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BioAssay: AID 588501

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
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 Tested Compounds
 Tested Compounds
All(354782)
 
 
Active(424)
 
 
Inactive(352449)
 
 
Inconclusive(1912)
 
 
 Tested Substances
 Tested Substances
All(355114)
 
 
Active(425)
 
 
Inactive(352777)
 
 
Inconclusive(1912)
 
 
AID: 588501
Data Source: NMMLSC (UNMCMD_Anthrax_LethalFactor_Protease_HTS_MLPCN)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2011-10-12
Modify Date: 2011-12-02

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 424
Related Experiments
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AIDNameTypeComment
588467Summary for High-throughput multiplex microsphere screening for inhibitors of toxin protease, specifically Botulinum neurotoxin light chain A proteaseSummarydepositor-specified cross reference
588469Summary of High-throughput multiplex microsphere screening for inhibitors of toxin protease, specifically Lethal Factor ProteaseSummarydepositor-specified cross reference
588470Summary of High-throughput multiplex microsphere screening for inhibitors of toxin protease, specifically Botulinum neurotoxin light chain F proteaseSummarydepositor-specified cross reference
588460High-throughput multiplex microsphere screening for inhibitors of toxin protease, specifically Botulinum neurotoxin light chain A protease, Validation Compound SetScreeningsame project related to Summary assay
588497High-throughput multiplex microsphere screening for inhibitors of toxin protease, specifically Botulinum neurotoxin light chain F protease, MLPCN compound setScreeningsame project related to Summary assay
588499High-throughput multiplex microsphere screening for inhibitors of toxin protease, specifically Botulinum neurotoxin light chain A protease, MLPCN compound setScreeningsame project related to Summary assay
602139High-throughput multiplex microsphere screening for inhibitors of toxin protease, specifically Botulinum neurotoxin light chain A protease, Cherry Pick 01Screeningsame project related to Summary assay
602176High-throughput multiplex microsphere dose response for inhibitors of toxin protease, specifically Botulinum neurotoxin light chain A protease, compounds from Cherry Pick 01Confirmatorysame project related to Summary assay
602325High-throughput multiplex microsphere screening for inhibitors of toxin protease, specifically Botulinum neurotoxin light chain A protease, Cherry Pick 02Screeningsame project related to Summary assay
602328High-throughput multiplex microsphere dose response for inhibitors of toxin protease, specifically Botulinum neurotoxin light chain A protease, compounds from Cherry Pick 02Confirmatorysame project related to Summary assay
624243High-throughput multiplex microsphere dose response for inhibitors of toxin protease, specifically Botulinum neurotoxin light chain A protease, compounds from Powder Set 01Confirmatorysame project related to Summary assay
624483Counterscreen of compound fluorescence effects on High-throughput multiplex microsphere screening for inhibitors of toxin proteaseScreeningsame project related to Summary assay
651691Confirmatory FRET assay using cleavage-site specific substrates for both Botulinum neurotoxin light chain A protease and lethal factor, Powder Set01Othersame project related to Summary assay
651879High-throughput multiplex microsphere dose response for inhibitors of toxin protease, specifically Botulinum neurotoxin light chain A protease, compounds from Powder Set 02Confirmatorysame project related to Summary assay
651901High-throughput multiplex microsphere dose response for inhibitors of toxin protease, specifically Lethal Factor protease, compounds from Powder Set 02Confirmatorysame project related to Summary assay
652102Confirmatory FRET assay using full length substrates for Botulinum neurotoxin light chain A protease, with Powder Set01Confirmatorysame project related to Summary assay
588461High-throughput multiplex microsphere screening for inhibitors of toxin protease, specifically Lethal Factor Protease, Validation compound setScreeningsame project related to Summary assay
602142High-throughput multiplex microsphere screening for inhibitors of toxin protease, specifically Lethal Factor Protease, Cherry Pick 01Screeningsame project related to Summary assay
602178High-throughput multiplex microsphere dose response for inhibitors of toxin protease, specifically Lethal Factor protease, compounds from Cherry Pick 01Confirmatorysame project related to Summary assay
602326High-throughput multiplex microsphere dose response for inhibitors of toxin protease, specifically Lethal Factor protease, compounds from Cherry Pick 02Confirmatorysame project related to Summary assay
602327High-throughput multiplex microsphere screening for inhibitors of toxin protease, specifically Lethal Factor Protease, Cherry Pick 02Screeningsame project related to Summary assay
624244High-throughput multiplex microsphere dose response for inhibitors of toxin protease, specifically Lethal Factor protease, compounds from Powder Set 01Confirmatorysame project related to Summary assay
624483Counterscreen of compound fluorescence effects on High-throughput multiplex microsphere screening for inhibitors of toxin proteaseScreeningsame project related to Summary assay
588459High-throughput multiplex microsphere screening for inhibitors of toxin protease, specifically Botulinum neurotoxin light chain F protease, Validation compound setScreeningsame project related to Summary assay
588497High-throughput multiplex microsphere screening for inhibitors of toxin protease, specifically Botulinum neurotoxin light chain F protease, MLPCN compound setScreeningsame project related to Summary assay
588499High-throughput multiplex microsphere screening for inhibitors of toxin protease, specifically Botulinum neurotoxin light chain A protease, MLPCN compound setScreeningsame project related to Summary assay
602140High-throughput multiplex microsphere screening for inhibitors of toxin protease, specifically Botulinum neurotoxin light chain F protease, Cherry Pick 01Screeningsame project related to Summary assay
602177High-throughput multiplex microsphere dose response for inhibitors of toxin protease, specifically Botulinum neurotoxin light chain F protease, compounds from Cherry Pick 01Confirmatorysame project related to Summary assay
602336High-throughput multiplex microsphere screening for inhibitors of toxin protease, specifically Botulinum neurotoxin light chain F protease, Cherry Pick 02Screeningsame project related to Summary assay
602338High-throughput multiplex microsphere dose response for inhibitors of toxin protease, specifically Botulinum neurotoxin light chain F protease, compounds from Cherry Pick 02Confirmatorysame project related to Summary assay
624245High-throughput multiplex microsphere dose response for inhibitors of toxin protease, specifically Botulinum neurotoxin light chain F protease, compounds from Powder Set 01Confirmatorysame project related to Summary assay
624483Counterscreen of compound fluorescence effects on High-throughput multiplex microsphere screening for inhibitors of toxin proteaseScreeningsame project related to Summary assay
651880High-throughput multiplex microsphere dose response for inhibitors of toxin protease, specifically Botulinum neurotoxin light chain F protease, compounds from Powder Set 02Confirmatorysame project related to Summary assay
651901High-throughput multiplex microsphere dose response for inhibitors of toxin protease, specifically Lethal Factor protease, compounds from Powder Set 02Confirmatorysame project related to Summary assay
Description:
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).
Protocol
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.
Comment
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.
Categorized Comment - additional comments and annotations
From BioAssay Depositor:
BAO: bioassay specification: assay biosafety level: bsl1
BAO: bioassay specification: assay footprint: microplate: 1536 well plate
BAO: bioassay specification: assay measurement throughput quality: single concentration single measurement
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: bioassay specification: assay stage: primary
BAO: bioassay specification: bioassay type: functional: enzyme activity
BAO: design: enzyme reporter: enzyme activity: enzyme inhibition
BAO: detection technology: fluorescence: flow cytometry
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
BAO: meta target detail: binding reporter specification: interaction: protein-protein
BAO: meta target: molecular target: protein target: enzyme: protease
BAO: version: 1.4b1080
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1PERCENT_INHIBITION (10μM**)Percent inhibition of substrate cleavage in the presence of 10 microM test compoundFloat%

** Test Concentration.
Additional Information
Grant Number: 1 R03 MH093184-01A1

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
Classification
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