Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues
Reexamination Certificate
2001-09-25
2004-07-13
Low, Christopher S. F. (Department: 1653)
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
C530S324000, C530S326000, C530S333000, C530S335000, C530S337000, C530S344000, C530S345000, C530S350000, C514S002600, C514S013800, C435S004000, C435S007100, C435S007710, C435S007720, C435S252400, C435S842000, C435S183000, C930S010000, C930S020000
Reexamination Certificate
active
06762280
ABSTRACT:
INTRODUCTION
The clostridial neurotoxins consist of tetanus toxin and the seven immunologically distinct serotypes of botulinum neurotoxin, elaborated by various strains of
Clostridium tetani
and
Clostridium botulinum
, respectively. They are among the most potent toxins known [Simpson, L. L. (1986) Ann. Rev. Pharmacol. Toxicol. 26: 427-453; Nieman, D. H. (1991) In: Sourcebook of Bacterial Protein Toxins (J. Alouf and J. Freer, Eds.) pp 303-348, Academic press, New York]. All references cited herein supra and infra are hereby incorporated in their entirety by reference thereto.
Nonetheless, these toxins have proven to be highly useful tools for research on the mechanisms of neurotransmitter release [Nieman, D. H. (1991) Trends Cell Biol. 4: 179-185; Schiavo, et al. (1994) Cell Biol. 5: 221-229], and are being used as clinical drugs in humans to treat a rapidly expanding group of muscle dysfunctions including strabismus, blepharospasm, cervical dystonia, and hemifacial spasm [Jancovic and Brin (1992) New Engl. J. Med. 324: 1186-1194; Kessler and Benecke (1997) Neurotoxicology 18(3): 761-770]. Although accidental botulinum intoxication is not considered a major public health threat, clostridial neurotoxins have long been recognized as potential biowarfare or bioterrorist agents [Arnon, S. S., et al. (2001) JAMA 285: 1059-1070].
The clostridial neurotoxins are synthesized by the bacteria as single-chain proteins of Mr~150,000, which are subsequently cleaved by endogenous proteases to yield a light chain (Mr~50,000) and a heavy chain (Mr~100,000), covalently linked to each other by a disulfide bond [Bandyopadhyay, et al. (1987) J. Biol. Chem. 262: 2660-2663]. The heavy chains contain receptor-binding and translocation domains, required for entry of neurotoxin into target cells. The light chains are zinc metalloproteases, highly specific for certain proteins involved in neurotransmitter release [Montecucco and Schiavo (1994) Mol. Microbiol. 13: 1-8]. Botulinum serotypes A and E cleave the protein SNAP-25, while tetanus toxin and botulinum serotypes B, D, F, and G cleave synaptobrevin (also called VAMP) [Pellizarri, R., et al. (1999) Philos. Trans. Royal Soc. London B. Biol. Sci. 354: 259-268]. Botulinum serotype C cleaves both syntaxin and SNAP-25 [Foran, P., et al. (1996) Biochemistry 35: 2630-2636]. Only one peptide bond is cleaved by each toxin within its substrate, but this is sufficient to inactivate the mechanism of neurotransmitter release. Toxicity is therefore a consequence of clostridial neurotoxin protease activity.
In view of the widespread applications for clostridial neurotoxins in neurological research and in medicine, and because of the possibilities for use as bioweapons, there is an urgent need for highly sensitive and reproducible assays that can be employed to detect the toxins in potentially contaminated food or environmental samples, to accurately quantify the toxins in research reagents or preparations intended for human clinical use, and in the search for anti-toxin drugs. Because botulinum neurotoxins are proteases, it follows that practical assays for this activity could form the basis for detection, quantification, and drug-screening systems. However, the development of such assays has been hampered by several factors: (1) As noted above, each botulinum neurotoxin will cleave only one peptide bond in a particular protein, raising the possibility that separate assays would be required for each toxin. (2) A considerable body of evidence has been published which indicates that the substrate recognition requirements of clostridial neurotoxin proteases are unusually large, compared to other proteases, and include discontinuous segments of their respective neuronal target proteins. Therefore, one would anticipate that only intact target proteins or very long polypeptides derived therefrom can function as substrates [for review, see Schiavo, G. et al. (1995) In: Clostridial Neurotoxins (C. Montecucco, Ed.) pp 257-274, Springer-Verlag, Berlin]. (3) The clostridial neurotoxin proteases do not hydrolyze short peptides spanning the cleavage sites, and the tertiary structures of the target proteins are critical elements in substrate recognition [Rossetto O. et al. (1994) Nature 372: 415-416; Schiavo, G. et al. (1995) supra; Washbourne, P. et al. (1997) FEBS Lett. 418: 1-5]. (4) Relatively minor changes in substrate structure, such as the replacement of only one amino acid with a similar one, even at some considerable distance from the cleavage site, can result in complete loss of substrate function [Yamasaki, S. et al. (1994) J. Biol. Chem. 269: 12764-12772; Shone and Roberts (1994) Eur. J. Biochem 225: 263-270; Schmidt and Bostian (1997) J. Prot. Chem. 16: 19-26]. Consequently, introduction of non-natural amino acids and/or bulky aromatic or fluorescent groups would be unlikely to result in a functional substrate.
Currently, the most commonly used methods for detecting botulinum toxins in food and for estimating concentrations in preparations for clinical use are the mouse lethality bioassay [Siegel and Metzger (1979) Appl. Environ. Micrbiol. 38: 606-611] and the antibody neutralization test [Siegel (1988) J. Clin. Microbiol. 26: 2351-2356]. Both require the use of animals, can take up to four days to complete, and are inherently inaccurate. Furthermore, determination of botulinum toxin concentration with the mouse bioassay cannot be used to predict pharmacological potency [Pearce, L. B. et al. (1997) Toxicon 35: 1373-1412].
Assays have been published which incorporate neurotoxin protease activity as one aspect of the overall method [Ekong, T. et al. (1997) Microbiology 143: 3337-3347; Wictome, M. et al. (1999) Appl. Environ. Microbiol. 65: 3787-3792; Keller, J. et al. (1999) J. Appl. Toxicol. 19: S13-S17]. Nonetheless, they are essentially immunoassays, because quantitation of results requires the production and use of specialized antibodies, capable of distinguishing between cleaved and uncleaved substrate, or between cleavage product and intact substrate. They have been developed only for botulinum serotypes A and B. They require multiple binding, elution, and washing steps, and are impractical for true high-throughput systems.
Other assays for the proteolytic activities of tetanus toxin and of serotypes A and B botulinum toxins have been reported [Shone, C. et al. (1993) Eur. J. Biochem. 217: 965-971; Cornille, F. et al. (1994) Eur. J. Biochem. 222: 173-181; Schmidt and Bostian (1995) J. Prot. Chem. 14: 703-708; Soleilhac, J.-M. et al. (1996) Anal. Biochem. 241: 120-127]. Although these assays could be adapted to high-throughput formats, they include high pressure liquid chromatography or solid-phase extraction steps, which add significant time, complexity, and expense to the procedures.
Recently, an assay for the proteolytic activity of type B botulinum neurotoxin has been published, which uses a fluoescence resonance energy transfer (FRET) substrate and does not require physical separation of products from reactants [Anne, C. et al. (2001) Analyt. Biochem. 291: 253-261]. The publication describes one substrate, suitable for use with botulinum serotype B only. Because of the extreme specificity of each clostridial neurotoxin for a particular peptide bond in a particular substrate, and the likelihood that structural modifications to any of the substrates will diminish or abolish cleavability (see discussion above), nothing may be inferred from these results with respect to the suitability of similar modifications to the substrates for the other clostridial neurotoxins, or to modifications of type B substrate other than those described in the publication.
An assay for the proteolytic activity of type A botulinum toxin has been described (U.S. Pat. No. 5,965,699) which can be conveniently used to quantitate, standardize, and compare different preparations of this toxin. The method is readily a
Schmidt James J.
Stafford Robert G.
Arwine Elizabeth
Kam Chih-Min
Low Christopher S. F.
The United States of America as represented by the Secretary of
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