CID 2745687

GPR35 (Ki = 12.8 nM)

使用 1 μM 双羟萘酸作为激动剂时，ERK1/2 磷酸化的 CID 2745687 (CID2745687) Ki 为 18 nM[1]。在 β-arrestin-2 相互作用实验中，CID 2745687 (CID-2745687) 是一种仅限于人 GPR35 的强拮抗剂 [2]。在激动剂浓度为 20 μM Zaprinast 和基于 BRET 的 GPR35-β-arrestin-2 相互作用测试中，CID 2745687 对人 GPR35 表现出中等效力和浓度依赖性，pIC50 为 6.70±0.09[2]。 CID 2745687 (pIC50=6.27±0.08) 完全抵消色甘酸二钠的激动作用 [2]。

---

通过高含量筛选鉴定CID2745687为GPR35拮抗剂。[1]
目前还没有公认的GPR35拮抗剂，但如果这些化合物可用，它们可能可用作抗肿瘤药物或工具化合物，用于在动物模型中描述GPR35的生理作用。为了鉴定GPR35拮抗剂，我们评估了化合物阻断10μM扎普利司特诱导的βarr2-GFP易位的能力。在桑福德伯纳姆研究所分子文库探针生产中心进行的一项筛选中，我们在基于图像的高含量初级筛选（PubChem AID 2508）中评估了293187种化合物，约150种化合物表现出低微摩尔或更好的活性。图6A显示了两个结构相关的命中，CID2745684和CID2745687，其验证剂量反应的结果低至0.5μM。使用高压液相色谱和质谱法确认了化合物的化学特性和纯度（光谱如补充图S3所示）。高通量屏幕的代表性图像如补充图S4所示。
两种药物中更有效的一种，CID2745687，是以干粉形式获得的，并进一步评估了其对抗1μM双羟萘酸的GPR35反应的能力。图6B是单独存在拮抗剂、单独存在双羟萘酸和存在拮抗剂时βarr2-GFP募集的代表性图像，其中CID2745687对募集的抑制作用明显。在βarr2-GFP转运测定中（图6C，左），来自三个独立实验的CID2745687显示Ki为12.8 nM（7.5-21.8）。对于以1μM双羟萘酸为激动剂的ERK1/2磷酸化（图6D，左），CID2745687 Ki为18 nM（9.1–35.7；n=3）。CID2745687的拮抗作用也是可逆的（图6C，右）和竞争性的（图6D，右）。为了证实双羟萘酸也能激活其他物种的GPR35，将HEK293细胞与未标记的小鼠GPR35和βarr2-GFP质粒瞬时共转染。通过与CID2745687共孵育，可以防止双羟萘酸（1μM）或扎普利司特（5μM）诱导的βarr2-GFP运输（补充图S5）的应用。

---

GPR35的拮抗剂也显示出明显的物种直系同源选择性。[2]
基于某些激动剂配体的显著物种选择性，我们接下来考虑了所报道的拮抗剂是否也具有物种选择性。CID2745687（图1）是迄今为止主要科学文献中唯一详述的GPR35拮抗剂（赵等人，2010）。使用基于BRET的GPR35-β-arrestin-2相互作用测定和EC80浓度的扎普利司特（2×10-5M）作为激动剂，CID-2745687在人类GPR35上表现为中等效力的浓度依赖性拮抗剂，pIC50=6.70±0.09（平均值±S.E.M.；n=9）（图5A）。相比之下，即使在1×10−4 M的浓度下，CID-2745687也没有实质上阻断EC80浓度的扎普利司特（4×10−于6 M）对小鼠GPR35的激动作用（图5A），并且用大鼠GPR35观察到类似的无法拮抗EC80浓度扎普利司特（4倍10−7 M）的作用（图5B）。这又是出乎意料的，因为赵等人（2010）报道了CID-2745687在小鼠疼痛模型中阻断扎普林司特的作用。用色甘酸二钠作为激动剂获得了等效的结果。再一次，仅在人直系同源物上观察到CID-2745687（pIC50=6.27±0.08，平均值±S.E.M.；n=9）具有浓度依赖性抑制活性，可完全逆转EC80浓度色甘酸二钠的激动作用（图5B）。尽管我们只能探索双羟萘酸盐在人类GPR35上抑制激动剂作用的能力，但CID2745687完全且以浓度依赖的方式做到了这一点，pIC50=7.16±0.12（平均值±S.E.M.；n=9）（图5C）。

---

CID2745687和ML-145都可以防止激动剂诱导的人GPR35内化。[2]
值得注意的是，CID-2745687和ML-145（1×10-5M）也完全阻断了人FLAG-GPR35-eYFP对不同浓度的扎普利司特、色甘酸二钠和双羟萘酸盐的内化反应（图7，A-C）。当在大鼠FLAG-GPR35-eYFP上对氮磷司特进行测试时，CID2745687或ML-145的情况并非如此（图7D）。在小鼠FLAG-GPR35-eYFP中，尽管ML-145对扎普利司特的效力或效果没有影响（图7E），但在1×10−5 M CID-2745687下，效力持续略有下降，但没有统计学意义，但扎普利司t的效果不是最大（图7E）。为了更全面地探索这一点，评估了不同浓度的ML-145或CID-2745687在应答EC80浓度的扎普利司特时防止人或小鼠GPR35直系同源物内化的能力。在这里，ML-145（图8A）和CID-2745687（图8B）在人类直系同源物上都是扎普利司特的有效抑制剂，但在小鼠直系同源物中都没有产生实质性影响（图8，C和D）。当使用EC80浓度的色甘酸二钠（人和小鼠GPR35）或双羟萘酸盐（人直系同源）时，ML-145（图8，A和C）和CID-2745687（图8、B和D）的结果是等效的。

---

CID2745687通过不同的机制抑制激动剂对人GPR35的作用。[2]
为了评估CID-2745687是否也起竞争性拮抗剂的作用，在人GPR35上还检查了该配体在一定浓度范围内改变扎普利司特、色甘酸二钠和双羟萘酸盐浓度反应曲线的能力。对于扎普利司特（图11D）和色甘酸二钠（图11E），这些研究都使激动剂的效力向更高浓度发生了可克服的变化。这些曲线的全局拟合分析也与竞争性拮抗模式一致，CID-2745687的pA2亲和力值为7.7至7.8（1.6-1.9×10-8M），斜率因子接近1.0。相比之下，对双羟萘酸盐的等效研究产生了截然不同的结果（图11F）。增加CID-2745687的浓度对双羟萘酸的EC50几乎没有影响，而是降低了激动剂的最大作用，这表明了非竞争性作用模式。
与观察到的扎普利司特和色甘酸二钠都被CID2745687和ML-145竞争性拮抗，并且是β-arrestin-2相互作用测定中的完全激动剂一致，添加一系列次最大有效浓度的色甘酸钠并没有改变扎普利司特人GPR35的EC50（图12），这表明这两种激动剂具有共同或重叠的结合位点（Jenkins等人，2011）。

一种特殊的 GPR35 拮抗剂 CID 2745687（CID2745687；1 mg/kg；过去 4 周每天口服）可抵消洛度沙胺的抗纤维化作用[3]。

---

碘草酰胺对CCl4诱导的肝纤维化的保护作用[3]
每周两次腹腔注射CCl4，持续八周，以诱导肝纤维化。在过去4周内，每天口服洛草酰胺和/或CID2745687。施用CCl4、洛草酰胺和CID2745687后，体重和肝脏重量没有变化（补充图1A、1B）。肝脏与体重的比值也没有变化（补充图1C）。CCl4治疗后，血清AST和ALT水平升高，这意味着肝损伤，尽管这些水平在洛多沙胺和CID2745687治疗后没有变化（图1）。

为了测量慢性肝纤维化引起的组织学变化，用H&E对肝组织进行染色（图2）。与正常组织（对照组）相比，CCl4诱导了严重损伤，表现为载玻片上的损伤区域。洛多沙胺可保护机体免受CCl4诱导的损伤，联合使用CID2745687可抑制洛多沙酰胺介导的保护作用（图2）。如前所述，使用0-5的主观量表对肝损伤进行半定量评估（Lim等人，2016）。肝损伤的定量评估证实了碘草酰胺的保护作用和CID2745687的抑制作用（图2E）。

为了确认肝纤维化，还进行了Masson三色染色。如图3所示，CCl4诱导了纤维化，如蓝色染色区域所示，而洛多沙胺治疗小鼠的肝组织纤维化较少。CID2745687抑制了洛草酰胺的保护作用（图3）。在Ishak阶段，使用0-5的主观量表对肝纤维化程度进行半定量评估（Lim等人，2016；Nallangula等人，2017）。结果清楚地显示了洛草酰胺的保护作用和CID2745687的抑制作用（图3）。

接下来，测量肝组织中促纤维化标志物mRNA水平的变化。通过RT-PCR测量CCl4处理的肝组织中促纤维化标志物Iα1、Iα2、IIIα1、αSMA、TIMP1、TGF-β1和纤维连接蛋白的mRNA表达（图4A）。CCl4处理的小鼠肝脏中Iα1型胶原、Iα2型胶原、TIMP1、TGF-β1和纤维连接蛋白的mRNA水平显著升高，碘草胺处理的小鼠中CCl4诱导的Iα1、Iα2中胶原和TGF-β1的增加受到抑制（图4B）。CID2745687联合治疗显著逆转了洛草酰胺治疗对TGF-β1的影响，但对其他药物没有影响。CCl4处理后，胶原IIIα1和αSMA没有变化（图4A）。

β-Arrestin测定受体反应性。[1]
转染后48小时，使用瞬时表达人GPR35b和βarr2-GFP的U2OS细胞或瞬时表达小鼠GPR35和βarr2-GFP的HEK 293细胞。大多数实验使用永久表达HA-GPR35a和βarr2-GFP（UGPR35β）的U2OS细胞。将细胞铺在盖玻片上，置于24孔板中，用0.02mg/ml聚-d-赖氨酸预处理1小时。细胞在5%CO2中保持在37°C，直到准备好进行实验（80-85%融合），并在给药前用HBSS洗涤一次，然后在HBSS中进行实验。在药物处理40分钟后，评估了激动剂刺激的βarr2-GFP再分布。对稳定的UGPR35β细胞和瞬时转染的小鼠GPR35 HEK293细胞进行了15分钟的拮抗剂预孵育实验。为了检查拮抗剂的可逆性，将细胞与100 nMCID2745687预孵育10分钟，然后用HBSS洗涤五次，每次5分钟，然后加入1μM双羟萘酸。然后在室温下用4%多聚甲醛固定细胞20分钟，然后用HBSS洗涤三次。将玻璃盖玻片安装在载玻片上，使用40倍油物镜和488nm GFP激发在荧光显微镜上成像。

---

ERK活性的细胞内蛋白质测定。[1]
细胞在96孔板中生长至融合，在测定前将血清饥饿过夜。药物处理后，去除培养基，加入4%多聚甲醛的PBS溶液，在室温下固定细胞20分钟。在药物处理之前，将200 ng/ml的PTX与细胞一起孵育3小时。CID2745687拮抗剂与激动剂联合使用。在竞争性拮抗剂试验中，将300 nMCID2745687与一系列稀释的双羟萘酸联合使用。然后用0.1%Triton X-100的PBS溶液对细胞进行五次洗涤，每次洗涤5分钟。加入LI-COR阻断缓冲液，在旋转器上摇动样品1小时。在冷藏室中过夜，施用抗磷酸-ERK1/2（1:100）的第一抗体，然后在室温下施用山羊抗兔800CW（1:800）的第二抗体2小时。将Sapphire700（1:1000；LI-COR）和DRAQ5（1:2000）与二抗一起加入进行标准化。将平板干燥，然后使用LI-COR Odyssey红外成像仪进行扫描，分辨率为169μM，焦点偏移为3，强度为4.5至6。

Animal/Disease Models: Sixweeks old male C57BL/6 mice[3]
Doses: 1 mg/kg
Route of Administration: Oral administration, every day for 4 weeks
Experimental Results: Inhibited Lodoxamide-mediated protective effects.

---

Induction of hepatic fibrosis in C57BL/6 mice [3]
Six-week-old male C57BL/6 mice were housed under standard laboratory conditions (22°C ± 2°C, 12-h light/dark cycles) with free access to food and water in the laboratory animal facility at PNU. In this study, seven-week-old male C57BL/6 mice were randomly divided into 4 groups : control (n=5), in which mice were intraperitoneal (i.p.) injected a vehicle for 8 weeks; CCl4 (n=5), in which mice were i.p. injected a CCl4 (5 ml/kg, CCl4:con oil=2:8) twice a week for 8 weeks; CCl4 plus lodoxamide (n=5), in which mice were i.p. injected a CCl4 two times a week for 8 weeks and oral administration with lodoxamide (1 mg/kg) every day of the last 4 weeks; and CCl4 plus lodoxamide and CID2745687 (n=5), in which mice were i.p. injected a CCl4 two times a week for 8 weeks, and oral administration of lodoxamide (1 mg/kg) and CID2745687 (1 mg/kg) every day of the last 4 weeks.

[1]. Targeting of the orphan receptor GPR35 by pamoic acid: a potent activator of extracellular signal-regulated kinase and β-arrestin2 with antinociceptive activity. Mol Pharmacol. 2010 Oct;78(4):560-8.

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

[3]. Lodoxamide Attenuates Hepatic Fibrosis in Mice: Involvement of GPR35. Biomol Ther (Seoul). 2019 Jun 13;28(1):92-97.

5-[[[(tert-butylamino)-sulfanylidenemethyl]hydrazinylidene]methyl]-1-(2,4-difluorophenyl)-4-pyrazolecarboxylic acid methyl ester is a ring assembly and a member of pyrazoles.

---

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 β-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)pyrazole-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. [1]

---

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]

---

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]

---

A previous pharmacogenomic analysis identified cromolyn, an anti-allergic drug, as an effective anti-fibrotic agent that acts on hepatocytes and stellate cells. Furthermore, cromolyn was shown to be a G protein-coupled receptor 35 (GPR35) agonist. However, it has not been studied whether anti-fibrotic effects are mediated by GPR35. Therefore, in this study, the role of GPR35 in hepatic fibrosis was investigated through the use of lodoxamide, another anti-allergic drug and a potent GPR35 agonist. Longterm treatment with carbon tetrachloride induced hepatic fibrosis, which was inhibited by treatment with lodoxamide. Furthermore, CID2745687, a specific GPR35 antagonist, reversed lodoxamide-mediated anti-fibrotic effects. In addition, lodoxamide treatment showed significant effects on the mRNA expression of collagen Iα1, collagen Iα2, and TGF-β1 in the extracellular matrix. However, a transforming growth factor α (TGF-α) shedding assay revealed lodoxamide not to be a potent agonist of mouse GPR35 in vitro. Therefore, these results showed anti-fibrotic effects of lodoxamide in mice and raise concerns how lodoxamide protects against liver fibrosis in vivo and whether GPR35 is involved in the action.[3]

---

CID2745687 was reported as a potent and effective antagonist on human GPR35 but not of rodent GPR35 orthologs (Jenkins et al., 2012). However, in other reports CID274568 was reported to inhibit agonist-induced activation of mouse GPR35 in HEK293 cells, mouse astrocytes, and mouse colon epithelial cells (Zhao et al., 2010; Berlinguer-Palmini et al., 2013; Tsukahara et al., 2017). Although inhibitory effect of CID2745687 on lodoxamide action was observed in mice. we cannot exclude the possibility of off-target effects of CID2745687. This may suggest that lodoxamide and CID2745687 act as an agonist and an antagonist, respectively, of the same unknown target in mice. In our previous study on hepatocytes, CID2745687 reversed the effects of lodoxamide when administrated at a dose of 10 times greater (10 mg/kg) than that of lodoxamide (1 mg/kg) in vivo (Nam et al., 2019). However, in the present study, the same dose of CID2745687 effectively reversed the effects of lodoxamide, suggesting different affinities between the two experimental models of hepatic steatosis and hepatic fibrosis. Further investigation is necessary to elucidate the different efficacies of CID2745687. In summary, lodoxamide attenuates CCl4-induced liver fibrosis in mice and CID2745687 reverses the lodoxamide’s effect. However, involvement of mouse GPR35 in the effects is questioned in AP-TGF-α shedding assay. Therefore, we reported the results to accelerate drug development for liver fibrosis and raise concerns how lodoxamide protects against liver fibrosis in vivo and whether GPR35 is involved in the action. [3]

C17H19F2N5O2S

395.43

395.122

C, 51.64; H, 4.84; F, 9.61; N, 17.71; O, 8.09; S, 8.11

264233-05-8

9581011

Off-white to light yellow solid powder

1.3±0.1 g/cm3

493.6±55.0 °C at 760 mmHg

252.3±31.5 °C

0.0±1.3 mmHg at 25°C

1.591

3.05

119.67

2

7

6

27

572

0

CC(C)(C)NC(=S)N/N=C/C1=C(C=NN1C2=C(C=C(C=C2)F)F)C(=O)OC

CYNLZIBKERMMOA-AWQFTUOYSA-N

InChI=1S/C17H19F2N5O2S/c1-17(2,3)22-16(27)23-20-9-14-11(15(25)26-4)8-21-24(14)13-6-5-10(18)7-12(13)19/h5-9H,1-4H3,(H2,22,23,27)/b20-9+

methyl 5-[(E)-(tert-butylcarbamothioylhydrazinylidene)methyl]-1-(2,4-difluorophenyl)pyrazole-4-carboxylate

264233-05-8; CID 2745687; ML194; MLS000834953; methyl 5-[(E)-(tert-butylcarbamothioylhydrazinylidene)methyl]-1-(2,4-difluorophenyl)pyrazole-4-carboxylate; CHEMBL1708510; CID 27456; SMR000461569;

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: 125 mg/mL (316.11 mM)

配方 1 中的溶解度: ≥ 2.08 mg/mL (5.26 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 20.8 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

---

请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。