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BioAssay: AID 504464

Gel-Based Assay for Inhibitors of HIV Integrase: Hit Validation Counterscreen for TDP1 Inhibitors

HIV Integrase enables the HIV virus to integrate its genetic material into host cells's DNA. This particular assay was utilized as a counterscreen for inhibitors of human tyrosyl DNA phosphodiesterase I (Tdp1). See probe development summary bioassay AID 485312 for further details on the tdp1 inhibitors project. ..more
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 Tested Compounds
 Tested Compounds
All(1)
 
 
Inactive(1)
 
 
 Tested Substances
 Tested Substances
All(1)
 
 
Inactive(1)
 
 
AID: 504464
Data Source: NCGC (TDP1756)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2011-03-16

Data Table ( Complete ):           View All Data
Target
Sequence: HIV Integrase
Description ..   

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Tested Compound:
Related Experiments
AIDNameTypeComment
485290qHTS Assay for Inhibitors of Tyrosyl-DNA Phosphodiesterase (TDP1)Confirmatorydepositor-specified cross reference: qHTS Assay for Inhibitors of Tyrosyl-DNA Phosphodiesterase (TDP1)
485312Probe Development Summary for Inhibitors of Tyrosyl-DNA Phosphodiesterase (TDP1)Summarydepositor-specified cross reference: Probe Development Summary for Inhibitors of Tyrosyl-DNA Phosphodiesterase (TDP1)
489007Confirmation Assay for Inhibitors of Tyrosyl-DNA Phosphodiesterase (TDP1)Confirmatorydepositor-specified cross reference: Confirmation Assay for Inhibitors of Tyrosyl-DNA Phosphodiesterase (TDP1)
492989Gel-Based Assay for Inhibitors of Tyrosyl-DNA Phosphodiesterase (TDP1)Confirmatorysame project related to Summary assay
492990Gel-Based Assay for Inhibitors of HIV Integrase: Counterscreen for TDP1Confirmatorysame project related to Summary assay
504474Gel-Based Assay for Inhibitors of Tyrosyl-DNA Phosphodiesterase (TDP1): Hit ValidationConfirmatorysame project related to Summary assay
Description:
HIV Integrase enables the HIV virus to integrate its genetic material into host cells's DNA. This particular assay was utilized as a counterscreen for inhibitors of human tyrosyl DNA phosphodiesterase I (Tdp1). See probe development summary bioassay AID 485312 for further details on the tdp1 inhibitors project.

This counterscreen assay was used to validate hits from tdp1 primary screen. Compounds that are active against Tdp1 but inactive against HIV integrase were ideal for followup in Tdp1 probe development project.
Protocol
HIV-1 integrase catalytic assay was carried out as previously described [Marchand et al. Methods in Enzymology 2001] by mixing 400 nM recombinant integrase with 20 nM [32P] radiolabeled DNA substrate in a buffer containing 50 mM MOPS, pH 7.2, 7.5 mM MgCl2, 14.3 mM 2-mercaptoethanol, and the drug of interest or 10% DMSO. Reactions were incubated at 37 degrees C for 1 h and quenched by the addition of an equal volume of gel loading dye (formamide with 0.25% bromophenol blue, 0.25% xylene cyanol and 5 mM EDTA). Reaction products were separated on a 16% polyacrylamide denaturing sequencing gel. Dried gels were exposed overnight and visualized using a Typhoon 8600 (GE Healthcare, Piscataway, NJ). Densitometry analyses were performed using the ImageQuant software from GE Healthcare.
Comment
Compounds that showed significant inhibition at any tested concentration were considered active. Compounds that gave basal response were considered as inactive. Inactive compounds were assigned a PUBCHEM_ACTIVITY_SCORE of 0. There were no active compounds in this counterscreen.
Categorized Comment - additional comments and annotations
From ChEMBL:
Assay Type: Functional
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
1PhenotypeIndicates type of activity observed: inhibitor, activator, fluorescent, cytotoxic, inactive, or inconclusive.String
2Potency*Concentration at which compound exhibits half-maximal efficacy, AC50. Extrapolated AC50s also include the highest efficacy observed and the concentration of compound at which it was observed.FloatμM
3Activity at 0.45 uM (0.45μM**)% Activity at given concentrationFloat%
4Activity at 1.4 uM (1.14μM**)% Activity at given concentrationFloat%
5Activity at 4.1 uM (4.1μM**)% Activity at given concentrationFloat%
6Activity at 12.3 uM (12.3μM**)% Activity at given concentrationFloat%
7Activity at 37 uM (37μM**)% Activity at given concentrationFloat%
8Activity at 111 uM (111μM**)% Activity at given concentrationFloat%
9Activity at 333 uM (333μM**)% Activity at given concentrationFloat%

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: MH089814-01

Data Table (Concise)
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