A High Content Imaging Assay for Identification of Botulinum Neurotoxin Inhibitors

Summary

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.

Abstract

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.

Introduction

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 (....

Protocol

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.

1. Prepare 10 mM stock solutions of each library compound in 100% DMSO in polypropylene 96 well plates. Use the 10 mM stock to prepare an intermediate dilution plate that is 10-fold greater than the desired screening concentration. Prepare 100 uM intermediate plate by dispensing 10....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Botulinum neurotoxin A	Metabiologics	NA	No catalog number
Microtitre plates	Greiner	655946	Poly-D-Lysine 96-well plates
BACs antibody	Lampire Biological	NA
Microchem	National	0255
Methanol	Thermo Scientific	A412-20
Formaldehyde	Thermo Scientific	28908
Horse serum	Invitrogen	16050
PE JANUS MDT Mini Automated Workstation	Perkin Elmer	AJMDT01
Opera	Perkin Elmer	OP-QEHS-01
Triton X-100	Sigma-Aldrich	9002-93-1
BIII tubulin antibody	R&D Systems	BAM1195
Tween 20	Sigma	P1379-1L
Hoechst 33342dye	Invitrogen	3570
Antimouse IgG	Invitrogen	A21236
Anti rabbit IgG	Invitrogen	A10042
Columbus Image analysis software	Perkin Elmer	Ver 2.4
Spotfire	Perkin Elmer	Ver 5.5
Clorox bleach	Fisher Scientific	18-861-284
PlateStack