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BioAssay: AID 540301

Inhibitors of DNA Polymerase Beta: Hit Validation in HIV-RT Assay

The base excision repair system in human cells is a target for therapeutic modulation of the response to irradiation treatment and DNA-damaging drugs. A key enzyme in this system is the bi-functional DNA polymerase known as DNA polymerase beta (pol b). Although our understanding of the dNMP incorporation reaction by human pol b includes crystal structures representing stages of the catalytic more ..
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 Tested Compounds
 Tested Compounds
All(254)
 
 
Inactive(233)
 
 
Inconclusive(21)
 
 
 Tested Substances
 Tested Substances
All(254)
 
 
Inactive(233)
 
 
Inconclusive(21)
 
 
AID: 540301
Data Source: NCGC (PolB704)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2011-07-26

Data Table ( Complete ):           View All Data
Target
Tested Compounds:
Related Experiments
AIDNameTypeComment
485311Probe Development Summary for Inhibitors of DNA Polymerase BetaSummarydepositor-specified cross reference: Summary AID of project
485314qHTS Assay for Inhibitors of DNA Polymerase BetaConfirmatorysame project related to Summary assay
540280Inhibitors of DNA Polymerase Beta: Hit validationConfirmatorysame project related to Summary assay
540300Inhibitors of DNA Polymerase Beta: Hit Validation in Klenow Fragment AssayConfirmatorysame project related to Summary assay
540325Inhibitors of DNA Polymerase Beta: Hit Validation in Radiolabeled Extension AssayOthersame project related to Summary assay
Description:
The base excision repair system in human cells is a target for therapeutic modulation of the response to irradiation treatment and DNA-damaging drugs. A key enzyme in this system is the bi-functional DNA polymerase known as DNA polymerase beta (pol b). Although our understanding of the dNMP incorporation reaction by human pol b includes crystal structures representing stages of the catalytic cycle, research with small molecule probes or inhibitors to date has failed to provide the information necessary to effectively target this enzyme. Using an integrated approach involving discovery of small molecule probes for pol b via qHTS and validation by crystallography and kinetics along with cell-based validation experiments, we will test the hypothesis that base excision repair can be strategically modulated in human cells by targeting pol b. Thus, we anticipate the pol b-specific probes emerging from this research will serve as starting points in the development of clinical agents for use in down regulation of DNA base excision repair during therapeutic interventions against cancer.

After the completion of the qHTS against the Molecular Libraries Small Molecular Repository (MLSMR), cherry picked compounds were confirmed in the primary qHTS pol beta assay and further characterized in several follow-up assays. To avoid compound promiscuity, we selected a completely unrelated viral polymerase to polymerase beta, HIV reverse transcriptase(RT) as a counter screen. There are distinct differences between the two polymerases, such as source of host and HIV RT is responsible for transcribing the virus' single stranded RNA into double-stranded DNA after entry into its host cell. Hence, if a compound were to inhibit both enzymes, it could be assumed that compounds probably inhibit wide class of DNA polymerases. To us, a favorable outcome in this assay is inactivity.

NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Probe Centers Network [MLPCN]

MLPCN Grant: MH090863-01
Assay Submitter (PI): Samuel Wilson, National Institute of Environmental Health Sciences (NIEHS), NIH
Protocol
A stepwise description of the 1536-well assay is shown in next Table. Three microliters of reagents (buffer in column 3 and 4 as negative control and 10 nM Pol beta in columns 1, 2, and 5-48) was dispensed into 1,536-well black solid-bottomed plate. Compounds (23 nL) were transferred via Kalypsys pin tool equipped with 1536-pin array (10 nL slotted pins, V&P Scientific, San Diego, CA). The plates were incubated for 15 min at room temperature, and 1 uL substrate (50 nM final concentration) were added to start the reaction and immediately read at 0 min read on Viewlux reader. The plate were further incubated for 10 min at room temperature, and then read on Viewlux again. Throughout the screen, reagent bottle and all liquid lines were chilled and made light-tight to minimize reagent degradation. All screening operations were performed on a fully integrated robotic system (Kalypsys, San Diego, CA) containing one RX-130 and two RX-90 anthropomorphic robotic arms (Staubli, Duncan, SC). Library plates were screened starting from the lowest and proceeding to the highest concentration, and a double-dipping step of the highest concentration was required to access higher concentrations of compounds. Vehicle-only plates, with DMSO being pin-transferred to the entire column 5-48 compound area, were inserted uniformly at the beginning and the end of each library in order to monitor for and record any shifts in the background, which can be affected by reagent dispensers or loss in enzyme activity overtime. Screening data were corrected, normalized, and concentration-effect relationships were derived by using publicly-available curve fitting algorithms developed in-house (http://ncgc.nih.gov/pub/openhts). A four parameter Hill equation was fitted to the concentration-response data by minimizing the residual error between the modeled and observed responses.
Comment
Compound Ranking:

1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Description". For this assay, apparent inhibitors are ranked higher than compounds that showed apparent activation.
2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.
Categorized Comment - additional comments and annotations
From ChEMBL:
Assay Type: Functional
Assay Cell Type: NULL
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
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
3EfficacyMaximal efficacy of compound, reported as a percentage of control. These values are estimated based on fits of the Hill equation to the dose-response curves.Float%
4Analysis CommentAnnotation/notes on a particular compound's data or its analysis.String
5Curve_DescriptionA description of dose-response curve quality. A complete curve has two observed asymptotes; a partial curve may not have attained its second asymptote at the highest concentration tested. High efficacy curves exhibit efficacy greater than 80% of control. Partial efficacies are statistically significant, but below 80% of control.String
6Fit_LogAC50The logarithm of the AC50 from a fit of the data to the Hill equation (calculated based on Molar Units).Float
7Fit_HillSlopeThe Hill slope from a fit of the data to the Hill equation.Float
8Fit_R2R^2 fit value of the curve. Closer to 1.0 equates to better Hill equation fit.Float
9Fit_InfiniteActivityThe asymptotic efficacy from a fit of the data to the Hill equation.Float%
10Fit_ZeroActivityEfficacy at zero concentration of compound from a fit of the data to the Hill equation.Float%
11Fit_CurveClassNumerical encoding of curve description for the fitted Hill equation.Float
12Excluded_PointsWhich dose-response titration points were excluded from analysis based on outlier analysis. Each number represents whether a titration point was (1) or was not (0) excluded, for the titration series going from smallest to highest compound concentrations.String
13Max_ResponseMaximum activity observed for compound (usually at highest concentration tested).Float%
14Activity at 0.0001441714 uM (0.000144171μM**)% Activity at given concentration.Float%
15Activity at 0.0003225714 uM (0.000322571μM**)% Activity at given concentration.Float%
16Activity at 0.0004325714 uM (0.000432571μM**)% Activity at given concentration.Float%
17Activity at 0.0009677143 uM (0.000967714μM**)% Activity at given concentration.Float%
18Activity at 0.00130 uM (0.00129771μM**)% Activity at given concentration.Float%
19Activity at 0.00290 uM (0.00290315μM**)% Activity at given concentration.Float%
20Activity at 0.00465 uM (0.00464851μM**)% Activity at given concentration.Float%
21Activity at 0.00871 uM (0.00870948μM**)% Activity at given concentration.Float%
22Activity at 0.012 uM (0.0116794μM**)% Activity at given concentration.Float%
23Activity at 0.019 uM (0.0186012μM**)% Activity at given concentration.Float%
24Activity at 0.026 uM (0.0261358μM**)% Activity at given concentration.Float%
25Activity at 0.077 uM (0.0771516μM**)% Activity at given concentration.Float%
26Activity at 0.254 uM (0.254μM**)% Activity at given concentration.Float%
27Activity at 0.705 uM (0.705467μM**)% Activity at given concentration.Float%
28Activity at 1.190 uM (1.18988μM**)% Activity at given concentration.Float%
29Activity at 2.116 uM (2.1164μM**)% Activity at given concentration.Float%
30Activity at 2.838 uM (2.8381μM**)% Activity at given concentration.Float%
31Activity at 5.777 uM (5.777μM**)% Activity at given concentration.Float%
32Activity at 8.514 uM (8.51429μM**)% Activity at given concentration.Float%
33Activity at 19.05 uM (19.0476μM**)% Activity at given concentration.Float%
34Activity at 25.54 uM (25.5429μM**)% Activity at given concentration.Float%
35Activity at 57.14 uM (57.1429μM**)% Activity at given concentration.Float%
36Compound QCNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: MH090863

Data Table (Concise)
Data Table ( Complete ):     View All Data
Classification
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