Substrate peptides and assays for detecting and measuring...

Details Substrate peptides and assays for detecting and measuring... Substrate peptides and assays for detecting and measuring...

2001-10-12

2003-01-07

Navarro, Mark (Department: 1645)

Chemistry: natural resins or derivatives; peptides or proteins;

Peptides of 3 to 100 amino acid residues

Peptides with at least one nonpeptide bond other than a...

C530S327000, C435S975000

Reexamination Certificate

active

06504006

ABSTRACT:

INTRODUCTION
The present invention generally relates to a group of fluorescent peptides for detecting the enzymatic activity of botulinum toxin type A.
Seven immunologically distinct botulinum neurotoxins, types A, B, C, D, E, F, and G are produced by the anaerobic bacterium
Clostridium botulinum
. Synthesized as a single 150 kD polypeptide chain in the bacteria, these neurotoxins are subsequently cleaved to produce two chains, a heavy chain and a light chain, which are linked by a disulfide bond. For each toxin, the 50 kD light (L) chain is a zinc-dependent protease, which cleaves a single target protein essential for synaptic vesicle membrane fusion during neurotransmission. Cleavage of the target protein inhibits neurotransmitter release among neurons, which leads to muscular paralysis [Bigalke and Shoer, (1999) In:Handbook of Experimental Pharmacology, Vol. 145 Bacterial Protein Toxins (K. Aktories and I. Just, Eds.) pp.407-443, Springer-Verlag, Berlin]. All references cited herein supra and infra are hereby incorporated in their entirety by reference thereto.
Clinically, there are three forms of this neuroparalytic disease: food borne, infant and wound botulism. As little as 30-100 ng of the neurotoxin is potentially lethal to humans. Thus, detection of these toxins requires a highly sensitive and reliable assay. In addition, botulinum neurotoxins are increasing useful clinically as an active ingredient in therapeutic agents. Again, sensitive and accurate potency assays are essential to assure toxin quantity, activity, and safety in any given therapeutic formulation. Currently the mouse bioassay is the “gold standard” for the measurement and standardization of these neurotoxins. While this in vivo assay has the prerequisite sensitivity with a detection limit of 10-20 pg neurotoxin for the most active toxin serotype, there are a number of major drawbacks. 1) The method requires the use of a large number of animals for initial detection for typing by neutralization tests and for toxin quantitation. 2) The method is costly largely due to the expense incurred for the maintenance of animal facilities. 3) The method is slow and taking up to 4 days for completion. 4) The method lacks specificity and is not reliable if the sample contains other lethal substances. 5) The method is performed by a limited number of laboratories.
Considerable efforts have been expended in the development of suitable alternative in vitro assays. Several laboratories have developed ELISA-type assays with sensitivities comparable to the mouse bioassay [Pearce et al (1997)
Toxicon
35:1373-1412; Szilagyi et al (2000)
Toxicon
38: 381-389]. However, these assays are based on immunodetection, and as such may not distinguish active from inactive toxin, a crucial limitation. Additionally, immunodetection may not reveal novel toxins constructed from the enzymatic portion of botulinum toxin and a binding domain of another protein. The discovery that all seven neurotoxin serotypes contain a zinc endoprotease and the identification of the specific protein substrates and cleavage sites recognized by each serotype, provides a potentially sensitive and direct means for monitoring the presence of toxin [Fujii et al (1992)
Toxicon
30:1486-1488; Schiavo et al (1993)
Trends Microbiol
1:170-174; Blasi et al (1993)
Nature
365:160-163]. Investigations using synthetic substrates derived from synaptosome-associated protein (SNAP-25) for botulinum toxin type A and derived from vesicle-associated membrane protein (VAMP-2) for type B, demonstrated that cleavage products could be observed using RP-HPLC [Shone et al (1993)
Eur. J. Biochem
. 217:965-971; Schmidt and Bostian (1997)
J. Prot. Chem
. 16:19-26]. This method, however, does not allow for detection of small quantities of toxin. An in vitro endopeptidase assay has been described that is as sensitive as the mouse bioassay for detection of botulinum toxin type A [Ekong et al (1997)
Microbiology
143:3337-3347]. The approach involves the use of antibodies directed to the neurotoxin-cleaved substrate. The assay is limited by the need for specific reagents and requires a multi step process consuming considerable time as is characteristic of ELISA assays.
A U.S. Pat. No. 5,965,699 (“'699 patent”) by Schmidt et al describes and claims a sensitive assay to detect cleavage of peptide substrates by botulinum toxin type A. The method uses fluorescamine to detect primary amines newly created by hydrolysis of the substrates. Using purified toxins, this method works well for tests, such as screening for potential inhibitors. However, if test samples involving food products or serum contain other sources of primary amines, such as contaminating proteins, the contaminating proteins may generate high background in the tests. The high background would hinder the detection of small quantities of toxin.
A classic approach for measuring the activity of proteolytic enzymes utilizes short, di-and tripeptide substrates which contain a chromophore or fluorophore moiety at the site of cleavage [Castillo et al (1979)
Anal. Biochem
. 99:53]. The chromophore or fluorophore is attached to the C-terminal of a short di- or tri-peptide through acylation which changes the electronic properties. Subsequent cleavage of the substrate and deacylation of the chromophore/fluorophore gives rise to a measurable spectrophotometric change proportional to the extent of hydrolysis. These assays do not require separation of the cleavage products. They are highly sensitive, generally relatively fast and simple to perform. However, because of the demanding substrate recognition requirements of the botulinum toxins, there are two formidable problems. First, if the neurotoxins require large peptide substrates for efficient cleavage, di- or tri-peptides would not be recognized by the neurotoxin. Secondly, and most importantly, hydrolysis of a given substrate only occurs for those peptides that contain appropriate amino acids spanning both sides of the cleavage site. Substitution of a chromophore in close proximity to the cleavage site, which is necessary when using this technique, would eliminate hydrolysis.
More recently, an alternative approach, based on intramolecularly quenched fluorescent substrates has been successfully utilized to characterize various peptidases [Szollosi et al (1998)
Cytometry
34:159-179]. These fluorogenic substrates contain a fluorescent group at one end and a suitable chromogenic group that can quench the fluorescence at the other. Substrates may be designed to include amino acids on either side of the enzymatically hydrolyzed bond. The fluorescent signal in the uncleaved substrate is quenched by fluorescence resonance energy transfer (FRET) between the fluorophore and quencher groups. Efficiency of quenching depends on i) the distance between the donor/acceptor groups, the shorter the distance the more efficient the quenching, and ii) the extent to which the emission spectrum of the fluorophore and the absorbance spectrum of the chromophore overlap [Yaron et al (1979)
Anal. Biochem
. 95:228-235]. The greater the spectral overlap a given donor/acceptor pair has, the more efficient the quenching of the signal will be. Full fluorescence is restored after cleavage of the peptide. Hence, enzymatic activity can be monitored continuously by recording the increase in fluorescence intensity with time.
A similar intramolecularly quenched fluorescent substrate is described and successfully used for determining botulinum toxin type B protease activity [Anne et al (2001) Anal. Biochem. 291:253-261]. The 34 amino acid substrate described is a contact quenched peptide. The mechanism of quenching is not by FRET, but is collisional. For these contact quenched substrates, the fluorophore and quencher must be in close proximity. In this substrate, they are separated by only four amino acids. Even though the modified amino acids are quite close to the cleavage site, this peptide substrate is still recogn

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