SB-674042

OX₁ Receptor ( IC50 = 3.76 nM ); OX₂ Receptor ( IC50 = 531 nM ); OX₁ Receptor ( Ki = 1.1 nM ); OX₂ Receptor ( Ki = 129 nM )
SB-674042 is a selective, nonpeptide antagonist of the human orexin-1 (OX₁) receptor. Its binding affinity (Ki) was not directly provided in competition assays, but saturation and kinetic studies determined its equilibrium dissociation constant (Kd). [1]
The radiolabeled form, [³H]SB-674042, binds to the human OX₁ receptor stably expressed in CHO cells with a Kd of 3.76 ± 0.45 nM in a membrane-based scintillation proximity assay (SPA) format and a Kd of 5.03 ± 0.31 nM in a whole-cell assay format. [1]
In functional calcium mobilisation assays, SB-674042 acts as a competitive antagonist at the OX₁ receptor with an inhibition constant (Kb) of 1.1 ± 0.1 nM. It shows >100-fold selectivity over the human orexin-2 (OX₂) receptor (Kb at OX₂ = 129 ± 15 nM). [1]

SB-674042 ([3H])（0.2-24 nM；2 h）具有高亲和力，可作为放射性配体，适合标记在 CHO 细胞中稳定表达的人 OX1 受体[1]。 SB-674042（5 μM；4 ℃ 30 分钟，37 ℃ 3 小时）可降低 CB1 受体激动剂在共表达 orexin-1 和 CB1 受体的 HEK293 细胞中磷酸化 ERK1/2 的效力[2]。 SB-674042（1 μM；24 小时）可消除 INS-1 细胞中 Orexin-A（1 nM-1 μM；24 小时）引起的 mTOR 磷酸化增加，表明由orexin-A 诱导的 mTOR 通路激活依赖于激活的 OX1 受体[3]。 Western Blot 分析[3] 细胞系：INS-1 细胞 浓度：1 μM 孵育时间：24 小时；与 1 μM Orexin-A 一起作用 24 小时 结果：降低 Orexin-A 诱导的 mTOR 磷酸化水平。

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[³H]SB-674042 对表达于CHO细胞中的人OX₁受体表现出可饱和的高亲和力结合，在全细胞中特异性结合占总结合的70%以上，在膜制剂中占80%以上。在野生型CHO细胞膜中未检测到特异性结合。[1]
在使用FLIPR技术的钙动员实验中，SB-674042 能够有效且竞争性地拮抗食欲素-A（10 nM）在表达OX₁受体的CHO细胞中诱导的细胞内钙升高，Kb为 1.1 ± 0.1 nM。[1]
SB-674042 对一系列其他受体（包括5-羟色胺能、多巴胺能、肾上腺素能和嘌呤能受体）在浓度高达10 µM时均未显示显著亲和力，表明其对OX₁受体具有高选择性。[1]

SB-674042（0.3 nM/0.3 μL；icv；单剂量）可减少小鼠压力替代模型 (SMA) 中动物的情境和提示恐惧冻结反应[4]。动物模型：应激小鼠模型（雄性C57BL/6NHsd小鼠，22-26 g）[4] 剂量：0.3 nM/0.3 μL 给药方式：脑室内注射；让小鼠遭受 4 天的社会攻击（第 1-4 天） 结果：小鼠中 39.4% 的逃避表型和 60.6% 的停留表型。

[3H]SB-674042全细胞结合试验[1]
在96孔Packard培养板中培养过夜后，丢弃培养基，在25°C下将细胞在含有150 mM NaCl、20 mM HEPES和0.5%牛血清白蛋白（pH 7.4）的缓冲液中孵育60分钟。通过用不同浓度的[3H]SB-674042（0.2-24 nM）孵育细胞进行饱和研究；总测定体积为250μl。通过用0.1M NaOH裂解细胞并使用Bradford法（Bradford，1976）以牛血清白蛋白（BSA）为标准来测定蛋白质含量。
通过在加入[3H]SB-674042后1-60分钟测量[3H]SB-674042（3 nM）的特异性结合来进行关联动力学研究。对于解离研究，细胞首先与[3H]SB-674042（3 nM）孵育60分钟。然后在加入3μM SB-408124后2-120分钟测量特异性结合。通过用[3H]SB-674042（3 nM）和不同浓度的测试化合物孵育细胞进行竞争研究。用250μl冰冷的磷酸盐缓冲盐水洗涤细胞三次，终止所有测定。向每个孔中加入100μl Microscint 40，将平板在室温下放置2小时。然后使用Packard Topcount测量细胞相关放射性，计数时间为2分钟孔-1。

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[3H]SB-674042基于膜的SPA结合测定[1]
CHO-K1_OX1细胞膜（75μg ml−1）通过与小麦胚芽凝集素-聚乙烯基甲苯（WGA-PVT）闪烁邻近试验（SPA）珠（5 mg ml−l）在含有25 mM HEPES、2.5 mM MgCl2、0.5 mM EDTA和0.025%杆菌肽（pH 7.4）的缓冲液中在4°C下振荡1小时进行预偶联。珠膜悬浮液在300×g下离心，并重新悬浮在相同体积的室温试验缓冲液中。在96孔Packard Optiplate中，将100μl的珠膜悬浮液与[3H]SB-674042（5 nM）一起孵育，总测定体积为200μl，使最终蛋白质浓度为7.5μg孔-1。非特异性结合是在3μM SB-408124存在下剩余的结合。将分析板摇动10分钟，然后在室温下孵育4小时，然后在Packard TopCount闪烁计数器上计数（计数时间2分钟，孔-1）。
通过在[3H]SB-674042（0.1-20 nM）的浓度范围内孵育珠膜（相当于7.5μg蛋白质孔-1和2.5 mg珠ml-1）进行饱和研究。使用Bradford法（Bradford，1976）以牛血清白蛋白为标准测定蛋白质含量。通过在添加珠膜（相当于7.5μg蛋白质孔-1和2.5 mg珠ml-1）后1-30分钟测量[3H]SB-674042（5 nM）的特异性结合来进行关联动力学研究。对于解离研究，首先将珠膜与[3H]SB-674042（5 nM）一起孵育30分钟。然后在加入3μM SB-408124后2-120分钟测量特异性结合。通过用[3H]SB-674042（5 nM）和不同浓度的测试化合物孵育珠膜（相当于7.5μg蛋白质孔-1和2.5 mg珠ml-1）进行竞争研究。

细胞系：INS-1细胞
浓度：1 μM
孵育时间：24小时；与 1 μM Orexin-A 一起作用 24 小时
结果：降低 Orexin-A 诱导的 mTOR 磷酸化水平。

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培养大鼠胰岛素瘤INS-1细胞，并用不同浓度的食欲素-A、有或没有OX1受体选择性拮抗剂SB-674042或磷脂酰肌醇3-激酶/mTOR拮抗剂PF-04691502处理。使用INS-1细胞进行胰岛素释放实验、蛋白质印迹分析和统计分析。
结果：我们的结果表明，用食欲素-A处理细胞会以浓度依赖的方式增加OX1受体的表达和mTOR的磷酸化。用食欲素-A处理的细胞也观察到胰岛素分泌的增加。我们通过使用OX1受体选择性拮抗剂SB-674042或磷脂酰肌醇3-激酶/mTOR拮抗剂PF-04691502进一步证明，胰岛素分泌的减少取决于OX1受体和mTOR信号通路的激活，这消除了食欲素-A治疗的影响。
结论：我们的研究结果表明，食欲素-A/OX1受体通过激活AKT及其下游靶点mTOR来刺激胰岛素分泌。因此，食欲素可能在mTOR的参与下调节细胞存活的能量平衡[3]。

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全细胞放射性配体结合实验： 将稳定表达人OX₁受体的CHO-K1细胞接种于96孔板。进行饱和实验时，将细胞与一系列浓度（0.2-24 nM）的[³H]SB-674042在测定缓冲液中孵育；进行竞争实验时，使用固定浓度（3 nM）。非特异性结合在3 µM SB-408124存在下定义。孵育后，用冰预冷的缓冲液洗涤细胞，添加闪烁液，并使用微板闪烁计数器定量细胞相关的放射性。[1]
基于膜的SPA结合实验： 将来自CHO-K1_OX₁细胞的膜与小麦胚芽凝集素包被的闪烁亲近测定（SPA）珠预偶联。将珠-膜复合物与[³H]SB-674042（饱和实验0.1-20 nM；竞争实验5 nM）在测定缓冲液中孵育。非特异性结合用3 µM SB-408124确定。振荡孔板，孵育，然后在闪烁计数器上计数。[1]
钙动员实验（FLIPR）： 将稳定表达人OX₁或OX₂受体的CHO-DG44细胞接种于96孔板，并用钙敏感荧光染料Fluo-3 AM加载。洗涤细胞后，与或不与SB-674042（或其他拮抗剂）孵育30分钟。使用荧光成像板读数仪（FLIPR）监测添加10 nM食欲素-A前后的荧光信号。根据对食欲素-A诱导的钙反应的抑制曲线计算拮抗剂的效价（Kb）。[1]

Stress-induced mice model (male C57BL/6NHsd mice, 22-26 g)
0.3 nM/0.3 μL
Intracerebroventricular injection; subjected mice to 4 days of social aggression (days 1-4)

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The primary treatments for these experiments is inhibition of BLA Orx1R, via the antagonist SB-674042 (0.3 nmol/0.3 μL delivered bilaterally intra-BLA, 1h prior to interaction on Day 3), contrasted with Orx1R stimulation (accomplished by OrxA + Orx2R antagonism), or short-hairpin knockdown (bilateral intra-BLA transfection beginning 30 days prior to SAM interaction). Considering the difference in timing of delivery, these treatments were done and analyzed separately, with a priori hypotheses. All behavioral measures were performed during the dark cycle when the animals are active, and included Escape (use of the apical tunnels), Stay (remaining in the SAM arena with the novel aggressor), time spent attentive to the escape hole, latency to escape (for Escape mice), fear conditioned freezing (measured in response to the tone [CS] and context, prior to the social interaction unconditioned stimulus [US], and as a conditioned response [CR on Day 5] in the absence of the US), and food intake. Thus, treatment groups included home cage controls, and intra-BLA SB-674042 (or vehicle, OrxA, OrxA + MK-1064, MK-1064) injection of Escape and Stay mice. In addition, transgenic treatment groups included home cage controls, intra-BLA AAV-Orx1R-shRNA injection, and intra-BLA AAV-scramble-shRNA injection. Brains and blood were collected for visual representations of gene expression (using RNAscope) of HCRTR1, HCRTR2, calbindin (CALB1), Ca++/Calmodulin Kinase type 2 alpha (CAMKIIα), Glutamate Decarboxylase (GAD1), and parvalbumin (PVALB) in BLA, as well as to measure plasma concentrations of the stress hormone corticosterone (by enzyme linked immunosorbent assay). Gene expression (using RT-qPCR) of HCRTR1, HCRTR2, PLCB1, MAPK1, MAPK3, BDNF, and GAPDH (housekeeping gene) were measured in BLA tissue. All experimental designs and statistical analyses were based on a priori hypotheses, using two-way repeated measures ANOVA, two-way ANOVA, one-way ANOVA, Regression analyses, and t-test, followed (where appropriate) by post hoc analyses.[4]

[1]. Characterisation of the binding of [³H]-SB-674042, a novel nonpeptide antagonist, to the human orexin-1 receptor. Br J Pharmacol. 2004 Jan;141(2):340-6.

[2]. Orexin-1 receptor-cannabinoid CB1 receptor heterodimerization results in both ligand-dependent and -independent coordinated alterations of receptor localization and function. J Biol Chem. 2006 Dec 15;281(50):38812-24.

[3]. Orexin-A Stimulates Insulin Secretion Through the Activation of the OX1 Receptor and Mammalian Target of Rapamycin in Rat Insulinoma Cells. Pancreas. 2019 Apr;48(4):568-573.

[4]. Orexin 1 Receptor Antagonism in the Basolateral Amygdala Shifts the Balance From Pro- to Antistress Signaling and Behavior. Biol Psychiatry. 2022 May 1;91(9):841-852.

1. This study characterized the binding of a novel non-peptide antagonist radioligand, [(3)H]SB-674042 (1-(5-(2-fluorophenyl)-2-methylthiazolyl-4-yl)-1-((S)-2-(5-phenyl-(1,3,4)oxadiazol-2-ylmethyl)-pyrrolidine-1-yl)-methyl ketone), to the human orexin-1 (OX(1)) receptor stably expressed in Chinese hamster ovary (CHO) cells. Two methods were employed: whole-cell assay and cell membrane-based scintillation proximity assay (SPA). 2. The specific binding of [(3)H]SB-674042 was saturated in both whole-cell and cell membrane assays. Analysis revealed the existence of a high-affinity binding site, with K_d values of 3.76±0.45 nM and 5.03±0.31 nM in the whole-cell and membrane-bound states, respectively, and corresponding B_max values of 30.8±1.8 pmol/mg protein and 34.4±2.0 pmol/mg protein, respectively. Kinetic studies also yielded similar K_d values. 3. Whole-cell competition experiments showed that natural orexin peptides have low affinity for the OX₁ receptor, with orexin A having an affinity approximately five times that of orexin B (K_i values of 318±158 nM and 1516±597 nM, respectively). 4. SB-334867, SB-408124 (1-(6,8-difluoro-2-methyl-quinoline-4-yl)-3-(4-dimethylaminophenyl)-urea) and SB-410220 (1-(5,8-difluoro-quinoline-4-yl)-3-(4-dimethylaminophenyl)-urea) all showed high affinity for the OX(1) receptor in whole-cell (K(i) values of 99±18, 57±8.3 and 19±4.5 nm, respectively) and membrane (K(i) values of 38±3.6, 27±4.1 and 4.5±0.2 nm, respectively). 5. Calcium mobilization studies have shown that SB-334867, SB-408124 and SB-410220 are functional antagonists of the OX(1) receptor, with efficacy consistent with their affinity measured in radioligand binding assays, and a selectivity of approximately 50-fold for the orexin-2 receptor. 6. These studies have shown that [(3)H]SB-674042 is a specific high-affinity radioligand for the OX(1) receptor. The emergence of such a radioligand will be a valuable tool for studying the physiological function of the OX(1) receptor. [1] After induction of expression in HEK293 cells, the human orexin-1 receptor is targeted to the cell surface, but undergoes endocytosis after exposure to the peptide agonist orexin A. In contrast, constitutive expression of the human cannabinoid CB1 receptor mainly results in its punctate distribution within the cell, consistent with spontaneous, non-agonist-dependent endocytosis. Expression of the orexin-1 receptor in the presence of the CB1 receptor resulted in both receptors exhibiting a spontaneous endocytic phenotype. Single-cell fluorescence resonance energy transfer imaging showed that both receptors existed in intracellular vesicles as heterodimers/oligomers. Addition of the CB1 receptor antagonist SR-141716A to cells expressing only the CB1 receptor led to the receptor's relocation to the cell surface. Although SR-141716A did not show significant affinity for the orexin-1 receptor, treatment with SR-141716A also induced the relocation of the orexin-1 receptor to the cell surface in cells co-expressing the CB1 receptor. Treatment of cells co-expressing both orexin-1 and CB1 receptors with the orexin-1 receptor antagonist SB-674042 also resulted in the relocation of both receptors to the cell surface. SR-141716A treatment reduced the potency of orexin A in activating mitogen-activated protein kinases ERK1/2 only in cells co-expressing both receptors. SB-674042 treatment also reduced the potency of the CB1 receptor agonist phosphorylation of ERK1/2 only when both receptors were co-expressed. These studies introduce a novel pharmacological paradigm in which ligands modulate the function of receptors with which they have no significant affinity by modulating receptor heterodimers. [2]

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Background: Stress produces different behavioral responses by selectively modifying specific neural circuit elements. The orexin (Orx) system targets key components of this neural circuit in the basolateral amygdala (BLA). Methods: We evaluated the role of Orx1 receptors (Orx1Rs) in stress-induced phenotypic expression in mice. We characterized the role of Orx1Rs in the basolateral amygdala (BLA) using a stress substitution model (a social stress paradigm that produces two behavioral phenotypes) with a strategy of acute pharmacological inhibition (SB-674042) and gene knockdown (AAV-U6-Orx1R-shRNA). Results: In the basolateral amygdala (BLA), we observed that Orx1R (Hcrtr1) mRNA was primarily expressed in CamKIIα+ glutamatergic neurons, but less so in GABAergic (γ-aminobutyric acid) cells. Although there was slight overlap in the expression of Hcrtr1 and Orx2 receptor (Hcrtr2) mRNAs in the BLA, we found that these receptors are typically expressed in different cells. Following phenotype formation, antagonism of Orx1R in the BLA shifted behavioral expression from stress-sensitive (staying) to stress-tolerant (escape) behavior, an effect mimicked by gene knockdown. Acute inhibition of Orx1R in the BLA also reduced situational fear and cue-induced fear freeze responses in staying animals. This phenotype-specific behavioral change was accompanied by biased molecular transcription, namely Hcrtr2 over Hcrtr1, Mapk3 over Plcb1, and elevated Bdnf mRNA levels. Conclusion: Antagonism of Orx1R leads to functional reorganization of gene expression within the BLA, thereby promoting increased expression of Hcrtr2, Mapk3, and Bdnf. These results collectively provide evidence for the receptor-driven mechanism of balanced stress and anti-stress responses in the basolateral amygdala (BLA). [4]

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SB-674042 was identified as 1-(5-(2-fluorophenyl)-2-methylthiazolyl-4-yl)-1-((S)-2-(5-phenyl-(1,3,4)oxadiazol-2-ylmethyl)-pyrrolidine-1-yl)-methyl ketone. [1]
The radiolabeled version [³H]SB-674042 (specific activity 27 Ci mmol⁻¹) is considered the first selective non-peptide OX₁ receptor antagonist radioligand. It is a valuable tool compound for studying the localization and function of OX₁ receptors. [1]
Kinetic studies showed that the binding of [³H]SB-674042 to the OX₁ receptor reached equilibrium within 30-60 minutes, and the dissociation induced by excess non-competitive agent was monophasic. [1]

C24H21FN4O2S

448.51254

448.137

C, 64.27; H, 4.72; F, 4.24; N, 12.49; O, 7.13; S, 7.15

483313-22-0

10204153

White to off-white solid powder

5.092

100.36

0

7

5

32

652

1

FC1=C(C2=C(C(N3[C@H](CC4=NN=C(C5=CC=CC=C5)O4)CCC3)=O)N=C(C)S2)C=CC=C1

HYBZWVLPALMACV-KRWDZBQOSA-N

InChI=1S/C24H21FN4O2S/c1-15-26-21(22(32-15)18-11-5-6-12-19(18)25)24(30)29-13-7-10-17(29)14-20-27-28-23(31-20)16-8-3-2-4-9-16/h2-6,8-9,11-12,17H,7,10,13-14H2,1H3/t17-/m0/s1

[5-(2-fluorophenyl)-2-methyl-1,3-thiazol-4-yl]-[(2S)-2-[(5-phenyl-1,3,4-oxadiazol-2-yl)methyl]pyrrolidin-1-yl]methanone

SB-674042; SB 674042; SB-674042; 483313-22-0; SB 674042; SB674042; [5-(2-fluorophenyl)-2-methyl-1,3-thiazol-4-yl]-[(2S)-2-[(5-phenyl-1,3,4-oxadiazol-2-yl)methyl]pyrrolidin-1-yl]methanone; CHEMBL2110363; DTXSID90436738; (S)-(5-(2-Fluorophenyl)-2-methylthiazol-4-yl)(2-((5-phenyl-1,3,4-oxadiazol-2-yl)methyl)pyrrolidin-1-yl)methanone; SB674042

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: ~25 mg/mL (~55.7 mM)

配方 1 中的溶解度: ≥ 1.43 mg/mL (3.19 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (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 14.3 mg/mL澄清DMSO储备液加入400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 1.43 mg/mL (3.19 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (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 14.3mg/mL澄清的DMSO储备液加入到900μL 20％SBE-β-CD生理盐水中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 1.43 mg/mL (3.19 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 14.3 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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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：
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10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
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