# US 10,246,492 B2 · Botulinum assay with synaptobrevin substrate moiety

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- Number: US 10,246,492 B2
- Document ID: US10246492B2
- Status: Granted
- Kind: B2
- Application number: 14/941,452
- Filed: 2015-11-13
- Published: 2016-03-10
- Granted: 2019-04-02
- Earliest claimed: 2009-10-16
- Assignees: BioMadison, Inc.
- Applicants: Biomadison, Inc
- Inventors: Ward C. Tucker; Francis Mark Dunning
- Web page: https://patentgrep.com/patent/US10246492B2

## CPC

- C07K 14/00: Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C12N 9/52: derived from bacteria {or Archaea}
- C12Q 1/37: involving peptidase or proteinase
- G01N 33/542: with steric inhibition or signal modification, e.g. fluorescent quenching
- G01N 2333/33: from Clostridium (G)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

Compositions and methods for analyzing intracellular BoNT protease activity, and especially BoNT/B, BoNT/G, BoNT/D, and/or BoNT/F protease activity are provided. Cells express at least two recombinant hybrid proteins each of which includes a fluorophore and a membrane anchoring peptide, and at least one of which includes a BoNT protease recognition and cleavage sequence positioned to release a fluorophore upon cleavage. Analysis is performed by monitoring fluorescence following exposure to a BoNT.

## Claims

### Claim 1

A recombinant nucleic acid comprising:
a sequence that encodes a first hybrid proteins having a structure of A-C-B and a second hybrid protein having a structure of A-C′ D;
wherein A is a transmembrane protein domain of synaptobrevin targeted to an intracellular vesicle membrane and is not cleavable by a Botulinum neurotoxin protease, B is a first fluorescent protein, C is a first linking domain comprising a Botulinum neurotoxin protease recognition sequence and a Botulinum neurotoxin protease cleavage sequence, C′ is a second linking domain comprising an analog of C that includes the Botulinum neurotoxin protease recognition sequence but not the Botulinum neurotoxin protease cleavage sequence, and D is a second fluorescent protein selected to form a Förster resonance energy transfer (FRET) pair with the first fluorescent protein, wherein said transmembrane domain, said peptide spacer, and said BoNT protease recognition and cleavage sequence are selected to support FRET between said first fluorescent protein and said second fluorescent protein when said first hybrid protein and said second hybrid protein are collocated with a vesicle.

### Claim 2 (depends on 1)

The recombinant nucleic acid of claim 1 wherein C comprises at least one of the group consisting of a Botulinum neurotoxin/B protease recognition and protease cleavage sequence, a Botulinum neurotoxin/G protease recognition and protease cleavage sequence, a Botulinum neurotoxin/D protease recognition and protease cleavage sequence, and a Botulinum neurotoxin/F protease recognition and protease cleavage sequence, and wherein C′ comprises at least one of the group consisting of a Botulinum neurotoxin/B protease recognition sequence absent the corresponding protease cleavage sequence, a Botulinum neurotoxin/G protease recognition sequence absent the corresponding protease cleavage sequence, a Botulinum neurotoxin/D protease recognition sequence absent the corresponding protease cleavage sequence, and a Botulinum neurotoxin/F protease recognition sequence absent the corresponding protease cleavage sequence.

### Claim 3

A transfected cell comprising:
a nucleic acid comprising a sequence that encodes a first hybrid protein having a structure of A-C-B and a second hybrid protein having a structure of A-C′-D;
wherein A is a transmembrane protein domain of synaptobrevin targeted to an intracellular vesicle membrane, and that is not cleavable by a Botulinum neurotoxin protease, B is a first fluorescent protein, C is a first linking region comprising a Botulinum neurotoxin protease recognition sequence and Botulinum neurotoxin protease cleavage sequence, C′ is a second linking region comprising an analog of C that includes the Botulinum neurotoxin protease recognition sequence but not the Botulinum neurotoxin protease cleavage sequence, and D is a second fluorescent protein selected to form a FRET pair with the first fluorescent protein, wherein said transmembrane domain, said peptide space, and said BoNT protease recognition and cleavage sequence are selected to support FRET between said first fluorescent protein and said second fluorescent protein when said first hybrid protein and said second hybrid protein are collated with a vesicle.

### Claim 4 (depends on 3)

The transfected cell of claim 3, wherein the cell is stably transfected with the nucleic acid.

### Claim 5 (depends on 3)

The transfected cell of claim 3, wherein the cell is a cell selected from the group consisting of a neuronal cell, a neuroendocrine tumor cell, a hybrid cell, and a stem cell.

### Claim 6 (depends on 3)

The transfected cell of claim 3, wherein the two hybrid proteins form at least a portion of a reporting construct complex, and wherein the first fluorescent protein and the second fluorescent protein demonstrate significant Förster resonance energy transfer within the reporting construct complex as compared to a reporting construct complex without said two hybrid proteins.

### Claim 7 (depends on 3)

A cell-based method of measuring protease activity of a BoNT protease, comprising
providing the transfected cell of claim 3;
contacting the transfected cell with a BoNT protease under conditions to take up the BoNT protease by the transfected cell; and
measuring FRET in said transfected cell, thereby measuring protease activity of said BoNT protease.

### Claim 8 (depends on 7)

The method of claim 7, wherein the transfected cell is a cell selected from the group consisting of a neuronal cell, a neuroendocrine tumor cell, a hybrid cell, and a stem cell.

### Claim 9 (depends on 7)

The method of claim 7, further comprising a step of contacting the transfected cell with a putative BoNT inhibitor prior to contacting the transfected cell with the BoNT protease.

### Claim 10 (depends on 1)

The recombinant nucleic acid of claim 1, wherein C and C′ are derived from synaptobrevin.

### Claim 11 (depends on 3)

The cell of claim 3, wherein C and C′ are derived from synaptobrevin.