Development Of High-throughput Screening Research Articles

APOBEC3G Subunits Self-associate via the C-terminal Deaminase Domain

Human APOBEC3G (hA3G) is a cytidine deaminase active on HIV single-stranded DNA. Small angle x-ray scattering and molecular envelope restorations predicted a C-terminal dimeric model for RNA-depleted hA3G in solution. Each subunit was elongated, suggesting that individual domains of hA3G are solvent-exposed and therefore may interact with other macromolecules even as isolated substructures. In this study, co-immunoprecipitation and in-cell quenched fluorescence resonance energy transfer assays reveal that hA3G forms RNA-independent oligomers through interactions within its C terminus. Residues 209-336 were necessary and sufficient for homoligomerization. N-terminal domains of hA3G were unable to multimerize but remained functional for Gag and viral infectivity factor (Vif) interactions when expressed apart from the C terminus. These findings corroborate the small angle x-ray scattering structural model and are instructive for development of high throughput screens that target specific domains and their functions to identify HIV/AIDS therapeutics.

Clinical Status of Anti-Cancer Agents Derived from Marine Sources

The chemical, biological and ecological diversity of the marine ecosystem has contributed immensely in the discovery of extremely potent compounds that have shown potent activities in antitumor, analgesia, antiinflammatory, immunomodulation, allergy, anti-viral etc. The compounds of marine origin are diverse in structural class from simple linear peptides to complex macrocyclic polyethers. The recent advances in the sophisticated instruments for the isolation and characterization of marine natural products and development of high-throughput screening, have substantially increased the rate of discovery of various compounds of biomedical application. Didemnin was the first marine peptide that entered in human clinical trials in US for the treatment of cancer and other compounds such as dolastatin-10, soblidotin, didemnin B, ecteinascidin 743, girolline, aplidine, cryptophycins (also arenastatin A), bryostatin 1, ILX 651, kahalalide F, E7389, discodermolide, ES-285 (spisulosine), HTI-286 (hemiasterlin derivative), squalamine, KRN-7000, vitilevuamide, Laulimalide, Curacin A, diazonamide, peloruside A, eleutherobin, sarcodictyin, thiocoraline, salicylihalimides A, ascididemnin, CGX-1160, CGX-1007dictyodendrins, GTS-21 (aka DMBX), manoalide, IPL-576,092 (aka HMR-4011A) have entered in the clinical trials. This article summarize clinical status and synthetic advances of some of these compounds.

Identification of an APOBEC3G Binding Site in Human Immunodeficiency Virus Type 1 Vif and Inhibitors of Vif-APOBEC3G Binding

The APOBEC3 cytidine deaminases are potent antiviral factors that restrict replication of human immunodeficiency virus type 1 (HIV-1). HIV-1 Vif binds APOBEC3G and APOBEC3F and targets these proteins for ubiquitination by forming an E3 ubiquitin ligase with cullin 5 and elongins B and C. The N-terminal region of Vif is required for APOBEC3G binding, but the binding site(s) is unknown. To identify the APOBEC3G binding site in Vif, we established a scalable binding assay in a format compatible with development of high-throughput screens. In vitro binding assays using recombinant proteins identified Vif peptides and monoclonal antibodies that inhibit Vif-APOBEC3G binding and suggested involvement of Vif residues 33 to 83 in APOBEC3G binding. Cell-based binding assays confirmed these results and demonstrated that residues 40 to 71 in the N terminus of Vif contain a nonlinear binding site for APOBEC3G. Mutation of the highly conserved residues His42/43 but not other charged residues in this region inhibited Vif-APOBEC3G binding, Vif-mediated degradation of APOBEC3G, and viral infectivity. In contrast, mutation of these residues had no significant effect on Vif binding and degradation of APOBEC3F, suggesting a differential requirement for His42/43 in Vif binding to APOBEC3G and APOBEC3F. These results identify a nonlinear APOBEC3 binding site in the N terminus of Vif and demonstrate that peptides or antibodies directed against this region can inhibit Vif-APOBEC3G binding, validating the Vif-APOBEC3 interface as a potential drug target.

Chemical Genetics: Elucidating Biological Systems with Small-Molecule Compounds

Chemical genetics employs diverse small-molecule compounds to elucidate biological processes in a manner analogous to the mutagenesis strategies at the core of classical genetics. Screening small-molecule libraries for compounds that induce a phenotype of interest represents the forward chemical genetic approach, whereas the reverse approach involves small molecules targeting a single protein. Here, we review key differences between the goals for small-molecule screening in industry versus academia, recent developments in high-throughput screening, and publicly available resources of compound collections, screening facilities, and databases. A particularly exciting outcome of a chemical genetic screen is the discovery of a previously unknown role for a protein in a pathway together with compounds that affect the function of that protein. In illustrative cases, such discoveries have led to progress toward therapeutic development and more commonly have increased the size of the small molecule "toolbox" available to the research community for the study of biological processes.

High Throughput Screening Assay for UDP-Glucuronosyltransferase 1A1 Glucuronidation Profiling

Development of high throughput screening (HTS) assays for evaluation of a compound's toxicity and potential for drug-drug interactions is a critical step towards production of better drug candidates and cost reduction in the drug development process. HTS assays for drug metabolism mediated by cytochrome P450s are now routinely used in compound library characterization and for computer modeling studies. However, development and application of HTS assays involving UDP-glucuronosyltransferases (UGTs) are lagging behind. Here we describe the development of a fluorescence-based HTS assay for UGT1A1 using recombinant enzyme and fluorescent substrate in the presence of an aqueous solution of PreserveX-QML (QBI Life Sciences, Madison, WI) polymeric micelles, acting as a stabilizer and a blocker of nonspecific interactions. The data include assay characteristics in 384-well plate format obtained with robotic liquid handling equipment and structures of hits (assay modifiers) obtained from the screening of a small molecule library at the University of Wisconsin HTS screening facility. The application of the assay for predicting UGT-related drug-drug interactions and building pharmacophore models, as well as the effects of polymeric micelles on the assay performance and compound promiscuity, is discussed.

Bioluminescent Assays for High-Throughput Screening

In the development of high throughput screening (HTS) as a central paradigm of drug discovery, fluorescence has generally been adopted as the favored methodology. Nevertheless, luminescence has maintained a prominent position among certain assay formats, most notably genetic reporters. Recently, there has been growing partiality for luminescent assays across a wider range of applications due to their sensitivity, broad linearity, and robustness to library compounds and complex biological samples. This trend has been fostered by the development of several new assay designs for diverse targets such as kinases, cytochrome p450s, proteases, apoptosis, and cytotoxicity. This review addresses recent progress made in the use of bioluminescent assays for HTS, highlighting new detection capabilities brought about by engineering luciferase genes, enzymes, and substrates. In genetic reporter applications, modifications to the luciferase genes have improved assay sensitivity by substantially increasing expression efficiency and enhanced response dynamics by reducing expression lifetime. The performance of assays based on detection of ATP and luciferin has been enhanced by modifications to the luciferase enzyme that increase its chemical and physical stability. Detection of ATP allows rapid analysis of cell metabolism and enzymatic processes coupled to ATP metabolism. Because luciferins are not naturally associated with mammalian physiology, assays for luciferin detection utilize synthetic derivatives designed to yield luminescence only when coupled with specific target enzymes. Finally, new methods for modulating the specific activity of luciferases are leading to the development of intracellular biosensors for dynamic detection of physiological processes.

High throughput screening in drug discovery

Drug discovery is a highly complex and multidisciplinary process which goal is to identify new antitumoral drugs. The screening attrition rate in the current drug discovery protocols suggests that one marketable drug emerges from approximately one million screened compounds. This leads to pressure to screen larger libraries in order to continue the pipeline and to the development of High Throughput Screening. HTS is only a name for specific developments in laboratory automation to collect a large amount of experimental data in a relatively short time. HTS can test hundreds of thousands of compounds per day, however, if fewer compounds could be tested without compromising the probability of success, the cost and time would be greatly reduced. To that end, new developments in large-scale cell biology and compound library design in silico have evolved to obtain data with higher predictability of clinical efficacy.

Predicting oral drug absorption and hepatobiliary clearance: Human intestinal and hepatic in vitro cell models

Membrane transport proteins control the uptake and efflux of many drugs in tissues including the intestine, liver and kidneys and thus play important roles in drug absorption, distribution and excretion. With the development of high throughput screening in an industrial environment, the importance of having appropriate in vitro systems to study drug transporter function, regulation, and interactions are invaluable. Cell lines are efficient tools in screening individual transport processes. In this review, we focus on the processes involved in the absorption and hepatobiliary clearance of drugs and the potential of cell lines to model such process, paying particular attention to the use of Caco-2 and HepG2 cells.

Simultaneous silencing of multiple genes in the apple scab fungus, Venturia inaequalis, by expression of RNA with chimeric inverted repeats

RNA-mediated gene silencing has been demonstrated in plants, animals, and more recently in filamentous fungi. Here, we report high frequency, RNA-mediated gene silencing in the apple scab fungus, Venturia inaequalis. The green fluorescent protein ( GFP) transgene was silenced in a GFP-expressing transformant. An endogenous gene, trihydroxynaphthalene reductase ( THN), involved in melanin biosynthesis, was also silenced. Silencing of these two genes resulted in obvious phenotypes in vitro. High frequency gene silencing was achieved using hairpin constructs for the GFP or the THN genes transferred by Agrobacterium (71 and 61%, respectively). THN-silenced transformants exhibited a distinctive light brown phenotype and maintained the ability to infect apple. Of significance was the simultaneous silencing of the two genes from a single chimeric, inverted repeat hairpin construct. Silencing of both genes with this construct occurred at a frequency of 51% of all the transformants. All 125 colonies silenced for the GFP gene were also silenced for THN. As THN and GFP silenced transformants have readily detectable phenotypes, the genes have utility as markers for gene silencing. Simultaneous, multiple gene silencing, utilising such marker genes, will enable the development of high through-put screening for functional genomics. This chimeric technology will be particularly valuable when linked with silenced genes that have no obvious phenotype in vitro.

Química combinatória: moderna ferramenta para a obtenção de candidatos a protótipos de novos fármacos

A Quimica Combinatoria (QC) e atualmente uma das mais promissoras ferramentas para a descoberta e o desenvolvimento de novas moleculas com potencialidades terapeuticas. Nesta metodologia de sintese os produtos sao formados simultaneamente e podem ser testados biologicamente em uma so vez, seja em forma de misturas ou separadamente. A principal meta desta nova metodologia e reduzir o tempo necessario para a obtencao de um novo farmaco. A sintese de quimiotecas pode ser realizada atraves da utilizacao de polimeros insoluveis, soluveis ou pela tradicional sintese em solucao, sendo que cada uma delas apresenta caracteristicas que podem ser vantajosas ou nao, dependendo da metodologia utilizada. O desenvolvimento de tecnicas de high throughput screening (HTS) devido as suas caracteristicas peculiares necessita de tempo, razao pela qual a sintese de quimiotecas pequenas e racionais e enfatizada.

Pharmacogenomics in Drug Discovery and Development Research.

In the last decade of the 20th century, the development of high throughput screening (HTS) and combinatorial chemistry technologies accelerated the drug discovery process. In the 21st century, emerging genomic technologies (i.e., bioinformatics, functional genomics, and pharmacogenomics) are shifting the paradigm of drug discovery and development. This review summaries recent advances in toxicogenomics and pharmacogenomics for drug discovery research. In particular, we describe genetic polymorphisms and single nucleotide polymorphisms (SNPs) of ATP-binding cassette (ABC) transporters which are critically involved in pharmacokinetics profiles of various drugs.

A Review of Automation Options to Support Plate Preparation, Cherry Picking, and Homogeneous Assays

Developments in high throughput screening (HTS) have led to new needs in automation to enable better handling of applications such as homogeneous assays and cherry picking. Software and hardware integration approaches for screening automation have been changing in concert with these new application needs. The result of this combination has been the production of robotic systems for drug discovery with improved stability and functionality. This review critically assesses some Zymark, Tecan, and Beckman solutions for current HTS requirements.

Gene expression systems in the development of high-throughput screens

Recent advances in the development of combinatorial automated chemical synthesis, robotic sample handling, and data collection and analysis have significantly increased the number of compounds available for screening against potential therapeutic targets. The implementation of highly sensitive in vitro biochemical and cell-based high-throughput screening assays is essential to facilitate the rapid identification of selective and potent lead molecules from compound libraries. The ability to easily produce functional proteins in sufficient quantities for in vitro biochemical assays and to devise useful cell-based systems is dependent on the successful application of a variety of gene expression systems.

Experimental Designs for Selecting Molecules from Large Chemical Databases

Recent developments in high-throughput screening and combinatorial chemistry have generated interest in experimental design methods to select subsets of molecules from large chemical databases. In this manuscript three methods for selecting molecules from large databases are described: edge designs, spread designs, and coverage designs. Two algorithms with linear time complexity that approximate spread and coverage designs are described. These algorithms can be threaded for multiprocessor systems, are compatible with any definition of molecular distance, and may be applied to very large chemical databases. For example, ten thousand molecules were selected using the maximum dissimilarity approximation to a spread design from a sixty-dimensional simulated molecular database of one million molecules in approximately 6 h on a UNIX workstation.