qHTS Assay for Anthrax Lethal Toxin Internalization
Lethal factor (LF, 83 kDa), edema factor (98 kDa) and protective antigen (PA, 83 kDa) are lethal toxins produced by bacillus anthracis, a Gram-positive and spore-forming bacterium responsible for anthrax. While LF and EF contribute the cytotoxic activity of anthrax bacteria, PA is required for internalization of LF an EF. ..more
BioActive Compounds: 466
NIH Molecular Libraries Screening Centers Network [MLSCN]
NIH Chemical Genomics Center [NCGC]
MLSCN Grant: 1 X01 MH082337-01
PI Name: Dr. Thomas Bugge
NCGC Assay Overview:
Lethal factor (LF, 83 kDa), edema factor (98 kDa) and protective antigen (PA, 83 kDa) are lethal toxins produced by bacillus anthracis, a Gram-positive and spore-forming bacterium responsible for anthrax. While LF and EF contribute the cytotoxic activity of anthrax bacteria, PA is required for internalization of LF an EF.
To enable translocation of anthrax toxins to the cytoplasm, PA has to be cleaved by cell surface furin or furin-like protease, forms a PA heptamer complex and binds to anthrax toxin receptor on cell-surface. Heptamerization of PA generates high-affinity binding sites for anthrax toxins, and upon binding to the toxin, is translocated to the cytoplasm. In addition, similar to many other bacterial toxins (e.g. diphtheria, botulinum, and tetanus toxins), a proton gradient is required for the anthrax toxin translocation. Low molecular weight molecules which interfere those internalization processes, may potentially lead to development of antidotes for the anthrax toxin.
By using LF-beta-lactamase fusion proteins, a quantitative high throughput assay was developed to screen small molecules potentially reducing or blocking internalization of the anthrax toxin. The fusion protein was generated by fusing the PA-binding region of LF N-terminal (1-254 a.a.) to the TEM-1 beta-lactamase (developed by Dr. Thomas Bugge's laboratory in NIH). In the presence of PA, LF-beta-lactamase fusion proteins will be internalized, and act on beta-lactamase substrate (CCF2/AM) trapped in cells after cleavage by cytoplasm esterases. An intact CCF2 fluoresces at 520 nm (green light) by FRET. CCF2 hydrolyzed by beta-lactamase releases acceptor fluorescence and fluoresces at 447 nm (blue light). EnVision plate reader (PerkinElmer, Boston, MA) was used to monitor fluoresce intensity with the following filter set, Lambda(EX)=405 nm, Lambda(EM)=460nm/530 nm. The data were plotted a ratio of the 460nm/530nm emissions.
NCGC Assay Protocol Summary
ME-180 cells were maintained in F12 medium (Invitrogen, Carlsbad, CA) at 37C under a humidified atmosphere containing 5% CO2 and 95% air. The medium contained 10% FBS, 2 mM L-glutamine, 50 U/ml gentamicin (Invitrogen). The cells were stored at -135 C for the assay. All compounds were screened as titrations from 0.6 nM to 46 uM final concentration. The assay was performed in 1536-well format plate (Kalypsys, treated black clear bottom) as follows:
1.2k/well frozen ME-180 was seed for overnight with 1% FBS at 37C under a humidified atmosphere containing 5% CO2 and 95% air.
2.Pinning 23 nL compounds. The final titration was 0.6 nM to 46 uM.
3.Incubation at 37C for 15 min.
4.Dispensing 1uL LF-beta-Lac (1 ug/ml final) with or without PA (2 ug/ml final).
5.Incubation at 37C for 2 hr.
6.Dispensing 1 uL of CCF2 in CCF2 detection buffer (CCF2, 0.5uM final)
7.Incubation at room temperature for 5 hr.
8.Reading plates by EnVision (PerkinElmer).
Keywords: Anthrax LF, LF internalization, beta-lactamase, CCF2, fluorescence, cytotoxicity, MLSMR, MLSCN, NIH Roadmap, qHTS and NCGC
1. Compounds are first classified as being probable inhibitors (compounds decreasing signal), activators (compounds increasing signal), fluorescent (artifactual results), cytotoxic (artifactual results), inactive, or inconclusive based on the ratio and component channel readouts.
2. Compounds are then classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Ratio-Curve Description".
3. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active inhibitor 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. Ratio-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)