CA2462686C

Fret protease assays for clostridial toxins

Abstract

CA2462686C, drawing sheet 1 — Sheet 1 of 37

Term

Term ended

Expired 22 August 2022, 4.1 years ago.

108 claims: 40 independent, 68 dependent

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CA 02462686 2008-12-24 117 We claim:1. A clostridial toxin substrate, comprising: (a) a donor fluorophore;(b) an acceptor having an absorbance spectrum overlapping the emission spectrum of said donor fluorophore;and (c) a clostridial toxin recognition sequence comprising a clostridial P5-P4-P3· PrPrPi'-Pa'-Pa’-PW cleavage site sequence, said clostridial P5-P4-P3-P2Ρι-Ρι-Ρί'-Ρβ'-ΡΑΡδ' cleavage site sequence intervening between said donor fluorophore and said acceptor;wherein resonance energy transfer is exhibited between said donor fluorophore and said acceptor.
4
A botulinum toxin serotype A (BoNT/A) substrate, comprising:(a) a donor fluorophore;(b) an acceptor having an absorbance spectrum overlapping the emission spectrum of said donor fluorophore;and (c) a BoNT/A recognition sequence comprising a BoNT/A P5-P4-P3-P2-P1-P11· Pa-Pa'-PZ-Pe' cleavage site sequence, said BoNT/A P5-p4-P3-P2-PrPi,-p2'Ρ3,“Ρ4,·Ρ5Ι cleavage site sequence intervening between said donor fluorophore and said acceptor;wherein resonance energy transfer is exhibited between said donor fluorophore and said acceptor. CA 02462686 2008-12-24 118
9
A botulinum toxin serotype B (BoNT/B) substrate, comprising:(a) a donor fluorophore;(b) an acceptor having an absorbance spectrum overlapping the emission spectrum of said donor fluorophore, wherein the acceptor is an acceptor fluorophore;and (c) a BoNT/B recognition sequence comprising a BoNT/B Ps-PrPyPrPrPi'· Ρζ'-Ρβ-ΡΛΡδ' cleavage site sequence, said BoNT/B Ps-P^Ps-Pz-Pi-Pi'-Pz'Pa'-Pi'-Ps' cleavage site sequence intervening between said donor fluorophore and said acceptor;wherein resonance energy transfer is exhibited between said donor fluorophore and said acceptor fluorophore.
14
A botulinum toxin substrate, comprising:(a) a donor fluorophore;(b) an acceptor having an absorbance spectrum overlapping ths emission spectrum of said donor fluorophore;and (c) a BoNT/A recognition sequence comprising a BoNT/A Ps-PrPrPz-Pi-Pi'Pa'-Ps'-P^-Ps cleavage site sequence, wherein BoNT/A P-i'-Pî cleavage site sequence intervenes between said donor fluorophore and said acceptor;wherein said donor fluorophore, said acceptor, or both said donor fluorophore and said acceptor is not positioned within said BoNT/A Ps-PcPa-Pa-Pi-Pi'-Pi'-Pa'P^'-Ps' cleavage site sequence;and wherein resonance energy transfer is exhibited between said donor fluorophore and said acceptor.
21
22. A botulinum toxin serotype A (BoNT/A) substrate, comprising:(a) a donor fluorophore;(b) an acceptor having an absorbance spectrum overlapping the emission spectrum of said donor fluorophore;and (c) a BoNT/A recognition sequence comprising a BoNT/A P5-P4-P3-P2-P1-P1'P2*-P3-P4’-Ps cleavage site sequence, said BoNT/A Ps-PrPa-Pa-PrPi-Pz’P3-P4-P5' cleavage site sequence intervening between said donor fluorophore and said acceptor;wherein either of said donor fluorophoe, said acceptor, or both said donor fluorophore and said acceptor are genetically encoded;and wherein resonance energy transfer is exhibited between said donor fluorophore and said acceptor.
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30. The substrate of any one of claims 1 to 29, wherein said substrate can be cleaved with an activity of at least 1 nanomoles/minute/milligram toxin.
30
31. The substrate of any one of claims 1 to 29, wherein said substrate can be cleaved with an activity of at least 20 nanomoles/minute/milligram toxin.
31
32. The substrate of any one of claims 1 to 29, wherein said substrate can be cleaved with an activity of at least 50 nanomoles/minute/milligram toxin.
32
33. The substrate of any one of claims 1 to 29, wherein said substrate can be cleaved with an activity of at least 100 nanomoles/minute/milligram toxin. CA 02462686 2008-12-24 123
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34. The substrate of any one of claims 1 to 29, wherein said substrate can be cleaved with an activity of at least 150 nanomoles/minute/milligram toxin.
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35. The substrate of any one of claims 1 to 29, which is a peptide or peptidomimetic having at most 400 residues.
35
36. The substrate of any one of claims 1 to 29, which is a peptide or peptidomimetic having at most 300 residues.
36
37. The substrate of any one of claims 1 to 29, which is a peptide or peptidomimetic having at most 200 residues.
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38. The substrate of any one of claims 1 to 29, which is a peptide or peptidomimetic having at most 100 residues.
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39. The substrate of any one of claims 1 to 29, which is a peptide or peptidomimetic having at most 50 residues.
39
40. The substrate of any one of claims 1 to 29, which is a peptide or peptidomimetic having at most 40 residues.
40
41. The substrate of any one of claims 1 to 29, which is a peptide or peptidomimetic having at most 20 residues.
41
42. The substrate of any one of claims 1 to 29, wherein said donor fluorophore and said acceptor are separated by at most forty residues.
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43. The substrate of any one of claims 1 to 29, wherein said donor fluorophore and said acceptor are separated by at most thirty residues.
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44. The substrate of any one of ciaims 1 to 29, wherein said donor fluorophore and said acceptor are separated by at most twenty residues. CA 02462686 2009-06-09 124
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45. The substrate of any one of claims 1 to 29, wherein said donor fluorophore and said acceptor are separated by at most fifteen residues.
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46. Theisubstrate of any one of claims 1 to 29, Wherein said donor fluorophore and said acceptor are separated by at most ten residues. 47· The substrate of any one of claims 1 to 29, Wherein said donor fluorophore and said acceptor are separated by at most eight residues.
46
48. The substrate of any one of claims 1 to 29, wherein said donor fluorophore and said acceptor are separated by at most six residues.
47
49. A method for determining clostridial toxin protease activity, comprising the steps of:(a) treating a sample, under conditions suitable for clostridial toxin protease activity, with a clostridial toxin substrate comprising said clostridial toxin substrate according to any one of claims 1 to 29, (b) exciting said donor fluorophore;and (c) determining resonance energy transfer of said treated substrate relative to a control substrate, wherein a difference In resonance energy transfer of said treated substrate as compared to said control substrate Is indicative of clostridial protease activity.
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67. A clostridial toxin substrate, comprising:(a) a lanttianide donor fluorophore;(b) an acceptor having an absorbance spectrum overlapping the emission spectrum of said lanthanide donor fluorophore;and (c) a clostridial toxin recognition sequence comprising a clostridial P5-P4-P3-P2-P1-P1'Ρΐ'-Ρβ'-Ρ^-Ρδ' cleavage site sequence, said clostridial Ρε-Ρί-Ρβ-Ρΐ-Ρι-ΡΪ-Ρΐ'-Ρ^-ΡΛΡδ' cleavage site sequence intervening between said lanthanide donor fluorophore and said acceptor;wherein, under the appropriate conditions, resonance energy transfer is exhibited between said lanthanide donor fluorophore and said acceptor.
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74. The substrate of claim. 67, wherein said lanthanide donor fluorophore is a terbium, europium, dysprosium and samarium. CA 02462686 2008-12-24 128
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75. The substrate of claim- 67, comprising a botulinum toxin recognition sequence.
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78. A botulinum toxin serotype A (BoNT/A) substrate, comprising:(a) a lanthanide donor fluorophore;(b) an acceptor having an absorbance spectrum overlapping the emission spectrum of said lanthanide donor fluorophore;and (c) a BoNT/A recognition sequence comprising a BoNT/A Ρδ-ΡΑ-Ρδ^νΡι-Ρι'-Ρζ'-Ρδ'Pa-Ps' cleavage site sequence, said BoNT/A Ρδ-Ρ^-Ρΐ-Ρι-Ρι'-Ρζ'-Ρ^-ΡΛΡδ' cleavage site sequence intervening between said lanthanide donor fluorophore and said acceptor;wherein, under the appropriate conditions, resonance energy transfer is exhibited between said lanthanide donor fluorophore and said acceptor. 79· The substrate of claim 78, wherein said BoNT/A Ρ5-Ρ4-Ρ3-Ρ2-Ρ1-ΡΓ-Ρ2'-Ρ3,-Ρ4'P5' cleavage site sequence comprises at least six consecutive residues of SNAP 25, said six consecutive residues comprising Gin-Arg, or a peptidomimetic thereof. 80- The substrate of claim 79, wherein said BoNT/A P5-P4-P3-P2-P1-P1'-P2'-P3,-P4'P5' cleavage site sequence comprises at least six consecutive residues of human SNAP 25, said six consecutive residues comprising Gln197-Arg198, or a peptidomimetic thereof.
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83. A botulinum toxin serotype B (BoNT/B) substrate, comprising:(a) a lanthanide donor fluorophore;(b) an acceptor having an absorbance spectrum overlapping the emission spectrum of said donor fluorophore, wherein the acceptor is an acceptor fluorophore;and (c) a BoNT/B recognition sequence comprising a BoNT/B Ρδ-Ρ^-Ρΐ-Ρι-Ρι'-Ρζ'-ΡβPAPs' cleavage site sequence, said BoNT/B Ρδ-Ρ^-Ρζ-Ρι-Ρι'-Ρζ'-Ρ^-ΡΛΡδ' cleavage site sequence intervening between said lanthanide donor fluorophore and said acceptor;wherein, under the appropriate conditions, resonance energy transfer is exhibited between said lanthanide donor fluorophore and said acceptor fluorophore.
80
88. The substrate of any one of claims 78-87, wherein said lanthanide donor fluorophore is genetically encoded.
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89. The substrate of any one of claims 78-87, wherein said acceptor is genetically encoded.
82
90. The substrate of any one of claims 78-87, wherein said lanthanide donor fluorophore and said acceptor are genetically encoded.
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91. The substrate of any one of claims 78-87, wherein said lanthanide donor fluorophore is a lanthanide chelate. CA 02462686 2008-12-24 131
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92. The substrate of any one of claims 78-87, wherein said lanthanide donor fluorophore is a lanthanide cryptate.
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93. The substrate of any one of claims 78-87, wherein said lanthanide donor fluorophore is a terbium, europium, dysprosium and samarium.
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94. A botulinum toxin serotype A (BoNT/A) substrate, comprising:(a) a lanthanide donor fluorophore;(b) an acceptor having an absorbance spectrum overlapping the emission spectrum of said donor fluorophore;and (c) a BoNT/A recognition sequence comprising a BoNT/A Ρ5-Ρ4-Ρ3-Ρ2-ΡγΡι'-Ρ2,-Ρ3ιΡΛΡδ' cleavage site sequence, said BoNT/A Ρδ-Ρ^Ρβ-ΡΣ-Ρι-Ρι'-Ρΐ'-Ρ^-Ρ^-Ρβ' cleavage site sequence intervening between said lanthanide donor fluorophore and said acceptor;wherein said lanthanide donor fluorophore, said acceptor, or both said lanthanide donor fluorophore and said acceptor is not positioned within said BoNT/A P5-P4-P3-P2Pi-Pi’-Pz'-Ps'-Pa-Ps' cleavage site sequence;and wherein, under the appropriate conditions, resonance energy transfer is exhibited between said lanthanide donor fluorophore and said acceptor. 95- The method of claim 94, wherein said lanthanide donor fluorophore is not positioned within said BoNT/A P5-P4-P3-P2-P1-P1'-P2'-P3'-P4'-P5' cleavage site sequence.
93
102. The substrate of any one of claims 67-101, wherein said substrate can be cleaved with an activity of at least 1 nanomoles/minute/milligram toxin, at least 20 nanomoles/minute/milligram toxin, at least 50 nanomoles/minute/milligram toxin, at least 100 nanomoles/minute/milligram toxin, or at least 150 nanomoles/minute/milligram toxin.
94
103. The substrate of any one of claims 67-101, which is a peptide or peptidomimetic having at most 400 residues, at most 300 residues, at most 200 residues, at most 100 residues, at most 50 residues, at most 40 residues, or at most 20 residues.
95
104. The substrate of any one of claims 67-101, wherein said lanthanide donor fluorophore and said acceptor are separated by at most forty residues, at most thirty residues, at most twenty residues, at most fifteen residues, at most ten residues, at most eight residues, or at most six residues.
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105. A method of determining clostridial toxin protease activity, comprising the steps of:(a) treating a sample, under conditions suitable for clostridial toxin protease activity, with a clostridial toxin substrate comprising, said Clostridial toxin substrate according to any one of Claims 1-39;(b) exciting said donor fluorophore;and (c) determining resonance energy transfer of said treated substrate relative to a control substrate, CA 02462686 2008-12-24 133 wherein a difference in resonance energy transfer of said treated substrate as compared to said control substrate is indicative of clostridial toxin protease activity.

Independent claims40

2,145 paragraphs in 317 sections, as filed

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FRET PROTEASE ASSAYS FOR CLOSTRIDIAL TOXINS BACKGROUND OF THE INVENTION

FIELD OF THE INVENTION

The present invention relates generally to fluorescence resonance energy transfer and protease assays, for example, assays for protease activity of clostridial toxins such botulinum toxins and tetanus toxins, and more specifically, to intramolecularly quenched substrates and methods for assaying for clostridial toxin protease activity.

BACKGROUND INFORMATION

The neuroparalytic syndrome of tetanus and the rare but potentially fatal disease, botulism, are caused by neurotoxins produced by bacteria of the genus Clostridium. These clostridial neurotoxins are highly potent and specific poisons of neural cells, with the human lethal dose of the botulinum toxins on the order of micrograms. Thus, the presence of even minute levels of botulinum toxins in foodstuffs represents a public health hazard that must be avoided through rigorous testing.

However, in spite of their potentially deleterious effects, low controlled doses of botulinum neurotoxins have been successfully used as therapeutics. These toxins have been used in the therapeutic management of a variety of focal and segmental dystonias, of strabismus and other conditions in which a reversible depression of a cholinergic nerve terminal activity is desired. Established therapeutic uses of botulinum neurotoxins in humans include, for example,

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PCT/US2002/027212 blepharospasm, hemifacial spasm, laringeal dysphonia, focal hyperhidrosis, hypersalivation, oromandibular dystonia, cervical dystonia, torticollis, strabismus, limbs dystonia, occupational cramps and myokymia (Rossetto et al, Toxicon 39:27-41 (2001)). Intramuscular injection of spastic tissue with small quantities of BoNT/A, for example, has been used effectively to treat spasticity due to brain injury, spinal cord injury, stroke, multiple sclerosis and cerebral palsy.

Additional possible clinical uses of clostridial neurotoxins currently are being investigated.

Given the potential danger associated with small quantities of botulinum toxins in foodstuffs and the need to prepare accurate pharmaceutical formulations, assays for botulinum neurotoxins presently are employed in both the food and pharmaceutical industry. The food industry requires assays for the botulinum neurotoxins to validate new food packaging methods and to ensure food safety. The growing clinical use of the botulinum toxins necessitates accurate assays for botulinum neurotoxin activity for product formulation as well as quality control. In both industries, a mouse lethality test currently is used to assay for botulinum neurotoxin activity. Unfortunately, this assay suffers from several drawbacks : cost due to the large numbers of laboratory animals required; lack of specificity; and the potential for inaccuracy unless large animal groups are used.

Thus, there is a need for new materials and methods for assaying for clostridial toxin activity. The present invention satisfies this need and provides related advantages as well.

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Those skilled in the art of fluorescence resonance energytransfer understand that a variety of donor fluorophore-acceptor combinations are useful in a BoNT/F substrate of the invention, including, as not limiting examples, fluorescein- tetramethylrhodamine;

DABCYL-EDANS; and Alexa Fluor® 488-QSY® 7.

The present invention also provides a botulinum toxin serotype G (BoNT/G) substrate containing a donor fluorophore; an acceptor having an absorbance spectrum overlapping the emission spectrum of the donor fluorophore; and a BoNT/G recognition sequence that includes a cleavage site, where the cleavage site intervenes between the donor fluorophore and the acceptor and where, under the appropriate conditions, resonance energy transfer is exhibited between the donor fluorophore and the acceptor. A BoNT/G substrate can have, for example, at least six consecutive residues of VAMP, the six consecutive residues including Ala-Ala, or a peptidomimetic thereof. Such a BoNT/<3 substrate can have, for example, at least six consecutive residues of human VAMP, the six consecutive residues including Ala83-Ala84, or a peptidomimetic thereof. In one embodiment, a BoNT/G substrate contains the amino acid sequence Glu-Thr-Ser-Ala-Ala-Lys-Leu-Lys (SEQ ID NO: 10), or a peptidomimetic thereof. As discussed above in regard to other clostridial toxin substrates, a variety of donor fluorophore-acceptor combinations are useful in a BoNT/G substrate of the invention.. Such donor fluorophore-acceptor combinations include, for example, fluorescein-tetramethylrhodamine; DABCYL-EDANS; and Alexa Fluor®488-QSY® 7.

Also provided by the invention is a tetanus toxin (TeNT) substrate containing a donor fluorophore; an

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PCT/US2002/027212 acceptor having an absorbance spectrum overlapping the emission spectrum of the donor fluorophore; and a TeNT recognition sequence that includes a cleavage site, where the cleavage site intervenes between the donor fluorophore and the acceptor and where, under the appropriate conditions, resonance energy transfer is exhibited between the donor fluorophore and the acceptor. A TeNT substrate of the invention can have, for example, at least six consecutive residues of VAMP, the six consecutive residues include Gln-Phe, or a peptidomimetic thereof. For example, such a TeNT substrate can have at least six consecutive residues of human VAMP-2, the six consecutive residues including Gln76-Phe77, or a peptidomimetic thereof. In one embodiment, a TeNT substrate contains the amino acid sequence Gly-Ala-SerGln-Phe-Glu-Thr-Ser (SEQ ID NO: 11), or a peptidomimetic thereof. In another embodiment, the TeNT substrate contains residues 33 to 94 of human VAMP-2 (SEQ ID NO: 4); residues 25 to 93 of human VAMP-2 (SEQ ID NO: 4); or residues 27 to 116 of rat VAMP-2 (SEQ ID NO: 7), or a peptidomimetic of one of these sequences. A variety of donor fluorophore-acceptor combinations are useful in a TeNT substrate of the invention, including, without limitation, fluorescein-tetramethylrhodamine; DABCYL-EDANS ; and Alexa Fluor® 488-QSY® 7.

In specific embodiments, the invention provides a BoNT/A, BoNT/B, BoNT/Cl, BoNT/D, BoNT/E, BoNT/F, BoNT/G or TeNT substrate that is cleaved with an activity of at least 1 nanomoles/minute/milligram toxin. In other embodiments, a BoNT/A, BoNT/B, BoNT/Cl, BoNT/D, BoNT/E, BoNT/F, BoNT/G or TeNT substrate of the invention is cleaved with an activity of at least 10 nanomoles/minute/ milligram toxin. In further embodiments, a BoNT/A, BoNT/B, BoNT/Cl, BoNT/D, BoNT/E, BoNT/F, BoNT/G or TeNT

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PCT/US2002/027212 substrate of the invention is cleaved with an activity of at least 20 nanomoles/minute/milligram toxin. In yet other embodiments, a BoNT/A, BoNT/B, BoNT/Cl, BoNT/D, BoNT/E, BoNT/F, BoNT/G or TeNT substrate of the invention is cleaved with an activity of at least 50, 100 or 150 nanomoles/minute/milligram toxin.

A variety of donor fluorophores and acceptors, including fluorescent and non-fluorescent acceptors, are useful in the clostridial toxin substrates of the invention. Donor fluorophores useful in the invention include, but are not limited to, fluorescein, Alexa Fluor® 488, DABCYL, and BODIPY. Acceptors useful in the invention include, but are not limited to, tetramethylrhodamine, EDANS and QSY® 7. Exemplary donor fluorophore-acceptor pairs useful in a clostridial toxin substrate of the invention include, without limitation, fluorescein-tetramethylrhodamine, Alexa Fluor® 488tetramethylrhodamine, DABCYL-EDANS, fluorescein-QSY® 7, and Alexa Fluor® 488-QSY® 7.

Clostridial toxin substrates of the invention encompass peptides and peptidomimetics of a variety of lengths and in which the donor fluorophore and acceptor are separated by different numbers of residues. In particular embodiments, a clostridial toxin substrate of the invention is a peptide or peptidomimetic having at most 20 residues, at most 40 residues, at most 50 residues, or at most 100 residues. In other embodiments, the donor fluorophore and the acceptor are separated by at most six residues, at most eight residues, at most ten residues or at most fifteen residues.

Further provided by the invention is a method of determining clostridial toxin protease activity. The

PCT/US2002/027212

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows a schematic of the deduced structure and postulated mechanism of activation of clostridial neurotoxins. Toxins can be produced as an inactive single polypeptide chain of 150 kDa, composed of three 50 kDa domains connected by loops. Selective proteolytic cleavage activates the toxins by generating two disulfide-linked chains: the L chain of 50 kDa and the H chain of 100 kDa, which is made up of two domains denoted HN and Hc. The three domains play distinct roles: the C-terminal domain of the heavy chain (Hc) functions in cell binding while the N-terminal domain of the heavy chain (HN) permits translocation from endosome to cell cytoplasm. Following reduction of the disulfide linkage inside the cell, the zinc-endopeptidase activity of the L chain is liberated.

Figure 2 shows a schematic of the four steps required for tetanus and botulinum toxin activity in central and peripheral neurons.

Figure 3 shows the subcellular localization at the plasma membrane and sites of cleavage of SNAP-25, VAMP and syntaxin. VAMP is bound to synaptic vesicle membrane, whereas SNAP-25 and syntaxin are bound to the target plasma membrane. BoNT/A and /E cleave SNAP-25 close to the carboxy-terminus, releasing nine or 26 residues, respectively. BoNT/B, /D, /F, /G and TeNT act on the conserved central portion of VAMP (dotted) and release the amino-terminal portion of VAMP into the cytosol. BoNT/Cl cleaves SNAP-25 close to the carboxy-terminus as well as cleaving syntaxin at a single site near the cytosolic membrane surface. The action of BoNT/B, /Cl, /D, /F, /G and TeNT results in release of a

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PCT/US2002/027212 large portion of the cytosolic domain of VAMP or syntaxin, while only a small portion of SNAP-25 is released by selective proteolysis by BoNT/A, /Cl or /E.

Figure 4 shows the neurotoxin recognition motif of VAMP, SNAP-25 and syntaxin. (A) Hatched boxes indicate the presence and positions of a motif common to the three targets of clostridial neurotoxins. (B) The recognition motif is composed of hydrophobic residues (h); negatively charged Asp or Glu residues (-) and polar residues (p); x represents' any amino acid. The motif is included in regions of VAMP, SNAP-25 and syntaxin predicted to adopt an α-helical conformation. (C) A top view of the motif in an α-helical conformation is shown. Negatively charged residues align on one face, while hydrophobic residues align on a second face.

Figure 5 shows an alignment of various SNAP-25 proteins and their BoNT/E, BoNT/A and BoNT/Cl cleavage sites. Human SNAP-25 (SEQ ID NO: 2; GenBank accession g4507099; see, also, related human SNAP-25 sequence g2135800); mouse SNAP-25 (SEQ ID NO: 12; GenBank accession G6755588); Drosophila SNAP-25 (SEQ ID NO: 13; GenBank accession g548941); goldfish SNAP-25 (SEQ ID NO: 14; GenBank accession g2133923); sea urchin SNAP-25 (SEQ ID NO: 15; GenBank accession g2707818) and chicken SNAP-25 (SEQ ID NO: 16; GenBank accession g481202) are depicted.

Figure 6 shows an alignment of various VAMP proteins and their BoNT/F, BoNT/D, BoNT/B, TeNT and

BoNT/G cleavage sites. Human VAMP-1 (SEQ GenBank accession

GenBank accession

GenBank accession gl35093); human VAMP-2 gl35094); mouse VAMP-2 g2501081); bovine VAMP

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GenBank accession g89782); frog VAMP (SEQ ID NO: 19; GenBank accession g6094391); and sea urchin VAMP (SEQ ID NO: 20; GenBank accession g5031415) are depicted.

Figure 7 shows an alignment of various syntaxin proteins and their BoNT/Cl cleavage sites. Human syntaxin 1A (SEQ ID NO: 21; GenBank accession gl5079184), human syntaxin 1B2 (SEQ ID NO: 22; GenBank accession gl5072437), mouse syntaxin 1A (SEQ ID NO: 23; GenBank accession gl5011853), Drosophila syntaxin 1A (SEQ ID NO: 24; GenBank accession g2501095); C. elegans syntaxin A (SEQ ID NO: 25; GenBank accession g7511662) and sea urchin syntaxin (SEQ ID NO: 26; GenBank accession gl3310402) are depicted.

PCI7US2002/027212 translocation. The L chain is responsible for the intracellular catalytic activity (Montecucco and Schiavo, supra, 1995).

The term donor fluorophore-acceptor pair, as used herein, means a donor fluorophore and an acceptor that has an absorbance spectrum overlapping the emission spectrum of the donor fluorophore. Where the first member of the pair is a donor fluorophore, the second member of the pair will be an acceptor. Where the first member of the pair is an acceptor, the second member of the pair will be a donor fluorophore.

In one embodiment, the first member of the donor fluorophore-acceptor pair is a donor fluorophore, and the second member is an acceptor. In another embodiment, the first member of the donor fluorophore-acceptor pair is an acceptor, and the second member is a donor fluorophore. A variety of donor fluorophores and acceptors are useful in the composite clostridial toxin substrates of the invention, including the donor fluorophores and acceptors described herein.

In one embodiment, the donor fluorophore is a lanthanide. Lanthanide donor fluorophores useful in a composite substrate of the invention include, without limitation, terbium, europium, dysprosium and samarium.

The term affinity couple, as used herein, means two molecules that are capable of forming a stable, non-covalent association. Affinity couples useful in a composite substrate of the invention include, without limitation, streptavidin-biotin; S peptide-S protein; histidine tag-nickel chelate; antibody-antigen, for example, FLAG and anti-FLAG antibody; and receptor-ligand.

In one embodiment, the affinity couple is streptavidin-biotin. In a further embodiment, the first partner of the affinity couple is streptavidin, and the

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PCT/US2002/027212 second partner is biotin. In another embodiment, the first partner of the affinity couple is biotin, and the second partner is streptavidin. In yet further embodiments, the affinity couple is streptavidin-biotin, and the donor fluorophore is terbium, europium, dysprosium or samarium.

Clostridial toxins have specific and distinct cleavage sites. BoNT/A cleaves a Gin-Arg bond; BoNT/B and TeNT cleaves a Gln-Phe bond; BoNT/Cl cleaves a Lys-Ala or Arg-Ala bond; BoNT/D cleaves a Lys-Leu bond; BoNT/E cleaves an Arg-Ile bond; BoNT/F cleaves a Gln-Lys bond; and BoNT/G cleaves an Ala-Ala bond (see Table 1). The scissile bond can be represented P^P/, and it is understood that a Px or Px ' site, or both, can be substituted with another amino acid or amino acid mimetic in place of the naturally occurring residue. For example, BoNT/A substrates have been prepared in which the Px position (Gin) is modified to be an alanine, 2-aminobutyric acid or asparagine residue; these substrates were hydrolyzed by BoNT/A at the Px-Arg bond (Schmidt and Bostian, J. Protein Chem. 16:19-26 (1997)). However, it is recognized that substitutions can be introduced at the Px position of the scissile bond, for example, a BoNT/A scissile bond, while conservation of the Px ' residue is more often important for detectable proteolysis (Vaidyanathan et al., J. Neurochem. 72:327337 (1999)). Thus, in one embodiment, the invention provides a clostridial toxin substrate in which the Px' residue is not modified or substituted relative to the naturally occurring residue in a target protein cleaved by the clostridial toxin. In another embodiment, the invention provides a clostridial toxin substrate in which the Px residue is modified or substituted relative to the

I naturally occurring residue in a target protein cleaved

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WO 2004/031773 by the clostridial toxin; such a substrate retains susceptibility to peptide bond cleavage between the Px and Pi' residues.

<td> 5</td><td colspan="3"> Table 1 Bond cleaved in human VAMP-2, SNAP-25 or syntaxin</td>

<td></td><td> Toxin</td><td> Target</td><td> P4-P3-P2-Px -- Ρ1'-Ρ2·-Ρ3·-Ρ4·</td>

<td> 10</td><td> BoNT/Cl</td><td> syntaxin</td><td> Asp-Thr-Lys-Lys—Ala*-Val-Lys-Tyr SEQ ID NO: 5</td>

* Scissile bond shown in bold

SNAP-25, VAMP and syntaxin share a short motif located within regions predicted to adopt an a-helical

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PCT/US2002/027212 conformation (see Figure 4). This motif is present in SNAP-25, VAMP and syntaxin isoforms expressed in animals sensitive to the neurotoxins. In contrast, Drosophila and yeast homologs that are resistant to these neurotoxins and syntaxin isoforms not involved in exocytosis contain sequence variations in the a-helical motif regions of these VAMP and syntaxin proteins.

Multiple repetitions of the a-helical motif are present in proteins sensitive to cleavage by clostridial toxins: four copies are naturally present in SNAP-25;

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PCT/US2002/027212 clostridial toxins: BoNT/A, BoNT/B, BoNT/Cl, BoNT/D, BoNT/E, BoNT/F, BoNT/G and TeNT.

It is understood that a clostridial toxin substrate of the invention can be cleaved at a reduced or enhanced rate relative to SNAP-25, VAMP or syntaxin or relative to a similar peptide or peptidomimetic that does not contain extrinsic fluorophores. A clostridial toxin substrate of the invention such as a BoNT/A, BoNT/B, BoNT/Cl, BoNT/D, BoNT/E, BoNT/F, BoNT/G or TeNT substrate, can be cleaved, for example, with an initial hydrolysis rate that is at least 5% of the initial hydrolysis rate, under otherwise identical conditions, of human SNAP-25, VAMP or syntaxin, where the clostridial toxin substrate and SNAP-25, VAMP or syntaxin each is present at a concentration of 1.0 mM.

Thus, a BoNT/A, BoNT/Cl or BoNT/E substrate of the invention can be cleaved, for example, with an initial hydrolysis rate that is at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, or 300% of the initial hydrolysis rate, under otherwise identical conditions, of human SNAP-25 by BoNT/A, BoNT/Cl or BoNT/E, respectively, where the substrate of the invention and human SNAP-25 each is present at a concentration of 1.0 mM. In other embodiments, a BoNT/A, BoNT/Cl or BoNT/E substrate of the invention is with an initial hydrolysis rate that is at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, or 300% of the initial hydrolysis rate, under otherwise identical conditions, of human SNAP-25 by BoNT/A, BoNT/Cl or BoNT/E, respectively, where the substrate of the invention and human SNAP-25 each is present at a concentration of 50 mM.

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Similarly, a BoNT/B, BoNT/D, BoNT/F or BoNT/G substrate of the invention can be cleaved, for example, with an initial hydrolysis rate that is at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, or 300% of the initial hydrolysis rate, under otherwise identical conditions, of human VAMP-2 by BoNT/B, BoNT/D, BoNT/F or BoNT/G, respectively, where substrate of the invention and human VAMP-2 each is present at a concentration of 1.0 mM. In other embodiments, a BoNT/B, BoNT/D, BoNT/F or BoNT/G substrate of the invention is cleaved with an initial hydrolysis rate that is at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, or 300% of the initial hydrolysis rate, under otherwise identical conditions, of human VAMP-2 by BoNT/B, BoNT/D, BoNT/F or BoNT/G, respectively, where substrate of the invention and human VAMP-2 each is present at a concentration of 50 mM.

The invention also provides a BoNT/Cl substrate of the invention that is cleaved with an initial hydrolysis rate that is at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, or 300% of the initial hydrolysis rate, under otherwise identical conditions, of human syntaxin by BoNT/Cl, where the BoNT/Cl substrate and human syntaxin each is present at a concentration of 1.0 mM. In other embodiments, the invention provides a BoNT/Cl substrate that is cleaved with an initial hydrolysis rate that is at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, or 300% of the initial hydrolysis rate, under otherwise identical conditions, of human syntaxin by BoNT/Cl, where the BoNT/Cl substrate and human syntaxin each is present at a concentration of 50 mM.

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As used herein, the term botulinum toxin serotype A recognition sequence is synonymous with BoNT/A recognition sequence and means a scissile bond together with adjacent or non-adjacent recognition elements sufficient for detectable proteolysis at the scissile bond by a BoNT/A under conditions suitable for clostridial toxin protease activity. A scissile bond cleaved by BoNT/A can be, for example, Gin-Ala.

A variety of BoNT/A recognition sequences are well known in the art. A BoNT/A recognition sequence can have, for example, residues 134 to 206 or residues 137 to 206 of human SNAP-25 (Ekong et al., supra, 1997; U.S. Patent No. 5,962,637). A BoNT/A recognition sequence also can include, without limitation, the sequence ThrArg-Ile-Asp-Glu-Ala-Asn-Gln-Arg-Ala-Thr-Lys-Met (SEQ ID NO: 27), or a peptidomimetic thereof, which corresponds to residues 190 to 202 of human SNAP-25; Ser-Asn-Lys-ThrArg-Ile-Asp-Glu-Ala-Asn-Gln-Arg-Ala-Thr-Lys (SEQ ID NO: 28), or a peptidomimetic thereof, which corresponds to residues 187 to 201 of human SNAP-25; Ser-Asn-Lys-ThrArg-Ile-Asp-Glu-Ala-Asn-Gln-Arg-Ala-Thr-Lys-Met (SEQ ID NO: 29), or a peptidomimetic thereof, which corresponds to residues 187 to 202 of human SNAP-25; Ser-Asn-Lys-ThrArg-Ile-Asp-Glu-Ala-Asn-Gln-Arg-Ala-Thr-Lys-Met-Leu (SEQ ID NO: 30), or a peptidomimetic thereof, which corresponds to residues 187 to 203 of human SNAP-25; Asp-Ser-Asn-Lys-Thr-Arg-Ile-Asp-Glu-Ala-Asn-Gln-Arg-AlaThr-Lys-Met (SEQ ID NO: 31), or a peptidomimetic thereof, which corresponds to residues 186 to 202 of human SNAP-25; or Asp-Ser-Asn-Lys-Thr-Arg-Ile-Asp-Glu-Ala-AsnCA 02462696 2004-02-26

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Gln-Arg-Ala-Thr-Lys-Met-Leu (SEQ ID NO: 32), or a peptidomimetic thereof, which corresponds to residues 186 to 203 of human SNAP-25. See, for example, Schmidt and Bostian, J. Protein Chem. 14:703-708 (1995); Schmidt and Bostian, supra, 1997; Schmidt et al., FEBS Letters 435:61-64 (1998); and Schmidt and Bostian, U.S. Patent No. 5,965,699). If desired, a similar BoNT/A recognition sequence can be prepared from a corresponding (homologous) segment of another BoNT/A-sensitive SNAP-25 isoform or homolog such as, for example, murine, rat, goldfish or zebrafish SNAP-25 or can l?e any of the peptides disclosed herein or described in the art, for example, in U.S. Patent No. 5,965,699.

A BoNT/A recognition sequence can correspond to a segment of a protein that is sensitive to cleavage by botulinum toxin serotype A, or can be substantially similar to a segment of a BoNT/A-sensitive protein. As illustrated in Table 2, a variety of naturally occurring proteins sensitive to cleavage by BoNT/A are known in the art and include, for example, human, mouse and rat SNAP-25; and goldfish SNAP-25A and SNAP-25B. Thus, a BoNT/A recognition sequence useful in a BoNT/A substrate of the invention can correspond, for example, to a segment of human SNAP-25, mouse SNAP-25, rat SNAP-25, goldfish SNAP-25A or 25B, or another naturally occurring protein sensitive to cleavage by BoNT/A. Furthermore, comparison of native SNAP-25 amino acid sequences cleaved by BoNT/A reveals that such sequences are not absolutely conserved (see Table 2 and Figure 5), indicating that a variety of amino acid substitutions and modifications relative to a naturally occurring BoNT/A-sensitive SNAP-25 sequence can be tolerated in a BoNT/A substrate of the invention.

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TABLE 2

Cleavage of SNAP-25 and related proteins3'

H H U

0) (0 0 « P Λ

H

O ·· Μ O cq S ra φ P ri CQ

Ü1 «J t> nJ

Φ H o

O

H tn φ •ri ft

CQ

d -H tn Ci fl

-Φ ΓΗ

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Q

H

Oi ·. Μ O

W ¢5

TABLE 2

Cleavage of SNAP-25 and related proteinsa,b,c'd ra

4) 4J rl m

0) > nJ <D ι—I o

44 ra H

ra 0) H

O <U nJ rd •rd Λ a

O ra O

Q

4=1

O

O cm Λ jj d ι -H a M a M ra o tu < o ra u >

0 0 4)

ο Λ u Ό iu

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A clostridial toxin substrate of the invention, such as a BoNT/A substrate, can have one or multiple modifications as compared to a naturally occurring sequence that is cleaved by the corresponding clostridial toxin. For example, as compared to a 17-mer corresponding to residues 187 to 203 of human SNAP-25, substitution of Aspl93 with Asn in the BoNT/A substrate resulted in a relative rate of proteolysis of 0.23; substitution of Glul94 with Gin resulted in a relative rate of 2.08; substitution of Alal95 with 2-aminobutyric acid resulted in a relative rate of 0.38; and substitution of Glnl97 with Asn, 2-aminobutyric acid or Ala resulted in a relative rate of 0.66, 0.25, or 0.19, respectively (see Table 3). Furthermore, substitution of Alal99 with 2aminobutyric acid resulted in a relative rate of 0.79; substitution of Thr200 with Ser or 2-aminobutyric acid resulted in a relative rate of 0.26 or 1.20, respectively; substitution of Lys201 with Ala resulted in a relative rate of 0.12; and substitution of Met202 with Ala or norleucine resulted in a relative rate of 0.38 or 1.20, respectively. See Schmidt and Bostian, supra, 1997.

These results indicate that a variety of residues can be substituted in a clostridial toxin substrate of the invention as compared to a naturally occurring toxinsensitive sequence. In the case of BoNT/A, these results indicate that residues including but not limited to G1U194, Alal95, Glnl97, Alal99, Thr200 and Met202, Leu203, Gly204, Ser205, and Gly206, as well as residues more distal from the Gin-Arg scissile bond can be substituted or can be conjugated to a donor fluorophore or acceptor to produce a BoNT/A substrate of the invention. Such a BoNT/A substrate is detectably proteolyzed at the scissile bond by BoNT/A under conditions suitable for clostridial toxin protease activity. Thus, a BoNT/A substrate of the invention can include, if desired, one or several amino

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TABLE 3

Kinetic parameters of BoNT/A synthetic peptide substrates

Peptide Sequence* SEQ ID NO: Relative

Rateb

<td> [1-15]</td><td> SNKTRIDEANQRATK</td><td> 28</td><td> 0.03</td>

<td> [1-16]</td><td> SNKTRIDEANQRATKM</td><td> 29</td><td> 1.17</td>

<td> [1-17]</td><td> SNKTRIDEANQRATKML</td><td> 30</td><td> 1.00</td>

<td> M16A</td><td> SNKTRIDEANQRATKAL</td><td> 44</td><td> 0.38</td>

<td> M16X</td><td> SNKTRIDEANQRATKXL</td><td> 45</td><td> 1.20</td>

<td> K15A</td><td> SNKTRIDEANQRATAML</td><td> 46</td><td> 0.12</td>

<td> T14S</td><td> SNKTRIDEANQRASKML</td><td> 47</td><td> 0.26</td>

<td> T14B</td><td> SNKTRIDEANQRABKML</td><td> 48</td><td> 1.20</td>

<td> A13B</td><td> SNKTRIDEANQRBTKML</td><td> 49</td><td> 0.79</td>

<td> QUA</td><td> SNKTRIDEANARATKML</td><td> 50</td><td> 0.19</td>

<td> Q11B</td><td> SNKTRIDEANBRATKML</td><td> 51</td><td> 0.25</td>

<td> QI IN</td><td> SNKTRIDEANNRATKML</td><td> 52</td><td> 0.66</td>

<td> N10A</td><td> SNKTRIDEAAQRATKML</td><td> 53</td><td> 0.06</td>

<td> A9B</td><td> SNKTRIDEBNQRATKML</td><td> 54</td><td> 0.38</td>

<td> E8Q</td><td> SNKTRIDQANQRATKML</td><td> 55</td><td> 2.08</td>

<td> D7N</td><td> SNKTRINEANQRATKML</td><td> 56</td><td> 0.23</td>

<td></td><td> Nonstandard amino</td><td colspan="2"> acid abbreviations are:</td>

<td></td><td> B, 2-aminobutyric (norleucine)</td><td> acid;</td><td> X, 2-aminohexanoic acid</td>

PCT/US2002/027212

I <44 o fl) tn rd > <0

0) r4 U

Q) s β

cd fl) 4J H co fl) tn (0 ί> <0 fl) r-4 u <w o cd H tn

0) H

Ü a)

m E-·

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TABLE 4 Cleavage of VAMP*

ta Φ •ri

CQ

O ta H

0) Φ Η

Ο Φ a w

η Η

ca Λ 4-1

co fl φ

Φ I—I

η ο φ φ ι—I

Sequence corrected in position 68 (t>s).

II II

Φ XI

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A variety of naturally occurring SNAP-25 proteins also are sensitive to cleavage by BoNT/Cl, including human, mouse and rat SNAP-25; goldfish SNAP-25A and 25B; and Drosophila and leech SNAP-25. Thus, a

BoNT/Cl recognition sequence useful in a BoNT/Cl substrate of the invention can correspond, for example, to a segment of human, mouse or rat SNAP-25, goldfish SNAP-25A or 25B, Torpedo SNAP-25, zebrafish SNAP-25, Drosophila SNAP-25, leech SNAP-25, or another naturally occurring protein sensitive to cleavage by BoNT/Cl. As discussed above in regard to variants of naturally occurring syntaxin sequences, comparison of native SNAP25 amino acid sequences cleaved by BoNT/Cl reveals significant sequence variability (see Table 2 and Figure

5 above), indicating that a variety of amino acid substitutions and modifications relative to a naturally occurring BoNT/Cl-sensitive SNAP-25 sequence can be tolerated in a BoNT/Cl substrate of the invention.

The present invention further provides a botulinum toxin serotype D (BoNT/D) substrate containing a donor fluorophore; an acceptor having an absorbance spectrum overlapping the emission spectrum of the donor fluorophore; and a BoNT/D recognition sequence that includes a cleavage site, where the cleavage site intervenes between the donor fluorophore and the acceptor and where, under the appropriate conditions, resonance energy transfer is exhibited between the donor fluorophore and the acceptor. A BoNT/D substrate of the invention can have, for example, at least six consecutive residues of VAMP, the six consecutive residues including Lys-Leu, or a peptidomimetic thereof. In one embodiment, a BoNT/D substrate contains at least six consecutive residues of human VAMP, the six consecutive residues including Lys59-Leu60, or a peptidomimetic thereof. In

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PCT/US2002/027212 another embodiment, a BoNT/D substrate of the invention contains the amino acid sequence Arg-Asp-Gln-Lys-Leu-SerGlu-Leu (SEQ ID NO: 6), or a peptidomimetic thereof. In a further embodiment, a BoNT/D substrate of the invention includes residues 27 to 116 of rat VAMP-2 (SEQ ID NO: 7), or a peptidomimetic thereof. It is understood that a variety of donor fluorophore-acceptor combinations are useful in a BoNT/D substrate of the invention; such donor fluorophore-acceptor pairs include, but are not limited to, fluorescein-tetramethylrhodamine; DABCYL-EDANS; and Alexa Fluor® 488-QSY® 7. Additional exemplary donor fluorophores and acceptors useful in a BoNT/D substrate of the invention are provided herein below.

The Forster radius (Ro) is the distance at which resonance energy transfer is 50% efficient, that is, 50% of excited donor fluorophores are deactivated by FRET. The magnitude of the Forster radius depends on the quantum yield of the donor fluorophore; the extinction coefficient of the acceptor; and the overlap between the donor fluorophore's emission spectrum and the acceptor's excitation spectrum.

Ro = [8.8 x 1023 · K2 · n’4 · QYD · J (λ)]1/6 Â where k2 = dipole orientation factor (range 0 to 4; k2 = 2/3 for randomly oriented donors and acceptors)

QYd = fluorescence quantum yield of the donor in the absence of the acceptor n = refractive index

J (λ) = spectral overlap integral = |θΑ(λ) · FD(À) · λ4άλ cm3M'1 where θΑ = extinction coefficient of acceptor

Fd = fluorescence emission intensity of donor as a fraction of the total integrated intensity

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PCT/US2002/027212 (Forster, Ann. Physik 2:55-75 (1948)).

Typical Forster radius values for various donor fluorophore/acceptor pairs are given in Table 6 below (see, also, Wu and Brand, Analytical Biochem. 218:1-13 (1994), which is incorporated herein by reference). Comprehensive lists of Forster radii also are known in the art (see, for example, Berlman, Energy Transfer Parameters of Aromatic Compounds Academic Press, New York 1973). Furthermore, those skilled in the art recognize that component factors of the Forster radius (Ro) are dependent upon the environment such that the actual value observed can vary from the listed value.

Any of a number of donor fluorophores and acceptors in various combinations can be useful in a clostridial toxin substrate of the present invention. A donor fluorophore generally is selected such that there is substantial spectral overlap between the emission spectrum of the donor fluorophore overlaps with the excitation spectrum of the acceptor. In addition, a donor fluorophore can be selected, for example, to have an excitation maximum near a laser frequency such as Helium-Cadmium 442 nm or argon 488 nm, whereby laser light serves as an effective means to excite the donor fluorophore. In one embodiment, the wavelength maximum of the emission spectrum of the acceptor moiety is at least 10 nm greater than the wavelength maximum of the excitation spectrum of the donor fluorophore. In a further embodiment, the acceptor is a fluorophore having an emission spectrum in the red portion of the visible spectrum. In an additional embodiment, the acceptor is a fluorophore having an emission spectrum in the infrared region of the spectrum. A variety of donor fluorophoreacceptor pairs, and their Forster radii, are provided

CA 02462686 2007-06-05 herein in Tables 6 and 7. See, also, Haugland, Handbook of Fluorescent Probes and Research Chemicals 6th Edition, Molecular Probes, Inc., Eugene, Oregon, 1996.

TABLE 6

<td> Donor fluorophore</td><td> Acceptor</td><td> Ro (Â)</td><td> Reference</td>

<td> Fluorescein</td><td> TMR</td><td> 49-54</td><td> Johnson et al., Biochemistry 32:6402 6410 (1993) ; Odom et al., Biochemistry 23:5069-5076 (1984)</td>

<td> Fluorescein</td><td> QSY® 7</td><td> 61</td><td> —</td>

<td> EDANS</td><td> DABCYL</td><td> 33</td><td></td>

<td rowspan="2"> Napthalene</td><td rowspan="2"> Dansyl</td><td> 22</td><td> Haas et al.. Proc . Natl. Acad. Sci. USA</td>

<td> IANBD</td><td> DDPM</td><td> 25</td><td> Kasprzyk et al., Biochemistry 22:1877 1882 (1983)</td>

<td> IAEDANS</td><td> DDPM</td><td> 25-29</td><td> Dalbey et al., Biochemistry 22:4696 4706 (1983); Cheung < al.. Bioohvs. Chem. 40:1-17 (1991)</td>

<td> DNSM</td><td> LY</td><td> 26-32</td><td> Nalin et al., Biochemistry 28:2318 2324 (1985)</td>

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<td colspan="4"> TABLE 6</td>

<td> Donor fluorophore</td><td> Acceptor</td><td> Ro (Â)</td><td> Reference</td>

<td> IAEDANS</td><td> IANBD</td><td> 27-51</td><td> Franzen et al., Biochemistry 19:60806089 (1980); First et</td>

<td> e-A</td><td> F2DNB</td><td> 29</td><td> al.. Biochemistry 28:3606-3613 (1989) Perkins et al., J. Biol. Chem. 259:8786-</td>

<td> Pyrene</td><td> Bimane</td><td> 30</td><td> 8793 (1984) Borochov-Neori and Montai, Biochemistry</td>

<td> ANAI</td><td> IPM</td><td> 30</td><td> 28:1711-1718 (1989) Peerce and Wright, Proc. Natl. Acad. Sci.</td>

<td> 5 IAANS</td><td> IAF</td><td> 31</td><td> USA 83:8092-8096 (1986) Grossman, Biochim. Biophvs. Acta</td>

<td> e-A</td><td> F2DPS</td><td> 31</td><td> 1040:276-280 (1990) Perkins et al., supra,</td>

<td> e-A</td><td> DDPM</td><td> . 31</td><td> 1984 Miki and Mihashi, Biochim. Biophys. Acta</td>

<td> IAEDANS</td><td> TNP</td><td> 31-40</td><td> 533:163-172 (1978) Takashi et al., Biochemistry 21:56615668 (1982); dos</td>

Remedios and Cooke,

Biochim. Biophys. Acta 788:193-205 (1984)

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<td colspan="4"> TABLE 6</td>

<td> Donor fluorophore</td><td> Acceptor</td><td> Ro (A)</td><td> Reference</td>

<td> MNA</td><td> DACM</td><td> 32</td><td> Amir and Haas, Biochemistry 26:2162-</td>

<td> PM</td><td> NBD</td><td> 32</td><td> 2175 (1987) Snyder and Hammes, Biochemistry 24:2324-</td>

<td> FITC</td><td> TNP-ATP</td><td> 32</td><td> 2331 (1985) Amler et al., Bioohvs.</td>

<td> DANZ</td><td> DABM</td><td> 34</td><td> J. 61:553-568 (1992) Albaugh and Steiner, J. Phvs. Chem.</td>

<td> 5 NCP</td><td> CPM</td><td> 34</td><td> 93:8013-8016 (1989) Mitra and Hammes, Biochemistry 28:3063-</td>

<td> NAA</td><td> DNP</td><td> 33-37</td><td> 3069 (1989) McWherter et al., Biochemistry 25:1951-</td>

<td> LY</td><td> TNP-ATP</td><td> 35</td><td> 1963 (1986) Nalin, supra, 1985</td>

<td> IAF</td><td> diI-C18</td><td> 35</td><td> Shahrokh et al., iL. Biol. Chem. 266:12082-</td>

<td> IAF</td><td> TMR</td><td> 37</td><td> 12089 (1991) Taylor et al., J. Cell Biol. 89:362-367</td>

<td> 10 FMA</td><td> FMA</td><td> 37</td><td> (1981) Dissing et al., Biochim. Bioohvs. Acta</td>

<td> PM</td><td> DMAMS</td><td> 38</td><td> 553:66-83 (1979) Lin and Dowben, J. Biol. Chem. 258:5142-</td>

5150 (1983)

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<td colspan="4"> TABLE 6</td>

<td> Donor fluorophore</td><td> Acceptor</td><td> Ro (Â)</td><td> Reference</td>

<td> mBBR</td><td> FITC</td><td> 38</td><td> Tompa and Batke, Biochem. Int. 20:487-</td>

<td> mBBR</td><td> DABM</td><td> 38</td><td> 494 (1990) Kasprzak et al., Biochemistry 27:4512-</td>

<td> eA</td><td> NBD</td><td> 38</td><td> 4523 (1988) Miki and Iio. Biochim. Bioohvs. Acta 790:201-</td>

<td> Pyrene</td><td> Coumarin</td><td> 39</td><td> 207 (1984) Borochov-Neori and</td>

<td> 5 IPM</td><td> FNAI</td><td> 39</td><td> Montai, supra, 1989 Peerce and Wright,</td>

<td> IAEDANS</td><td> DABM</td><td> 40</td><td> supra, 1986 Tao et al. Biochemistry 22:3059-</td>

<td> IAEDANS</td><td> TNP-ATP</td><td> 40</td><td> 3066 (1983) Tao et al., supra,</td>

<td> e-A</td><td> IANBD</td><td> 40</td><td> 1983 Miki and Wahl, Biochim. Bioohvs. Acta</td>

<td> NBD</td><td> SRH</td><td> 40-74</td><td> 786:188-196 (1984) Wolf et al., Biochemistry 31:2865-</td>

<td> 10 ISA</td><td> TNP</td><td> 42</td><td> 2873 (1992) Jacobson and Colman, Biochemistry 23:3789-</td>

<td> Dansyl</td><td> ODR</td><td> 43</td><td> 3799 (1984) Lu et al., J. Biol. Chem. 264:12956-12962</td>

(1989)

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<td colspan="4"> TABLE 6</td>

<td> Donor fluorophore</td><td> Acceptor</td><td> Ro (Â)</td><td> Reference</td>

<td> DANZ</td><td> IAF</td><td> 44-49</td><td> Cheung et al., Biochemistrv 21:5135-</td>

<td> FNAI</td><td> EITC</td><td> 45</td><td> 5142 (1983) Peerce and Wright,</td>

<td> NBD</td><td> LRH</td><td> 45-70</td><td> supra, 1986 Wolf et al., supra,</td>

<td> IAF</td><td> EIA</td><td> 46</td><td> 1992 Taylor et al., supra,</td>

<td> FITC</td><td> ENAI</td><td> 46</td><td> 1981 Peerce and Wright,</td>

<td> Proflavin</td><td> ETSC</td><td> 46</td><td> supra, 1986 Robbins et al., Biochemistrv 20:5301-</td>

<td> CPM</td><td> TNP-ATP</td><td> 46</td><td> 5309 (1981) Snyder and Hammes,</td>

<td> IAEDANS</td><td> IAF</td><td> 46-56</td><td> supra, 1985 Franzen, supra, 1985;</td>

<td> CPM</td><td> Fluorescein</td><td> 47</td><td> Grossman, supra, 1990 Thielen et al., Biochemistrv 23:6668-</td>

<td> IAEDANS</td><td> FITC</td><td> 49</td><td> 6674 (1984) Jona et al., Biochim. Biobhvs. Acta 1028:183-199 (1990);</td>

Birmachu et al. ,

Biochemistry 28:39403947 (1989)

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TABLE 6

Donor

Acceptor fluorophore R°

Reference (Â)

<td></td><td></td><td></td><td> Shahrokh et al., J.</td>

<td> CF</td><td> TR</td><td> 51</td><td> Johnson et al., supra, 1993</td>

<td> CPM</td><td> TRS</td><td> 51</td><td> Odom et al., supra, 1984</td>

<td> e-A</td><td> TNP-ATP</td><td> 51</td><td> dos Remedios and Cooke, supra, 1984</td>

<td> CPM</td><td> FM</td><td> 52</td><td> Odom et al., supra, 1984</td>

<td> LY</td><td> EM</td><td> 53</td><td> Shapiro et al., J. Biol. Chem. 266:17276- 17285 (1991)</td>

<td> FITC</td><td> EITC</td><td> 54</td><td> Carraway et al., J. Biol. Chem. 264:8699- 8707 (1989)</td>

<td> IAEDANS</td><td> Di0-C14</td><td> 57</td><td> Shahrokh et al., supra, 1991</td>

<td> IAF</td><td> ErlTC</td><td> 58</td><td> Amler et al., supra, 1992 Kosk-Kosicka et al.,</td>

<td> FITC</td><td> ETSC</td><td> 61-64</td><td> Robbins et al., supra, 1981</td>

<td> FITC</td><td> ErlTC</td><td> 62</td><td> Amler et al., supra, 1992</td>

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TABLE 6

Donor Ro

Acceptor Reference fluorophore (A)

Ozinskas et al., Anal.

<td> BPE</td><td> CY5</td><td> 72</td><td> Biochem. 213:264-270 (1993)</td>

<td> Fluorescein</td><td> Fluorescein</td><td> 44</td><td> —</td>

<td> BODIBY FL</td><td> BODIPY FL</td><td> 57</td><td> — — — -</td>

ANAI, 2-anthracence N-acetylimidazole;

BPE, B-phycoerythrin;

CF, carboxyfluorescein succinimidyl ester;

CPM, 7-diethylamino-3-(4'-maleimidylphenyl)4-methylcoumarin;

CY5, carboxymethyl indocyanine-Ν’hydroxy succinimidyl ester;

diI-C18, 1,1' -dioctadecyl-3-3,3,3 ' , 3 ' tetramethyl-indocarbocyanine;

diO-C14, 3,3' -ditetradecyloxacarbocyanine;

DABM, 4-dimethylaminophenylazo-phenyl-4'maleimide;

DACM, (7-(dimethylamino)coumarin-4-yl)-acetyl ;

DANZ, dansylaziridine; DDPM, N- (4dimethylamino-3,5-dinitrophenyl)maleimide;

DMAMS, dimethylamino-4-maleimidostilbene;

DSMN, N-(2,5'-dimethoxystiben-4-yl)-maleimide;

DNP, 2,4-dinitrophneyl;

e-A, l,N6-ethenoadenosine;

EIA, 5-(iodoacetetamido)eosin;

EITC, eosin-5-isothiocyanate;

ENAI, eosin N-acETYLIMIDAZOLE;

EM, eosin maleimide;

ErlTC, erythrosin-5'-isothiocyanate;

ETSC, eosin thiosemicarazide;

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FjDNB, 1,5-difluro-2,41-dinitrobenzene;

F2DPS, 4,4'-difluoro-3,3'-dinitrophenylsulfone;

FITC, fluorescein thiosemicarbazide;

IAANS, 2-(41-iodoacetamido)anilino)napthalene6-sulfonic acid;

IAEDANS, 5-(2-((iodoacetyl)amino)ethyl)amino)napthlene-1-sulfonic acid;

IAF, 5-iodoacetamidofluorescein;

IANBD, N- ( (2- (iodoacetoxy) ethyl) -Ν'methyl) amino-7-nitrobenz-2-oxa-l,3diazole;

IPM, 3(4-isothiocyanatophenyl)7-diethyl-4amino-4-methylcoumarin;

ISA, 4-(iodoacetamido)salicylic acid;

LRH, 1i s saminerhodamine ;

LY, Lucifer yellow;

mBBR, monobromob i amane;

MNA, (2-methoxy-1-naphthyl)-methyl;

NAA, 2-napthoxyacetic acid;

NBD, 7-nirto-2,1,3-benzoxadiazol-4-yl;

NCP, N-cyclohexyl-Ν'-(1-pyrenyl)carbodiimide;

ODR, octadecylrhodamine;

PM, N- (1-pyrene)-maleimide;

SRH, sulforhodamine;

TMR, tetramethylrhodamine;

TNP, trinitrophenyl; and

TR, Texas Red

An aromatic amino acid such as tryptophan or tyrosine also can be a donor fluorophore useful in a clostridial toxin substrate of the invention. Exemplary donor fluorophore-acceptor pairs in which tryptophan or tyrosine is the donor fluorophore and relevant Forster distances are shown in Table 7 below. Modified amino acids also can be useful as donor fluorophores or

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WO 2004/031773 acceptors in a clostridial toxin substrate of the invention. Such fluorescent or quenching modified amino acids are known in the art and include, for example, the fluorescent amino acid L-pyrenylalanine (Pya) and the non-fluorescent acceptor p-nitrophenylalanine (Nop), as described, for example, in Anne et al., Analytical Biochem. 291:253-261 (2001) .

TABLE 7

Forster Distances Using Trp as a Donor

Donor Acceptor Ro (Â) Reference

<td> Trp</td><td> Ru(III) (NH3)5</td><td> 12-16</td><td> Recchia et al.. Biochim.</td>

<td> Trp</td><td> Nitrobenzoyl</td><td> 16</td><td> Bioohvs. Acta 702:105-111 (1982) Wiczk et al., J. Fluo</td>

<td> Trp</td><td> Dansyl</td><td> 21</td><td> 1:273-286 (1991) Steinberg. Annu. Rev.</td>

<td> Trp</td><td> IAEDANS</td><td> 22</td><td> Biochem. 40:83-114 (1971) Matsumoto and Hammes,</td>

<td> 15 Trp</td><td> ANS</td><td> 23</td><td> Biochemistry 14:214-224 (1975) Conrad and Brand,</td>

<td> Trp</td><td> Anthroyloxy</td><td> 24</td><td> Biochemistry 7:777-787 (1968) Wiczk et al., supra, 1991</td>

<td> Trp</td><td> TNB</td><td> 24</td><td> Wu and Brand, Biochemistry</td>

<td> Trp</td><td> Anthroyl</td><td> 25</td><td> 31:7939-7947 (1992) Burqun et al.. Arch.</td>

<td> Trp</td><td> Tyr-NO2</td><td> 26</td><td> Biochem. Biochvs. 286:394- 401 (1991) Steiner et al., J. Fluo.</td>

1:15-22 (1991)

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TABLE 7

Forster Distances Using Trp as a Donor

Donor Acceptor Ro (Â) Reference

<td> Trp</td><td> Pyrene</td><td> 28</td>

Vekshin, Mol. Biol. 17:827832 (1983)

Ladokhin et al., Proc. SPIE 1640:562-569 (1992)

Wiczk et al., supra, 1991

PCT/US2002/027212

It is understood that donor-acceptor pairs having well-separated emission maxima can be useful in the substrates and methods of the invention;

well-separated emission maxima allow altered acceptor emission to be detected without donor emission contamination. A donor fluorophore, or acceptor, or both, can emit, for example, in the far-red, for example, greater than 650 nm. Such far-red emitting donor fluorophores and acceptors include cyanine dyes such as Cy5, Cy5.5 and Cy7 (Selvin, supra, 2000). In one embodiment, the invention provides a clostridial toxin substrate containing Cy3 and Cy5 as the donor fluorophore-acceptor pair; Cy3 emits maximally as 570 nm and Cy5 emits maximally at 670 nm. Such cyanine dyes can be prepared by straightforward synthesis, as described, for example, in Gruber et al., Bioconj. Chem. 11:161-166 (2000).

A donor fluorophore useful in a clostridial toxin substrate of the invention also can be, for example, a lanthanide atom, also known as a rare-earth element. Lanthanides such as terbium (Tb), europium (Eu), dysprosium (Dy) and samarium (Sm) have sharply spiked wavelengths, millisecond lifetimes following an excitation pulse, are unpolarized, and have high quantum yields. A lanthanide donor fluorophore suçh as a terbium or europium chelate can be combined with a variety of acceptors including organic dye acceptor. A Eu-chelate donor fluorophore can be combined, for example, with allophycocyanin (APC), and a Tb-chelate donor fluorophore can be combined, for example, with tetramethylrhodamine. Background fluorescence due to direct excitation is eliminated temporally; the lifetimes of organic acceptors generally are in the nanosecond range, while the sensitized emission follows the lifetime of the donor

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PCT/US2002/027212 fluorophore and is on the order of microseconds to milliseconds (see Selvin, supra, 2000) . Thus, determination of resonance energy transfer can be initiated relatively late following excitation, after non-specific interfering fluorescence has faded away. Lanthanide chelates are well known in the art and are commercially available, for example, from EG&G® Wallac (Turku, Finland).

A donor fluorophore useful in the invention also can be the well known fluorophore (7-methoxycoumarin-4-yl)acetyl (Mca), which can be combined with an acceptor such as the quencher 2,4-dinitrophenyl (Dnp). See, for example, Kakiuchi et al., J. Virol. Methods 80:77-84 (1999). When Mca is combined with the appropriate quencher such as Dnp in a clostridial toxin substrate of the invention, increased donor emissiqn fluorescence from Mca (XEm 393 nm) is detected upon cleavage at the clostridial toxin cleavage site and is indicative of toxin protease activity.

A donor fluorophore useful in a clostridial toxin substrate of the invention also can be, for example, a 2-aminobenzoyl (Abz) group, which can be combined, if desired, with a quencher such as

2,4-dinitrophenyl (Dnp). In an intact clostridial toxin substrate, the Dnp group quenches, by resonance energy transfer, the fluorescence of the Abz group; proteolytic cleavage of the substrate relieves quenching and results in an increase in fluorescence proportional to the concentration of the released Abz fragment. A clostridial toxin substrate containing, for example, Abz at the amino-terminus and a Dnp-dérivâtized residue such as lysine can be prepared by routine methods as

Methods for modifying proteins, peptides and peptidomimetics to contain a donor fluorophore or acceptor are well known in the art (Fairclough and Cantor, Methods Enzvmol. 48:347-379 (1978); Glaser et al., Chemical Modification of Proteins Elsevier Biochemical Press, Amsterdam (1975); Haugland, Excited States of Biopolymers (Steiner Ed.) pp. 29-58, Plenum Press, New York (1983); Means and Feeney, Bioconjugate Chem. 1:2-12 (1990); Matthews et al., Methods Enzvmol. 208:468-496 (1991); Lundblad, Chemical Reagents for Protein Modification 2nd Ed., CRC Press, Boca Ratan, Florida (1991); Haugland, supra, 1996). A variety of groups can be used to couple a donor fluorophore or acceptor, for example, to a peptide or peptidomimetic

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PCT/US2002/027212 containing a clostridial toxin recognition sequence. A thiol group, for example, can be used to couple a donor fluorophore or acceptor to the desired position in a peptide or peptidomimetic to produce a clostridial toxin substrate of the invention. Haloacetyl and maleimide labeling reagents also can be used to couple donor fluorophores or acceptors in preparing a substrate of the invention (see, for example, Wu and Brand, supra, 1994.

Donor fluorophores and acceptors including proteins such as GFP and allophycocyanin (APC) can be attached to a clostridial toxin recognition sequence by a variety of means. A donor fluorophore or acceptor can be attached by chemical means via a cross-linker moiety. Cross-linkers are well known in the art, including homoor hetero-bifunctional cross-linkers such as BMH and SPDP. Where the donor fluorophore or acceptor is a protein, well known chemical methods for specifically linking molecules to the amino- or carboxy-terminus of a protein can be employed. See, for example, Chemical Approaches to Protein Engineering in Protein Engineering- A Practical Approach Rees et al. (Eds) Oxford University Press, 1992.

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103 embodiment, the conditions include DTT in the range of 0.01 mM to 50 mM; in other embodiments, the conditions include DTT in the range of 0.1 mM to 20 mM, 1 to 20 mM, or 5 to 10 mM. If desired, an isolated clostridial toxin or sample can be pre-incubated with a reducing agent, for example, with 10 mM dithiothreitol (DTT) for about 30 minutes prior to addition of clostridial toxin substrate.

Clostridial toxins are zinc metalloproteases, and a source of zinc, such as zinc chloride or zinc acetate, typically in the range of 1 to 500 μΜ, for example, 5 to 10 μΜ can be included, if desired, as part of the conditions suitable for clostridial toxin protease activity. One skilled in the art understands that zinc chelators such as EDTA generally are excluded from a buffer for assaying clostridial toxin protease activity.

Conditions suitable for clostridial toxin protease activity also can include, if desired, bovine serum albumin (BSA). When included, BSA typically is provided in the range of 0.1 mg/ml to 10 mg/ml. In one embodiment, BSA is included at a concentration of 1 mg/ml. See, for example, Schmidt and Bostian, supra, 1997.

The amount of clostridial toxin substrate can be varied in a method of the invention. Peptide substrate concentrations useful in a method of the invention include concentrations, for example, in the range of 5 μΜ to 3.0 mM. A peptide substrate can be supplied at a concentration, for example, of 5 μΜ to

500 μΜ, 5 μΜ to 50 μΜ, 50 μΜ to 3.0 mM, 0.5 mM to 3.0 mM, 0.5 mM to 2.0 mM, or 0.5 mM to 1.0 mM. The skilled artisan understands that the concentration of clostridial toxin substrate or the amount of sample can be limited,

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104 if desired, such that the assay is linear. At increasingly high concentrations of substrate or toxin, linearity of the assay is lost due to the inner filter effect, which involves intermolecular energy transfer. Thus, in one embodiment, a method of the invention relies on a clostridial toxin substrate concentration which is limited such that intermolecular quenching does not occur. In another embodiment, a method of the invention relies on a clostridial toxin substrate concentration of less than 100 μΜ. In further embodiments, a method of the invention relies on a clostridial toxin substrate concentration of less than 50 μΜ or less than 25 μΜ. If desired, a linear assay also can be performed by mixing clostridial toxin substrate with corresponding, unlabeled substrate which lacks the donor fluorophore and acceptor of the clostridial toxin substrate. The appropriate dilution can be determined, for example, by preparing serial dilutions of clostridial toxin substrate in the corresponding unlabeled substrate.

The concentration of purified or partially purified clostridial toxin assayed in a method of the invention generally is in the range of about 0.0001 to 5000 ng/ml toxin, for example, about 0.001 to 5000 ng/ml, 0.01 to 5000 ng/ml, 0.1 to 5000 ng/ml, 1 to 5000 ng/ml, or 10 to 5000 ng/ml toxin, which can be, for example, purified recombinant light chain or dichain toxin or formulated clostridial toxin product containing human serum albumin and excipients. Generally, the amount of purified toxin used in a method of the invention is in the range of 0.1 pg to 10 pg. It is understood that purified, partially purified or crude samples can be diluted to within a convenient range for assaying for clostridial toxin protease activity against a standard curve. Similarly, one skilled in the art understands

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105 that a sample can be diluted, if desired, such that the assay for toxin protease activity is linear.

EXAMPLE I

ANALYSIS OF BoNT/A ACTIVITY USING FLUORESCENCE RESONANCE ENERGY TRANSFER

This example describes the use of a FRET assay to analyze proteolytic activity of a botulinum toxin.

The FRET substrate Xl-Asp-Ser-Asn-Lys-Thr-ArgIle-Asp-Glu-Ala-Asn-Gln-Arg-Ala-Thr-Lys-Met-Leu-Z2-NH2 (SEQ ID NO: 85) was synthesized by Alpha Diagnostics International (San Antonio, TX). This substrate contains a recognition sequence for BoNT/A flanked by a fluorescein-modified lysine residue (XI) and a tetramethylrhodamine-modified lysine residue (Z2) followed by a carboxy-terminal amide. Following proteolysis by botulinum toxin serotype A, the cleavage products Xl-Asp-Ser-Asn-Lys-Thr-Arg-Ile-Asp-Glu-Ala-AsnCA 02462686 2004-02-26

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115

Gin (SEQ ID NO: 86) and Arg-Ala-Thr-Lys-Met-Leu-Z2-NH2 (SEQ ID NO: 87) are produced.

Additional FRET substrates also are synthesized: Xl-Asp-Ser-Asn-Lys-Thr-Arg-Ile-Asp-Glu-Ala5 Asn-Gln-Arg-Ala-Thr-Lys-Met-Leu-Gly-Ser-Gly-Z2-NH2 (SEQ ID NO: 88); Xl-Ala-Asp-Ser-Asn-Lys-Thr-Arg-Ile-Asp-Glu-AlaAsn-Gln-Arg-Ala-Thr-Lys-Met-Leu-Z2~NH2 (SEQ ID NO: 89) ; Xl-Ala-Asp-Ser-Asn-Lys-Thr-Arg-Ile-Asp-Glu-Ala-Asn-GlnArg-Ala-Thr-Lys-Met-Leu-Gly-Ser-Gly-Z2-NH2 (SEQ ID

NO: 90); Xl-Thr-Arg-Ile-Asp-Glu-Ala-Asn-Gln-Arg-Ala-ThrLys-Met-Leu-Z2-NH2 (SEQ ID NO: 91); Xl-Thr-Arg-Ile-AspGlu-Ala-Asn-Gln-Arg-Ala-Thr-Lys-Met-Leu-Gly-Ser-Gly-Z2-NH2 (SEQ ID NO: 92); Xl-Met-Glu-Lys-Thr-Arg-Ile-Asp-Glu-AlaAsn-Gln-Arg-Ala-Thr-Lys-Met-Leu-Gly-Ser-Gly-Z2-NH2 (SEQ ID

NO: 93), in each of which XI is a fluorescein-modified lysine residue and Z2 is a tetramethylrhodamine-modified lysine residue; X3-Thr-Arg-Ile-Asp-Glu-Ala-Asn-Gln-ArgAla-Thr-Lys-Met-Leu-Z4-NH2 (SEQ ID NO: 94) , in which X3 is a DABCYL modified lysine residue and Z4 is a EDANS modified glutamate residue; and X3-Thr-Arg-Ile-Asp-GluAla-Asn-Gln-Arg-Ala-Thr-Lys-Met-Leu-Gly-Ser-Gly-ZS-NHa (SEQ ID NO: 95), in which X3 is a DABCYL modified lysine residue and Z5 is a EDANS modified lysine residue.

Purified BoNT/A light chain (LC/A) or cellular extract containing LC/A is diluted in assay buffer (0.05 M HEPES (pH 7.4); 1% FBS ; 10 μΜ ZnCl2; and 10 mM DTT). Dichain BoNT/A is incubated with 10 mM dithiothreitol (DTT) for about 30 minutes prior to analysis. Reactions contain various concentrations of

LC/A, dichain toxin or formulated BOTOX® product, from

0.1 ng to 10 pg. Toxin is assayed as follows: FRET substrate is added to a final concentration of 10 μΜ in a final volume of 100 pL assay buffer. The reaction is

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116 incubated at 37°C for 30 minutes, and is subsequently terminated by addition of 50 pL 2M H2SO4.

Fluorescence is measured in a fluorimeter microplate reader (Molecular Devices SPECTRA^* GEMINI XS) with Xex = 488 nM, = 520 nM and Xem = 585 nm. A reduction of at least about 5% in the Àem = 585 nm is indicative of BoNT/A protease activity. An increase of about 5% in the Aem = 520 nm also is indicative of BoNT/A protease activity of the dichain or light chain botulinum 10 toxin.

Kinetic assays are performed as follows.

Several reactions containing the same amount of LC/A or dichain toxin are initiated in the buffer and under the conditions described above. Different reactions are then 15 stopped at two or five minute intervals, and fluorescence detected as described above.

These results demonstrate that botulinum toxin proteolytic activity can be assayed with an intramolecularly quenched FRET substrate.

Although the invention has been described with reference to the examples provided above, it should be understood that various modifications can be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the claims.

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SEQUENCE LISTING <110> Allergan Incorporated

Steward, Lance E.

Fernandez-Salas, Ester

Aoki, Kei Roger <120> Fret Protease Assays For clostridial

Toxi ns <130> 17451(BOT) <150> US 09/942,098 <151> 2001-08-28 <160> 96 <170> FastSEQ for windows version 4.0 <210> 1 <211> 8 <212> PRT <213> Artificial Sequence <220>

<223> synthetic construct <400> 1

Glu Ala Asn Gin Arg Ala Thr Lys

5 <210> 2 <211> 206 <212> PRT <213> Homo sapiens <400> 2

116-1

CA 02462686 2004-05-14

<td> <210> <211> <212> <213></td><td> 3 8 PRT Artificial</td><td> sequence</td>

<td> <220> <223></td><td> synthetic</td><td> construct</td>

<400> 3

Gly Ala Ser Gin Phe Glu Thr ser

5 <210> 4 <211> 116 <212> PRT <213> Homo sapiens <400> 4

<td> <213></td><td> Artificial</td><td> Sequence</td>

<td> <223></td><td> synthetic</td><td> construct</td>

Asp Thr Lys Lys Ala val Lys Trp

<td> <210> <211> <212> <213></td><td> 6 8 PRT Artifi ci al</td><td> Sequence</td>

<td> <22O> <223></td><td> syntheti c</td><td> construct</td>

Arg Asp Gin Lys Leu Ser Glu Leu 1 5

116-2

CA 02462686 2004-05-14 <210> 7 <211> 206 <212> PRT <213> Rattus sp.

<400> 7

<210> 8 <211> 8 <212> PRT <213> Artificial Sequence <220>

<223> synthetic construct <400> 8

Gin lie Asp Arg lie Met Glu Lys

5 <210> 9 <211> 8 <212> PRT <213> Artificial Sequence <220>

<223> synthetic construct <400> 9

Glu Arg Asp Gin Lys Leu Ser Glu

5 <210> 10 <211> 8 <212> PRT <213> Artificial Sequence

116-3

CA 02462686 2004-05-14 <22Ο>

<223> synthetic construct <400> 10

Glu Thr Ser Ala Ala Lys Leu Lys

5 <210> 11 <211> 8 <212> PRT <213> Artificial sequence <220>

<223> synthetic construct <400> 11

Gly Ala Ser Gin Phe Glu Thr Ser

5 <210> 12 <211> 206 <212> PRT <213> Mus musculus <400> 12

<210> 13 <211> 212 <212> PRT <213> Drosophila sp.

<400> 13

116-4

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210 <210> 14 <211> 203 <212> PRT <213> Carassius auratus <400> 14

<210> 15 <211> 212 <212> PRT <213> Strongylocentrotus purpuratus <400> 15

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40 45

210 <210> 16 <211> 249 <212> PRT <213> Gallus gall us <400> 16

<210> 17 <211> 116 <212> PRT <213> Mus musculus

116-6

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<210> 18 <211> 116 <212> PRT <213> Bos taurus <400> 18

<210> 19 <211> 114 <212> PRT <213> Xenopus Taevis <400> 19

Ser Thr

116-7 <210> 20

CA 02462686 2004-05-14 <211> 104 <212> PRT <213> strongylocentrotus purpuratus <400> 20

<210> 21 <211> 288 <212> PRT <213> Homo sapiens <400> 21

<210> 22 <211> 288 <212> PRT <213> Homo sapiens

116-8

CA 02462686 2004-05-14 <400> 22

<210> 23 <211> 288 <212> PRT <213> Mus musculus <400> 23

116-9

CA 02462686 2004-05-14

<210> 24 <211> 291 <212> PRT <213> Drosophila sp.

<400> 24

<210> 25 <211> 291 <212> PRT <213> Caenorhabditis elegans <400> 25

Met Thr Lys Asp Arg Leu Ser Ala Leu

Lys Ala Ala Gin

116-10

Ser Glu Asp

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Glu Gin

Glu Glu

Ile Ala

Ser Asn

Met Ala

Leu île

Asp Leu

Val Glu

130

Arg Cys

145

Val Gly val Phe Leu Al a Ser île

210

Glu Ser

225

Al a Lys Gin Tyr Gly val Lys Val

290

ASp Asp

Phe Phe

Asn Asn Pro val val île Glu Asn

100

Arg Ile

115 val Met

Lys Gly

Asp Glu

Thr Gin

180

Asp île

195

Arg Glu

Gin Gly

Glu Phe

Gin Ser

260 île Leu

275

Leu

Asp

Glu val

Asn

Lys 85 Ala

Arg

Thr

Arg

Asp

165

Gly

Glu

Leu

Glu val

245

Lys île

Met Hi s

Gin val

Glu Glu

Asp Gin

Arg Al a

Ile Asp

Lys Thr

Asp Tyr

135 Ile Gin 150 Leu Glu

Ile Ile

Al a Arg

Hi s Asp

215

Met val

230

Asp Arg

Al a Arg

Thr Gly

Met Asp

Glu Glu val Lys Lys Thr Al a Asn Hi s Asp

105

Gin Hi s

120

Asn Lys

Arg Gin Glu Met

Thr Asp

185 Hi s Asn

200

Met Phe

Asp Arg

Al a Val

Arg Lys

265

Leu île

280

Thr Gly

Ile Arg

Lys Lys

Lys Glu

Lys val

Glu Gin

Ser Thr

Thr Gin

Leu Asp

155 île Glu

170

Thr Gin

Asp Ile

Met Asp île Glu

235

Ala Asp

250

Lys Ile île Phe

Asn Ala

Gly Ser

His Ser 60 Glu Leu

Arg Gly

Gly Ala

Leu Ser

125 Thr Asp 140 île Ala

Ser Gly

Gin Ala

Met Lys

205

Met Ala

220

Tyr Asn

Thr Lys

Cys île île Leu

285

Gin val

Ala

Asp

Lys

Gly

110

Arg

Tyr

Gly

Asn

Lys

190

Leu

Met val

Lys

Leu

270

Phe

Tyr Met

Asp île île Leu

Glu Leu

Leu Lys

Asn Ala

Arg Phe

Arg Glu

Lys Gin

160

Pro Gly

175

Gin Thr

Glu Ser

Leu val

Glu His

240

Ala val

255

Val Thr

Tyr Ala <210> 26 <211> 288 <212> PRT <213> Strongylocentrotus purpuratus <400> 26

Glu

Phe

Asp

Ile

Glu

Leu

Asn

Arg

125

Tyr

Gly

Asn

Lys

Leu

Asp

Met

Lys

Leu

Lys

Ser

110

Lys

Arg

Lys

Pro

Gin

190

Glu

Asp val 15

Glu Glu île ser

Ser Ala

Met ser

Met Met

Ala Asp

Phe val

Glu Arg ser Thr

160 Ala Ile 175 Thr Leu ser ser

116-11

CA 02462686 2004-05-14

<210> 27 <211> 13 <212> PRT <213> Homo sapiens <400> 27

Thr Arg lie Asp Glu Ala Asn Gin Arg Ala Thr Lys Met

5 10 <210> 28 <211> 15 <212> PRT <213> Homo sapiens <400> 28

Ser Asn Lys Thr Arg lie Asp Glu Ala Asn Gin Arg Ala Thr

5 10

Lys <210> 29 <211> 16 <212> PRT <213> Homo sapiens <400> 29

Ser Asn Lys Thr Arg

5 lie Asp Glu Ala Asn Gin Arg Ala Thr

Lys Met <210> 30 <211> 17 <212> PRT <213> Homo sapiens <400> 30 ser Asn Lys Thr Arg lie Asp Glu Ala Asn Gin Arg Ala Thr Lys Met 15 10 15

Leu <210> 31 <211> 17 <212> PRT <213> Homo sapiens <400> 31

Asp Ser Asn Lys Thr Arg He Asp Glu Ala Asn Gin Arg Ala Thr Lys 15 10 15

Met <210> 32 <211> 18

116-12

CA 02462686 2004-05-14 <212> PRT <213> Homo sapiens <400> 32

Asp Ser Asn Lys Thr Arg lie Asp Glu Ala Asn Gin Arg Ala Thr Lys 15 10 15

Met Leu

<td colspan="4"> <210> 33 <211> 33 <212> PRT <213> Mus musculus</td>

Thr Arg lie Asp Glu Ala Asn Gin Arg Ala Thr Lys Met Leu Gly Ser

25 30

Gly <210> 34 <211> 32 <212> PRT <213> Homo sapiens <400> 34

<210> 35 <211> 32 <212> PRT <213> Mus musculus <400> 35

<210> 36 <211> 34 <212> PRT <213> Gallus gal lus

<210> 37 <211> 33 <212> PRT <213> Carassius auratus <400> 37

Gin Asn Arg Gin lie Asp Arg lie Met Asp Met Ala Asp Ser Asn Lys 116-13

CA 02462686 2004-05-14

<td> <213></td><td colspan="2"> Carassi us</td><td colspan="2"> auratus</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

Gly <210> 39 <211> 30 <212> PRT <213> Torpedo sp.

<400> 39

<210> 40 <211> 33 <212> PRT <213> Strongylocentrotus purpuratus

Lys <210> 41 <211> 31 <212> PRT <213> caenorhabditis elagans <400> 41

<td> <213></td><td colspan="3"> Drosophila sp</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

116-14

CA 02462686 2004-05-14

<td> <213></td><td> Hi rudinida</td><td> 1 sp.</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<210> 44 <211> 17 <212> PRT <213> Artificial Sequence <220>

<223> synthetic peptide <400> 44

Ser Asn Lys Thr Arg lie Asp Glu Ala Asn Gin Arg Ala Thr Lys Ala 15 10 15

Leu <210> 45 <211> 17 <212> PRT <213> Artificial Sequence <220>

<223> synthetic peptide <221> MOD_RES <222> 16 <223> xaa=Nle <400> 45

Ser Asn Lys Thr Arg lie Asp Glu Ala Asn Gin Arg Ala Thr

5 10

Leu

Lys Xaa <210> 46 <211> 17 <212> PRT <213> Artificial Sequence <220>

<223> synthetic peptide <400> 46

Ser Asn Lys Thr Arg He Asp Glu Ala Asn Gin Arg Ala Thr Ala Met 15 10 15

Leu <210> 47 <211> 17 <212> PRT <213> Artificial sequence <220>

116-15

CA 02462686 2004-05-14 <223> synthetic peptide <400> 47

Ser Asn Lys Thr Arg lie Asp Glu Ala Asn Gin Arg Ala Ser Lys Met 15 10 15

Leu <210> 48 <211> 17 <212> PRT <213> Artificial Sequence <220>

<223> synthetic peptide <221> MOD_RES <222> 14 <223> xaa=Abu <400> 48

Ser Asn Lys Thr Arg lie Asp Glu Ala Asn Gin Arg Ala xaa

5 10

Leu

Lys Met <210> 49 <211> 17 <212> PRT <213> Artificial Sequence <220>

<223> synthetic peptide <221> MOD_RES <222> 13 <223> xaa=Abu <400> 49

Ser Asn Lys Thr Arg lie Asp Glu Ala Asn Gin Arg xaa Thr Lys Met

10 15

Leu <210> 50 <211> 17 <212> PRT <213> Artificial Sequence <220>

<223> synthetic peptide <400> 50

Ser Asn Lys Thr Arg lie Asp Glu Ala Asn Ala Arg Ala Thr Lys Met 15 10 15

Leu <210> 51 <211> 16 <212> PRT <213> Artificial Sequence <220>

<223> synthetic peptide

116-16

CA 02462686 2004-05-14 <221> MOD_RES <222> 11 <223> xaa=Abu <400> 51

<td colspan="2"> <210> 52 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> synthetic peptide <400> 52 Ser Asn Lys Thr Arg lie Asp</td><td> Glu Ala Asn Asn Arg</td><td> Ala Thr Lys Met</td>

<210> 53 <211> 17 <212> PRT <213> Artificial Sequence <220>

<223> synthetic peptide <400> 53

Ser Asn Lys Thr Arg lie Asp Glu Ala Ala Gin Arg Ala Thr Lys Met 15 10 15

Leu <210> 54 <211> 17 <212> PRT <213> Artificial Sequence <220>

<223> synthetic peptide <221> MOD_RES <222> 9 <223> xaa=Abu <400> 54 ser Asn Lys Thr Arg lie Asp Glu xaa Asn Gin Arg Ala Thr Lys Met 15 10 15

Leu <210> 55 <211> 17 <212> PRT <213> Artificial Sequence <220>

<223> synthetic peptide <400> 55

Ser Asn Lys Thr Arg lie Asp Gin Ala Asn Gin Arg Ala Thr Lys Met 116-17

CA 02462686 2004-05-14

Leu <210> 56 <211> 17 <212> PRT <213> Artificial sequence <22O>

<223> synthetic peptide <400> 56

Ser Asn Lys Thr Arg lie Asn

Leu

Glu Ala Asn Gin Arg Ala Thr

Lys Met <210> 57 <211> 40 <212> PRT <213> Homo sapiens <400> 57

<210> 58 <211> 40 <212> PRT <213> Bos taurus <400> 58

<210> 59 <211> 40 <212> PRT <213> Rattus sp.

<400> 59

<210> 60 <211> 40 <212> PRT <213> Rattus sp.

<400> 60

Asp Lys val Leu Glu Arg Asp Gin Lys Leu ser Glu Leu Asp Asp Arg 116-18

CA 02462686 2004-05-14

<210> 61 <211> 40 <212> PRT <213> Rattus sp.

<400> 61

<210> 62 <211> 40 <212> PRT <213> Rattus sp.

<400> 62

<210> 63 <211> 32 <212> PRT <213> Gallus gallus <400> 63

<td colspan="4"> <210> 64 <211> 32 <212> PRT <213> Gallus gallus</td>

Gin Ala Gly Ala Ser Gin Phe Glu Thr Ser Ala Ala Lys Leu Lys Arg

25 30 <210> 65 <211> 40 <212> PRT <213> Torpedo sp.

<400> 65

116-19

CA 02462686 2004-05-14

Lys Leu Lys Arg Lys Tyr Trp Trp

40 <210> 66 <211> 40 <212> PRT <213> Strongylocentrotus purpuratus <400> 66

<210> 67 <211> 40 <212> PRT <213> Aplysia sp.

<400> 67

<210> 68 <211> 40 <212> PRT <213> Teuthoida sp.

<400> 68

<210> 69 <211> 40 <212> PRT <213> Caenorhabditis elegans <400> 69

<210> 70 <211> 40 <212> PRT <213> Drosophila sp.

<400> 70

116-20

CA 02462686 2004-05-14

25 30

Lys Leu Lys Arg Lys Gin Trp Trp

40 <210> 71 <211> 40 <212> PRT <213> Drosophila sp.

<400> 71

<210> 72 <211> 39 <212> PRT <213> Hirudinida sp.

<400> 72

<td colspan="4"> <210> 73 <211> 18 <212> PRT <213> Homo sapiens</td>

<td> <213></td><td> Bos taurus</td><td></td><td></td>

<td> <213></td><td> Rattus</td><td> sp.</td><td></td><td></td><td></td>

116-21

CA 02462686 2004-05-14 <210> 76 <211> 18 <212> PRT <213> Rattus sp.

<400> 76

<td> <210> 77 <211> 18 <212> PRT <213> Rattus sp.</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> <213></td><td> Gallus</td><td> gall us</td><td></td><td></td>

<210> 79 <211> 18 <212> PRT <213> strongylocentrotus purpuratus <400> 79

Val Arg Arg Gin Asn Asp Thr Lys Lys Ala Val Lys Tyr Gin Ser Lys 15 10 15

Ala Arg <210> 80 <211> 18 <212> PRT <213> Aplysia sp.

<400> 80

Glu Thr Ala Lys Met Asp Thr Lys Lys Ala val

5 10

Ala Arg

Lys Tyr Gin Ser Lys <210> 81 <211> 18 <212> PRT <213> Teuthoida sp.

<400> 81

Glu Thr Ala Lys val Asp Thr Lys Lys Ala val Lys Tyr Gin Ser Lys 15 10 15

Ala Arg

116-22

CA 02462686 2004-05-14 <210> 82 <211> 18 <212> PRT <213> Drosophila sp.

<400> 82

Gin Thr Ala Thr Gin Asp Thr Lys Lys Ala Leu Lys Tyr Gin ser Lys 15 10 15

Ala Arg

<td> <213></td><td> Hi rudinida</td><td> sp.</td><td></td><td></td>

<210> 84 <211> 5 <212> PRT <213> Artificial Sequence <220>

<223> synthetic construct <400> 84

Gly Gly Gly Gly Ser

5 <210> 85 <211> 19 <212> PRT <213> Artificial Sequence <220>

<223> synthetic construct <221> MOD_RES <222> 1 <223> xaa=fluorescein-modified lysine <221> MOD_RES <222> 20 <223> Xaa=tetramethylrhodamine-modified lysine <221> AMIDATION <222> (0)...(0) <223> at the C-terminal <400> 85 xaa Asp Asn Lys Thr Arg lie Asp Glu Ala Asn Gin Arg Ala Thr Lys 15 10 15

Met Leu xaa <210> 86 <211> 13

116-23

CA 02462686 2004-05-14 <212> PRT <213> Artificial Sequence <22O>

<223> synthetic construct <221> MOD_RES <222> 1 <223> xaa=fluorescein-modified lysine <400> 86

Xaa Asp Ser Asn Lys Thr Arg lie Asp Glu Ala Asn Gin 15 10 <210> 87 <211> 7 <212> PRT <213> Artificial Sequence <220>

<223> synthetic construct <221> MOD_RES <222> 7 <223> xaa=tetramethylrhodamine-modified lysine <221> AMIDATION <222> (0)...(0) <223> at the C-terminal <400> 87

Arg Ala Thr Lys Met Leu xaa

5 <210> 88 <211> 23 <212> PRT <213> Artificial Sequence <220>

<223> synthetic peptide <221> MOD_RES <222> 1 <223> xaa=fluorescein-modified lysine <221> MOD_RES <222> 23 <223> xaa=tetramethylrhodamine-modified lysine <221> AMIDATION <222> (0)...(0) <223> at the c-terminal <400> 88 xaa Asp Ser Asn Lys Thr Arg lie Asp Glu Ala Asn Gin Arg Ala Thr 15 10 15

Lys Met Leu Gly Ser Gly xaa <210> 89 <211> 21 <212> PRT <213> Artificial Sequence <220>

116-24

CA 02462686 2004-05-14 <223> synthetic peptide <221> MOD—RES <222> 1 <223> Xaa=fluorescein-modified lysine <221> MOD-RES <222> 21 <223> Xaa=tetramethylrhodamine-modified lysine <221> AMIDATION <222> (0)...(0) <223> at the C-terminal <400> 89 xaa Ala Asp Ser Asn Lys Thr Arg lie Asp Glu Ala Asn Gin Arg Ala 15 10 15

Thr Lys Met Leu xaa <210> 90 <211> 24 <212> PRT <213> Artificial sequence <22O>

<223> synthetic peptide <221> MOD_RES <222> 1 <223> xaa=fluorescein-modified lysine <221> MOD-RES <222> 24 <223> Xaa=tetramethylrhodamine-modified lysine <221> AMIDATION <222> (0)...(0) <223> at the c-terminal <400> 90 xaa Ala Asp

Thr Lys Met

Ser Asn Lys Thr Arg lie Asp Glu Ala Asn Gin Arg Ala 5 10 15

Leu Gly Ser Gly Xaa <210> 91 <211> 16 <212> PRT <213> Artificial Sequence <220>

<223> synthetic peptide <221> MOD-RES <222> 1 <223> xaa=fluorescein-modified lysine <221> MOD-RES <222> 16 <223> Xaa=tetramethylrhodamine-modified lysine <221> AMIDATION <222> (0)...(0) <223> at the C-terminal <400> 91

116-25

CA 02462686 2004-05-14

<td> <213></td><td> Artificial sequence</td><td></td><td></td><td></td><td></td><td></td>

<td> <223></td><td> synthetic peptide</td><td></td><td></td><td></td><td></td><td></td>

<td> <223></td><td> xaa=fluorescei n-modifi ed</td><td> lysine</td><td></td><td></td><td></td><td></td>

<td> <223></td><td> xaa=tetramethylrhodami ne-</td><td> modi fi ed</td><td> lysine</td><td></td><td></td><td></td>

<td> <221></td><td> AMIDATION</td><td></td><td></td><td></td><td></td><td></td>

<td> <223></td><td> at the c-terminal</td><td></td><td></td><td></td><td></td><td></td>

<210> 93 <211> 22 <212> PRT <213> Artificial Sequence <220>

<223> synthetic peptide <221> MOD_RES <222> 1 <223> xaa=fluorescein-modified lysine <221> MOD_RES <222> 22 <223> Xaa=tetramethylrhodamine-modified lysine <221> AMIDATION <222> (0)...(0) <223> at the C-terminal <400> 93 xaa Met Glu Lys Thr Arg lie Asp Glu Ala Asn Gin Arg Ala Thr Lys 15 10 15

Met Leu Gly ser Gly xaa <210> 94 <211> 16 <212> PRT <213> Artificial Sequence <220>

<223> synthetic peptide <221> MOD_RES <222> 1

116-26

CA 02462686 2004-05-14 <22 3> Xaa-DABCYL modified lysine <221> MOD_RES <222> 16 <223> Xaa=EDANS modified glutamate <221> AMIDATION <222> (0)...(0) <223> at the C-terminal <400> 94 xaa Thr Arg lie Asp Glu Ala Asn Gin Arg Ala Thr Lys Met Leu xaa

10 15 <210> 95 <211> 19 <212> PRT <213> Artificial sequence <220>

<223> synthetic peptide <221> MOD_RES <222> 1 <223> xaa=DABCYL modified lysine <221> MOD—RES <222> 19 <223> xaa=EDANS modified lysine <221> AMIDATION <222> (0)...(0) <223> at the c-terminal <400> 95 xaa Thr Arg lie Asp Glu Ala Asn

Ser Gly Xaa

Gin Arg Ala Thr Lys Met Leu Gly

15 <210> 96 <211> 118 <212> PRT <213> Homo sapiens <400> 96

116-27

Contents317

71 members in 10 offices

This recordMember, by publication datePriority claim

Priority claims9

Priority claims
Document	Office	Kind	Date
09942098	United States of America	–	2001-08-28
94209801	United States of America	A	2001-08-28
94209801	United States of America	A	2001-08-28
0227212	United States of America	W	2002-08-22
0227212	United States of America	W	2002-08-22
09942098	–	–	–
PCTUS2002027212	–	–	–
US20010942098	–	–	–
WO2002US27212	–	–	–

Members71

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US2003143651A1	United States of America	A1	2003-07-31
CA2462686A1	Canada	A1	2004-04-15
WO2004031773A1	World Intellectual Property Organization (WIPO)	A1	2004-04-15
AU2002368109A1	Australia	A1	2004-04-23
EP1438586A1	European Patent Office (EPO)	A1	2004-07-21
EP1438586A4	European Patent Office (EPO)	A4	2005-03-16
US2005100973A1	United States of America	A1	2005-05-12
JP2005520000A	Japan	A	2005-07-07
AU2005272796A1	Australia	A1	2006-02-23
WO2006020748A2	World Intellectual Property Organization (WIPO)	A2	2006-02-23
CA2576354A1	Canada	A1	2006-03-23
US2006063221A1	United States of America	A1	2006-03-23
AU2005287262A1	Australia	A1	2006-03-30
CA2581102A1	Canada	A1	2006-03-30
WO2006033843A2	World Intellectual Property Organization (WIPO)	A2	2006-03-30
WO2006033843A3	World Intellectual Property Organization (WIPO)	A3	2006-05-26
US2006154314A9	United States of America	A9	2006-07-13
WO2006020748A3	World Intellectual Property Organization (WIPO)	A3	2006-10-05
BR0212140A	Brazil	A	2007-01-09
EP1776380A2	European Patent Office (EPO)	A2	2007-04-25
EP1792185A2	European Patent Office (EPO)	A2	2007-06-06
KR20070085274A	Republic of Korea	A	2007-08-27
US2007243565A1	United States of America	A1	2007-10-18
US2008032318A1	United States of America	A1	2008-02-07
US2008038756A1	United States of America	A1	2008-02-14
US7332567B2	United States of America	B2	2008-02-19
US2008064054A1	United States of America	A1	2008-03-13
EP1901069A1	European Patent Office (EPO)	A1	2008-03-19
AU2002368109B2	Australia	B2	2008-04-03
JP2008509667A	Japan	A	2008-04-03
AU2002368109B8	Australia	B8	2008-04-24
JP2008513037A	Japan	A	2008-05-01
US7374896B2	United States of America	B2	2008-05-20
AU2008201932A1	Australia	A1	2008-05-22
BRPI0514318A	Brazil	A	2008-06-10
US2008166739A1	United States of America	A1	2008-07-10
US2008171348A1	United States of America	A1	2008-07-17
US2008176249A1	United States of America	A1	2008-07-24
AU2008202928A1	Australia	A1	2008-07-31
JP2008174567A	Japan	A	2008-07-31
JP2008201786A	Japan	A	2008-09-04
US2008213796A1	United States of America	A1	2008-09-04
US2008220456A1	United States of America	A1	2008-09-11
US2009042231A1	United States of America	A1	2009-02-12
US2010075346A1	United States of America	A1	2010-03-25
US2010075357A1	United States of America	A1	2010-03-25
US2010075358A1	United States of America	A1	2010-03-25
US2010081157A1	United States of America	A1	2010-04-01
US2010081158A1	United States of America	A1	2010-04-01
US2010151494A1	United States of America	A1	2010-06-17
CA2462686CThis record	Canada	C	2010-07-27
US7838260B2	United States of America	B2	2010-11-23
US7846722B2	United States of America	B2	2010-12-07
US8003753B2	United States of America	B2	2011-08-23
US8013113B2	United States of America	B2	2011-09-06
US8022172B2	United States of America	B2	2011-09-20
AU2008201932B2	Australia	B2	2011-10-20
US8048643B2	United States of America	B2	2011-11-01
US8053208B2	United States of America	B2	2011-11-08
US8053209B2	United States of America	B2	2011-11-08
EP1901069B1	European Patent Office (EPO)	B1	2012-01-04
EP1438586B1	European Patent Office (EPO)	B1	2012-01-11
AT540317T	Austria	T	2012-01-15
AT541213T	Austria	T	2012-01-15
ATE540317T1	Austria	T1	2012-01-15
ATE541213T1	Austria	T1	2012-01-15
DK1901069T3	Denmark	T3	2012-03-12
DK1438586T3	Denmark	T3	2012-03-19
BR0212140B1	Brazil	B1	2014-11-11
BRPI0212140B1	Brazil	B1	2014-11-11
BRPI0212140B8	Brazil	B8	2021-07-27

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	Event	Code
2022-10-24	ExpiryMKEX	MKEX
2004-08-10	Examination requestEEER	EEER