Fluorescent Biochemical Primary HTS to Identify Inhibitors of P. aeruginosa PvdQ acylase Measured in Biochemical System Using Plate Reader - 2091-02_Inhibitor_Dose_DryPowder_Activity_Set4
Bacteria typically require total iron concentrations in the micromolar range to support growth. Many pathogens such as P. aeruginosa produce siderophores (e.g. pyoverdine) with molecular weights below 1500 Da that bind to iron with remarkably high affinities. The PvdQ acylase is responsible for removal of the fatty acid on pyoverdine. Interestingly, mutant strains of P. aeruginosa with a deletion more ..
BioActive Compounds: 23
Keywords: Pyoverdine, laurate, PvdQ
Assay Overview: Fluorometric assay (4-methylumbelliferone).
Bacteria typically require total iron concentrations in the micromolar range to support growth. Many pathogens such as P. aeruginosa produce siderophores (e.g. pyoverdine) with molecular weights below 1500 Da that bind to iron with remarkably high affinities. The PvdQ acylase is responsible for removal of the fatty acid on pyoverdine. Interestingly, mutant strains of P. aeruginosa with a deletion in the PvdQ gene do not make pyoverdine and cannot grow on iron limiting media. This primary screen aims to selectively identify small molecules inhibitors of the PvdQ enzymatic activity. The small molecules bind to PvdQ and block the deceylation of 4-methylumbelliferone laurate. The enzymatic activity of PvdQ will be measured through fluorescence emission mediated by the dissociation of 4-methylumbellerone to the fatty acid laurate. The PvdQ enzyme is pre-incubated with the compounds 30 minutes before the substrate 4-MU laurate is added.
Expected outcome: Identification of probe(s) inhibiting PvdQ enzymatic activity. Compounds reducing the fluorescence mediated by PvdQ and 4-methylumbelliferone with an IC50 below 10 uM will be considered hits.
2091 Pyoverdine Inhibitors (Pre-incubation PvdQ Primary assay PROTOCOL)
TNT assay buffer preparation:
-Tris 50 mM (pH8.0)
-NaCl 50 mM
-Tris (2-carboxyethyl) phosphine hydrochloride (TCEP) 0.2 mM (Sigma, C4706)
We diluted 50 mL of Tris 1M pH8.0 (stock solution), 10 mL of NaCl 5M and 50 ml TCEP 4mM with 890 mL water. The TNT assay buffer was filtered using a filtering unit (Nalgene/Nunc 0.9 mm filter unit, catalog number 151-4020).
4-methylumbelliferone laurate (4-MU laurate) substrate preparation:
162 mg (16 mM) of 4-MU laurate (Research Organics, 0183M, MW 358.5 g/mol) was dissolved in 25 ml isopropanol and was mixed with 2.5 ml of Triton X-100(Sigma, T8787)(0.1 volume), vortexed for 10 sec and gently mixed with 375 ml TNT buffer (15 volumes).
The P. aeruginaosa PvdQ was provided by Dr. Andrew Gulick, Hauptman-Woodward Medical Research Institute, Buffalo, NY at a concentration of 10.8 mg/ml (135.9 uM stock concentration). The PvdQ preparation came as 40 ul droplets. We added 40ul of the PvdQ preparation in 54.3 ml TNT buffer (final concentatration of 0.1 uM) with 250 uL triton X-100 (0.5%).
The PvdQ (1.5 ul) was dispensed using BioRaptrTM FRD microfluid workstation (Beckman Coulter) at a final concentration 0.02uM (PvdQ) and 0.8 mM (4-MU laurate) at room temperature in 1536-well high base black barcoded plate square well assay plate (Aurora Biotechnologies, cat number 00019180BK) where 7.5 nl (8 concentrations, 3 fold dilution (starting final concentration 10 uM)) of the MLPCN compound library was already dispensed in the assay plate. The positive control Isopropyl Dodecylfluorophosphonate (IDFP)(Cayman chemicals, cat. number 10215) was added in 24 selective wells (1.7 mM stock solution in TNT buffer to a 200 uM final concentration). After 30 minutes pre-incubation, 4-MU laurate (6 ul) was then added and the assay plates incubated for an additional 60 minutes at room temperature.
The assay plates were read at time 0 and 60 min using the Viewlux (Perkin Elmer) with 303-367 nm excitation filter and 440-460 nm emission filter. The fluorescence mediated by the enzymatic reaction was calculated the difference between the read at 60 minutes and 0 minute.
PRESENCE OF CONTROLS: Neutral control wells (NC; n=176) and positive control wells (PC; n=24) were included on every plate.
EXPECTED OUTCOME: Active compounds result in decreasing readout signal.
ACTIVE CONCENTRATION LIMIT:
For each sample, the highest valid tested concentration (Max_Concentration) was determined and the active concentration limit (AC_limit) was set to equal (2)(Max_Concentration).
The raw signals of the plate wells were normalized using the 'Neutral Controls Minus Inhibitors' method in Genedata Assay Analyzer (v7.0.3):
The median raw signal of the intraplate neutral control wells was set to a normalized activity value of 0.
The median raw signal of the intraplate positive control wells was set to a normalized activity value of -100.
Experimental wells values were scaled to this range.
PATTERN CORRECTION: No plate pattern correction algorithm from Genedata Condoseo (v.7.0.3) was applied.
MEASUREMENT USED TO DETERMINE ACTIVE CONCENTRATION (AC): AC50
AC values were calculated using the curve fitting strategies in Genedata Screener Condoseo (7.0.3).
AC values were calculated up to the active concentration limit described for each sample.
pAC was set to equal -1*log10(AC)
Activity_Outcome = 1 (inactive) when:
a) compound shows activity but in a direction opposite to the expected outcome
in these cases, values describing curve fitting parameters (Sinf, S0, Hill Slope, log_AC50, log_AC50_SE) are set to null
b) curve fit is constant where activity is > -30% and < 30% at all tested concentrations, or
c) AC > AC_limit
Activity_Outcome = 2 (active) when:
AC <= AC_limit
Activity_Outcome = 3 (inconclusive) when:
a) Curve fitting strategy resulted in a constant fit with activity >= -70% but <= -30%, or
b) The fit was deemed not valid due to poor fit quality.
If PUBCHEM_ACTIVITY_OUTCOME = 1 (inactive) or 3 (inconclusive),
then PUBCHEM_ACTIVITY_SCORE = 0
If PUBCHEM_ACTIVITY_OUTCOME = 2 (active)
then PUBCHEM_ACTIVITY_SCORE = (10)(pAC)
Scores relate to AC in this manner:
120 = 1 pM
90 = 1 nM
60 = 1 uM
30 = 1 mM
0 = 1 M
When the active concentration (AC) is calculated to be greater than the highest valid tested concentration (Max_Concentration), the PUBCHEM_ACTIVITY_SCORE is calculated using Max_Concentration as the basis.
When the active concentration (AC) is calculated to be less than the lowest tested concentration, the PUBCHEM_ACTIVITY_SCORE is calculated using the lowest tested concentration as the basis.
The individual dose data point columns ('Activity_at_xxuM') reported here represent the median of valid (unmasked) replicate observations at each concentration. These values are the inputs to a curve fitting algorithm.
All other data columns represent values which are derived during the curve fitting algorithm; this may sometimes include automatic further masking of some replicate data points.
Occasionally this results in perceived inconsistencies: for example, between the derived 'Maximal_Activity' and the apparent most active data point.
Categorized Comment - additional comments and annotations
* Activity Concentration. ** Test Concentration.
Data Table (Concise)