Methods of identifying a compound that increases or decreases the interaction of Hn-33 with synaptotagmin II
This application is based, inter alia, on the discovery of a binding interaction between the Hn-33 hemagglutinin polypeptide of the type A Clostridium botulinum neurotoxin complex and synaptosomal proteins, including synaptotagmin II (Syt II). Methods of screening for compounds that modulate, e.g., increase or decrease, this interaction are provided. Also provided are compositions and methods for targeting compounds to neuronal and cancer cells by coupling the compounds to Hn-33 or biologically active Hn-33 variants.
1. A method of identifying a compound that increases the interaction of a hemagglutinin polypeptide from the type A Clostridium botulinum neurotoxin complex (Hn-33) with a synaptotagmin II polypeptide, wherein the Hn-33 polypeptide is capable of binding to the synaptotagmin II polypeptide, the method comprising:
providing an Hn-33 polypeptide comprising amino acids 174 to 290 of SEQ ID NO: 1 and at least 95% identical to SEQ ID NO: 1;
providing a synaptotagmin II polypeptide comprising the sequence of SEQ ID NO: 2;
contacting the Hn-33 polypeptide and the synaptotagmin II polypeptide in the presence of a test compound; and
determining a change in the binding between the Hn-33 polypeptide and the synaptotagmin II polypeptide in the presence of the test compound compared to the binding in the absence of the test compound, wherein an increase in the binding indicates that the compound increases the interaction of the Hn-33 polypeptide with the synaptotagmin II polypeptide.
2. The method of claim 1 , wherein the synaptotagmin II polypeptide is expressed on the surface of a cell, contacting the Hn-33 polypeptide and the synaptotagmin II polypeptide in the presence of a test compound comprises contacting the cell with the Hn-33 polypeptide in the presence of a test compound, and wherein determining a change in the binding between the Hn-33 polypeptide and the synaptotagmin II polypeptide comprises determining a change in the binding between the Hn-33 polypeptide and the cell in the presence of the test compound.
3. The method of claim 1 , wherein the Hn-33 polypeptide comprises the sequence of SEQ ID NO: 1.
4. The method of claim 1 , wherein the Hn-33 polypeptide is a fusion protein.
5. The method of claim 2 , wherein the Hn-33 polypeptide comprises the sequence of SEQ ID NO: 1.
6. The method of claim 2 , wherein the Hn-33 polypeptide is a fusion protein.
7. A method of identifying a compound that decreases the interaction of a hemagglutinin polypeptide from the type A Clostridium botulinum neurotoxin complex (Hn-33) with a synaptotagmin II protein, wherein the Hn-33 polypeptide is capable of binding to the synaptotagmin II polypeptide, the method comprising:
providing an Hn-33 polypeptide comprising amino acids 174 to 290 of SEQ ID NO: 1 and at least 95% identical to SEQ ID NO: 1;
providing a synaptotagmin II polypeptide comprising the sequence of SEQ ID NO: 2;
contacting the Hn-33 polypeptide and the synaptotagmin II polypeptide in the presence of a test compound; and
determining a change in the binding between the Hn-33 polypeptide and the synaptotagmin II polypeptide in the presence of the test compound compared to the binding in the absence of the test compound, wherein a decrease in the binding indicates that the compound decreases the interaction of the Hn-33 polypeptide with the synaptotagmin II polypeptide.
8. The method of claim 7 , wherein the Hn-33 polypeptide comprises the sequence of SEQ ID NO: 1.
9. The method of claim 7 , wherein the Hn-33 polypeptide is a fusion protein.
10. The method of claim 7 , wherein the synaptotagmin II polypeptide is expressed on the surface of a cell, contacting the Hn-33 polypeptide and the synaptotagmin II polypeptide in the presence of a test compound comprises contacting the cell with the Hn-33 polypeptide in the presence of a test compound, and wherein determining a change in the binding between the Hn-33 polypeptide and the synaptotagmin II polypeptide comprises determining a change in the binding between the Hn-33 polypeptide and the cell in the presence of the test compound.
11. The method of claim 10 , wherein the Hn-33 polypeptide comprises the sequence of SEQ ID NO: 1.
12. The method of claim 10 , wherein the Hn-33 polypeptide is a fusion protein.