MOL #66746

Supporting materials for “Targeting of the Orphan Receptor GPR35 by Pamoic Acid: a Potent Activator of ERK and βarrestin2 with Antinociceptive Activity” by Pingwei Zhao, Haleli Sharir, Ankur Kapur, Alan Cowan, Ellen B. Geller, Martin W. Adler, Herbert H. Seltzman, Patricia H. Reggio, Susanne Heynen-Genel, Michelle Sauer, Thomas D.Y. Chung, Yushi Bai, Wei Chen, Marc G. Caron, Larry S. Barak and Mary E. Abood, Mol Pharmacol

Fig. S1. GPR35 isoforms ‘a’ and ‘b’ differ by a 31 amino acid insert at the N-terminus. Blue color shows the addition of 31 AA at the N-terminus to form the GPR35b. Red color shows the locations of the seven transmembrane domains (TM).



Figure S2 – Pamoic acid High Pressure Liquid Chromatography (HPLC) Elution Profile. HPLC data determining the purity of the pamoic acid lot used in these studies was obtained directly from Sigma. (A) A 20 μL representative sample, Sigma Chemicals Lot number 1360006 and sample number 187344_001_2, of 1.5 mg pamoic acid in a 10 ml solution of 3 parts acetonitrile and 7 parts 0.01M NaOH was eluted on a Supelco Discovery C18 HPLC (15 cm x 4.6 mm ID, 5 μm particle size) column in a gradient mobile phase consisting of %A = Acetonitrile and %C = Phosphoric Acid (0.01 M) at room temperature with a flow rate of 2 ml/minute using UV monitoring at 240 nm. The retention time of material on the column is given by the abscissa and the relative magnitude of sample is shown on the ordinate in mV. (B) The same sample as in (A) presented on an expanded scale to demonstrate the impurity content as minor peaks. The integrated relative area under the major pamoic acid peak at 12.5 minutes retention time was 99.39% of the combined area under all the different peaks indicating that the sample consisted of pamoic acid to greater than 99% purity.

Figure S3 – High Pressure Liquid Chromatography Elution Profile of GPR35 Antagonist Compounds CID2745684 and CID2745687. HPLC and mass spectrometry profiles of (A-B) CID2745684 (MW: 353.347126 g/mol | MF: C14H13F2N5O2S) and (C-D) CID2745687 (MW: 395.426866 g/mol | MF: C17H19F2N5O2S) shown at scales sufficient to highlight impurities. For HPLC an injection volume of 5 µL with a 1.0 mL/min flow rate was used. Solvent A was 5% (v/v) formic acid in water, and B was 100% acetonitrile (JT Baker). Separation was achieved on a reversed-phase 5 µm Kromasil 100 C18 column (2.1 mm × 50 mm, Peeke Scientific, CA) by using a gradient mobile phase of 10–95% B over 4.5 min. Spectral data over the wavelength range of 200–300 nm were collected. The samples CID274568 (4/7) were computed as 95% and 98% pure (+/- 0.5 %) respectively.

Fig. S4. Compounds CID2745684 and CID2745687 inhibited barr2-GFP response in high content screen of GPR35 antagonists. Blue DAPI staining shows the nucleus of UGPR35β cells. βarr2-GFP is shown as green in the images. Representative images from the high content screen showed no βarr2-GFP translocation when treated with vehicle (upper left panel), the response with 10 µM zaprinast (ZAP, upper right panel), and inhibition of the zaprinast response by 1 µM CID2745684 and 1 µM CID2745687 (bottom panels).


Fig S5. Pamoic acid and zaprinast induce βarr2-GFP response in HEK293 cells expressing mouse GPR35 which is blocked by CID2745687. Pamoic acid (1 µM) and zaprinast (5 µM) induced trafficking of βarr2-GFP in HEK293 cells (top panels). CID2745687 (1 µM) alone did not induce βarr2-GFP trafficking in HEK293 cells expressing mouse GPR35, but CID2745687 (1 µM) inhibited the pamoic acid (1 µM) and zaprinast (5 µM) induced response (bottom panels). Scale bar, 10µm.

Table S1 – GPR55 and Cannabinoid receptor ligands that do not activate GPR35. There was no response of βarrestin2-GFP trafficking to the plasma membrane up to 30 µM for these compounds.

Anandamide / AM4056 / Cannabidiol
2-arachidonyl-glycerol / HU-210 / Cannabinol
Delta-9-tetrahydrocannabinol / O-1602 / JWH-015
WIN55212-2 / SR144528 / PIMSR1
CP55,940 / SR141716A / Lysophosphatidylinositol
AM251 / VCHSR1 / Lysophosphatidic acid

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