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BioAssay: AID 493162

A screen for compounds that inhibit processive DNA synthesis of vaccinia virus

Variola virus, the causative agent of smallpox, is a potential bio-weapon. A high-throughput screen of 49,663 compounds was conducted to identify novel small chemical inhibitors that target DNA synthesis of vaccinia, the prototypical poxvirus. This screen and follow-up studies identified inhibitors of vaccinia DNA synthesis with IC50 values as low as 0.5 microM. Cell-based assays showed that 16 more ..
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 Tested Compounds
 Tested Compounds
All(45652)
 
 
Active(427)
 
 
Inactive(45227)
 
 
 Tested Substances
 Tested Substances
All(45756)
 
 
Active(431)
 
 
Inactive(45325)
 
 
 Related BioAssays
 Related BioAssays
AID: 493162
Data Source: ICCB-Longwood/NSRB Screening Facility, Harvard Medical School (HMS517)
Depositor Category: Other
Deposit Date: 2011-02-08

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 427
Description:
Variola virus, the causative agent of smallpox, is a potential bio-weapon. A high-throughput screen of 49,663 compounds was conducted to identify novel small chemical inhibitors that target DNA synthesis of vaccinia, the prototypical poxvirus. This screen and follow-up studies identified inhibitors of vaccinia DNA synthesis with IC50 values as low as 0.5 microM. Cell-based assays showed that 16 inhibitors effectively blocked vaccinia infection with minimal cytotoxicity, and three inhibitors had selectivity indexes that approximate that of cidofovir. These new non-nucleoside inhibitors are expected to interfere with components of the vaccinia DNA synthesis apparatus that are distinct from cidofovir. Based on the high sequence similarity between the proteins of vaccinia and variola viruses, these new inhibitors are anticipated to be equally effective against smallpox.
Protocol
DNA synthesis reactions were performed in 384-well plates coated with streptavidin (SigmaScreen plates, Sigma-Aldrich cat # S8686). Two pmoles of biotinylated primer-template dissolved in 30 microL PBS were immobilized on streptavidin-coated wells. The unbound primer-template was removed, the wells were washed twice with 50 microL PBS and loaded with 20 microL reaction buffer (20 mM Tris-Cl pH7.5).
100 nL of compound were transferred from library plates to assay plates using an Epson transfer robot (Epson Robots, Carson, CA) fitted with a 384-pin array. DNA synthesis was initiated by loading 10 microL of a 3x reaction mixture. In each well, reactions were conducted with 0.5 uL vaccinia virus extract containing 0.2 mU DNA polymerase activity in 20 mM Tris-Cl pH7.5, 100 mM ammonium sulfate, 5 mM MgCl2, 0.1 mM EDTA, 0.5 mM DTT, 4% glycerol, 40 microg/mL BSA, 5 microM of each dATP, dCTP, and dGTP, 4 microM dTTP, 0.5 microM DIG-11-dUTP, in a total volume of 30 microL. After brief centrifugation, plates were incubated at 37 C for 30 minutes. The DNA synthesis reaction was stopped with 30 microL of a solution containing 50 mM EDTA and 2% SDS in 10 mM Tris pH8.
Incorporation of DIG-11-dUTP in the newly synthesized DNA strand was detected with peroxidase-conjugated anti-digoxigenin antibody (anti-DIG-POD). Wells were flow-washed with 400 microL PBS containing 0.1% Tween-20 at the lowest dispensing speed and loaded with 4.5 mU of anti-DIG-POD in 30 microL PBS/blocking solution. After gently rocking at room temperature for 1h, the antibody solution was removed and wells were flow-washed as above. Thirty uL of 2, 2'-azino-bis(3-ethylbenzthiazoline)-sulfonate (ABTS) peroxidase substrate dissolved in citrate buffer was added and plates were gently rocked at room temperature for 1h. Color development was stopped with 10 microL of 4% SDS, and absorbance at 405 nm was measured.
Comment
Absorbance readings from each well were divided by the plate median absorbance of experimental wells for each replicate and multiplied by 100 to derive normalized % absorbance. The replicate average normalized % absorbance was used to score activity, with wells having average % absorbance <= 90% scored as active. Note that not all wells with average % absorbance less than this threshold were considered active. Average normalized % absorbance values between 0 and 100 were subtracted from 100 to generate activity scores. Wells with average % absorbance >= 100 were scored as 0 for activity.
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Absorbance_AOptical density at 405 nmFloat
2Absorbance_BOptical density at 405 nmFloat
3Normalized % Absorbance_AOptical density at 405 nm, normalized to plate median absorbanceFloat%
4Normalized % Absorbance_BOptical density at 405 nm, normalized to plate median absorbanceFloat%
5Normalized % Absorbance_AvgAverage normalized optical density at 405 nmFloat%

Data Table (Concise)
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