GPR40ant39

Protein target name:

FFAR1

Mechanism of action:

AntagonistAllosteric (predicted)

Recommended Concentration for use in cells:

16 nM - 1.6 uM

Probe Information

Target and Probe Information

Target Details

Target class:

GPCR

Subclass:

Free fatty acid receptors

HUGO ID:

HGNC:4498

Entrez gene ID:

2864

UNIPROT ID:

O14842

Target alias:

Free fatty acid receptor 1, FFA1R, GPCR40, GPR40

Probe Details

Chemical Probes Portal ID:

CPP1503

PubChem CID:

126961699

SMILES string:

C1=CC(=C(C(=C1F)F)F)OCC2=CC(=NC(=N2)C3=CC=CC=N3)N4CC(C4)(C)C#N

InChi Key:

QKPUKWQKXUUTOK-UHFFFAOYSA-N

Orthogonal probe:

FasiglifamTUG-469AMG-837

Validation in Cells and Model Organisms

In vitro validation

On target activity

Potency:

IC50

160 nM

Potency assay:

HEK cells were stably transfected with GPR40, stimulated with elaidic acid and measuring the secondary messenger IP1 by time resolved fluorescence resonance energy transfer as an endpoint, full details are in the ESI of the paper.

Structure-activity relationship:

SAR is published, series shows a strong lipophilicity correlation but this compound shows a better balance of the two properties.

Off target activity

Potency (off target):

IC50

Off target protein and potency:

Na+/K+ ATPase (pig): 790 nM

Potency assay (off target):

The compound had no activity in CEREP panel, including 144 GPCRs, at 1 uM. The above is the only target that had a potency > 1 uM. GRP40ant39 had no measurable activity against FFAR2 (GPR43).

Probe Selectivity in Vitro:

hERG pIC50=5.0

In cell validation

On target activity in cells

Potency in cells:

IC50

160 nM

Potency assay (cells):

Target engagement assay (cells):

This is difficult to assess for GPCRs. An orthogonal assay using calcium flux gives the same results, giving confidence that the compound doesn't hit the assay technology.

In vivo validation

Organism:

male Zucker fa/fa rat

Dose:

80 mg/kg

Route of delivery:

Oral

Plasma half life:

69 min

Systemic clearance:

30 mL/min/kg

Fraction of probe bound to plasma protein:

0.95

Organ of Interest

Target engagement assay:

Indirect. In beta-3 agonist challenge assays (lipolysis), GPR40ant39 inhibited nonesterified fatty acid (NEFA)-driven insulin resistance.

Primary Reference

Reference (doi):

10.1039/C5MD00037H

SAB Rating

Rating for use in Cells

3 review(s)

Rating for use in Model Organisms

3 review(s)

SAB Members

SAB Comments

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.

Jun 7 2017 - 12:53pm

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.

Jul 17 2017 - 12:39pm

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.

Aug 10 2017 - 8:52pm

GPR40ant39

Probe Information

Target Details

Free fatty acid receptor 1, FFA1R, GPCR40, GPR40

Probe Details

In vitro validation

In cell validation

In vivo validation

Primary Reference

Supporting Organizations

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GPR40ant39

Probe Information

Target Details

Free fatty acid receptor 1, FFA1R, GPCR40, GPR40

Probe Details

In vitro validation

In cell validation

In vivo validation

Primary Reference

Supporting Organizations

Interested in supporting the portal?