetic acid small cryogel columns was used for library screening. For the first time we were able to simultaneously screen a metal binding peptide tags library obtained from E. coli against different metal ions. From screening 25 different tags, three clones were able to bind to all metal ions studied (Ni2+, Zn2+, Co2+ and Cd2+). It was clearly demonstrated that the new construct could facilitate the screening of large peptide libraries.

Affinity Labels↗
PubMed2006

Drug-target identification in Drosophila cells: combining high-throughout RNAi and small-molecule screens.

RNA interference (RNAi) and small-molecule approaches are synergistic on multiple levels, from technology and high-throughput screen development to target identification and functional studies. Here, we describe the RNAi screening platform that we have established and made available to the community through the Drosophila RNAi Screening Center at Harvard Medical School. We then illustrate how the combination of RNAi and small-molecule HTS can lead to effective identification of targets in drug discovery.

Animals↗
PubMed2006

High-throughput synthesis and screening of ternary metal alloys for electrocatalysis.

We report the application of a new method for the high-throughput synthesis and screening of thin film materials and its application to the discovery of electrocatalysts. Results are presented for the PtPdAu ternary alloy system with respect to activity for oxygen reduction. The results reveal an enhancement in activity for a range of PtPd alloy compositions over either of the pure elements. An optimum composition range of ternary alloys with significant activity was also identified. A correlation was also investigated between the surface reduction potential and the activity for oxygen reduction in both binary and ternary alloys. The results demonstrate the potential of the methodology for the discovery and optimization of electrocatalysts for a wide range of applications.

Journal Article↗
PubMed2003

Comparison of assay technologies for a nuclear receptor assay screen reveals differences in the sets of identified functional antagonists.

Many assay technologies currently exist to develop high-throughput screening assays, and the number of choices continues to increase. Results from a previous study comparing assay technologies in our laboratory do not support the common assumption that the same hits would be found regardless of which assay technology is used. To extend this investigation, a nuclear receptor antagonist assay was developed using 3 assay formats: AlphaScreen, time-resolved fluorescence (TRF), and time-resolved fluorescence resonance energy transfer (TR-FRET). Compounds ( approximately 42000) from the Novartis library were evaluated in all 3 assay formats. A total of 128 compounds were evaluated in dose-response experiments, and 109 compounds were confirmed active from all 3 formats. The AlphaScreen, TRF, and TR-FRET assay technologies identified 104, 23, and 57 active compounds, respectively, with only 18 compounds active in all 3 assay formats. A total of 128 compounds were evaluated in a cell-based functional assay, and 35 compounds demonstrated activity in this cellular assay. Furthermore, 34, 11, and 16 hits that were originally identified in the dose-response experiment by AlphaScreen, TRF, and TR-FRET assay technologies, respectively, were functionally active. The results of the study indicated that AlphaScreen identified the greatest number of functional antagonists.

Animals↗
PubMed2004

Microarrayed compound screening (microARCS) to identify activators and inhibitors of AMP-activated protein kinase.

A novel and innovative high-throughput screening assay was developed to identify both activators and inhibitors of AMP-activated protein kinase (AMPK) using microarrayed compound screening (microARCS) technology. Test compounds were arrayed at a density of 8640 on a polystyrene sheet, and the enzyme and peptide substrate were introduced into the assay by incorporating them into an agarose gel followed by placement of the gels onto the compound sheet. Adenosine triphosphate (ATP) was delivered via a membrane, and the phosphorylated biotinylated substrate was captured onto a streptavidin affinity membrane (SAM trade mark ). For detection, the SAM trade mark was removed, washed, and imaged on a phosphor screen overnight. A library of more than 700,000 compounds was screened using this format to identify novel activators and inhibitors of AMPK.

AMP-Activated Protein Kinases↗
PubMed2004

Molecular diversity management strategies for building and enhancement of diverse and focused lead discovery compound screening collections.

This publication describes processes for the selection of chemical compounds for the building of a high-throughput screening (HTS) collection for drug discovery, using the currently implemented process in the Discovery Technologies Unit of the Novartis Institute for Biomedical Research, Basel Switzerland as reference. More generally, the currently existing compound acquisition models and practices are discussed. Our informatics, chemistry and biology-driven compound selection consists of two steps: 1) The individual compounds are filtered and grouped into three priority classes on the basis of their individual structural properties. Substructure filters are used to eliminate or penalize compounds based on unwanted structural properties. The similarity of the structures to reference ligands of the main proven druggable target families is computed, and drug-similar compounds are prioritized for the following diversity analysis. 2) The compounds are compared to the archive compounds and a diversity analysis is performed. This is done separately for the prioritized, regular and penalized compounds with increasingly stringent dissimilarity criterion. The process includes collecting vendor catalogues and monitoring the availability of samples together with the selection and purchase decision points. The development of a corporate vendor catalogue database is described. In addition to the selection methods on a per single molecule basis, selection criteria for scaffold and combinatorial chemistry projects in collaboration with compound vendors are discussed.

Computational Biology↗
PubMed1998

Functional Genomics Conference: From Identifying Proteins to Faster Drug Discovery. March 10-11, 1998, Washington DC, USA.

The massive effort to sequence the human, mouse, rat, nematode (Caenorhabditis elegans), fruit fly (Drosophila), zebra fish, yeast (Saccharomyces cerevisiae), fungal (Candida albicans and Aspergillus fumigatus) and several bacterial genomes has produced a flood of sequence data. Of the more than 100,000 human genes and thousands from other organisms, many partial sequences and several completed microbial genomes are available in both public and private databases. However, elucidation of function has been achieved for only a very small portion and an even smaller percentage have been validated as drug targets. Many companies interested in identifying new drug targets also see this bounty of opportunity as a major challenge. The raw sequence data say little about the importance of the gene and nothing about its potential as a target for drug discovery. Since 1994, a new term, 'functional genomics', has entered our lexicon. Functional genomics, which in effect is 'high-throughput biology', was originally focused on understanding gene function by studying the genes of simpler organisms, such as the nematode, C. elegans. As the genes from a number of organisms are highly conserved across species, it is believed that studying these basic systems can yield valuable insights for drug companies interested in targeting therapeutics for the higher organisms. More recently, the approach to functional genomics has expanded to include study of gene function in organisms to be targeted for therapeutic intervention. This new approach was the theme of the Functional Genomics Conference: From Identifying Proteins to Faster Drug Discovery held in Washington DC on March 10 and 11, 1998. The organisers (NMHCC) hoped that the breadth of the conference topics would reflect the complexities of the modern drug discovery process and covered technologies from gene chips, bioinformatics, disease models, protein discovery and expression, target validation, high-throughput screening for genes of unknown function, to integration of the drug discovery process. The two day conference placed emphasis on cutting edge technology solutions and the development of high-throughput tools to address the emerging opportunities in genome-based drug discovery.

Journal Article↗
PubMed2000

Privileged molecules for protein binding identified from NMR-based screening.

A statistical analysis of NMR-derived binding data on 11 protein targets was performed to identify molecular motifs that are preferred for protein binding. The analysis indicates that compounds which contain a biphenyl substructure preferentially bind to a wide range of proteins and that high levels of specificity (>250-fold) can be achieved even for these small molecules. These results suggest that high-throughput screening libraries that are enriched with biphenyl-containing compounds can be expected to have increased chances of yielding high-affinity ligands for proteins, and they suggest that the biphenyl can be utilized as a template for the discovery and design of therapeutics with high affinity and specificity for a broad range of protein targets.

Biphenyl Compounds↗
PubMed2000

Discovering novel ligands for macromolecules using X-ray crystallographic screening.

The need to decrease the time scale for clinical compound discovery has led to innovations at several stages in the process, including genomics/proteomics for target identification, ultrahigh-throughput screening for lead identification, and structure-based drug design and combinatorial chemistry for lead optimization. A critical juncture in the process is the identification of a proper lead compound, because a poor choice may generate costly difficulties at later stages. Lead compounds are commonly identified from high-throughput screens of large compound libraries, derived from known substrates/inhibitors, or identified in computational prescreeusing X-ray crystal structures. Structural information is often consulted to efficiently optimize leads, but under the current paradigm, such data require preidentification and confirmation of compound binding. Here, we describe a new X-ray crystallography-driven screening technique that combines the steps of lead identification, structural assessment, and optimization. The method is rapid, efficient, and high-throughput, and it results in detailed crystallographic structure information. The utility of the method is demonstrated in the discovery and optimization of a new orally available class of urokinase inhibitors for the treatment of cancer.

Administration, Oral↗
PubMed2006

Isolation of cell specific peptide ligands using fluorescent bacterial display libraries.

Methods for identifying and producing cell specific affinity reagents are critical in cell detection, separation, and therapeutic delivery applications, yet remain difficult and time consuming. To address these limitations, a rapid and quantitative screening approach was developed using intrinsically fluorescent bacterial display peptide libraries and fluorescence-activated cell sorting (FACS). High-throughput screening of fluorescent libraries yielded a panel of peptide ligands mediating specific recognition of human breast cancer tumor cells. Clonal populations of fluorescent, peptide-displaying bacteria enabled single-step, fluorescent labeling of the target cells for cytometry and microscopy analysis. Isolated peptides could be categorized into several distinct groups possessing strong consensus sequences with as many as six identities. Importantly, individual clones exhibited high specificity target cell binding, with more than 80-fold increased binding to tumor cells (ZR-75-1) relative to cell lines derived from healthy tissue (HMEC, MCF-10A). Fluorescent display libraries thus provide a powerful new methodology for parallel identification of cell specific affinity ligands.

Affinity Labels↗
PubMed1999

A Screening Strategy Based on Differential Binding of Ligand to Receptor and to Binding Proteins: Screening for Compounds Interacting with Corticotrophin-Releasing Factor-Binding Protein.

The ligand-receptor interaction has been commonly used in development of high throughput screening assays for new drugs. In some cases, an endogenous ligand interacts not only with membrane receptors but also with soluble binding proteins. Corticotrophin-releasing factor (CRF) is an important stress neurotransmitter/hormone involved in both the central and peripheral nervous systems. CRF exerts its function by interacting with CRFR1 and CRFR2 receptors. In addition, CRF-binding protein (CRF-BP) binds CRF with high affinity. Accordingly, CRF-BP has been suggested to play an important role in modulating CRF function. Based on the potential involvement of CRF-BP in many neurological disorders, it is desirable to develop a screening assay to look for drugs that either mimic or interfere with CRF binding to CRF-BP. An assay was developed to monitor the interactions of radiolabeled CRF with human/rat CRF-BP and the mouse CRFR1 (mCRFR1) receptor. By carefully examining the binding characteristics of radiolabeled CRF to mCRFR1, the assay was able to identify compounds that bind to CRF-BP with high affinity and have little or no affinity for mCRFR1 receptors. Based on a mathematical model, we have verified the screening system with several well-characterized CRF ligands that all have different affinities for CRF receptors and CRF-BP.

Journal Article↗
PubMed2000

Circular dichroism as a detection method in the screening of enantioselective catalysts.

The combination of liquid chromatography (HPLC), UV/Vis-spectroscopy and circular dichroism (CD) can be used to construct a high-throughput screening system to determine the enantioselectivity of enzyme- or metal-catalyzed reduction of acetophenone with formation of (S)- and (R)-1-phenylethanol. Prerequisite for the viability of this system is the experimental finding that the anisotropy factor g is linearly related to the enantiomeric excess (ee) and that it is independent of concentration, thereby excluding possible aggregation effects.

Acetonitriles↗
PubMed2001

Development of a yeast stop codon assay readily and generally applicable to human genes.

We established a yeast-based method to screen chain-terminating mutations that is readily applicable to any gene of interest. Based on the finding that 18- to 24-base-long homologous sequences are sufficient for gap repair in vivo in yeast, we used a strategy to amplify a test-gene fragment with addition of 24-bp sequences homologous to both cut-ends of a yeast expression vector, pMT18. After co-transformation with the amplified fragment and the linearized pMT18, each yeast (Saccharomyces cerevisiae) cell automatically forms a single-copy circular plasmid (because of CEN/ARS), which expresses a test-gene::ADE2 chimera protein. When the reading frame of the test-gene contains a nonsense or frameshift mutation, truncation of the chimera protein results in lack of ADE2 activity, leading to formation of a red colony. By using a nested polymerase chain reaction using proofreading Pfu polymerase to ensure specificity of the product, the assay achieved a low background (false positivity). We applied the assay to BRCA1, APC, hMSH6, and E-cadherin genes, and successfully detected mutations in mRNA and genomic DNA. Because this method--universal stop codon assay--requires only 4 to 5 days to screen a number of samples for any target gene, it may serve as a high-throughput screening system of general utility for chain-terminating mutations that are most prevalent in human genetic diseases.

Adenomatous Polyposis Coli Protein↗
PubMed1998

Fluorescent-protein biosensors: new tools for drug discovery.

Recent improvements in target discovery and high-throughput screening have increased the pressure at key points along the drug-discovery pipeline. High-content screening was developed to ease the bottlenecks formed at the target-validation and lead-optimization points, and a new generation of reagents that report on specific molecular processes in living cells (fluorescent-protein biosensors) have been important in its development. Creative designs of fluorescent-protein biosensors have emerged and been used to measure the molecular dynamics of macromolecules, metabolites and ions. Recent applications of fluorescent-protein biosensors to biological problems have provided a foundation for their use in biotechnology.

Biosensing Techniques↗
PubMed2002

Epitope-mapping of transglutaminase with parallel label-free optical detection.

The gastrointestinal disorder coeliac disease (CD) is induced by the ingestion of wheat gluten and is characterized by damage of the typical structure of the intestinal mucosa. The enzyme tissue transglutaminase (tTGase) was identified as the major target of disease-specific antibodies in-patients. We performed an epitope fine-mapping with a series of pentadecapeptides synthesized using parallel multiple peptide synthesis. For the detection of biomolecular interactions a label-free parallel method, reflectometric interference spectroscopy (RIfS), was used. This is the first optical label-free method adapted to a high throughput screening (HTS) format and the experimental results demonstrate its applicability as a biological screening device. A high titer of anti-tTGase antibodies is found in the serum of coeliac patients. We have taken the first step towards a fast non-surgical test for the detection of these antibodies. In order to identify and characterize a continuous epitope with high affinity against the anti-tTGase antibody a screening of 21 pentadecapeptides has been accomplished with the parallel RIfS system. A single channel RIfS-system with high resolution was used to determine binding constants of identified peptides with high affinity.

Animals↗
PubMed2003

High throughput electrophysiology: new perspectives for ion channel drug discovery.

Proper function of ion channels is crucial for all living cells. Ion channel dysfunction may lead to a number of diseases, so-called channelopathies, and a number of common diseases, including epilepsy, arrhythmia, and type II diabetes, are primarily treated by drugs that modulate ion channels. A cornerstone in current drug discovery is high throughput screening assays which allow examination of the activity of specific ion channels though only to a limited extent. Conventional patch clamp remains the sole technique with sufficiently high time resolution and sensitivity required for precise and direct characterization of ion channel properties. However, patch clamp is a slow, labor-intensive, and thus expensive, technique. New techniques combining the reliability and high information content of patch clamping with the virtues of high throughput philosophy are emerging and predicted to make a number of ion channel targets accessible for drug screening. Specifically, genuine HTS parallel processing techniques based on arrays of planar silicon chips are being developed, but also lower throughput sequential techniques may be of value in compound screening, lead optimization, and safety screening. The introduction of new powerful HTS electrophysiological techniques is predicted to cause a revolution in ion channel drug discovery.

Animals↗
PubMed2004

Screening for genotoxicity using the DRAG assay: investigation of halogenated environmental contaminants.

The DRAG test is a rapid high-throughput screening assay for detection of repairable adducts by growth inhibition of Chinese hamster ovary cells (CHO) characterized by different defects in DNA repair. A more pronounced growth inhibition caused by a certain DNA-reactive substance in a repair-deficient cell line (EM9, UV4 and UV5) as compared to wild-type cells (AA8) is interpreted as a consequence of their inability to repair induced DNA lesions. Thus, the use of such cell lines in the DRAG test may provide information of the type of DNA lesions induced by a certain genotoxic substance. To select optimal assay conditions, as well as to provide a mechanistic basis for interpreting the results, the model compounds benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE), ethyl methanesulfonate (EMS), mitomycin C (MMC) and hydrogen peroxide (H2O2) were used. These agents can induce bulky adducts, alkyl adducts, cross-links and oxidative damage, respectively. The specificity of the DRAG test constitutes an important prerequisite for its practical use in a broader context. To assess this aspect, we have investigated the genotoxic and cytotoxic properties of a selection of metabolites of and isomers from polychlorinated biphenyls (PCB) and polybrominated diphenyl ethers (PBDE), along with a few other halogenated compounds. All these compounds have been detected as pollutants in the external environment, and for most of them there is no convincing evidence of mutagenicity from conventional assays. As could be predicted from their mode of action, BPDE, MMC, and EMS were all found to be more toxic in the repair-deficient cell lines compared with wild-type cells. The results with H2O2 were inconclusive, and the PCB metabolite 4,4'-diOH-CB80 only exhibited borderline activity, while all other halogenated compounds, or their metabolites, were found to be inactive. In conclusion, the DRAG assay could provide a robust and useful tool when screening large numbers of potentially genotoxic agents, while in addition providing mechanistic information. However, the usefulness of the selected cell lines to detect oxidative damage may be limited.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗