Fluorescent Biochemical Primary HTS to Identify Inhibitors of P. aeruginosa PvdQ acylase Measured in Biochemical System Using Plate Reader and Imaging Combination - 2091-01_Inhibitor_SinglePoint_HTS_Activity
Bacteria typically require total iron concentrations in the uM 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 more ..
BioActive Compounds: 213
Keywords: Pyoverdine, laurate, PvdQ
Assay Overview: Fluorometric assay (4-methylumbelliferone).
Bacteria typically require total iron concentrations in the uM 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.
Expected outcome: Identification of probe(s) inhibiting PvdQ enzymatic activity. Compounds (10 uM final concentration) reducing the fluorescence mediated by PvdQ and 4-methylumbelliferone by 20% in duplicate will be considered hits.
2091 Pyoverdine Inhibitors (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 4 mM 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-methylumbelliferyl laurate (4-MU laurate) substrate preparation (volume of substrate required to fill up about 30 1536-well black square well assay plates):
144 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 10.06 mg/ml (126.6 uM stock concentration). The PvdQ preparation came as 40 ul droplets. We added 80 ul of the PvdQ preparation in 50.2 ml TNT buffer (final concentatration of 0.2 uM).
The PvdQ (0.75 ul) and 4-MU laurate (6.75 ul) solutions were dispensed using BioRaptrTM FRD microfluid workstation (Beckman Coulter) at a final concentration 0.02 uM (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 0.75 nl (10 mM compound concentration) 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).
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 as the difference between the read at 60 minutes and 0 minute.
PRESENCE OF CONTROLS: Neutral control wells (NC) and positive control wells (PC) were included on every plate.
EXPECTED OUTCOME: Active compounds result in decreasing readout signal.
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: The plate pattern correction algorithm 'Runwise Pattern (Multiplicative)' in Genedata (v7.0.3) was applied to the normalized plate data.
This was set as equal to the mean of the normalized and corrected sample replicate activities, rounded to the nearest integer .
The minimum PUBCHEM_ACTIVITY_SCORE required for a compound to be called a hit (the activity threshold, or AT) was set at 30.
PERCENTAGE OF ACTIVE REPLICATES:
For each sample, the percentage of replicates (PCT_ACTIVE_REP) which had activity scores >= AT was determined.
The minimum percentage of replicates required for a compound to be called a hit (PAR_T) was set at 100.
Samples passing BOTH threshold criteria were assigned an outcome of 2 (active):
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP >= PAR_T
Samples passing NEITHER threshold criteria were assigned an outcome of 1 (inactive):
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP < PAR_T
Samples passing AT only were assigned an outcome of 3 (inconclusive) :
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP < PAR_T
Samples passing PAR_T only were assigned an outcome of 3 (inconclusive) :
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP >= PAR_T
Categorized Comment - additional comments and annotations
** Test Concentration.
Data Table (Concise)