Methods for identifying insulin mimetics
Methods are provided for identifying and selecting candidate molecules that activate glucose transport through binding of the insulin receptor at a site other than the insulin binding site. The methods include analyzing the properties of one or more candidate molecules in terms of the ability to bind the insulin receptor and activate glucose transport. Optionally the methods include, competitive assays in the presence of the glucose receptor, a candidate molecule, and one or more of insulin, alpha PGG and beta PGG.
1. A method of identifying a candidate molecule that activates glucose transport, comprising:
a.) combining an insulin receptor molecule and insulin under conditions suitable for specific interaction between said insulin receptor molecule and insulin resulting in an equilibrium binding state;
b.) further combining said candidate molecule with said combined insulin receptor molecule and insulin under conditions suitable for specific interaction between said candidate molecule and combined insulin receptor molecule and insulin; and
c.) determining the ability of said candidate molecule to bind to said combined insulin receptor molecule and insulin;
d.) determining whether said candidate molecule activates glucose transport;
e.) determining whether said candidate molecule influences maximal binding (B max ) of insulin to said insulin receptor in a dose dependent manner without materially altering insulin's binding affinity (K d ) for said insulin receptor; and
f.) selecting a candidate molecule that binds to said combined insulin receptor molecule and insulin, activates glucose transport, and decreases maximal binding (B max ) of insulin to said insulin receptor in a dose dependent manner without materially altering insulin's binding affinity (K d ) for said insulin receptor.
2. The method of claim 1 , wherein the step of determining the ability of said candidate molecule to bind to said combined insulin receptor molecule and insulin results in a displacement of insulin from said insulin receptor molecule in a non-competitive manner.
3. The method of claim 2 , wherein said candidate molecule displays 50% of its ability to displace insulin from said insulin receptor molecule (IC 50 ) at a concentration equal to or less than approximately 10 μM.
4. The method of claim 1 , wherein said insulin receptor molecule comprises at least one alpha subunit and at least one beta subunit.
5. The method of claim 4 , wherein the step of determining the ability of said candidate molecule to bind to said combined insulin receptor molecule and insulin involves determining the ability of said candidate molecule to bind to said at least one alpha subunit of said insulin receptor molecule.