Fluorescence Cell-Free Homogeneous Secondary Screen to Identify Non-Covalent Inhibitors of RecA-Intein Splicing Activity
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 screening, the RecA intein splicing activity was followed by GFP more ..
BioActive Compounds: 1380
Keywords:GFP-RecA intein, protein splicing, refolding, reducing reagent, GFP fluorescence, covalent
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 screening, the RecA intein splicing activity was followed by GFP fluorescence. This current MOA assay is to distinguish covalent inhibitors and candidates for non-covalent inhibition. Specifically, The GFP-RecA intein was incubated first with compounds in 1536-well plates, the RecA splicing activity was initiated by addition of 5mM DTT, which is known to abrogate covalent inhibition by electrophiles. GFP fluorescence, produced as a result of protein splicing, was read with fluorescence plate reader.
Expected Outcome: Active wells will show a reduced GFP fluorescence intensity due to non-covalent inhibition of RecA-Intein splicing activity.
GFP-RecA Splicing Assay with DTT in Dose(for mode of action)
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 GFP-RecA Intein
Add 3 uL/well of GFP-recA intein(2uM) using Combi nl(Thermo), incubate at room temperature for 30 minutes.
3) Dispense DTT to initiate the reaction
Add 3ulL/well of DTT(10mM) 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/Em510nm)
20 mM NaPi 7.0
500 mM NaCl
500 mM L-Arginine.HCL
ZnCl2 (in H2O, Sigma 229997-50G)
DTT (in intein buffer, Sigma 43816)
10 mM DTT
GFP-RecA intein (in intein buffer, provided by Henry Paulus's lab)
2 uM GFP-RecA Intein
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.
AC50 values were calculated using the curve fitting strategies in Genedata
Screener Condoseo (7.0.3). AC50 values were extrapolated up to 1 log
over the highest tested concentration.
PubChem Activity Score and Outcome
Inactive compounds = 0
Active compounds = -10*Log(AC50)
Superactive compounds (full inhibition at all concentrations; constant curve fit) = 100
Activity_Outcome = 1 (inactive) when
AC50 > 1 log over the highest tested concentration
Activity_Outcome = 2 (active) when
AC50 <= 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)