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BioAssay: AID 493033

A screen for compounds that inhibit the bacterial siderophore biosynthetic enzyme MbtI

A simple steady-state kinetic high-throughput assay was developed for the salicylate synthase MbtI from Mycobacterium tuberculosis, which catalyzes the first committed step of mycobactin biosynthesis. The mycobactins are small-molecule iron chelators produced by M. tuberculosis, and their biosynthesis has been identified as a promising target for the development of new antitubercular agents. The more ..
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 Tested Compounds
 Tested Compounds
All(104200)
 
 
Active(927)
 
 
Inactive(103286)
 
 
 Tested Substances
 Tested Substances
All(104781)
 
 
Active(931)
 
 
Inactive(103850)
 
 
 Related BioAssays
 Related BioAssays
AID: 493033
Data Source: ICCB-Longwood/NSRB Screening Facility, Harvard Medical School (HMS832)
Depositor Category: Other
Deposit Date: 2011-01-18

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 927
Description:
A simple steady-state kinetic high-throughput assay was developed for the salicylate synthase MbtI from Mycobacterium tuberculosis, which catalyzes the first committed step of mycobactin biosynthesis. The mycobactins are small-molecule iron chelators produced by M. tuberculosis, and their biosynthesis has been identified as a promising target for the development of new antitubercular agents. The assay was miniaturized to a 384-well plate format, and high-throughput screening identified three classes of compounds: benzisothiazolones (class I), diarylsulfones (class II), and benzimidazole-2-thiones (class III). Each compound series was further pursued to investigate their biochemical mechanism and structure-activity relationships. Benzimidazole-2-thione 4 emerged as the most promising inhibitor owing to its potent reversible inhibition.
Protocol
The reaction master mix containing all components except MgCl2 (100 mM Tris-HCl pH 8.0; 0.0025% w/v Igepal CA-630; 415 nM MbtI; 65 microM chorismate) was prepared freshly and dispensed into 384-well assay plates at 25 microL per well using a Matrix WellMate.

Test compounds (5 mg/mL stocks in 100% DMSO) were transferred to assay plates at 100 nL per compound, and fluorescence intensity was measured in an EnVision plate reader to detect potential autofluorescent compounds (330/420 nm excitation/emission, LANCE/DELFIA dichroic mirror with 400 nm cutoff). The plates were shaken for 30 s, and 5 microL of 6 mM MgCl2 solution in 100 mM Tris-HCl pH 8.0, 0.0025% w/v Igepal CA-630 were dispensed to all wells in columns 1-23. 5 microL of 100 mM Tris-HCl pH 8.0, 0.0025% w/v Igepal CA-630 were dispensed to all wells in columns 24. Plates were incubated at room temperature for 3 h.

Reactions were quenched by addition of 5 microL of 0.5 M EDTA pH 8.0 to all wells. Fluorescence intensity was then measured again.
Comment
The fluorescence intensity (FI) measured before starting the enzymatic reaction was subtracted from the final FI reading, after EDTA quenching, for each well of all plates. Outliers within the positive and negative controls, i.e., wells that exhibited >=10% deviation for the respective FI average in each plate, were discarded. Normalized percent inhibition (NPI) was then calculated for each well, plate by plate, based on the respective controls (well FI was subtracted from the negative control plate average FI, divided by the difference between negative and positive control plate average FI, and multiplied by 100). An active list was extracted by selecting compounds that exhibited NPI >50% in both assay replicates and for which the intrinsic FI (measured before the enzymatic reaction) did not differ by >30% of that of the controls. Compounds that showed NPI >80% and moderate intrinsic FI (130 to 250% of the controls FI) were also considered active at this stage. Activity scores were based on average NPI of the two well replicates when average NPI >=0 and <=100. Average NPI <0 was scored as 0 for activity; average NPI >100 was scored as 100 for activity.
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Intensity_Background_ABackground FI for replicate A, measured before enzymatic reactionFloat
2Intensity_Background_BBackground FI for replicate B, measured before enzymatic reactionFloat
3Intensity_Adjusted_AFinal FI for replicate A, read after EDTA quenching and adjusted for background intensityFloat
4Intensity_Adjusted_BFinal FI for replicate B, read after EDTA quenching and adjusted for background intensityFloat
5NPI_APercent inhibition, normalized to plate average FI of positive and negative controls for replicate A.Float%
6NPI_BPercent inhibition, normalized to plate average FI of positive and negative controls for replicate B.Float%
7% Background IntensityBackground FI, normalized to plate average control background FIFloat%

Data Table (Concise)
Classification
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