qHTS Assay for Activators of ClpP
The Clp protease proteolytic subunit (ClpP) is the core unit of the Clp complex belonging to the peptidase family S14 that hydrolyzes protein into small peptides in the presence of ATP and magnesium. It is ubiquitously expressed and is the major protease in bacteria. Studies showed that ClpP is a target for a novel class of acyldepsipeptides (ADEP) in bacteria that are resistant to current more ..
BioActive Compounds: 6438
The Clp protease proteolytic subunit (ClpP) is the core unit of the Clp complex belonging to the peptidase family S14 that hydrolyzes protein into small peptides in the presence of ATP and magnesium. It is ubiquitously expressed and is the major protease in bacteria. Studies showed that ClpP is a target for a novel class of acyldepsipeptides (ADEP) in bacteria that are resistant to current antibiotic treatment. Unlike common antibiotics that simply interfere with the essential enzyme activity, ADEP binding activates the ClpP core in the absence of the regulatory Clp-ATPases leading to uncontrolled proteolysis and eventually cell death. Activators binding to the docking site on ClpP can also block formation of the ClpXP and ClpA/C-P holoenzyme complexes interfering with the normal biological functions of ClpP and thus have the potential to become new anti-microbials.
To identify activators of ClpP, a FRET based quenching reversal assay was developed using a purified B. subtilis ClpP and a decapeptide substrate (FRET-FV). The FRET-FV is tagged with a fluorescent molecule amino benzoic acid (donor) at the N-terminus and a nitrotyrosine quencher (acceptor) at the C-terminus. FRET-FV cleaves upon activation of ClpP with ADEP treatement and reverses the quenching effect of the nitrotyrosine causing an increase in fluorescence emission signal at 430nm. This assay was used to screen the Molecular Libraries Small Molecule Repository (MLSMR).
NIH Molecular Libraries Probe Production Centers Network [MLPCN]
NIH Chemical Genomics Center [NCGC]
MLPCN Grant: MH095569
Assay Submitter (PI): Michael Maurizi, National Cancer Institute
Two and a half microL/well of purified enzyme solution (final concentrations 10 microg/mL B. subtilis ClpP in 100 mM Tris/HCl pH8, 100 mM KCl, 0.05% low protease BSA, 2.5% glycerol, 0.01% Tween-20 buffer) was dispensed into 1536-well assay plates (Greiner, black solid, medium-binding plates) with Aurora Discovery BioRAPTR Flying Reagent Dispenser (FRD, Beckton-Dickenson). Compound solution (23 nL) was transferred to the assay plate using Kalypsys pin tool equipped with a 1536-pin tool. One and a half microL/well substrate solution (final concentrations 3 microM FRET-FV substrate in 100 mM Tris/HCl pH8, 100 mM KCl, 0.05% low protease BSA, 2.5% glycerol, 0.01% Tween-20 buffer) was then added for a total of 4 microL final reaction volume. FRET signals at time 0 and after 20 min room temperature incubation were obtained using the PerkinElmer Envision plate reader with 320nm excitation and 430nm emission filters. Delta FRET value (Time 20 - Time 0) was calculated and normalized to the delta FRET value of positive control ADEP (5.8 microM final concentration) and negative control DMSO and no enzyme wells.
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
* Activity Concentration. ** Test Concentration.
Data Table (Concise)