Method to identify compounds useful to treat dysregulated lipogenesis, diabetes, and related disorders
View Patent ↗Provided herein are compounds, compositions, and methods of identifying compounds that neutralize the ability of the cannabinoid receptor type-1 (CB1) agonist 2-arachidonylglycerol (2-AG) in complex with its obligate binding partner adipocyte lipid binding protein (aP2) from agonizing CB1 signaling in peripheral tissues. Further provided herein are methods of treating a disorder associated with dysregulated or abnormal hepatic de novo lipogenesis and/or hepatic selective insulin resistance by inhibiting cannabinoid receptor type-1 (CB1) agonist 2-arachidonylglycerol (2-AG) in complex with its obligate binding partner adipocyte lipid binding protein (aP2) from binding and agonizing CB1.
1 . A method of selecting a compound capable of binding 2-arachidonylglycerol/adipocyte binding protein complex (2-AG/aP2) for use in treating a human subject with a disorder mediated by the dysregulation of de novo lipogenesis (DNL) comprising:
i. contacting the compound with 2-AG/aP2, wherein the compound is an antibody, antibody fragment, or antigen binding agent that has been humanized;
ii. determining whether the compound binds to 2-AG/aP2 with a KD of less than or equal to 10 −9 M; and
iii. the compound binds to the 2-AG/aP2 complex with a KD of less than or equal to 10 −9 M, selecting the compound.
2 . The method of claim 1 , further comprising:
i. introducing the compound selected in claim 1 , into a first cellular assay in the presence of 2-AG and aP2, and/or 2-AG/aP2, wherein the first cellular assay includes a population of cells expressing cannabinoid receptor type-1 (CB1);
ii. measuring the biological activity of CB1 in the first cellular assay;
iii. measuring the biological activity of CB1 in a second cellular assay in the presence of 2-AG and aP2, and/or 2-AG/aP2, wherein the second cellular assay includes a population of cells expressing CB1;
iv. comparing the biological activity of CB1 in the first cellular assay and second cellular assay, wherein the CB1 biological activity is selected from suppression of downstream AMPK phosphorylation, phosphorylation of mitogen-activated protein kinases (MAPK), increased de novo lipogenesis (DNL) production, increased very low-density lipoprotein (VLDL) production, inhibition of cyclic AMP formation, or a combination thereof; and,
v. if CB1 biological activity is reduced in the first cellular assay compared to the second cellular assay, selecting the compound.
3 . The method of claim 2 , wherein the cell population expressing CB1 is a population of human hepatocytes.
4 . A method of selecting a compound capable of neutralizing 2-AG/aP2 agonism of CB1 for use in treating a human subject with a disorder mediated by the dysregulation of de novo lipogenesis (DNL) comprising:
i. contacting the compound with 2-AG/aP2, wherein the compound is an antibody, antibody fragment, or antigen binding agent that has been humanized;
ii. determining whether the compound binds to 2-AG/aP2 with a KD of less than or equal to 10 −9 M;
iii. if the compound binds to the 2-AG/aP2 complex with a KD of less than or equal to 10 −9 M, introducing the compound into an assay with 2-AG and aP2, or 2-AG/aP2, and CB1;
iv. determining whether 2-AG/aP2 binds to CB1; and,
v. if non-binding of 2-AG/aP2 to CB1 is observed, selecting the compound.
5 . The method of claim 4 , further comprising:
i. introducing the compound selected in claim 4 into a first cellular assay in the presence of 2-AG and aP2, and/or 2-AG/aP2, wherein the cellular assay includes a population of cells expressing CB1;
ii. measuring the biological activity of CB1 in the first cellular assay;
iii. measuring the biological activity of CB1 in a second cellular assay in the presence of 2-AG and aP2, and/or 2-AG/aP2, wherein the second cellular assay includes a population of cells expressing CB1;
iv. comparing the biological activity of CB1 in the first cellular assay and second cellular assay, wherein the CB1 biological activity is selected from suppression of downstream AMPK phosphorylation, phosphorylation of mitogen-activated protein kinases (MAPK), increased de novo lipogenesis (DNL) production, increased very low-density lipoprotein (VLDL) production, inhibition of cyclic AMP formation, or a combination thereof; and,
v. if CB1 biological activity is reduced in the first cellular assay compared to the second cellular assay, selecting the compound.
6 . The method of claim 5 , wherein the cell population expressing CB1 is human hepatocytes.