uHTS identification of APOBEC3G DNA Deaminase Inhibitors via a fluorescence-based single-stranded DNA deaminase assay
The human APOBEC3G (A3G) protein is a DNA deaminase that has potent activity against HIV-1 and a variety of other retroelements. Although HIV encodes a natural inhibitor of A3G called Vif, it was hypothesized that the virus still benefits from the mutagenic activity of A3G (immune escape and drug resistance). ..more
BioActive Compounds: 931
Data Source: Sanford-Burnham Center for Chemical Genomics (SBCCG)
Source Affiliation: Sanford-Burnham Medical Research Institute (SBMRI, San Diego, CA)
Network: NIH Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Number: 1 R03 MH089432-01
Assay Provider: Dr. Reuben S Harris, Regents of the University of Minnesota, Minneapolis, MN
The human APOBEC3G (A3G) protein is a DNA deaminase that has potent activity against HIV-1 and a variety of other retroelements. Although HIV encodes a natural inhibitor of A3G called Vif, it was hypothesized that the virus still benefits from the mutagenic activity of A3G (immune escape and drug resistance).
The purpose of the screening is to identify A3G inhibitor by using a fluorescence-based single-strand DNA deaminase assay. In this assay, an A3G protein, a single-strand DNA oligonucleotide with a 5'-CCC target site, and Uracil DNA Glycosylase (UDG) are all mixed. After incubation at room temperature, A3G deaminates C-to-U and UDG removes the U. 1N sodium hydroxide is added to break the oligonucleotide at the abasic site. Cleavage releases the 3' TAMRA quenching molecule from the 5' 6-FAM fluorophore, which results in fluorescence
1- Harris, R. S., S. K. Petersen-Mahrt, and M. S. Neuberger. 2002. RNA editing enzyme APOBEC1 and some of its homologs can act as DNA mutators. Molecular Cell 10:1247-53.
2- Longerich, S., A.M. Galloway, R.S. Harris, C. Wong & S.M. Rosenberg (1995) Adaptive mutation sequences reproduced by mismatch repair-deficiency. Proceedings of the National Academy of Sciences USA, 92,12017-20.
3- Rosenberg, S.M., C. Thulin & R.S. Harris (1998) Transient and heritable mutators in adaptive evolution in the lab and in nature. Genetics, 148, 1559-66.
4- Petersen-Mahrt, S.K., R.S. Harris & M.S. Neuberger (2002) AID mutates E. coli suggesting a DNA deamination mechanism for antibody diversification. Nature, 418, 99-103.
5- Beale, R.C.L.B., S.K. Petersen-Mahrt, I.N. Watt, R.S. Harris, C. Rada & M.S. Neuberger (2004) Comparison of the differential context-dependence of DNA deamination by APOBEC enzymes: correlation with mutation spectra in vivo. Journal of Molecular Biology, 337, 585-96.
Protein dilution buffer: Tris (pH 7.4) at 15 mM, NaCl at 150 mM, Triton X-100 at 0.5%, glycerol at 10%
Oligo dilution buffer: TE at 20 mM
Oligo: 5' d FAM-AAATATCCCAAAGAGAGA-TAMRA 3': BioSearch Technologies
UDG: New England Biolabs
A3G-MycHis: University of Minnesota
2M Tris (pH 7.9)
Assay plate: Corning 1536 Well Black Solid Bottom Plate (Catalogue # 3724)
I. Compound Addition:
1- Using LabCyte Echo, transfer 40 nL from 2 mM compound source plate into assay plate Columns 5-48 (final concentration of test compounds is 20 uM, 1.0% DMSO), and 40 nL of DMSO to control wells in Columns 1-4.
II. Preparing reagents:
2- Prepare protein dilution buffer
3- Prepare oligo dilution buffer
4- Prepare intermediate A3G at 5 ng/ul. The final concentration of A3G in the plate is 1.25 ng/ul.
5- Prepare Oligo/UDG intermediate with 1 uM for Oligo and 0.002U/ul for UDG. The final concentration in the plate is 0.5 uM for Oligo and 0.001U/ul for UDG.
III. Reagent Addition
6- Add 2 ul of protein dilution buffer in columns 1 & 2
7- Add 2 ul of A3G in columns 3 to 48
8- Add 2 ul of Oligo/UDG to all columns
9- Cover the plate with Kalypsis metal lid; incubate the plate at room temperature for 45 minutes.
10- Add 1 ul of 1N NaOH to all columns
11- Cover the plate with Kalypsis metal lid; incubate the plate at room temperature for 30 minutes.
12- Add 1 ul of 2M Tris (pH 7.9) to all columns
13- Cover the plate with Kalypsis metal lid; incubate the plate at room temperature overnight.
IV. Reading plates:
14- Read the plate on PerkinElmer-EnVision plate reader (485/535)
Compounds that demonstrated an activity of >= 40% at 20 uM concentration and <= 30% at 20 uM in "uHTS identification of APOBEC3A DNA Deaminase Inhibitors via a fluorescence-based single-stranded DNA deaminase assay" (AID TBD)are defined as actives in this assay.
To simplify the distinction between the inactives of the primary screen and of the confirmatory screening stage, the Tiered Activity Scoring System was developed and implemented.
Activity scoring rules were devised to take into consideration compound efficacy, its potential interference with the assay and the screening stage that the data was obtained. Details of the Scoring System will be published elsewhere. Briefly, the outline of the scoring system utilized for the assay is as follows:
1) First tier (0-40 range) is reserved for primary screening data. The score is correlated with % activity in the assay:
a. If outcome of the primary screen is inactive, then the assigned score is 0
b. If outcome of the primary screen is inconclusive, then the assigned score is 10
c. If outcome of the primary screen is active, then the assigned score is 20
Scoring for Single concentration confirmation screening is not applicable to this assay.
d. If outcome of the single-concentration confirmation screen is inactive, then the assigned score is 21
e. If outcome of the single-concentration confirmation screen is inconclusive, then the assigned score is 25
f. If outcome of the single-concentration confirmation screen is active, then the assigned score is 30
This scoring system helps track the stage of the testing of a particular SID. For the primary hits which are available for confirmation, their scores will be greater than 20. For those which are not further confirmed, their score will stay under 21.
2) Second tier (41-80 range) is reserved for dose-response confirmation data and is not applicable in this assay
3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues and is not applicable in this assay
** Test Concentration.
Data Table (Concise)