User:Brittany Stankavich/Sandbox 1

Introduction

Human GPR40 receptor, hGPR40, is a free fatty-acid receptor that binds to long chain free fatty acids, inducing insulin secretion. It is a G protein highly expressed in human pancreatic β cells, brain, and endocrine cells of gastrointestinal tract ^[1]. However, what makes this receptor significant is that the secretion of insulin is glucose dependent. Thus, there needs to be an agonist bound, in addition to presence of glucose in the blood in order for insulin secretion to occur. This glucose-dependence makes GPR40 a target for type-2 diabetes because it allows for increased glycemic control and therefore, low risk of hypoglycemia.

Function

Signal Transduction

The natural substrate of hGPR40 are free fatty acids (FFAs) which bind to the G-protein and enhance glucose-stimulated insulin secretion. FFAs bind to GPR40 which then couples with the G-protein Gq leading to increased phospholipase C (PLC) activity. PLC catalyzes the hydrolysis of the phospholipid phosphatidylinositol-4,5-biphosphate (PIP2) resulting in the formation of diacylglycerol (DAG) and inositol 1,4,5-triphosphate (IP3). DAG can activate protein kinase C (PKC) to enhance insulin secretion. IP3 on the other hand is soluble and diffuses to the endoplasmic reticulum where it is able to bind to a receptor on a ligand-gated Ca2+ channel. This binding triggers the opening of the channel causing stored Ca²⁺ to be released into the cytoplasm. Upon this large increase in intracellular free Ca²⁺, there is also an increase in glucose-dependent insulin secretion suggesting that insulin release can be contributed in part to the changes in Ca²⁺ concentration resulting from activated GPR40 ^[2].

Ligand Binding

Free Fatty Acids

GPR40’s natural substrate are FFAs in which a free carboxyl group is required to bind. However, GPR40 can be activated by a wide variety of fatty acids with chain lengths ranging from saturated fatty acids with 8 carbons to 23 carbons. In addition, various mono (i.e. palmitoleic (C16:1) and oleic (C18:1) acids) and poly-unsaturated fatty acids (i.e. linoleic (C18:2) and eicosatrienoic (C20:3) acids) can activate GPR40 (Morgan et al. 2009). The agonists potency varies according to the carbon-chain length however. The activity of GPR40 increases when the chain is increased from C6 to C15 but then decreased when the chain was extended beyond C15. One explanation for this is that as alkyl chain increased, so did the hydrophobic interactions with the protein within the binding pocket. However, for FFAs with carbon chains longer than C15, the molecular size is too large for the binding pocket. This causes the alkyl chain to extend beyond the binding pocket and destabilize the binding ^[3].

FFAs bind to hGPR40 by coordinating its free carboxyl group to three amino acids, Arg183, Asn244, and Arg258, which are located close to the of hGPR40 on TM5, 6 and 7. Because of the close proximity of these residues to the extracellular domain and the dominantly hydrophobic nature of FFA’s, it is possible that ligand binding occurs close to, or within, the plane of the membrane ^[2].

TAK-875

Tak-875 is known to be a partial agonist of GPR40. The bonding of this ligand to the bonding site is fairly unique, as it is proposed that the ligand must enter through the membrane bilayer. This is performed via a method similar to ligand binding to sphingosine 1-phosphate receptor 1 3v2w, retinal loading of opsin 4j4q and the entry of anandamide in cannabinoid receptors, in which extracellular loops block the binding from the extracellular matrix ^[4]. In contrast, delta opioid receptor binding 4ej4 allow for binding directly from the extracellular matrix. The binding mechanism through the bilayer may be selectively favoring the free fatty acid because of the non-polar regions of the ligand.

The carboxylic acid terminus of TAK-875 is buried within a very hydrophobic region where it interacts with polar residues Arg 183, Arg 258, Tyr 91, and Tyr 240.

Binding Pockets

Medical Relevance

While undergoing clinical trials, the use of TAK-875 in the treatment of diabetes mellitus was terminated in the III phase. This was due to observed liver toxicity. The liver toxicity is believed to be due to its effects on the bile acids, achieved through the inhibition of the efflux of bile acids into bile ^[5]. Other molecules are currently undergoing development in an effort to activate hGPR40 in a way that does not adversely affect the liver. The leading research suggests a molecule similar to that of 3-ethoxypropanoic acid ^[6]. However, this molecule must be modified before it could be conceivably used, as its half-life is extremely short, due to the rapid oxidation at the benzyl position during metabolism ^[6]