Fluorescence Cell-Free Homogeneous Counter Screen to Identify Inhibitors of GFP Chromophore Formation
M. tuberculosis harbors three inteins, which interrupt the DnaB, RecA, and SufB proteins. GFP-RecA intein fusion protein, which is purified as the 4-thiopyridine derivative in 8 M urea, can be refolded by dilution and retains full competence to undergo protein splicing upon activation by a thiol reducing reagent. In primary screenning, the RecA intein splicing activity was followed by GFP more ..
BioActive Compounds: 1764
Keywords: GFP, refolding, reducing reagent, GFP fluorescence
M. tuberculosis harbors three inteins, which interrupt the DnaB, RecA, and SufB proteins. GFP-RecA intein fusion protein, which is purified as the 4-thiopyridine derivative in 8 M urea, can be refolded by dilution and retains full competence to undergo protein splicing upon activation by a thiol reducing reagent. In primary screenning, the RecA intein splicing activity was followed by GFP fluorescence. The inhibition of GFP fluorescence could arise either from inhibition from the inhibition of protein splicing or from the inhibition of GFP chromophore formation. In this counter screen, cpds were tested with denatured GFP protein purified as the 4-thiopyridine derivative in 8 M urea to rule out false positives which interupt GFP chromophore formation. Specifically, 4-thiopyridine modified GFP was incubated with compounds first in 1536 well plates. the GFP chromophore formation was initiated by addition of reducing reagent TCEP. GFP fluorescence was read with fluorescence plate reader
Expected Outcome: Active wells will show a reduced GFP fluorescence intensity due to inhibition of GFP chromophore formation.
GFP refolding assay in Dose (counter screen)
1) Dispense positive control
Dispense 100 nL of positive control ZnCl2(60mM) using Combi nl in respective wells according to plate design to 1536-well assay ready plates (Aurora 00027830) that contain 22.5 nL/well of 10 mM compound in 8 doses with 3fold dilution(generated by Echo acoustic dispenser).
2) Dispense denatured GFP
Add 3 uL/well of denatured GFP(0.6uM) using Combi nl(Thermo), incubate at room temperature for 30 minutes.
3) Dispense TCEP to initiate the reaction
Add 3ulL/well of TCEP(2mM) with Combi nL (Thermo), incubate at room temperature for ~18 hours
Centrifuge the plates at 1000 rpm x1min
5) Read on Envision for Fluorescence(Ex.405nm/Em.510nm)
20mM NaPi 7.0
ZnCl2 (in H2O, Sigma 229997-50G)
TCEP (in intein buffer, Sigma 646547)
denatured GFP- (in intein buffer, provided by Henry Paulus's lab)
Dose Data Analysis
Neutral control wells (NC) and positive controls wells (PC) were included on every plate.
Active compounds result in decreased readout signal.
Normalization and Pattern Correction
The raw signal was normalized using the 'Neutral Controls Minus Inhibitors' method in Genedata Assay Analyzer (v7.0.3):
The median raw signal of the intraplate PC wells is set to a normalized activity value of -100.
The median raw signal of the intraplate NC wells is set to a normalized activity of 0.
The raw signal of each compound well is then scaled to these two reference points to obtain a normalized activity value.
No plate pattern correction algorithm method was applied.
EC50 values were calculated using the curve fitting strategies in Genedata
Screener Condoseo (7.0.3). EC50 values were extrapolated up to 1 log
over the highest tested concentration.
PubChem Activity Score and Outcome
Inactive compounds = 0
Active compounds = -10*Log(EC50)
Activity_Outcome = 1 (inactive) when
EC50 > 1 log over the highest tested concentration
Activity_Outcome = 2 (active) when
EC50 <= 1 log over the highest tested concentration
Activity_Outcome = 3 (inconclusive) when
Curve fitting strategy resulted in a constant fit with activity >30% but <70%
The fit was not valid due to poor fit quality.
Activity_Outcome = 4 (unspecified) when
no usable data was obtained from the tested compound
* Activity Concentration. ** Test Concentration.
Data Table (Concise)