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. Compounds that are active against Tdp1 but inactive against HIV integrase were ideal for followup in Tdp1 probe development project. ..more
Target
Tested Compounds:
Depositor Specified Assays
| AID | Name | Type | Comment |
|---|---|---|---|
| 485312 | Probe Development Summary for Inhibitors of Tyrosyl-DNA Phosphodiesterase (TDP1) | summary | Probe Development Summary for Inhibitors of Tyrosyl-DNA Phosphodiesterase (TDP1) |
| 489007 | Confirmation Assay for Inhibitors of Tyrosyl-DNA Phosphodiesterase (TDP1) | confirmatory | Confirmation Assay for Inhibitors of Tyrosyl-DNA Phosphodiesterase (TDP1) |
| 485290 | qHTS Assay for Inhibitors of Tyrosyl-DNA Phosphodiesterase (TDP1) | confirmatory | qHTS Assay for Inhibitors of Tyrosyl-DNA Phosphodiesterase (TDP1) |
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. 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.
Result Definitions
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| TID | Name | Description | Histogram | Type | Unit | |
|---|---|---|---|---|---|---|
| Outcome | The BioAssay activity outcome | Outcome | ||||
| 1 | Phenotype | Indicates type of activity observed: inhibitor, activator, fluorescent, cytotoxic, inactive, or inconclusive. | String | |||
| 2 | Potency* | 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. |  | Float | μM | |
| 3 | Activity at 0.45 uM (0.45μM**) | % Activity at given concentration | | Float | % | |
| 4 | Activity at 1.4 uM (1.14μM**) | % Activity at given concentration | | Float | % | |
| 5 | Activity at 4.1 uM (4.1μM**) | % Activity at given concentration | | Float | % | |
| 6 | Activity at 12.3 uM (12.3μM**) | % Activity at given concentration | | Float | % | |
| 7 | Activity at 37 uM (37μM**) | % Activity at given concentration | | Float | % | |
| 8 | Activity at 111 uM (111μM**) | % Activity at given concentration | | Float | % | |
| 9 | Activity at 333 uM (333μM**) | % Activity at given concentration | | Float | % |
* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: MH089814-01
Data Table (Concise)
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