Methyl 5-{2-[(tert-butylamino)carbothioyl]carbohydrazonoyl}-1-(2,4-difluorophenyl)-1H-pyrazole-4-carboxylate

pIC50: 6.70 for human GPR35

CID-2745687 is a GPR35 antagonist. GPR35 is a poorly characterized member of the rhodopsinlike, class A subfamily of G protein-coupled receptors (GPCRs). GPCRs, based on the expression pattern, has been considered as a possible therapeutic target in conditions including diabetes, cardiovascular disease, as well as inflammation and pain.

In vitro: Previous study indicated that both CID-2745687 and ML-145 could competitively inhibit the effects of cromolyn disodium and zaprinast (two agonists sharing an overlapping binding site) on human GPR35. In contrast, though ML-145 antagonized the effects of pamoate competitively, CID-2745687 showed a noncompetitive fashion. Additionally, neither ML-145 nor CID-2745687 was able to antagonize the agonist effects at rodent ortholog of GPR35 [1].

In vivo: To test whether GPR35 contributes to the metabolic effect of Zaprinast, the retina from Cngb1/ mice was preincubated with a GPR35 antagonist, CID-2745687, followed by an additional Zaprinast treatment. Results showed that CID-2745687 did not block the effect of Zaprinast on glutamate and aspartate. Moreover, pamoic acid, the GPR35 agonist, did not change aspartate or glutamate levels [1].

Clinical trial: N/A

References:
[1] Jenkins L,Harries N,Lappin JE,MacKenzie AE,Neetoo-Isseljee Z,Southern C,McIver EG,Nicklin SA,Taylor DL,Milligan G.  Antagonists of GPR35 display high species ortholog selectivity and varying modes of action. J Pharmacol Exp Ther.2012 Dec;343(3):683-95.
[2] Du J,Cleghorn WM,Contreras L,Lindsay K,Rountree AM,Chertov AO,Turner SJ,Sahaboglu A,Linton J,Sadilek M,Satrústegui J,Sweet IR,Paquet-Durand F,Hurley JB.  Inhibition of mitochondrial pyruvate transport by zaprinast causes massive accumulation of aspartate at the expense of glutamate in the retina. J Biol Chem.2013 Dec 13;288(50):36129-40.

Profiling of hydroxycinnamoylquinic acids in plant extracts using in-source CID fragmentation.[Pubmed:27591562]

J Mass Spectrom. 2016 Dec;51(12):1130-1145.

Hydroxycinnamoylquinic acids (HCQAs) are a major class of phenolic plant secondary metabolites, belonging to the chlorogenic acid family. Various health-beneficial properties of HCQAs have been shown, which has drawn interest for HCQA profiling in plants of human consumption. However, this task remains challenging, because several isomeric HCQAs can be present in the sample with identical molecular formulae and the limited availability of reference standards poses additional challenges to their identification. In the present work, a high performance liquid chromatography-electrospray ionization-quadrupole time-of-flight-mass spectrometry (HPLC-ESI-Q/TOF-MS) method accompanied with an effective data filtering protocol is presented, which is shown to be suitable for the identification of HCQAs in plant materials in a non-targeted manner. Both collision-induced dissociation (CID) fragmentation performed in a collision cell and in-source (CID) fragmentation were used to produce accurate mass fragments. It was shown that fragmentation characteristics required for identification of regio-isomers of HCQAs can be achieved with in-source CID fragmentation, enabling the use of a single-stage MS system with in-source fragmentation for convincing identification of HCQAs. Based on a thorough validation of identified HCQA compounds using coffee bean extracts as reference samples, comprehensive profiling of HCQAs in two apricot (Prunus armeniaca L.) genotypes ('Preventa' and 'Gonci magyarkajszi') was carried out for the first time and the following 10 HCQAs were shown to be present in apricot fruit: 3-caffeoylquinic acid (CQA), cis-3-CQA, 4-CQA, 5-CQA, cis-5-CQA, 3,5-diCQA, 3-p-coumaroylquinic acid (pCoQA), 4-pCoQA, 3-feruloylquinic acid (FQA) and cis-3-FQA. Copyright (c) 2016 John Wiley & Sons, Ltd. HIGHLIGHTS: An HPLC-ESI-Q/TOF-MS method suitable for the identification of hydroxycinnamoyilquinic acids (HCQAs) in plant material in a non-targeted manner was developed. Single-stage, high-resolution MS system with in-source fragmentation was shown to be suitable for convincing identification of HCQAs. Comprehensive profiling of HCQAs in two apricot (Prunus armeniaca L.) genotypes was carried out for the first time. Copyright (c) 2016 John Wiley & Sons, Ltd.

Analysis of matrix-assisted laser desorption/ionization quadrupole time-of-flight collision-induced dissociation spectra of simple precursor ions and isobaric oligosaccharide ion mixtures based on product ion intensities and pattern recognition.[Pubmed:28299859]

Rapid Commun Mass Spectrom. 2017 May 30;31(10):873-885.

RATIONALE: Qualitative analysis of glycomic tandem mass spectrometry (MS/MS) data based on m/z values of product ions alone is widely used, and often sufficient for analysis of single analytes. However, most complex glycomic mixtures contain multiple isobaric oligosaccharides, in which case this approach is often limited. Here we show how ion intensity information can be used in order to enhance MS/MS data analysis, and extract both qualitative and semiquantitative information from complex glycomic MS/MS datasets. METHODS: A matrix-assisted laser desorption/ionization quadrupole time-of-flight (MALDI QTOF) mass spectrometer was used in this study. We compared the intensities of product ions within a single product ion series, determined by their length, across the whole glycomic MS/MS dataset. In order to detect discernable patterns, the intensity data was normalized to the intensity of each product ion within the series. In most cases, normalized data yielded easily discernable patterns, relevant either for analysis of specific glycomic structure types, or mechanistic MS studies. RESULTS: We used our approach on a glycomic sample consisting of human milk oligosaccharides. The approach yielded useful results for both qualitative and semiquantitative analyses. All normalizations performed were not equally rich in information and the information content of generated tables was not possible to predict. These analyses were shown to be independent of instrument manufacturer. CONCLUSIONS: Our approach enabled more detailed qualitative analysis of MS/MS spectra of precursor ions containing isobaric oligosaccharide structures. While limited semiquantitative information could be extracted from the raw data as well, the accuracy of this method should be significantly enhanced when standard calibration mixtures can be prepared. Copyright (c) 2017 John Wiley & Sons, Ltd.

Gas-Phase Fragmentation Behavior of Oxidized Prenyl Peptides by CID and ETD Tandem Mass Spectrometry.[Pubmed:27785692]

J Am Soc Mass Spectrom. 2017 Apr;28(4):704-707.

Farnesylation and geranylgeranylation are the two types of prenyl modification of proteins. Prenylated peptides are highly hydrophobic and their abundances in biological samples are low. In this report, we studied the oxidized prenylated peptides by electrospray ionization mass spectrometry and identified them by collision-induced dissociation (CID) and electron-transfer dissociation (ETD) tandem mass spectrometry. Modified prenyl peptides were generated utilizing strong and low strength oxidizing agents to selectively oxidize and epoxidize cysteine sulfur and prenyl side chain. We selected three peptides with prenyl motifs and synthesized their prenylated versions. The detailed characteristic fragmentations of oxidized and epoxidized farnesylated and geranylgeranylated peptides were studied side by side with two popular fragmentation techniques. CID and ETD mass spectrometry clearly distinguished the modified version of these peptides. ETD mass spectrometry provided sequence information of the highly labile modified prenyl peptides and showed different characteristic fragmentations compared with CID. A detailed fragmentation of modified geranylgeranylated peptides was compared by CID and ETD mass spectrometry for the first time. Graphical Abstract .

Modeling collision energy transfer in APCI/CID mass spectra of PAHs using thermal-like post-collision internal energy distributions.[Pubmed:27802636]

J Chem Phys. 2016 Oct 28;145(16):164311.

The internal energy transferred when projectile molecular ions of naphthalene collide with argon gas atoms was extracted from the APCI-CID (atmospheric-pressure chemical ionization collision-induced dissociation) mass spectra acquired as a function of collision energy. Ion abundances were calculated by microcanonical integration of the differential rate equations using the Rice-Ramsperger-Kassel-Marcus rate constants derived from a UB3LYP/6-311G+(3df,2p)//UB3LYP/6-31G(d) fragmentation mechanism and thermal-like vibrational energy distributions pME,Tchar. The mean vibrational energy excess of the ions was characterized by the parameter Tchar ("characteristic temperature"), determined by fitting the theoretical ion abundances to the experimental breakdown graph (a plot of relative abundances of the ions as a function of kinetic energy) of activated naphthalene ions. According to these results, the APCI ion source produces species below Tchar = 1457 K, corresponding to 3.26 eV above the vibrational ground state. Subsequent collisions heat the ions up further, giving rise to a sigmoid curve of Tchar as a function of Ecom (center-of-mass-frame kinetic energy). The differential internal energy absorption per kinetic energy unit (dEvib/dEcom) changes with Ecom according to a symmetric bell-shaped function with a maximum at 6.38 +/- 0.32 eV (corresponding to 6.51 +/- 0.27 eV of vibrational energy excess), and a half-height full width of 6.30 +/- 1.15 eV. This function imposes restrictions on the amount of energy that can be transferred by collisions, such that a maximum is reached as kinetic energy is increased. This behavior suggests that the collisional energy transfer exhibits a pronounced increase around some specific value of energy. Finally, the model is tested against the CID mass spectra of anthracene and pyrene ions and the corresponding results are discussed.

Antagonists of GPR35 display high species ortholog selectivity and varying modes of action.[Pubmed:22967846]

J Pharmacol Exp Ther. 2012 Dec;343(3):683-95.

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 beta-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)p yrazole-4-carboxylate (CID-2745687) and 2-hydroxy-4-[4-(5Z)-5-[(E)-2-methyl-3-phenylprop-2-enylidene]-4-oxo-2-sulfanylide ne-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.

Targeting of the orphan receptor GPR35 by pamoic acid: a potent activator of extracellular signal-regulated kinase and beta-arrestin2 with antinociceptive activity.[Pubmed:20826425]

Mol Pharmacol. 2010 Oct;78(4):560-8.

Known agonists of the orphan receptor GPR35 are kynurenic acid, zaprinast, 5-nitro-2-(3-phenylproplyamino) benzoic acid, and lysophosphatidic acids. Their relatively low affinities for GPR35 and prominent off-target effects at other pathways, however, diminish their utility for understanding GPR35 signaling and for identifying potential therapeutic uses of GPR35. In a screen of the Prestwick Library of drugs and drug-like compounds, we have found that pamoic acid is a potent GPR35 agonist. Pamoic acid is considered by the Food and Drug Administration as an inactive compound that enables long-acting formulations of numerous drugs, such as the antihelminthics oxantel pamoate and pyrantel pamoate; the psychoactive compounds hydroxyzine pamoate (Vistaril) and imipramine pamoate (Tofranil-PM); and the peptide hormones triptorelin pamoate (Trelstar) and octreotide pamoate (OncoLar). We have found that pamoic acid induces a G(i/o)-linked, GPR35-mediated increase in the phosphorylation of extracellular signal-regulated kinase 1/2, recruitment of beta-arrestin2 to GPR35, and internalization of GPR35. In mice, it attenuates visceral pain perception, indicating an antinociceptive effect, possibly through GPR35 receptors. We have also identified in collaboration with the Sanford-Burnham Institute Molecular Libraries Probe Production Center new classes of GPR35 antagonist compounds, including the nanomolar potency antagonist methyl-5-[(tert-butylcarbamothioylhydrazinylidene)methyl]-1-(2,4-difluorophenyl)p yrazole-4-carboxylate (CID2745687). Pamoic acid and potent antagonists such as CID2745687 present novel opportunities for expanding the chemical space of GPR35, elucidating GPR35 pharmacology, and stimulating GPR35-associated drug development. Our results indicate that the unexpected biological functions of pamoic acid may yield potential new uses for a common drug constituent.