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BioAssay: AID 547

HTS for inhibitors of bacterial DnaK

Escherichia coli DnaK, a homolog of heat shock protein 70, has been shown to protect denature proteins from aggregation and promote their refolding by ATP hydrolysis. DnaK, along with its two co-cohort proteins DnaJ and GrpE, forms a microbial chaperone system that shelters microorganisms from environmental stresses such as temperature, osmotic, and pH changes, carbon and/or nitrogen starvation. more ..
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 Tested Compounds
 Tested Compounds
All(1274)
 
 
Active(11)
 
 
Inactive(1263)
 
 
 Tested Substances
 Tested Substances
All(1280)
 
 
Active(11)
 
 
Inactive(1269)
 
 
AID: 547
Data Source: Emory University Molecular Libraries Screening Center
Depositor Category: NIH Molecular Libraries Screening Center Network
BioAssay Version:
Deposit Date: 2006-12-04
Modify Date: 2007-11-06

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 11
Depositor Specified Assays
AIDNameTypeComment
1462Dose Response Confirmation for DnaK Inhibitorsconfirmatory
Description:
NIH Molecular Libraries Screening Centers Network [MLSCN]
Emory Chemical Biology Discovery Center in MLSCN
MLSCN Grant Number: none

Escherichia coli DnaK, a homolog of heat shock protein 70, has been shown to protect denature proteins from aggregation and promote their refolding by ATP hydrolysis. DnaK, along with its two co-cohort proteins DnaJ and GrpE, forms a microbial chaperone system that shelters microorganisms from environmental stresses such as temperature, osmotic, and pH changes, carbon and/or nitrogen starvation. Seeking inhibitors against bacterial DnaK chaperone systems will lead to a new direction for the development of antimicrobial agents, as current antimicrobial compounds have shown fewer efficacies due to the rapid emergence of resistant strains involving both Gram-positive and Gram-negative pathogens.

An in vitro refolding assay was developed by Chaperone Technologies to detect and quantify the activity of DnaK. Firefly luciferase was chemically denatured and used as a substrate for the chaperone system. Luminescence readout was then used to detect the refolding of luciferase enzyme. The Emory Center has optimized and adapted the assay to a 384-well format for HTS and was able to consistently achieve a signal to noise ratio of above 8 and a Z prime factor of above 0.5.
Protocol
Escherichia coli DnaK, DnaJ and GrpE proteins were purchased commercially through Assay Designs (Ann Arbor, Michigan), Luciferase enzyme was from Sigma. Promega's Steady-Glo Luciferase Assay System was used for the luminescence detection. DnaK refolding assay was screened against LOPAC 1280 library from Sigma at 10 uM with 2% DMSO. Screening for DnaK inhibitors was carried out in three steps: chemical denaturation of firefly luciferase, chaperone induced refolding, and detection of luciferase levels.

23 uM of firefly luciferase stock in glycine buffer (1 M glycine, 0.1 M of MgSO4, 0.01 M of EDTA, pH7) was denatured in denaturing buffer containing 6 M guanidinium HCl, 30 mM Tris HCl, and 5 mM DTT at pH7.5 for 30min at room temperature and 60 min at 5 degrees C. Denatured luciferase was then diluted to working concentration (20 nM) with cold refolding buffer plus (10 mM MOPS, 50 mM KCl, 5 mM MgCl2, 5 mM DTT and 1 mM ATP, pH7.5). The sample was furthered incubated at 5 degrees C for at least 60 min before use. At the same time, chaperone protein mixture of DnaK, DnaJ and GrpE was prepared in working concentrations of 200, 100 and 400 nM, respectively, in refolding buffer (10 mM MOPS, 50 mM KCl and 5 mM MgCl2, pH 7.5) and incubated for at least 3 hr at room temperature.

HTS was carried out in white, opaque bottom, 384-well polystyrene plates (Costar) with 12.5 uL of denatured luciferase and 12.5 uL of chaperone protein mixture using Wellmate dispenser by Matrix Technologies. For each plate, columns 1, 2 and 23, 24 contain controls: denatured luciferase only, refolding buffer only, chaperone proteins only and CHP105 at 100 ug/mL as positive controls. 0.5 uL of compounds (0.5 mM stock in DMSO) were then added to assay plates using Caliper's Sciclone liquid handler. In order to rule out compound induced refolding, a separate set of plates with only denatured luciferase plus compounds were set up parallel with the assay. All plates were incubated in the dark at room temperature for 3 hr. The luciferase activity was measured by adding 25 uL of diluted Steady-Glo (18.75 uL of refolding buffer, 6.25uL of Steady-Glo) before reading with Perkin Elmer's Envision.

HTS results were analyzed using Cambridgesoft. Positives were defined as signals less than 3SD of the assay. Assay signal = [luminescence signal (compound + denatured luciferase + protein mixture)] - [luminescence signal (compound + denatured luciferase)].
Comment
Positives identified may be targeting luciferase, rather than DnaK. Secondary assay is mandatory to determine DnaK specific inhibitions.

Activity score was calculated as follows:
Activity Score = 100 - (((luminescence value - minimum luminescence
value) / luminescence value range) * 100)
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Luminescence Valueluminescence value in counts per second was measured with an Envision ReaderIntegercps

Data Table (Concise)
Classification
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