Remedy for diabetes
A method of screening a compound having a hypoglycemic effect (hereinafter referred to as “hypoglycemic compound”), a remedy for diabetes which contains a compound having a novel function mechanism, etc. More specifically speaking, a method of screening a hypoglycemic compound capable of binding to the β subunit of a trimeric GTP-binding protein, a remedy for diabetes comprising a hypoglycemic compound, which is characterized by being capable of binding to the β subunit of a trimeric GTP-binding protein, as the active ingredient, etc.
1. A method for screening a hypoglycemic compound which enhances enzymatic activity of phosphoinositide 3-kinase β (PI3-kinase β) comprising
1) reacting a Gβ protein which is selected from the group consisting of Gβ1 subtype, Gβ2 subtype, Gβ3 subtype, Gβ4 subtype, and Gβ5 subtype with PI3-kinase β and at least two substrates,
wherein the Gβ protein comprises a Gβ monomer, a dimer with Gγ, or a trimer with Gα and Gγ, which may optionally contain an amino acid sequence insertion selected from the group consisting of a poly-His, c-Myc, HA-tag, Flag-tag, GST, and MBP,
wherein the PI3-kinase β comprises a PI3-kinase β which is extracted from a cell or organ expressing said P13-kinase β, a recombinant PI3-kinase β having substantial enzyme activity, or a heterodimer which contains a PI3-kinase β and a regulatory subunit selected from p85 and p55 in combination with an enzymatically active subunit p110 β,
wherein a first substrate is selected from phosphatidylinositol-[4,5]-bisphosphate and phosphadidylinositol, and a second substrate is ATP which may be labeled with a radioisotope;
2) measuring activity of the PI3-kinase β by at least one method selected from the group consisting of:
a) detecting radioisotope labeled phosphatidylinositol-[3,4,5]-bisphosphate or radioisotope labeled phosphadidylinositol-[3]-phosphate which is generated from the reaction of 1) using ATP which is labeled with a radioisotope as a substrate,
b) detecting phosphatidylinositol-[3,4,5]-trisphosphate using a protein which specifically binds to phosphatidylinositol-[3,4,5]-trisphosphate, and
c) determining a quantity of ADP which is generated by the reaction of 1);
3) repeating 1) and 2) in the presence of a test compound;
4) comparing the activity of the PI3-kinase β measured in 2) with the activity of the PI3-kinase β measured in 3);
5) determining whether the test compound has an effect of enhancing the enzymatic activity of PI3-kinase β wherein the activity in 3) is greater than the activity in 2); and
6) determining the hypoglycemic effect of the test compound which is found to enhance enzymatic activity of PI3-kinase β by administering the test compound to an animal and determining whether said test compound enhances the glucose uptake activity of a adipocyte.
2. A method for screening a hypoglycemic compound which enhances binding activity between a Gβ protein and phosphoinositide 3-kinase β (PI3-kinase β) comprising
1) reacting the Gβ protein which is selected from the group consisting of Gβ1 subtype, Gβ2 subtype, Gβ3 subtype, Gβ4 subtype, and Gβ5 subtype with PI3-kinase β,
wherein the Gβ protein comprises a Gβ monomer, a dimer with Gγ, or a trimer with Gα and Gγ, which may optionally contain an amino acid sequence insertion selected from the group consisting of a poly-His, c-Myc, HA-tag, Flag-tag, GST, and MBP,
wherein the PI3-kinase β comprises a PI3-kinase β which is extracted from a cell or organ expressing said P13-kinase β, a recombinant PI3-kinase β having substantial enzyme activity, or a heterodimer which contains a PI3-kinase β and a regulatory subunit selected from p85 and p55 in combination with an enzymatically active subunit p110 β,
2) measuring the binding activity of the Gβ protein and the PI3-kinase β by at least one method selected from the group consisting of:
a) a Western blot method for measuring an amount of the other protein contained in the precipitate after immunoprecipitation using an antibody against one of the proteins of Gβ and PI3-kinase β,
b) a sandwich assay using antibodies against the proteins, wherein said assay includes a solid-phase enzyme immunoassay, and a radioimmunoassay,
c) a scintillation proximity assay (SPA) method, a fluorescence resonance energy transfer (FRET) method, or an AlphaScreen assay method;
3) repeating 1) and 2) in the presence of a test compound;
4) comparing binding activity of the Gβ protein and the PI3-kinase β measured in 2) with binding activity of the Gβ protein and the PI3-kinase β measured in 3);
5) determining whether the test compound has an effect of enhancing the binding activity of the Gβ protein and the PI3-kinase β wherein the binding activity in 3) is greater than the binding activity in 2); and
6) determining the hypoglycemic effect of the test compound which is found to enhance binding activity of the Gβ protein and the PI3-kinase β by administering the test compound to an animal and determining whether said test compound enhances the glucose uptake activity of a adipocyte.
3. The method of claim 1 , wherein said Gβ protein is the Gβ1 subtype.
4. The method of claim 1 , wherein said Gβ protein is the Gβ2 subtype.
5. The method of claim 1 , wherein said Gβ protein is the Gβ3 subtype.
6. The method of claim 1 , wherein said Gβ protein is the Gβ4 subtype.
7. The method of claim 1 , wherein the first substrate is phosphatidylinositol-[4,5]-bisphosphate.
8. The method of claim 1 , wherein the first substrate is phosphadidylinositol.
9. The method of claim 1 , wherein said measuring activity is by a) detecting radioisotope labeled phosphatidylinositol-[3,4,5]-bisphosphate or radioisotope labeled phosphadidylinositol-[3]-phosphate which is generated from the reaction of 1) using ATP which is labeled with a radioisotope as a substrate.
10. The method of claim 1 , wherein said measuring activity is by b) detecting phosphatidylinositol-[3,4,5]-trisphosphate using a protein which specifically binds to phosphatidylinositol-[3,4,5]-trisphosphate.
11. The method of claim 1 , wherein said measuring activity is by c) determining a quantity of ADP which is generated by the reaction of 1).
12. The method of claim 2 , wherein said Gβ protein is the Gβ1 subtype.
13. The method of claim 2 , wherein said Gβ protein is the Gβ2 subtype.
14. The method of claim 2 , wherein said Gβ protein is the Gβ3 subtype.
15. The method of claim 2 , wherein said Gβ protein is the Gβ4 subtype.
16. The method of claim 2 , wherein said measuring binding activity is by a) a Western blot method for measuring an amount of the other protein contained in the precipitate after immunoprecipitation using an antibody against one of the proteins of Gβ and PI3-kinase β.
17. The method of claim 2 , wherein said measuring binding activity is by b) a sandwich assay using antibodies against the proteins, wherein said assay includes a solid-phase enzyme immunoassay, and a radioimmunoassay.
18. The method of claim 2 , wherein said measuring binding activity is by c) a scintillation proximity assay (SPA) method, a fluorescence resonance energy transfer (FRET) method, or an AlphaScreen assay method.