Bookmark and Share
BioAssay: AID 584

Promiscuous and Specific Inhibitors of AmpC Beta-Lactamase (assay with detergent)

This aggregation profiling approach exploits the sensitivity of aggregate formation to detergent. Inhibition of b-lactamase is measured in the presence and absence of 0.01% Triton X-100. This particular assay had the prescence of 0.01% Triton X-100. See Pubchem assay "Promiscuous and Specific Inhibitors of AmpC Beta-Lactamase (assay without detergent)" for related screen. Compounds that inhibit only in the absence of detergent are considered likely promiscuous aggregators. ..more
_
   
 Tested Compounds
 Tested Compounds
All(69666)
 
 
Active(64)
 
 
Inactive(69575)
 
 
Inconclusive(30)
 
 
 Tested Substances
 Tested Substances
All(70699)
 
 
Active(65)
 
 
Inactive(70604)
 
 
Inconclusive(30)
 
 
AID: 584
Data Source: NCGC (AMPC374)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
BioAssay Version:
Deposit Date: 2007-02-06
Modify Date: 2007-11-01

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 64
Depositor Specified Assays
AIDNameTypeComment
585Promiscuous and Specific Inhibitors of AmpC Beta-Lactamase (assay without detergent)confirmatory
485341qHTS Inhibitors of AmpC Beta-Lactamase (assay without detergent)confirmatory
1003Confirmation Cuvette-Based Assay for Inhibitors of AmpC Beta-Lactamase (assay with detergent)confirmatory
485294qHTS Inhibitors of AmpC Beta-Lactamase (assay with detergent)confirmatory
1002Confirmation Concentration-Response Assay for Inhibitors of AmpC Beta-Lactamase (assay with detergent)confirmatory
485291Probe Development Summary for Inhibitors of AmpC Beta Lactamasesummary
Description:
NIH Molecular Libraries Screening Centers Network [MLSCN]
NIH Chemical Genomics Center [NCGC]

MLSCN Grant: 1 X01 MH079825-01
PI Name: Shoichet, Brian K.

NCGC Assay Overview:
This aggregation profiling approach exploits the sensitivity of aggregate formation to detergent. Inhibition of b-lactamase is measured in the presence and absence of 0.01% Triton X-100. This particular assay had the prescence of 0.01% Triton X-100. See Pubchem assay "Promiscuous and Specific Inhibitors of AmpC Beta-Lactamase (assay without detergent)" for related screen. Compounds that inhibit only in the absence of detergent are considered likely promiscuous aggregators.
A screen of 1,030 compounds was performed in Brian Shoichet's lab in 96-well format [1]. AmpC activity was measured by following the absorbance at 480 nm. Nitrocefin is a chromogenic substrate: while nitrocefin is yellow (lmax 390 nm), the product of its hydrolysis is red (lmax 486 nm, 17,000 M-1). AmpC beta lactamase was provided by Brian Shoichet and Brian Feng, UCSF.
Protocol
NCGC Assay Protocol Summary:

Buffer:
20 mM phosphate buffer, pH 7.0
Triton X-100: No Detergent screen: 0.0001%, Detergent screen: 0.01%.

Reagents/Controls:
Buffer in columns 3 and 4 as negative control (no enzyme).
5.3 nM AmpC in columns 1, 2, 5-48. Titration of UCSF compound 9 control (from 10 mM, then 1:2) pin-transferred to column 2. Column 1 is neutral (100% activity).

Assay Steps:
Six uL of reagents were dispensed to 1536-well Greiner black clear bottom plates. Compounds and controls (23 nL) were transferred via Kalypsys PinTool. The plates were incubated for 15 min at room temperature, and then 2 uL of 800 uM solution of nitrocefin was added to start the reaction. The plates were immediately transferred to and read every 20 seconds for 4 min on ViewLux (Perkin-Elmer) High-throughput CCD imager using 480 nm absorbance protocol. During dispense, reagent bottles were kept submerged into 4 deg C water bath.

Keywords: NIH Roadmap, MLSCN, MLI, MLSMR, qHTS, NCGC, AmpC Beta-Lactamase, Aggregation
Comment
References:

1. Feng, et al. Nature Chem. Biol. 1, 146-148 (2005).

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 Classification". 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, within each curve class as defined above, compounds were ranked by logAC50 where -10 * logAC50 was used as a score and adjusted according to curve class.
Result Definitions
Show more
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Activity DirectionIndicates direction of observed activity: inactive, decreasing, increasing.String
2Curve 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 signficant, but below 80% of control.String
3Log of AC50Log of AC50.Float
4Hill CoefficientHill slope of fitted curve.Float
5Curve R2R^2 fit value of curve. Closer to 1.0 equates to better Hill equation fit.Float
6Curve Infinite ActivityHill Equation Infinite ActivityFloat
7Curve Zero ActivityHill Equation Zero ActivityFloat
8Curve Excluded 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
9Curve ClassNumerical encoding of curve description for the fitted Hill equation.Float
10Activity at 0.4nM% Activity at given concentration.Float%
11Activity at 0.8nM% Activity at given concentration.Float%
12Activity at 1.8nM% Activity at given concentration.Float%
13Activity at 4.1nM% Activity at given concentration.Float%
14Activity at 9.2nM% Activity at given concentration.Float%
15Activity at 20.6nM% Activity at given concentration.Float%
16Activity at 0.046uM% Activity at given concentration.Float%
17Activity at 0.103uM% Activity at given concentration.Float%
18Activity at 0.23uM% Activity at given concentration.Float%
19Activity at 0.15uM% Activity at given concentration.Float%
20Activity at 1.15uM% Activity at given concentration.Float%
21Activity at 2.57uM% Activity at given concentration.Float%
22Activity at 5.75uM% Activity at given concentration.Float%
23Activity at 12.85uM% Activity at given concentration.Float%
24Activity at 28.73uM% Activity at given concentration.Float%
25Compound TypeNCGC designation for compound stage: 'qHTS ECL (Exploratory)', 'NIHSMR (MLSCN)', 'Compound Followup', 'Compound Verification', 'Probe Optimization'String

Data Table (Concise)
PageFrom: