GPR40ant39

A TR-FRET assay was employed, which indirectly demonstrated target antagonism (IC₅₀=160 nM). Off-target activity was demonstrated on a panel of 144 GPCRs, including two family members, GPR43 (FFAR2) and GPR41 (FFAR3). The probe showed activity <1 mM on only 1 (pig Na+/K+ ATPase 0.79 mM) out of 144 other GPCRs. It was demonstrated that there was no activity on two close family members, but both of these family members are activated by short-chain fatty acids (FAs), while GPR40 is activated by medium- to long-chain FAs. Activity was not shown for other family members (GPR120 and GPR84), which are also activated by medium- to long-chain FAs. Also, given the fact that early hits carried some weak activity against lipid kinases such as phosphatidyl inositol-3-kinase (pIC₅₀ 4.2 and 4.8 for the α and β subtypes respectively), the activity of the probe should be evaluated against a panel of relevant kinases. Orally dosing rats at 80 mg/kg resulted in ~70% reduction of glucose-stimulated insulin secretion (GSIS). It has been shown that non-esterified FA driven GSIS accounts for ~50% of total GSIS, which suggests GPR40ant39 can completely inhibit NEFA-driven GSIS. From the data shown, this compound appears to be a good probe, but for the reason outlined above and the fact that other GPR40 antagonists exist (e.g., GW1100), more data are required.

Essentially this probe has been demonstrated to have selectivity for FFAR1 in in vitro studies with transfected cells. There is limited off-target profiling at GPCRs and, at high concentrations, some activity at several other targets (binding but no functional data reported): k-opioid receptor, angiotensin receptor (guinea pig), GABA-A receptor and Na+/K+ ATP-ase. No real functional activity is reported for these so that the use of this reagent in non-transfected cells is not recommended without using knock-out cells as control for off-target actions. The mode of action of the compound is not reported in the paper (e.g., competitive vs. non-competitive antagonism), and it is conceivable it is a negative allosteric modulator. The potency is quite weak (pIC50=6.6), so without further profiling, it is recommended only for use in transfected cells with untransfected cells as control. The in vivo data shows some favorable pharmacokinetics, although there was little information regarding metabolites and their activity.

GPR40ant39 is the result of a high-throughput screen in HEK cells transfected with GPR40 and stimulated with elaidic acid. Levels of the secondary messenger IP1 were measured as an endpoint. A medicinal chemistry effort to optimize potency and lipophilicity (logD7.4) gave GPR40ant39 as the compound with the best balance of properties. It is orally bioavailable and showed activity in a B3-agonist challenge model in Zucker rats. A shortcoming is that there is no experimental structural evidence for interaction of the probe with GPR40. Docking studies were done by computational modeling using a crystal structure of GPR40 bound to an agonist (TAK-875). The computational model hypothesized that GPR40ant39 bound to the same binding site as the agonist.