High-throughput multiplex microsphere screening for inhibitors of toxin protease, specifically Lethal Factor Protease, MLPCN compound set
Specialized Chemistry Center: Vanderbilt Specialized Chemistry Center For Accelerated Probe Development ..more
BioActive Compounds: 424
University of New Mexico Assay Overview:
Assay Support: 1 R03 MH093184-01A1
Project Title: High-throughput multiplex microsphere screening for toxin protease inhibitors
Assay Provider: Steven Graves Ph.D.
Screening Center/PI: UNMCMD/ Larry Sklar Ph.D.
Lead Biologist: Bruce Edwards Ph.D.,
Screening Operations Team: Jingshu Zhu, Mark Carter MS, Kristine Gouveia MS, Matthew Garcia
Chemistry Center PI: Craig W. Lindsley
Chemistry Lead: Kyle Emmitte
Specialized Chemistry Center: Vanderbilt Specialized Chemistry Center For Accelerated Probe Development
Proteases regulate many biological pathways that include: coagulation, immune system activation, metastasis, and viral life cycles. Within the larger set of proteases, pharmaceutical development for the proteases of the two-part bacterial toxins of Clostridium botulinum and Bacillus anthracis is of great interest due to their role in natural disease and biothreat scenarios (1-4).
Anthrax most commonly occurs in cutaneous form where it is treatable with a variety of antibiotics. Other forms, inhalational and gastrointestinal, rarely occur naturally. Inhalational anthrax is the most deadly form of the disease and is not effectively treated with antibiotics after the very early stages of infection. This form has a high mortality rate generally attributed to toxemia and sepsis. Inhibition of B. antharacis lethal factor (LF) is a potential path to effective treatment of late stage anthrax. The potential use of anthrax spores as bioterror agent makes the development of such treatments increasingly important.
The purpose of this proposal is to discover novel small molecule compounds that inhibit B. anthracis Lethal factor (LF). We are seeking to identify compounds that could affect protease activity through alteration of interactions with required domains distal from the cleavage site. Identification of such a novel allosteric inhibitor would truly be state-of-the-art over current active site LF protease inhibitors. Therefore, we have developed a screening approach that uses full-length substrates attached to microspheres that can detect molecules that either inhibit at the catalytic site or at known required sites distal to the cleavage site.
This project will be run in a multiplex HTS with Botulinum neurotoxin types A and F light chain proteases(BoNTALC and BoNTFLC).
This assay will be used to identify small molecule inhibitors of Botulinum toxin type A and F light chain proteases (BoNTALC and BoNTFLC) and inhibitors of Bacillus anthracis Lethal Factor protease (LF) in a multiplex format. Proteases that are not inhibited will cleave their substrates and produce a loss of signal effect. If the proteases are inhibited by a small molecule cleavage will not occur, thus the signal should stay the same. Full length protease substrates are used in order to detect inhibitors that act at either the protease catalytic site or at sites of protease interaction with substrate distal to the cleavage site.
Protease inhibition assays were performed as previously described (5), but with modifications. Biotinylated GFP protease substrates for LF, BoNTALC, BoNTFLC, and a protease-resistant substrate (pinpointGFP) were prepared and loaded on color-coded streptavidin microspheres (Spherotech Blue Array Particle kit, 5.1 microm diameter) as previously described (5-7). Additions to wells were in sequence as follows: 1st, 4 microL protease buffer (50 mM HEPES, 100 mM NaCl, 1 mg/ml bovine serum albumin, 0.025% Tween-20, pH 7.4); 2nd, 2 microL of a mixture of 1.5 microM LF, 5 nM BoNTALC and 375 nM BoNTFLC in protease buffer; 3rd, 100 nL of test compounds (1 mM in DMSO); and 4th, 4 microL containing 2x10;5/ml of each set of microspheres. Plates were sealed and incubated at 24 degreesC overnight (16-18 h), rotating continuously from inverted to upright position until analyzed in 1536 well plate format with the HyperCyt Cluster Cytometer platform the following day.
The assay response range was defined by replicate control wells containing protease buffer alone (PCntrl, positive control) or the protease mixture alone (NCntrl, negative control). Additional positive controls included Ebselen, VAMP peptide and IN-2-LF (selective inhibitors of BoNTALC, BoNTFLC and LF, respectively), which were added separately and together as a mixture to validate protease inhibition. In each well the median fluorescence intensity (MFI) was determined for each set of substrate-bearing microspheres. A fifth streptavidin microsphere set included in each well had no GFP moiety attached and was used to quantify the contribution of innate test compound fluorescence to the assay readout. All MFI values for substrate-bearing microspheres were corrected by subtraction of the MFI value for the substrate-free microspheres in the same well.
Test compound inhibition of substrate cleavage by protease was then calculated as 100 x (MFI:Test - Mean MFI:NCntrl)/(Mean MFI:PCntrl - Mean MFI:NCntrl) in which MFI:Test represents corrected MFI in the
presence of test compound, and Mean MFI:PCntrl and MFI:NCntrl represent means for replicate MFI determinations in control wells.
Pubchem Activity Score equals the percent compound inhibition.
Development of the HyperCyt Cluster Cytometer Platform for processing of 1536 well plates by high throughput flow cytometry was supported by NIH/NIMH Grant 1R01HG005066 to Bruce Edwards.
BAO: version: 1.4b1080
BAO: bioassay specification: assay stage: primary
BAO: bioassay specification: assay biosafety level: bsl1
BAO: bioassay specification: assay measurement type: endpoint assay
BAO: bioassay specification: assay readout content: assay readout method: regular screening
BAO: bioassay specification: assay readout content: content readout type: multiplexed readout
BAO: meta target: molecular target: protein target: enzyme: protease
BAO: meta target detail: binding reporter specification: interaction: protein-protein
BAO: design: enzyme reporter: enzyme activity: enzyme inhibition
BAO: detection technology: fluorescence: flow cytometry
BAO: bioassay specification: bioassay type: functional: enzyme activity
BAO: bioassay specification: assay footprint: microplate: 1536 well plate
BAO: bioassay specification: assay measurement throughput quality: single concentration single measurement
BAO: format: biochemical format: protein format: protein complex format
BAO: format detail: assay phase characteristic: homogeneous assay
BAO: format detail: reagent: buffer: hepes buffer
BAO: format detail: reagent: detergent: tween 20
** Test Concentration.
Data Table (Concise)