Une plate-forme à base de FRET haut-débit compatible pour l'identification et la caractérisation de la chaîne neurotoxine botulique de lumière modulateurs

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 cardiac and/or respiratory failure. Because of its extreme potency, ease of production, and long-term effects within the host, the CDC has labeled all BoNT serotypes as high-risk bioterrorism agents.

The mechanism of action of the toxin involves numerous steps, including binding to neuronal surface receptors, cellular uptake via receptor-mediated endocytosis, and translocation into the neuron cytosol. BoNT/A is comprised of two chains, a heavy and light chain, and both chains are required for toxicity. The heavy chain (HC) contains binding and translocation domains, while the light chain (LC) is a zinc-dependent metalloprotease that translocates from the endosome to the cytoplasm. Once inside the cytosol, the LC/A metalloprotease localizes to the inner cytoplasmic membrane and cleaves the membrane-bound host protein SNAP-25. SNAP-25 is a member of the SNARE (Soluble N-Ethylmaleimide-Sensitive Factor Attachment Protein Receptor) protein family, which plays a crucial role in acetylcholine exocytosis (reviewed in reference³). LC/A cleavage of SNAP-25 impairs SNARE complex function, which inhibits acetycholine neurotransmitter release and impairs muscle contraction.

Currently, treatment for botulism is limited and often includes administration of an equine neutralizing antibody⁴; however, because the toxin is rapidly internalized into neurons, the antibody has the antibody has a narrow time window of administration. Thus, many researchers believe that the BoNT/A LC may be a better therapeutic target. Because the LC is a zinc-dependent metalloprotease, one approach to inhibit LC/A activity has been to develop compounds that chelate the active-site zinc ion. For example, hydroxamate compounds chelate the active-site zinc and have excellent in vitro potency (K_i of the best BoNT/A LC small molecule inhibitor to date is 77 nM)⁵. However, many small molecules fail to advance as therapeutics due to various problems ex vivo or in vivo, including poor aqueous solubility, rapid metabolism, and/or high cytotoxicity. Therefore, new compounds with improved pharmacological and pharmacokinetic properties are needed. Small molecule compound identification often involves high-throughput screening (HTS) to identify novel scaffolds. Initial methods for BoNT/A LC activity screening were based on HPLC detection of short peptide substrate cleavage, which is time-consuming and not amenable to HTS applications^6-8. Subsequently, Schmidt and colleagues⁹ developed a high-throughput BoNT/A LC activity assay that utilizes a fluorescein-labeled peptide substrate covalently attached to a microtiter plate. The BoNT/A LC cleaves the substrate and releases fluorescein, which can be quantified with a fluorometer. The plate format of this assay allows numerous compounds to be screened simultaneously; however, the assay requires labeling synthetic peptides with fluorescein and coating the assay plates with derivatized substrate molecules, which are cumbersome techniques. A much simpler method for detecting BoNT/A LC activity at low concentrations was later described by Schmidt et al., where a series of fluorogenic substrates were utilized to monitor BoNT LC activity in real time¹⁰. Additional techniques described in the literature include a depolarization after resonance energy transfer-based assay to detect and quantify BoNT activity in crude extracts; this method can be used for high-throughput applications^10,11, although it requires sophisticated equipment to measure fluorescence resonance energy transfer (FRET) and polarization signals. Finally, several cell-based models for BoNT intoxication have been reported (reviewed in reference¹¹) that will enable researchers to study the often limiting properties of compounds previously mentioned, including cytotoxicity, cell permeability, and stability. However, most of the existing cell-based assays are not amenable to HTS, and are labor and time intensive.

Herein, we describe a detailed protocol for a HTS method that utilizes the commercially available FRET-based BoNT/A LC substrate. The substrate is based on the SNAP-25 cleavage sequence and is a synthetic 13-mer peptide that contains a terminal fluorophore and quencher. BoNT/A cleavage separates the fluorophore and quencher, abolishing FRET and increasing measured fluorescence, which can be continually measured in a fluorometer plate reader. The assay is used routinely in our, as well as other laboratories, to identify new classes of BoNT/A LC inhibitors or to determine the relative potency of previously identified compounds^5,12-15. This assay is suitable for HTS because of its simplicity, automation potential, low cost of materials, and ability to screen numerous compounds simultaneously (see reference¹⁶; Caglič et al., submitted; Bompiani et al., in preparation). In addition to HTS, this assay can be used to compare the relative potency of compounds by determining the IC₅₀ value (concentration required to inhibit 50% of BoNT/A LC activity) of a compound. The assay can either be performed manually in a 96-well format (Manual Screening section of the Protocol Text) or can be automated in a 384-well format for HTS (Automated Operation section of the Protocol Text).

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 Prepare 50 ml of assay buffer (40 mM HEPES, pH 7.4 and 0.01% Tween-20) and filter sterilize….

Representative 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 – &#956…

Materials

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