Counterscreen of compound fluorescence effects on High-throughput multiplex microsphere screening for inhibitors of toxin protease
Specialized Chemistry Center: Vanderbilt Specialized Chemistry Center For Accelerated Probe Development ..more
BioActive Compounds: 10930
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).
Botulinum Neurotoxin A Light Chain (BoNTALC) is also an active component of Botulinum Neurotoxin type A (BoNTA), which has become a widely used pharmaceutical (BoTox) that has important uses in the treatment of many diseases. Thus, discovery of BoNTALC inhibitors is also of interest due to the potential of both accidental and intentional misuse of BoTox.
The use of BoNT as a potential biological weapon and the role that BoNTA has in naturally caused botulism has driven much effort in pharmaceutical development targeting BoNTALC. In the USA, serotype A causes >50% of all cases of naturally occurring botulism. While naturally occurring food borne botulism is usually not fatal, symptoms can last for several months. Advances in hygiene and food processing have largely eliminated food borne botulism as a major public health concern but outbreaks still occur in the U.S. No effective pharmaceutical treatment currently exists for any type of botulinum toxin poisoning. The currently approved treatment for adult BoNTA poisoning is administration of equine antibodies to BoNTA, which cause serious side effects and have a short window of application. In the specialized case of infant botulism, caused by colonization of the large intestine by C. botulinum, a human anti-sera treatment has been approved for use in patients less than 1 year old. Though the available antitoxins provide some effective treatments for natural disease, they are expensive, require refrigeration, and would be difficult to administer in large-scale outbreaks. Therefore, an effective, stable, and inexpensive small molecule inhibitor is very desirable.
The purpose of this screening project is to discover novel small molecule compounds that inhibit BoNTALC protease. 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 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.
A potential side effect from this screening method is that compounds with innate fluorescent characterisitics could be registered as active compounds. Hence, here we report the results of a counterscreen to identify compounds that increase fluorescence (fluorescent compounds). These compounds have been filtered out as false positives from the primary screen of multiplex HTS with Botulinum Neurotoxin A Light Chain proteases (BoNTALC), Botulinum neurotoxin type F light chain proteases (BoNTFLC), and Bacillus anthracis lethal factor (LF).
The counterscreen assay for potential fluorescent compounds utilized the same protocol as the primary screen 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.
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 mum diameter) as previously described (5-7). In addition to these four different bead set, there was also a non-GFP labeled bead set added to the multiplex. This particular bead set was the one utilized to evaluate compound fluorescence.
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 nanoL 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. In each well the median fluorescence intensity (MFI) was determined for each set of substrate-bearing microspheres as well as for the fifth streptavidin microsphere set with no GFP moiety attached.
Column entitled "FLUORESCENCE_INTENSITY" represents the MFI of blank beads that had no GFP. A compound was scored as "active" if it exceeded the mean MFI by 3SD or more. This represented ~11,000 compounds or ~3% of total. Mean MFI was 5176 and SD was 3650. Only wells with >10 beads were included in Mean/SD calculation. Maximum MFI was 114208.
Activity score was calculated as 100 * (Bead MFI - Mean MFI)/(Max MFI - Mean MFI). If bead count was 10 or less the compound was assigned an outcome of 3, activity score of 0 and no MFI values were entered. If bead MFI was <= mean MFI, activity score was set to 0.
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.
Data Table (Concise)