GPR35 ( IC50 = 20.1 nM ); GPR55 ( EC50 = 21.7 μM )

ML-145 (10 μM) completely prevents human FLAG-GPR35-eYFP from internalizing in response to different dosages of zaprinast, cromolyn disodium, and pomoate in addition[2].
ML-145 either has no effect (mouse) or exhibits a negligible, seemingly noncompetitive inhibitory effect (rat) at the rodent orthologs[2].
ML-145 functions as a competitive antagonist for several agonists at human GPR35 and has an IC50 value of about 20 nM against the EC80 concentrations of different GPR35 agonists[1].

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ML-145, identified in a β-arrestin-dependent high content screen, has also been described as an antagonist of GPR35 (Heynen-Genel et al., 2010). Like CID-2745687, ML-145 fully inhibited the agonist effects of EC80 concentrations of zaprinast (Fig. 6A), cromolyn disodium (Fig. 6B), and pamoate (Fig. 6C) at human GPR35 in a concentration-dependent manner. Consistent with the reported higher affinity of ML-145 (Heynen-Genel et al., 2010), in each case this ligand displayed higher potency than CID-2745687 (Table 2). Once again, however, ML-145 was either without effect (mouse) or displayed only a small and apparently noncompetitive inhibitory effect (rat) at the rodent orthologs (Fig. 6, A and B). [2]

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Both CID2745687 and ML-145 Can Also Prevent Agonist-Induced Internalization of Human GPR35. [2]
It is noteworthy that both CID-2745687 and ML-145 (1 × 10−5 M) also fully blocked internalization of human FLAG-GPR35-eYFP in response to varying concentrations of zaprinast, cromolyn disodium, and pamoate (Fig. 7, A–C). This was not the case for either CID2745687 or ML-145 when tested against zaprinast at rat FLAG-GPR35-eYFP (Fig. 7D). At mouse FLAG-GPR35-eYFP, although ML-145 was without effect on the potency or effect of zaprinast (Fig. 7E), at 1 × 10−5 M CID-2745687 consistently produced a modest, but not statistically significant, decrease in potency but not a maximal effect of zaprinast (Fig. 7E). To explore this more fully, the ability of varying concentrations of ML-145 or CID-2745687 to prevent internalization of the human or mouse orthologs of GPR35 in response to an EC80 concentration of zaprinast was assessed. Here, both ML-145 (Fig. 8A) and CID-2745687 (Fig. 8B) were effective inhibitors of zaprinast at the human ortholog but neither produced a substantial effect at the mouse ortholog (Fig. 8, C and D). Equivalent results for both ML-145 (Fig. 8, A, and C) and CID-2745687 (Fig. 8, B and D) were produced when EC80 concentrations of cromolyn disodium (at both human and mouse GPR35) or pamoate (at the human ortholog) were used.

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As anticipated from the other assays, ML-145 inhibited the effect of EC80 concentrations of zaprinast (3 × 10−7 M), cromolyn disodium (3 × 10−6 M), and pamoate (1 × 10−8 M) at human GPR35 in the IP1 accumulation assays with pIC50 close to 7.4 in these conditions (Fig. 10A). Furthermore, ML-145 also decreased basal IP1 production in a concentration-dependent manner (Fig. 10A), consistent with this ligand behaving as an inverse agonist and suppressing the constitutive activity of human GPR35. By contrast, ML-145 displayed little capacity to inhibit the agonist action of an EC80 concentration of zaprinast at either mouse or rat GPR35 in this assay (Fig. 10A). Although it was necessary to use pamoate at 3 × 10−6 M to generate a substantial signal via rat GPR35, ML-145 was entirely without effect at concentrations up to 1 × 10−5 M (Fig. 10A). CID-2745687 also inhibited the effect of zaprinast, cromolyn disodium, and pamoate at human GPR35 in a concentration-dependent manner but was more than 10-fold less potent than ML-145 (Fig. 10B). As for ML-145, CID-2745687 also acted as an inverse agonist and was able to reduce basal IP1 generation in a concentration-dependent manner (Fig. 10B). As noted for ML-145, CID-2745687 was without significant effect at either rat or mouse GPR35 in these assays (Fig. 10B) [2].

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ML-145 Is a Competitive Antagonist at Human GPR35. [2]
To define the mode of action of ML-145 at human GPR35 we returned to the BRET-based β-arrestin-2 interaction assay and performed a series of agonist concentration-response curves to zaprinast (Fig. 11A), cromolyn disodium (Fig. 11B), and pamoate (Fig. 11C) in the presence of concentrations of ML-145 ranging from 1 to 50 nM. In all cases, global analysis of the data was consistent with ML-145 acting as a competitive and surmountable antagonist, although the relatively low potency of cromolyn disodium meant that sufficient agonist could not be used to fully overcome the effect of the highest concentration of ML-145 used. Such studies produced estimates of the pA2 for ML-145 between 8.67 and 8.83 (1.48–2.13 × 10−9 M).

Cell Culture and Transfection. [2]
Flp-In TREx 293 cells were maintained in Dulbecco's modified Eagle's medium without sodium pyruvate, supplemented with 10% (v/v) fetal calf serum, 1% penicillin/streptomycin mixture, and 10 μg/ml blasticidin at 37°C in a 5% CO2 humidified atmosphere. HEK293T cells were maintained in Dulbecco's modified Eagle's medium supplemented with 0.292 g/liter l-glutamine and 10% (v/v) newborn calf serum at 37°C in a 5% CO2 humidified atmosphere. Transfection was performed by using 1 mg/ml PEI (linear MW-25000). Cells were plated until 60 to 80% confluent then transfected with 5 μg of required plasmid DNA and PEI (ratio 1:6 DNA/PEI), diluted in 150 mM NaCl, pH 7.4. After incubation at room temperature for 10 min, the mixture was added to HEK293T cells. Cells were incubated 24 h then transferred to 96-well plates coated with poly-d-lysine. In all experiments, the total amount of DNA transfected was equalized between constructs by the addition of the empty expression vector pcDNA3.

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Generation of Flp-In TREx 293 Cells Stably Expressing Forms of GPR35. [2]
To generate Flp-In TREx 293 cells able to inducibly express human FLAG-GPR35-eYFP, rat FLAG-GPR35-eYFP, or mouse FLAG-GPR35-eYFP, the cells were transfected with a mixture containing the desired cDNA in pcDNA5/FRT/TO vector and the pOG44 vector (1:9) by using Effectene, according to the manufacturer's instructions. After 48 h, the medium was changed to medium supplemented with 200 μg/ml hygromycin B to initiate the selection of stably transfected cells. After isolation of resistant cells, expression of the appropriate construct from the Flp-In TREx locus was induced by treatment with up to 100 ng/ml doxycycline for 24 h.

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BRET. [2]
HEK293T cells were cotransfected with the required ortholog of FLAG-GPR35 tagged with eYFP and β-arrestin-2-Renilla luciferase 6 (ratio 4:1), using PEI. An additional set of transfections used only the Renilla luciferase construct and empty expression vector pcDNA3. From 10-cm dishes, 50,000 cells were seeded per well into poly-d-lysine-coated 96-well plates. After 24 h cells were washed twice with Hanks' balanced salt solution, pH 7.4, and coelentrazine-h was added to a final concentration of 5 μM. Cells were incubated in darkness for 10 min at 37°C before the addition of ligands. Cells were incubated an additional 5 min at 37°C before being read on a PHERAstar FS reader. The BRET ratio was then calculated as emission at 530 nm/emission at 485 nm. Net BRET was defined as the 530/485-nm ratio of cells coexpressing the Renilla luciferase and eYFP constructs minus the BRET ratio of cells expressing only the Renilla luciferase construct in the same experiment. This value was multiplied by 1000 to obtain mBRET units.

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Internalization and Live Cell Epifluorescence Microscopy. [2]
Cells expressing human or rat FLAG-GPR35-eYFP were grown on poly-d-lysine-coated coverslips. The coverslips were placed into a microscope chamber containing physiological saline solution (130 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 20 mM HEPES, and 10 mM d-glucose, pH 7.4). For internalization studies, ligands were added to the microscope chamber, and fluorescent images were acquired at 5-min intervals by using a spinning disk structured illumination Viva Tome device attached to the bottom port of a Zeiss Axio Observer.Z1 invert microscope. Narrow-band 490/20-nm excitation light was reflected through a 63×, oil immersion Plan-Apochromat objective lens to excite eYFP, and the resulting emitted light was detected at 536/40 nm by using an Axiocam MRm charge-coupled device camera.

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ArrayScan High Content Analysis of GPR35 Internalization. [2]
Flp-In T-Rex 293 cells harboring human FLAG-GPR35-eYFP, rat FLAG-GPR35-eYFP, or mouse FLAG-GPR35-eYFP were seeded into poly-d-lysine-coated, clear-view 96-well plates at a density of 60,000 cells per well and treated with up to 100 ng/ml doxycycline to induce receptor construct expression. After 24 h cells were washed twice with Hanks' balanced salt solution and incubated with ligands for 1 h at 37°C. Cells were then incubated for another 30 min with ligands and 10 μg/ml Hoechst nuclear stain at 37°C. Images were acquired immediately by using a Cellomics ArrayScan II high content imager, and internalized receptors were quantified by using a proprietary algorithm designed to identify the number of endosomal recycling compartments per cell.

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Inositol Phosphate Accumulation Assays. [2]
HEK293T cells were transiently cotransfected with the human, rat, or mouse orthologs of FLAG-GPR35-eYFP and a chimeric G protein (either Gαqi5 or Gαq135) by using PEI. After 16- to 24-h incubation at 37°C + 5% CO2, cells were resuspended in IP-One stimulation buffer (10 mM HEPES, 1 mM CaCl2, 0.5 mM MgCl2, 4.2 mM KCl, 146 mM NaCl, 5.5 mM glucose, and 50 mM LiCl, pH 7.4) and seeded at 10,000 cells/well in white, solid-bottom, 384-well plates. Ligands were diluted in IP-One stimulation buffer according to the manufacturer's instructions. Antagonist compounds were preincubated with cells for 15 min at 37°C before the addition of agonist. Cells were incubated with agonist for 2 h at 37°C, before the addition of d2-conjugated inositol monophosphate (IP1) and anti-IP1 Lumi4-Tb cryptate diluted in lysis buffer according to the manufacturer's instructions. After incubation at room temperature for 1 h, homogeneous time-resolved fluorescence was measured by using a PHERAstar FS plate reader.

[1]. Selective GPR35 Antagonists - Probes 1 & 2. National Center for Biotechnology Information (US); 2010-2010 Feb 28.

[2]. Antagonists of GPR35 display high species ortholog selectivity and varying modes of action. J Pharmacol Exp Ther. 2012 Dec;343(3):683-95.

Although many known receptors that regulate addiction have been pharmacologically and biochemically well characterized, some orphan receptors with homology to known receptors of abuse (i.e. GPR35) remain uncharacterized. GPR35 is a G-protein coupled receptor, first identified in 1998 after a screen of a human genomic library. More recent RT-PCR studies have now confirmed the presence of GPR35 in dorsal root ganglion, the cerebellum and brain, as well as GPR35b, which was cloned from a human whole brain cDNA library. Thus, GPR35 regulation appears to have profound physiological and pathophysiological implications. In line with the specific aim of this project, the identified probes ML-145 (CID-2286812) and ML144 (CID-1542103) represent selective antagonists for GPR35, but not for the related GPR55 orphan receptor, supporting the hypothesis that they do not produce non-specific interference with signaling directly at or downstream of the β-arrestin signaling pathway. These probes will serve as novel tools to delineate the biochemistry of GPR35 as potential therapeutics to selectively target pathways underlying pain and to enhance our understanding of the molecular basis of addiction. [1]

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Variation in pharmacology and function of ligands at species orthologs can be a confounding feature in understanding the biology and role of poorly characterized receptors. Substantial selectivity in potency of a number of GPR35 agonists has previously been demonstrated between human and rat orthologs of this G protein-coupled receptor. Via a bioluminescence resonance energy transfer-based assay of induced interactions between GPR35 and β-arrestin-2, addition of the mouse ortholog to such studies indicated that, as for the rat ortholog, murine GPR35 displayed very low potency for pamoate, whereas potency for the reference GPR35 agonist zaprinast was intermediate between the rat and human orthologs. This pattern was replicated in receptor internalization and G protein activation assays. The effectiveness and mode of action of two recently reported GPR35 antagonists, methyl-5-[(tert-butylcarbamothioylhydrazinylidene)methyl]-1-(2,4-difluorophenyl)pyrazole-4-carboxylate (CID2745687) and 2-hydroxy-4-[4-(5Z)-5-[(E)-2-methyl-3-phenylprop-2-enylidene]-4-oxo-2-sulfanylidene-1,3-thiazolidin-3-yl]butanoylamino)benzoic acid (ML-145), were investigated. Both CID-2745687 and ML-145 competitively inhibited the effects at human GPR35 of cromolyn disodium and zaprinast, two agonists that share an overlapping binding site. By contrast, although ML-145 also competitively antagonized the effects of pamoate, CID-2745687 acted in a noncompetitive fashion. Neither ML-145 nor CID-2745687 was able to effectively antagonize the agonist effects of either zaprinast or cromolyn disodium at either rodent ortholog of GPR35. These studies demonstrate that marked species selectivity of ligands at GPR35 is not restricted to agonists and considerable care is required to select appropriate ligands to explore the function of GPR35 in nonhuman cells and tissues. [2]

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Although it is also possible that nonreceptor accessory proteins might alter the pharmacology of GPR35 in a species-dependent manner, the current studies re-emphasize the need to perform standard but insightful pharmacological analyses to fully understand both the potential and the potential limitations of novel ligands identified via various screens and their detailed mode of action. The current studies define that, despite their high affinity at human GPR35, neither CID2745687 nor ML-145 are useful pharmacological antagonists for probing the functions of GPR35 in either mouse or rat models of physiology and disease, and pamoate is not a high-potency agonist at rodent orthologs of this receptor.[2]

C24H22N2O5S2

482.571883678436

482.097

C, 59.74; H, 4.60; N, 5.81; O, 16.58; S, 13.29

1164500-72-4

2286812

Yellow to brown solid powder

1.5±0.1 g/cm3

1.719

4.38

3

7

8

33

835

0

C1=CC=C(/C=C(/C=C2\SC(=S)N(CCCC(NC3=CC=C(C(=O)O)C(O)=C3)=O)C\2=O)\C)C=C1

COFMYJWNXSFLKQ-QIROLCGISA-N

InChI=1S/C24H22N2O5S2/c1-15(12-16-6-3-2-4-7-16)13-20-22(29)26(24(32)33-20)11-5-8-21(28)25-17-9-10-18(23(30)31)19(27)14-17/h2-4,6-7,9-10,12-14,27H,5,8,11H2,1H3,(H,25,28)(H,30,31)/b15-12+,20-13-

2-hydroxy-4-[4-[(5Z)-5-[(E)-2-methyl-3-phenylprop-2-enylidene]-4-oxo-2-sulfanylidene-1,3-thiazolidin-3-yl]butanoylamino]benzoic acid

ML145; ML-145; ML 145; 1164500-72-4; 2-hydroxy-4-[4-[(5Z)-5-[(E)-2-methyl-3-phenylprop-2-enylidene]-4-oxo-2-sulfanylidene-1,3-thiazolidin-3-yl]butanoylamino]benzoic acid; MLS000575039; CHEMBL1384502; SMR000208964; 2-hydroxy-4-({4-[5-(2-methyl-3-phenyl-2-propen-1-ylidene)-4-oxo-2-thioxo-1,3-thiazolidin-3-yl]butanoyl}amino)benzoic acid; ML 145

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: ~50 mg/mL (~103.6 mM)

Solubility in Formulation 1: 5 mg/mL (10.36 mM) in 10% DMSO + 40% PEG300 +5% Tween-80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 50.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 + to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)