Type 2 Diabetes Treatment Gains Momentum from GPR40 Breakthroughs.

Since 1980, the prevalence of diabetes cases worldwide has doubled, with more than 422 million adults having the disease(1). According to the CDC, type 2 diabetes accounts for approximately 90-95% of these diagnosed cases(2). Thus, the need for new therapeutics for the treatment of type 2 diabetes is of pressing need. 

A promising new target for therapeutics for the treatment of type 2 diabetes is the G-protein coupled receptor (GPCR), GPR40(3). This receptor is overexpressed in pancreatic
ß-cells and upon activation by long chain free fatty acids, glucose-stimulated insulin secretion is increased. Based on these data, targeting the GPR40 receptor with synthetic agonists could be a potential therapeutic option for the treatment of type 2 diabetes(4). In 2012, a group from Amgen, Inc. disproved the previous assumption that a single binding site existed on GPR40 for both endogenous free fatty acids and synthetic agonists(5). In this study, the authors show there exists at least three allosterically linked binding sites on the protein, with each site showing varying specificity for full and partial agonists. However, the exact location of these binding sites remained to be resolved(6)

A major breakthrough came in 2014, when a group from Takeda Pharmaceuticals determined the crystal structure of GPR40 bound to the partial agonist TAK-875(7). This structure showed the agonist bound to a novel pocket located between TM3 and TM4, a non-canonical GPCR binding site. Additionally, analysis of this structure also identified two additional putative binding sites for ligands which allowed for the hypothesis of how multiple ligands can bind to GPR40 to amplify the agonist signal. Comparison of this structure to other GPCR structures shows that GPR40 is in the agonist bound inactive-like state.

The latest advance in this field occurred last month (June, 2017) when a group from Merck Research Laboratories published two additional structures of the GPR40 protein(8). The first structure is of the ternary complex of GPR40 bound to the partial agonist MK-8666 and the full allosteric agonist (Ago-PAM) AP8, while the second structure is of the binary complex of GPR40 and MK-8666. Analysis of these structures show that AP8 binds in a novel pocket outside of the transmembrane helix bundle formed by TM3, TM4, TM5, and ICL2, while MK-8666 binds at the same site as TAK-874. Comparison of the ternary complex with other active-state GPCR structures suggest that upon binding of AP8, ICL2 adopts an ordered structure, and the arrangement of the seven transmembrane helices adopts an active-state-like conformation; however, there is no conformational change in TM6 (the hallmark of GPCR activation).

Phase 3 clinical trials of TAK-875 by Takeda were discontinued in late 2013 due to concerns about liver toxicity(9); however, in addition to Merck, GPR40-specific agonists are still in development by other companies, such as Bristol-Myers Squibb, Conexios Life Sciences, Japan Tobacco, Eli Lilly, and Piramal(6).

Anatrace Detergents
Both the Takeda and Merck studies used similar methods for the solubilization, purification, and crystallization of GPR40(7,8). In these studies, GPR40 was solubilized in a 10:1 mixture of DDM : CHS. Purification was carried out using a mixture of LMNG and CHS.  Crystallization of GPR40 in complex with the various ligands was done using the LCP method with a 9:1 (w/w) monoolein : cholesterol mixture. The LCP crystallization experiments were performed in Laminex glass sandwich plates.


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