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) has a high affinity and can be used as a radioligand to identify human OX1 receptors that are consistently expressed in CHO cells[1].
SB-674042 (5 μM; 4 ℃ for 30 min, and 37 ℃ for 3 h) decreases the ability of the CB1 receptor agonist (HY-14137) to phosphorylate ERK1/2 in HEK293 cells co-expressing the orexin-1 and CB1 receptors[2].
SB-674042 (1 μM; 24 h) eliminates the rise in mTOR phosphorylation in INS-1 cells in response to Orexin-A (HY-106224) (1 nM-1 μM; 24 h), suggesting that the activated OX1 receptor was necessary for the mTOR pathway to be activated by Orexin-A[3].

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[³H]SB-674042 exhibited saturable, high-affinity binding to human OX₁ receptors expressed in CHO cells, with specific binding representing >70% of total binding in whole cells and >80% in membrane preparations. No specific binding was detected in wild-type CHO cell membranes. [1]
In calcium mobilisation assays using FLIPR technology, SB-674042 potently and competitively antagonised orexin-A (10 nM)-induced intracellular calcium increases in CHO cells expressing the OX₁ receptor, with a Kb of 1.1 ± 0.1 nM. [1]
SB-674042 displayed no significant affinity (up to 10 µM) for a panel of other receptors including serotonergic, dopaminergic, adrenergic, and purinergic receptors, indicating high selectivity for the OX₁ receptor. [1]

SB-674042 (0.3 nM/0.3 μL; icv; single dose) decreases contextual and cues fear freezing responses in Stay animals in the Stress Alternatives Model (SMA) in mice[4].

[3H]SB-674042 whole cell binding assays[1]
After overnight culture in 96-well Packard Cultur plates, the medium was discarded and cells were incubated in buffer containing 150 mM NaCl, 20 mM HEPES and 0.5% bovine serum albumin (pH 7.4) for 60 min at 25°C. Saturation studies were carried out by incubating cells with a range of concentrations of [3H]SB-674042 (0.2–24 nM); the total assay volume was 250 μl. Protein content was assayed by lysing cells with 0.1 M NaOH and using the Bradford method (Bradford, 1976) with bovine serum albumin (BSA) as a standard.
Association kinetic studies were performed by measuring the specific binding of [3H]SB-674042 (3 nM) at 1–60 min after addition of [3H]SB-674042. For dissociation studies, cells were first incubated with [3H]SB-674042 (3 nM) for 60 min. Specific binding was then measured at 2–120 min after the addition of 3 μM SB-408124. Competition studies were performed by incubating cells with [3H]SB-674042 (3 nM) and a range of concentrations of the test compound. All assays were terminated by washing the cells three times with 250 μl ice-cold phosphate-buffered saline. A volume of 100 μl of Microscint 40 was added to each well and the plate was left at room temperature for 2 h. Cell-associated radioactivity was then measured using a Packard Topcount, with a count time of 2 min well−1.

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[3H]SB-674042 membrane-based SPA binding assays[1]
CHO-K1_OX1 cell membranes (75 μg ml−1) were precoupled by shaking with wheatgerm-agglutinin polyvinyltoluene (WGA-PVT) scintillation proximity assay (SPA) beads (5 mg ml−1) in buffer containing 25 mM HEPES, 2.5 mM MgCl2, 0.5 mM EDTA and 0.025% bacitracin (pH 7.4) at 4°C for 1 h. The bead-membrane suspension was centrifuged at 300 × g and resuspended in the same volume of room temperature assay buffer. A volume of 100 μl of bead-membrane suspension was incubated with [3H]SB-674042 (5 nM) in a total assay volume of 200 μl in a 96-well Packard Optiplate to give a final protein concentration of 7.5 μg well−1. Nonspecific binding was measured as that remaining in the presence of 3 μM SB-408124. Assay plates were shaken for 10 min and then incubated at room temperature for 4 h before being counted on a Packard TopCount scintillation counter (count time 2 min well−1).
Saturation studies were carried out by incubating bead-membranes (equivalent to 7.5 μg protein well−1 and 2.5 mg beads ml−1) with a range of concentrations of [3H]SB-674042 (0.1–20 nM). Protein content was assayed using the Bradford method (Bradford, 1976) using bovine serum albumin as a standard. Association kinetic studies were performed by measuring specific binding of [3H]SB-674042 (5 nM) at 1–30 min after addition of bead-membranes (equivalent to 7.5 μg protein well−1 and 2.5 mg beads ml−1). For dissociation studies, bead-membranes were first incubated with [3H]SB-674042 (5 nM) for 30 min. Specific binding was then measured at 2–120 min after the addition of 3 μM SB-408124. Competition studies were performed by incubating bead-membranes (equivalent to 7.5 μg protein well−1 and 2.5 mg beads ml−1) with [3H]SB-674042 (5 nM) and a range of concentrations of the test compound.

Cell Line: INS-1 cells
Concentration: 1 μM
Incubation Time: 24 hours; accompanied with 1 μM Orexin-A for 24 hour
Result: Decreased the phosphorylation level of mTOR induced by Orexin-A

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Rat insulinoma INS-1 cells were grown and treated with various concentrations of orexin-A, with or without OX1 receptor-selective antagonist SB-674042 or the phosphatidylinositol 3-kinase/mTOR antagonist PF-04691502. Insulin release experiments, Western blot analysis, and statistical analysis were conducted using INS-1 cells.
Results: Our results showed that treating cells with orexin-A increased the expression of the OX1 receptor and the phosphorylation of mTOR in a concentration-dependent manner. An increase in insulin secretion was also observed for cells treated with orexin-A. We further demonstrated that the increase in insulin secretion was dependent on the activation of the OX1 receptor and mTOR signaling pathway by using the OX1 receptor-selective antagonist SB-674042 or the phosphatidylinositol 3-kinase/mTOR antagonist PF-04691502, which abolished the effects of orexin-A treatment.
Conclusions: Our results concluded that orexin-A/OX1 receptor stimulates insulin secretion by activating AKT and its downstream target, mTOR. Therefore, orexins may regulate the energy balance for cell survival with the involvement of mTOR in this process[3].

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Whole-Cell Radioligand Binding Assay: CHO-K1 cells stably expressing the human OX₁ receptor were seeded in 96-well plates. Cells were incubated in assay buffer containing a range of concentrations of [³H]SB-674042 (0.2-24 nM) for saturation studies, or with a fixed concentration (3 nM) for competition studies. Nonspecific binding was defined in the presence of 3 µM SB-408124. After incubation, cells were washed with ice-cold buffer, scintillation fluid was added, and cell-associated radioactivity was quantified using a microplate scintillation counter. [1]
Membrane-Based SPA Binding Assay: Membranes from CHO-K1_OX₁ cells were precoupled to wheatgerm agglutinin-coated scintillation proximity assay (SPA) beads. The bead-membrane complex was incubated with [³H]SB-674042 (0.1-20 nM for saturation; 5 nM for competition) in assay buffer. Nonspecific binding was determined with 3 µM SB-408124. Plates were shaken, incubated, and counted on a scintillation counter. [1]
Calcium Mobilisation Assay (FLIPR): CHO-DG44 cells stably expressing human OX₁ or OX₂ receptors were seeded in 96-well plates and loaded with the calcium-sensitive fluorescent dye Fluo-3 AM. Cells were washed and incubated with or without SB-674042 (or other antagonists) for 30 minutes. Fluorescence was monitored before and after the addition of 10 nM orexin-A using a fluorometric imaging plate reader (FLIPR). The antagonist's potency (Kb) was calculated from the inhibition curve of the orexin-A-induced calcium response. [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

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)

Solubility in Formulation 1: ≥ 1.43 mg/mL (3.19 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 14.3 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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Solubility in Formulation 2: ≥ 1.43 mg/mL (3.19 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 14.3 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 1.43 mg/mL (3.19 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 14.3 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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