scholarly journals Evaluation of a System to Screen for Stimulators of Non-Specific DNA Nicking by HIV-1 Integrase: Application to a Library of 50,000 Compounds

2011 ◽  
Vol 22 (2) ◽  
pp. 67-74 ◽  
Author(s):  
Malgorzata Sudol ◽  
Jennifer L Fritz ◽  
Melissa Tran ◽  
Gavin P Robertson ◽  
Julie B Ealy ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Solution Phase ◽  
The Novel ◽  
Endonuclease Activity ◽  
Viral Dna ◽  
Hit Rate ◽  
Dna Nicking ◽  
Hiv 1 ◽  
Stimulation Of

Background: In addition to activities needed to catalyse integration, retroviral integrases exhibit non-specific endonuclease activity that is enhanced by certain small compounds, suggesting that integrase could be stimulated to damage viral DNA before integration occurs. Methods: A non-radioactive, plate-based, solution phase, fluorescence assay was used to screen a library of 50,080 drug-like chemicals for stimulation of non-specific DNA nicking by HIV-1 integrase. Results: A semi-automated workflow was established and primary hits were readily identified from a graphic output. Overall, 0.6% of the chemicals caused a large increase in fluorescence (the primary hit rate) without also having visible colour that could have artifactually caused this result. None of the potential stimulators from this moderate-size library, however, passed a secondary test that included an inactive integrase mutant that assessed whether the increased fluorescence depended on the endonuclease activity of integrase. Conclusions: This first attempt at identifying integrase stimulator compounds establishes the necessary logistics and workflow required. The results from this study should encourage larger scale high-throughput screening to advance the novel antiviral strategy of stimulating integrase to damage retroviral DNA.

scholarly journals Development and Application of a High-Throughput Screening Assay for HIV-1 Integrase Enzyme Activities

2005 ◽  
Vol 10 (6) ◽  
pp. 606-614 ◽  
Author(s):  
Sinu John ◽  
Thomas M. Fletcher ◽  
Colleen B. Jonsson
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Strand Transfer ◽  
Hiv Integrase ◽  
Chromosomal Dna ◽  
Screening Assay ◽  
Viral Dna ◽  
High Throughput Screening Assay ◽  
Hiv 1

Integrase (IN) mediates the covalent insertion of the retroviral genome into its host chromosomal DNA. This enzymatic activity can be reconstituted in vitro with short DNA oligonucleotides, which mimic a single viral DNA end, and purified IN. Herein we report a highly efficient and sensitive high-throughput screen, HIV Integrase Target SRI Assay (HITS™), for HIV-1 IN activity using 5′ biotin-labeled DNA (5′ BIO donor) and 3′ digoxygenin-labeled DNA (3′ DIG target). Following 3′ processing of the 5′ BIO donor, strand transfer proceeds with integration of the 5′ BIO donor into the 3′ DIG target. Products were captured on a streptavidin-coated microplate and the amount of DIG retained in the well was measured. The end point values, measured as absorbance, ranged from 0.9 to 1.5 for IN-mediated reactions as compared with background readings of 0.05 to 0.12. The Z factor for the assay ranged from 0.7 to 0.85. The assay was used to screen drugs in a high-throughput format, and furthermore, we adapted the assay to study mechanistic questions regarding the integration process. For example, using variations of the assay format, we showed high preference of E strand of the long terminal repeat (LTR) viral DNA as a target strand compared with its complementary A strand. The E strand is the strand processed by IN. Furthermore, we explored the reported inhibitory effect of reverse transcriptase on integration.

scholarly journals Human Immunodeficiency Virus Type 1 Latency Model for High-Throughput Screening

2005 ◽  
Vol 49 (12) ◽  
pp. 5185-5188 ◽  
Author(s):  
Sofiya Micheva-Viteva ◽  
Annmarie L. Pacchia ◽  
Yacov Ron ◽  
Stuart W. Peltz ◽  
Joseph P. Dougherty
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
High Throughput ◽  
High Throughput Screening ◽  
Immunodeficiency Virus ◽  
Latently Infected ◽  
A Cell ◽  
Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) is not eliminated from patients even after years of antiretroviral therapy, apparently due to the presence of latently infected cells. Here we describe the development of a cell-based system of latency that can be used for high-throughput screening aimed at novel drug discovery to eradicate HIV-1 infection.

scholarly journals Toward the Development of a Virus-Cell-Based Assay for the Discovery of Novel Compounds against Human Immunodeficiency Virus Type 1

2003 ◽  
Vol 47 (2) ◽  
pp. 501-508 ◽  
Author(s):  
Martin E. Adelson ◽  
Annmarie L. Pacchia ◽  
Malvika Kaul ◽  
Robert F. Rando ◽  
Yacov Ron ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Drug Discovery ◽  
Human Immunodeficiency Virus Type ◽  
High Throughput ◽  
High Throughput Screening ◽  
Single Cycle ◽  
Vector Systems ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT The emergence of human immunodeficiency virus type 1 (HIV-1) strains resistant to highly active antiretroviral therapy necessitates continued drug discovery for the treatment of HIV-1 infection. Most current drug discovery strategies focus upon a single aspect of HIV-1 replication. A virus-cell-based assay, which can be adapted to high-throughput screening, would allow the screening of multiple targets simultaneously. HIV-1-based vector systems mimic the HIV-1 life cycle without yielding replication-competent virus, making them potentially important tools for the development of safe, wide-ranging, rapid, and cost-effective assays amenable to high-throughput screening. Since replication of vector virus is typically restricted to a single cycle, a crucial question is whether such an assay provides the needed sensitivity to detect potential HIV-1 inhibitors. With a stable, inducible vector virus-producing cell line, the inhibitory effects of four reverse transcriptase inhibitors (zidovudine, stavudine, lamivudine, and didanosine) and one protease inhibitor (indinavir) were assessed. It was found that HIV-1 vector virus titer was inhibited in a single cycle of replication up to 300-fold without affecting cell viability, indicating that the assay provides the necessary sensitivity for identifying antiviral molecules. Thus, it seems likely that HIV-1-derived vector systems can be utilized in a novel fashion to facilitate the development of a safe, efficient method for screening compound libraries for anti-HIV-1 activity.

scholarly journals Long-Term Storage of Compound Solutions for High-Throughput Screening by Using a Novel 1536-Well Microplate

2009 ◽  
Vol 14 (5) ◽  
pp. 492-498 ◽  
Author(s):  
Martin Joseph Pfeifer ◽  
Guenther Scheel
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Cost Effective ◽  
The Novel ◽  
Long Term Storage ◽  
Plate Design ◽  
Minor Losses ◽  
Storage Times ◽  
Term Storage

This report describes the features and the performance of a new and significantly improved 1536-well microplate design. The design allows for simple, automation-friendly, and cost-effective storage of compound solutions for high-throughput screening. The plate design is based on Society for Biomolecular Sciences standards for microplates and can be molded from polystyrene or cycloolefin copolymer, thus making the plate suitable for use with acoustic dispensing as well as other conventional liquid dispensing in the nanoliter range. For a 9:1 DMSO/water mix as solvent, the novel plate design has shown to perform over 4 months with only minor losses in solvent. Thus, this novel plate design creates the basis for further reductions in compound storage volumes and allows for an increase in the storage times for microliter volumes for up to a year or more. The high protection against solvent evaporation is also visible for aqueous solutions, thus allowing for reduced edge effects during screening campaigns.

scholarly journals High-Throughput Screening Method of Inhibitors that Block the Interaction between 2 Helical Regions of HIV-1 gp41

2005 ◽  
Vol 10 (1) ◽  
pp. 13-19 ◽  
Author(s):  
Bong-Suk Jin ◽  
Won-Kyu Lee ◽  
Kwangseog Ahn ◽  
Myung Kyu Lee ◽  
Yeon Gyu Yu
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Screening Method ◽  
Assay Method ◽  
Enzyme Linked Immunosorbent Assay ◽  
Compound Library ◽  
Elisa Method ◽  
Helical Bundle ◽  
Fusion Inhibitors ◽  
Hiv 1

The HIV-1 envelope glycoprotein transmembrane subunit, gp41, mediates the fusion of viral and target cell membranes. The 2 helical regions in the ectodomain of gp41, the N-helix and the C-helix, form a helical bundle complex that has been suggested as a fusion-active conformation. Previously, an enzyme-linked immunosorbent assay (ELISA) method had been established to measure the interaction of 2 helical regions of gp41. In this study, the ELISA method was modified to apply high-throughput screening (HTS) of an organic compound library. A few compounds had been identified to prevent the interaction between 2 helical regions of gp41, and they were further shown to inhibit the gp41-mediated viral infection. In addition, they specifically quenched the fluorescence of tryptophan in the N-helix region, indicating that these compounds bound to the N-helix rather than the C-helix of gp41. These results suggested that this assay method targeting gp41 could be used for HTS of HIV fusion inhibitors. ( Journal of Biomolecular Screening 2005:13-19)

Identification by high throughput screening of small compounds inhibiting the nucleic acid destabilization activity of the HIV-1 nucleocapsid protein

Biochimie ◽  
2009 ◽  
Vol 91 (7) ◽  
pp. 916-923 ◽  
Author(s):  
Volodymyr Shvadchak ◽  
Sarah Sanglier ◽  
Sandrine Rocle ◽  
Pascal Villa ◽  
Jacques Haiech ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
High Throughput ◽  
Nucleocapsid Protein ◽  
High Throughput Screening ◽  
Hiv 1

High Throughput Screening for Cyanovirin-N Mimetics Binding to HIV-1 gp41

2002 ◽  
Vol 7 (2) ◽  
pp. 105-110 ◽  
Author(s):  
John A. Beutler ◽  
James B. McMahon ◽  
Tanya R. Johnson ◽  
Barry R. O'Keefe ◽  
Randy A. Buzzell ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Hiv 1

Validation of a High-Throughput Calcium Mobilization Assay for the Human Trace Amine-Associated Receptor 1

2020 ◽  
Vol 26 (1) ◽  
pp. 140-150
Author(s):  
Ann M. Decker ◽  
Kelly M. Mathews ◽  
Bruce E. Blough ◽  
Brian P. Gilmour
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Therapeutic Potential ◽  
Calcium Mobilization ◽  
Hit Rate ◽  
Wide Range ◽  
Trace Amine ◽  
The Central Nervous System ◽  
Calcium Mobilization Assay

The human trace amine-associated receptor 1 (hTAAR1) is a G protein-coupled receptor (GPCR) that is widely expressed in monoaminergic nuclei in the central nervous system and has therapeutic potential for multiple diseases, including drug addiction and schizophrenia. Thus, identification of novel hTAAR1 ligands is critical to advancing our knowledge of hTAAR1 function and to the development of therapeutics for a wide range of diseases. Herein we describe the development of a robust, 3-addition high-throughput screening (HTS) calcium mobilization assay using stable CHO-Gαq16-hTAAR1 cells, which functionally couple hTAAR1 to the promiscuous Gαq16 protein and thus allow signal transduction to occur through mobilization of internal calcium. Our previously established 96-well hTAAR1 assay was first miniaturized to the 384-well format and optimized to provide an assay with a Z′ factor of 0.84, which is indicative of a robust HTS assay. Using the 3-addition protocol, 22,000 compounds were screened and yielded a ~1% agonist hit rate and a ~0.2% antagonist hit rate. Of the antagonist hits, two confirmed hits are the most potent hTAAR1 antagonists identified to date (IC50 = 206 and 281 nM). While scientists have been studying hTAAR1 for years, the lack of suitable hTAAR1 antagonists has been a major roadblock for studying the basic pharmacology of hTAAR1. Thus, these new ligands will serve as valuable tools to study hTAAR1-mediated signaling mechanisms, therapeutic potential, and in vivo functions.

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