Funding was provided by the Joint Science and Technology Office &#8211; Chemical Biological Defense (JSTO-CBD) Defense Threat Reduction Agency (DTRA) under sponsor project number CCAR# CB3675 and National Institutes of Health (1 R21 AI101387-01 and 5 U01AI082051-05).

Plate 20,000 differentiated mouse ES cells (MES) / well in a 96 well Poly-D lysine coated plates and maintain in motor neuron terminal differentiation media for 5-7 days.
1. Compound Administration and Intoxication with BoNT/A
Perform all of the following work in a BSL2 enclosure to maintain compliance with CDC/NIH guidelines.
<ol>
	<li>Prepare 10 mM stock solutions of each library compound in 100% DMSO in polypropylene 96 well plates. Use the 10 mM stock to prepa...

<ol>
	<li>Lamanna, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+most+poisonous+poison.">The most poisonous poison.</a> Science. 130, 763-772 (1959).</li><li>Dover, N., Barash, J. R., Hill, K. K., Xie, G., Arnon, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Characterization+of+a+Novel+Botulinum+Neurotoxin+Type+H+Gene.">Molecular Characterization of a Novel Botulinum Neurotoxin Type H Gene.</a> J. Infect. Dis. , (2013).</li><li>Hakami, R. M., Ruthel, G., Stahl, A. M., Bavari, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gaining+ground:+assays+for+therapeutics+against+botulinum+neurotoxin.">Gaining ground: assays for therapeutics against botulinum neurotoxin.</a> Trends in microbiology. 18, 164-172 (2010).</li><li>Yersin, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+between+synaptic+vesicle+fusion+proteins+explored+by+atomic+force+microscopy.">Interactions between synaptic vesicle fusion proteins explored by atomic force microscopy.</a> Proceedings of the National Academy of Sciences of the United States of America. 100, 8736-8741 (2003).</li><li>Dong, M., Tepp, W. H., Johnson, E. A., Chapman, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+fluorescent+sensors+to+detect+botulinum+neurotoxin+activity+in+vitro+and+in+living+cells.">Using fluorescent sensors to detect botulinum neurotoxin activity in vitro and in living cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 101, 14701-14706 (2004).</li><li>Stahl, A. M., Adler, M., and, C. B. M., Gilfillan, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accelerating+botulism+therapeutic+product+development+in+the+Department+of+Defense.">Accelerating botulism therapeutic product development in the Department of Defense.</a> Drug Development Research. 70, 303-326 (2009).</li><li>Nuss, J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+cell-based+assays+to+measure+botulinum+neurotoxin+serotype+A+activity+using+cleavage-sensitive+antibodies.">Development of cell-based assays to measure botulinum neurotoxin serotype A activity using cleavage-sensitive antibodies.</a> Journal of biomolecular screening. 15, 42-51 (2010).</li><li>Opsenica, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+chemotype+that+inhibits+three+unrelated+pathogenic+targets:+the+botulinum+neurotoxin+serotype+A+light+chain,+P.+falciparum+malaria,+and+the+Ebola+filovirus.">A chemotype that inhibits three unrelated pathogenic targets: the botulinum neurotoxin serotype A light chain, P. falciparum malaria, and the Ebola filovirus.</a> Journal of medicinal chemistry. 54, 1157-1169 (2011).</li><li>Opsenica, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+synthesis+of+2,5-bis(4-amidinophenyl)thiophene+derivatives+providing+submicromolar-range+inhibition+of+the+botulinum+neurotoxin+serotype+A+metalloprotease.">The synthesis of 2,5-bis(4-amidinophenyl)thiophene derivatives providing submicromolar-range inhibition of the botulinum neurotoxin serotype A metalloprotease.</a> European journal of medicinal chemistry. 53, 374-379 (2012).</li><li>Nuss, J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacophore+Refinement+Guides+the+Rational+Design+of+Nanomolar-Range+Inhibitors+of+the+Botulinum+Neurotoxin+Serotype+A+Metalloprotease.">Pharmacophore Refinement Guides the Rational Design of Nanomolar-Range Inhibitors of the Botulinum Neurotoxin Serotype A Metalloprotease.</a> ACS medicinal chemistry letters. 1, 301-305 (2010).</li><li>Kiris, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+stem+cell-derived+motoneurons+provide+a+highly+sensitive+cell+culture+model+for+botulinum+neurotoxin+studies,+with+implications+for+high-throughput+drug+discovery.">Embryonic stem cell-derived motoneurons provide a highly sensitive cell culture model for botulinum neurotoxin studies, with implications for high-throughput drug discovery.</a> Stem cell research. 6, 195-205 (2011).</li><li>Pellett, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+targeting,+internalization,+and+biological+activity+of+a+recombinant+atoxic+derivative+of+botulinum+neurotoxin+A.">Neuronal targeting, internalization, and biological activity of a recombinant atoxic derivative of botulinum neurotoxin A.</a> Biochemical and biophysical research communications. 405, 673-677 (2011).</li><li>McNutt, P., Celver, J., Hamilton, T., Mesngon, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+stem+cell-derived+neurons+are+a+novel,+highly+sensitive+tissue+culture+platform+for+botulinum+research.">Embryonic stem cell-derived neurons are a novel, highly sensitive tissue culture platform for botulinum research.</a> Biochemical and biophysical research communications. 405, 85-90 (2011).</li><li>Fischer, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bimodal+modulation+of+the+botulinum+neurotoxin+protein-conducting+channel.">Bimodal modulation of the botulinum neurotoxin protein-conducting channel.</a> Proceedings of the National Academy of Sciences of the United States of America. 106, 1330-1335 (2009).</li></ol>

Krishna P Kota is an employee of Perkin Elmer Inc. Waltham, MA, that produces instruments and software used in this manuscript. The content of this publication does not necessarily reflect the views or policies of the U.S. Department of Health and Human Services, the U.S. Department of Defense, the U.S. Department of the Army, or the institutions and companies affiliated with the authors.

The high potency of Botulinum neurotoxins and the relative ease of their weaponization has resulted in their classification as Category A (highest priority) biothreat agents by the U.S. Centers for Disease Control and Prevention. Unfortunately, there are no FDA approved therapeutics to counter BoNT intoxication after the toxin has been internalized by the motor neurons. Any druggable mechanism that promotes neuronal recovery from BoNT intoxication could lead toward the development of a potential therapy to protect both t...

The bacterium Clostridium botulinum produces Botulinum neurotoxin, one of the most potent biological toxins known to man1. There are 7 distinct BoNT serotypes (BoNT/A-G). BoNT/A-Gall induce paralysis at the neuromuscular junction due to SNARE complex proteolysis 2,3. SNARE proteolysis prevents neurotransmitter vesicle-membrane fusion and therefore blocks neurotransmitter exocytosis4. The specific SNARE target depends upon the particular BoNT serotype involved in the intoxication process. BoNT/A and BoNT/E cleave SNAP-25 whereas BoNT/C cleaves both SNAP-25 and syntaxin5. The remaining serotypes cleave synaptobrevin (also called Vesicle-associated membrane protein (VAMP). BoNT/A was chosen for assay development as it is responsible for a high proportion of naturally occurring botulism and has the longest duration of action6. Development of small molecule therapeutics against BoNT/A is a major goal for our drug discovery program and has utilized traditional target-based methods to identify active site proteolytic inhibitors7,8-10. However, the creation of active site inhibitors with broad spectrum activity against multiple serotypes and post-exposure efficacy will likely be challenging.
We have therefore implemented an innovative, phenotypic drug discovery approach that uses BoNT SNAP-25 cleavage as a functional endpoint to identify small molecules that can block BoNT-mediated motor neuron intoxication. SNAP-25 is required for neurotransmitter release, as degradation of SNAP-25 is predictive of paralysis and lethality in vivo. For example, cell-based screening could lead to discovery of new modulators of cellular factors responsible for toxin inactivation or inhibition of toxin pathways inside targeted cells. An important factor in phenotypic assay development is the selection of physiologically relevant biological models. We and others have described mouse embryonic stem (ES) cell-derived motor neurons that recapitulate the immunologic character of primary motor neurons, including the expression of SNAP-2511-13. Importantly, these cellular systems are highly sensitive to BoNT/A intoxication and demonstrate dose-dependent cleavage of SNAP-25 in response to increasing concentrations of toxin11,12. The differentiated motor neurons are also produced in quantities that are sufficient for high throughput plate based analysis and allowed the design of an array of cellular assays.
The phenotypic assay is an immunofluorescence method utilizing two distinct antibodies to quantify cleavage of endogenously expressed full length SNAP-25 during BoNT/A intoxication of mouse motor neuron culture. A carboxyl terminal BoNT/A cleavage-sensitive (BACS) antibody that recognizes only full length SNAP-25 allows the assessment of BoNT/A mediated proteolysis of SNAP-25 expression in mouse motor neurons10. A schematic diagram of the HCI assay is depicted in Figure 1.

<table border="0" cellpadding="0" cellspacing="0" width="834">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Name of Reagent/ Equipment</td>
			<td style="width:180px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:280px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Botulinum neurotoxin A&#160;</td>
			<td style="width:180px;">Metabiologics</td>
			<td style="width:119px;">NA</td>
			<td style="width:280px;">No catalog number</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Microtitre plates</td>
			<td style="width:180px;">Greiner&#160;</td>
			<td style="width:119px;">655946</td>
			<td style="width:280px;">Poly-D-Lysine 96-well plates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">BACs antibody</td>
			<td style="width:180px;">Lampire Biological</td>
			<td style="width:119px;">NA</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Microchem</td>
			<td style="width:180px;">National&#160;</td>
			<td style="width:119px;">0255</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Methanol</td>
			<td style="width:180px;">Thermo Scientific</td>
			<td style="width:119px;">A412-20</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Formaldehyde</td>
			<td style="width:180px;">Thermo Scientific</td>
			<td style="width:119px;">28908</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Horse serum</td>
			<td style="width:180px;">Invitrogen</td>
			<td style="width:119px;">16050</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">PE JANUS MDT Mini Automated Workstation</td>
			<td style="width:180px;">Perkin Elmer</td>
			<td style="width:119px;">AJMDT01</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Opera</td>
			<td style="width:180px;">Perkin Elmer</td>
			<td>OP-QEHS-01</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Triton X 100</td>
			<td style="width:180px;">Sigma-Aldrich</td>
			<td style="width:119px;">9002-93-1</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">BIII tubulin antibody</td>
			<td style="width:180px;">R&#38;D Systems</td>
			<td style="width:119px;">BAM1195</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Tween 20</td>
			<td style="width:180px;">Sigma</td>
			<td style="width:119px;">P1379-1L</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hoechst 33342dye&#160;</td>
			<td style="width:180px;">Invitrogen</td>
			<td style="width:119px;">3570</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Antimouse IgG</td>
			<td style="width:180px;">Invitrogen</td>
			<td style="width:119px;">A21236</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Anti rabbit IgG</td>
			<td style="width:180px;">Invitrogen</td>
			<td style="width:119px;">A10042</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Columbus Image analysis software</td>
			<td style="width:180px;">Perkin Elmer</td>
			<td style="width:119px;">Ver 2.4</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Spotfire</td>
			<td style="width:180px;">Perkin Elmer</td>
			<td style="width:119px;">Ver 5.5</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Clorox bleach</td>
			<td style="width:180px;">Fisher Scientific</td>
			<td style="width:119px;">18-861-284</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">PlateStack</td>
			<td style="width:180px;"></td>
			<td style="width:119px;"></td>
			<td style="width:280px;"></td>
		</tr>
	</tbody>
</table>

a high content imaging assay for identification of botulinum neurotoxin inhibitors

Synaptosomal-associated protein-25 (SNAP-25) is a component of the soluble NSF attachment protein receptor (SNARE) complex that is essential for synaptic neurotransmitter release. Botulinum neurotoxin serotype A (BoNT/A) is a zinc metalloprotease that blocks exocytosis of neurotransmitter by cleaving the SNAP-25 component of the SNARE complex. Currently there are no licensed medicines to treat BoNT/A poisoning after internalization of the toxin by motor neurons. The development of effective therapeutic measures to counter BoNT/A intoxication has been limited, due in part to the lack of robust high-throughput assays for screening small molecule libraries. Here we describe a high content imaging (HCI) assay with utility for identification of BoNT/A inhibitors. Initial optimization efforts focused on improving the reproducibility of inter-plate results across multiple, independent experiments. Automation of immunostaining, image acquisition, and image analysis were found to increase assay consistency and minimize variability while enabling the multiparameter evaluation of experimental compounds in a murine motor neuron system.

Data from high and low controls created two distinct populations with the difference of two medians exceeding 3 standard deviations (Figure 7A). The goal of the screening process is to find the compounds within the sample population with values closer to positive control population, assuming a normal distribution within the sample population (Figure 7B, (i)). Data points that are 3 standard deviations beyond the mean are considered statistically different from the noise and classified as...

Watch this Scientific Journal Video about A High Content Imaging Assay for Identification of Botulinum Neurotoxin Inhibitors at JoVE.com

A High Content Imaging Assay for Identification of Botulinum Neurotoxin Inhibitors

Botulinum neurotoxin is one of the most potent toxins among Category-A biothreat agents, yet a post-exposure therapeutic is not available. The high content imaging approach is a powerful methodology for identifying novel inhibitors as it enables multiparameter screening using biologically relevant motor neurons, the primary target of this toxin.

Synaptosomal-associated protein-25 (SNAP-25) is a component of the soluble NSF attachment protein receptor (SNARE) complex that is essential for synaptic ...

a-high-content-imaging-assay-for-identification-botulinum-neurotoxin

Research

JoVE Journal

Neuroscience

8.5K visualizações.  Perkin Elmer Inc.. Botulinum neurotoxin is one of the most potent toxins among Category-A biothreat agents, yet a post-exposure therapeutic is not available. The high content imaging approach is a powerful methodology for identifying novel inhibitors as it enables multiparameter screening using biologically relevant motor neurons, the primary target of this toxin.