A High-throughput-compatible FRET-based Platform for Identification and Characterization of Botulinum Neurotoxin Light Chain Modulators

Summary

The botulinum neurotoxin type A light chain (BoNT/A LC) is a metalloprotease that enters motor neurons, cleaves its substrate SNAP-25, and disrupts neurotransmission, thereby resulting in flaccid paralysis. Utilizing a high-throughput-compatible FRET-based assay, large libraries of small molecules can be screened for their impact on BoNT/A LC enzymatic activity.

Abstract

Botulinum neurotoxin (BoNT) is a potent and potentially lethal bacterial toxin that binds to host motor neurons, is internalized into the cell, and cleaves intracellular proteins that are essential for neurotransmitter release. BoNT is comprised of a heavy chain (HC), which mediates host cell binding and internalization, and a light chain (LC), which cleaves intracellular host proteins essential for acetylcholine release. While therapies that inhibit toxin binding/internalization have a small time window of administration, compounds that target intracellular LC activity have a much larger time window of administrations, particularly relevant given the extremely long half-life of the toxin. In recent years, small molecules have been heavily analyzed as potential LC inhibitors based on their increased cellular permeability relative to larger therapeutics (peptides, aptamers, etc.). Lead identification often involves high-throughput screening (HTS), where large libraries of small molecules are screened based on their ability to modulate therapeutic target function. Here we describe a FRET-based assay with a commercial BoNT/A LC substrate and recombinant LC that can be automated for HTS of potential BoNT inhibitors. Moreover, we describe a manual technique that can be used for follow-up secondary screening, or for comparing the potency of several candidate compounds.

Introduction

Botulinum neurotoxin A (BoNT/A), the most potent toxin currently known (LD₅₀ ~1 ng/kg)¹, is a potent neurotoxin that is produced by the bacterium Clostridium botulinum. Within an affected host, BoNT/A disrupts neurotransmission at the neuromuscular junction by binding motor neurons, internalizing into the cytosol, and ultimately cleaving neuronal proteins that are essential for acetylcholine exocytosis. Once inside a neuron, BoNT/A can persist for as long as several months². Long-term inhibition of acetylcholine release hampers normal muscle contraction and results in flaccid paralysis, which, in severe cases, may result in ca....

Protocol

Manual Screening or IC₅₀ Determination

This protocol can be used to determine the relative potency of a compound (IC₅₀ value) by preparing a dilution series of the compound, or to manually screen for small-molecule inhibitors at a single concentration.

1. Preparation of Buffers, Reagents, and Required Instrumentation

1. Prepare 50 ml of assay buffer (40 mM HEPES, pH 7.4 and 0.01% Tween-20) and filter sterilize. The buffer can be stored at room temperature (RT) for several months.
2. Prepare a 70 nM working dilution of recombinant botulinum neurotoxin/....

Results

The FRET-based BoNT/A LC assay can be performed manually in a low-throughput manner to characterize known inhibitors or screen small libraries; alternatively, the protocol can be scaled-down and automated for high-throughput screening (HTS) with large libraries with the aim of identifying novel BoNT/A LC inhibitors. Regardless of the approach taken, an increase in fluorescence should be observed over time when BoNT/A LC is incubated with the substrate (Figure 1A shows a representative plot of fluorescenc.......

Discussion

The FRET-based BoNT/A LC assay described here represents an attractive method for identifying and characterizing small molecules that modulate BoNT/A LC activity. The solution-based nature of the assay makes this protocol amenable to high-throughput screening described in the Automated Operation section of the Protocol Text. The Z-factor is often used to determine the suitability of an assay for HTS campaigns¹⁸. Determined as Z = 1 – 3 (σ_p + σ_n)/(μ_p - μ.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
HEPES	Teknova	H1021
Tween-20	Fisher Scientific	BP337-100
Methanol (HPLC-grade)	Sigma-Aldrich	34860
Isopropanol (HPLC-grade)	Sigma-Aldrich	650447
96-well Black assay plate	Costar	3915
384-well Low-volume black assay plate	Greiner	788076
SNAPtide FITC/Dabcyl substrate	List Biological Laboratories	521	FRET-based BoNT/A LC substrate
Pin cleaning solution	V&P Scientific	VP 110
Lint-free blotting paper	V&P Scientific	VP 540DB
Biomek Seal and Sample Aluminum foil lids	Beckman Coulter	538619