Methods for identifying anti clostridial neurotoxin compounds
AssigneeState of Israel, Israel Institute for Biological Research
InventorsAlon Ben David, Ada Barnea, Ran Zichel
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Abstract
The presently claimed subject matter concerns methods and kits for identifying agents that reduce binding of a clostridial neurotoxin to synaptic vesicle glycoprotein 2 (SV2).
Claims20
A method for identifying an agent that reduces binding of a clostridial neurotoxin to synaptic vesicle glycoprotein 2 (SV2), wherein said method comprises the following steps:
- (a) bringing into contact a first fusion protein, said first fusion protein comprising a receptor binding domain of Botulinum neurotoxin (BoNT HC fragment) fused to a reporter protein, with a second protein, said second protein comprising the fourth luminal loop of the synaptic vesicle glycoprotein 2 (SV2) or fragment thereof optionally fused to an expression facilitating domain;
- (b) measuring the binding of said first fusion protein to said second protein in the presence of a sample;
- and
- (c) comparing the measured binding to the binding performed in a control measurement in the absence of said sample, wherein a lower binding than that of the control measurement indicates that said agent reduces binding of said clostridial neurotoxin to SV2.
A method for identifying at least one agent that reduces binding of Botulinum neurotoxin A (BoNT/A) to the synaptic vesicle glycoprotein 2C (SV2C), wherein said method comprises the following steps:
- (a) contacting a first fusion protein, said first fusion protein comprising BoNT/A HC fragment and a beta-galactosidase domain with at least one sample, thereby obtaining a mixture;
- (b) contacting said mixture obtained in step (a) with a second protein optionally attached to a surface, said second protein being a fusion protein comprising the fourth luminal loop of the synaptic vesicle glycoprotein 2C (SV2C) and a glutathione-s-transferase domain (GST);
- (c) adding at least one substrate of the beta-galactosidase domain of said first fusion protein;
- and
- (d) determining the signal;
- wherein a signal lower than a threshold signal indicates that said agent reduces binding of BoNT/A to SV2C.
A kit comprising:
- (a) a first fusion protein, said first fusion protein comprising a receptor binding domain of Botulinum neurotoxin A (BoNT/A HC fragment) fused to a reporter protein or a composition comprising said first fusion protein and a suitable carrier or excipient;
- (b) a second protein, said second protein comprising the fourth luminal loop of the synaptic vesicle glycoprotein 2 (SV2) or fragment thereof optionally fused to an expression facilitating domain or a composition comprising said second protein and a suitable carrier or excipient;
- optionally, said second protein is attached to a solid surface, wherein said second protein comprises a BoNT receptor which is synaptic vesicle glycoprotein 2C (SV2C);
- and optionally
- (c) instructions for use.
An isolated fusion protein comprising the receptor binding domain of Botulinum neurotoxin (BoNT HC fragment) and a reporter protein, wherein said isolated fusion protein comprises the BoNT/A HC fragment having an amino acid sequence which is at least 70% identical to the sequence denoted by SEQ ID NO. 2 and wherein said reporter protein is a beta-galactosidase domain having the amino acid sequence which is at least 70% identical to the sequence denoted by SEQ ID NO. 1.
Dependencies are inferred from the claim text.