Non-peptide neuropeptide Y (NPY) Y2 receptor (IC50 = 15 nM)

在用大鼠Y2受体cDNA转染的HEK 293细胞中，受体结合测试表明BIIE-0246以高亲和力竞争特定的[125I]PYY3-36结合位点(IC50=15±3nM)。另一方面，在转染的 HEK 293 细胞中，即使浓度高达至 10 μM。它们是大鼠 Y1、Y4 或 Y5 受体的 cDNA [1]。

当喂食饲料时，遗传性肥胖的 NPY 小鼠具有较高的体重和肥胖水平。在两种基因型中，BIIE-0246 治疗在 4.5 周和 2 周后均导致体重增加。对于脂肪量的增加，BIIE-0246 没有明显的效果。 BIIE-0246 对 DIO 中的体重和成分的影响因基因型而异（治疗 × 基因型相互作用，体重 P < 0.05，脂肪量 P < 0.001，瘦肉量 P < 0.05）。事后分析显示，DIO-WT 组的去脂体重增加较少，体重和脂肪量增加较多的趋势。 BIIE-0246 阻止 DIO-NPY 脂肪量增加 (P=0.05)。有趣的是，在 4.5 周的队列中没有观察到较高的胆固醇水平，但在给予 BIIE-0246 两周的 WT 小鼠中却出现了较高的胆固醇水平。在两个 DIO-NPY 治疗组中（DIO-NPY 媒介物 P<0.01；DIO-NPY BIIE-0246 P<0.001），胆固醇水平与体脂量呈正相关；在任何其他组中都没有观察到这种关系。此外，用 BIIE-0246 治疗的 DIO-NPY 组的脂肪量和胆固醇的回归曲线显着低于用媒介物治疗的组 [2]。

结合分析[1]
所有结合试验都是通过在最终体积为500μl的KRP中加入100μl的膜或细胞制剂来启动的，KRP含有0.1%（w v−1）BSA、0.05%（w v-1）杆菌肽放射性标记探针和未标记的肽或竞争对手。在放射性标记探针浓度增加的情况下进行等温饱和，而在存在和不存在各种竞争对手的情况下，使用30-35pM的放射性标记探针进行竞争结合实验，浓度范围为10-12-10-6M。在大鼠脑匀浆中，分别使用[125I[Leu31，Pro34]PYY和[125I]PYY3-36研究Y1样和Y2样受体，如前所述（Dumont等人，1995）。[125I]GR231118和[125I][Leu31，Pro34]PYY分别用于转染大鼠Y1和Y5受体cDNA的HEK 293细胞。分别使用[125I]PYY3-36和[125I]hPP在转染有大鼠Y2和Y4受体cDNA的HEK 293细胞中进行结合。在1μM pNPY存在下测定非特异性结合。孵育2小时后，使用细胞收获器过滤装置通过Schleicher和Schuell#32玻璃过滤器（之前浸泡在1.0%聚乙烯亚胺中）快速过滤终止结合反应。用3ml冷KRP冲洗过滤器三次，使用效率为85%的伽马计数器对过滤器上残留的放射性进行定量。[1]
所有结合实验重复三到六次，每次重复三次，结果以特异性结合（竞争）或fmol（饱和）的百分比表示。Kd值（即占据总受体群体50%所需的放射性配体浓度）是使用GraphPad Prism软件从饱和等温线结合实验中获得的数据计算出来的，并拟合为单位点双曲线。使用GraphPad Prism软件根据竞争结合测定数据计算各种肽和拮抗剂的IC50值（即竞争50%的放射性配体特异性结合所需的未标记肽的浓度），并拟合到S形剂量反应曲线。

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定量受体放射自显影术[1]
受体放射自显影如其他地方详细描述的那样进行（Dumont等人，1996；1998a，1998b；Jacques等人，1997）。所有切片（20μm）均使用-17°C的冷冻切片机获得，安装在明胶-铬-明矾涂层载玻片上，在4°C的干燥器中干燥过夜，然后保持在-80°C直至使用。 [1]
在实验当天，相邻的冠状切片在室温下在pH 7.4的KRP缓冲液中预孵育60分钟，然后在含有0.1%BSA、0.05%杆菌肽、30 pM[125I]PYY3-36和不同浓度的BIIE0246（10-10-10-5M）的新鲜KRP缓冲溶液中孵育120分钟。孵育2小时后，将切片在冰冷的KRP缓冲液中洗涤四次，每次2分钟，然后浸入去离子水中去除盐分并快速干燥。使用1μM NPY测定非特异性结合。将孵育切片与3H Hyperfilms一起与放射性标准物放置6天。胶片的显影和定量如其他地方详细描述的那样（Dumont等人，1996；1998a）。

体外生物测定[1]
兔（Cadieux等人，1993）和狗（Pheng等人，1997）的隐静脉、大鼠输精管（Martel等人，1990；Dumont等人，1994）、大鼠结肠（Pheng等，1999）和人脑动脉（Abounader等人，1995）的制备如其他地方详细描述的。通过累积添加兔隐静脉和人脑动脉（NPY诱导的收缩）以及大鼠输精管（NPY抑制电刺激抽搐反应）的肽，生成了对NPY的浓度反应曲线，而对大鼠结肠（NPY诱发的收缩）则采用非累积方式。在这些组织中，通过在NPY前10分钟施加不同浓度或单一最大浓度（1μM）的BIIE0246，研究了BIIE0246的拮抗特性。在犬隐静脉中，通过在NPY预收缩组织上累积添加BIIE0246，研究了BIIE0246阻断NPY收缩作用的能力。[1]
通过绘制NPY的摩尔浓度与以最大响应百分比表示的响应的关系图，构建了浓度响应曲线。从这些图中，通过非线性回归分析（S形剂量反应曲线）计算EC50值。根据每条曲线计算EC50值，并根据这些数据计算大鼠输精管、兔隐静脉和人脑动脉的平均值±s.e.mean。对于每种使用的拮抗剂浓度，浓度比是通过将拮抗剂存在下NPY的EC50值除以阻断剂不存在时获得的EC50来计算的。构建Schild图，并使用线性回归确定x截距（pA2值）。对于犬隐静脉，pA2值被确定为将NPY的收缩作用降低50%所需的BIIE0246浓度。

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转染细胞[1]
HEK 293细胞在添加了10%胎牛血清和抗生素（青霉素G钠、硫酸链霉素和两性霉素B）的Dulbecco改良Eagle培养基（D-MEM）中维持。使用磷酸钙法用大鼠Y1、Y2、Y4或Y5受体cDNA中的任一种转染培养的细胞（Tong等人，1995）。简要地，将125μl 2.5 M磷酸钙加入到1.125 ml水中，水中含有50μg大鼠Y1、Y2、Y4或Y5受体cDNA，该cDNA先前插入表达pcDNA3载体中，并与1.25 ml pH 7.05的2×HEPES缓冲液缓慢混合，在室温下放置20分钟。将混合物加入到含有30%融合的HEK 293细胞的150 mm培养皿中，然后返回培养箱。第二天早上换了媒介。48小时后，用pH 7.4的KRP缓冲液洗涤细胞并刮除。然后将分离的细胞在400×g下离心10分钟，用KRP缓冲液（pH 7.4), 再次离心两次并重新悬浮在8ml pH 7.4的KRP缓冲液中并用于受体结合测定。

Drug treatment was studied at the age of 20 weeks. Prior to treatments the mice were habituated for 2 weeks to the handling stress with daily saline injections (i.p.). Half of the chow-fed mice (n = 10–13/group) were treated for 4.5 weeks, and half were sacrificed and tissues collected already after 2-week treatment (n = 7–14/group). DIO mice (n = 7–12/group) were treated for 2 weeks. (The study protocol is presented in Supplementary Figure 1). Mice received 1.3 mg/kg of Y2-receptor antagonist (BIIE0246, Tocris Bioscience, Bristol, United Kingdom) or vehicle (DMSO, Tween® 80) (Fisher Scientific, Fair Lawn, NJ, United States and 0.9% NaCl, 1:1:18, respectively) with daily intraperitoneal (i.p.) injections. BIIE0246 with a dose of 2 mg/kg has previously been used in a study with acute administration (Forbes et al., 2012), which supports rationality of the dose (1.3 mg/kg) being used with repeated dosing in the present study. The half-life of BIIE0246 in mouse is less than 3 h, but markedly longer than the half-life of the other Y2-receptor antagonists, thus making it the most suitable Y2-receptor antagonist for chronic administration (Brothers et al., 2010). [2]

[1]. BIIE0246, a potent and highly selective non-peptide neuropeptide Y Y(2) receptor antagonist. Br J Pharmacol. 2000 Mar;129(6):1075-88.

[2]. Peripherally Administered Y2-Receptor Antagonist BIIE0246 Prevents Diet-Induced Obesity in Mice With Excess Neuropeptide Y, but Enhances Obesity in Control Mice. Front Pharmacol. 2018; 9: 319.

1. BIIE0246, a newly synthesized non-peptide neuropeptide Y (NPY) Y(2) receptor antagonist, was able to compete with high affinity (8 to 15 nM) for specific [(125)I]PYY(3 - 36) binding sites in HEK293 cells transfected with the rat Y(2) receptor cDNA, and in rat brain and human frontal cortex membrane homogenates. 2. Interestingly, in rat brain homogenates while NPY, C2-NPY and PYY(3 - 36) inhibited all specific [(125)I]PYY(3 - 36) labelling, BIIE0246 failed to compete for all specific binding suggesting that [(125)I]PYY(3 - 36) recognized, in addition to the Y(2) subtype, another population of specific NPY binding sites, most likely the Y(5) receptor. 3. Quantitative receptor autoradiographic data confirmed the presence of [(125)I]PYY(3 - 36)/BIIE0246-sensitive (Y(2)) and-insensitive (Y(5)) binding sites in the rat brain as well as in the marmoset monkey and human hippocampal formation. 4. In the rat vas deferens and dog saphenous vein (two prototypical Y(2) bioassays), BIIE0246 induced parallel shifts to the right of NPY concentration-response curves with pA(2) values of 8.1 and 8.6, respectively. In the rat colon (a Y(2)/Y(4) bioassay), BIIE0246 (1 microM) completely blocked the contraction induced by PYY(3 - 36), but not that of [Leu(31), Pro(34)]NPY (a Y(1), Y(4) and Y(5) agonist) and hPP (a Y(4) and Y(5) agonist). Additionally, BIIE0246 failed to alter the contractile effects of NPY in prototypical Y(1) in vitro bioassays. 5. Taken together, these results demonstrate that BIIE0246 is a highly potent, high affinity antagonist selective for the Y(2) receptor subtype. It should prove most useful to establish further the functional role of the Y(2) receptor in the organism.[1]

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In summary, we have demonstrated using several receptor binding assays and in vitro bioassays that BIIE0246 is a potent and selective Y2 receptor antagonist devoid of high affinity for the Y1, Y4 and Y5 subtypes. To our knowledge, BIIE0246 represents the first potent and selective tool to precisely establish the potential roles of the Y2 receptor in various tissues and to molecularly dissect features of agonist vs antagonist recognition sites on this receptor. The availability of BIIE0246, in addition to Y2 knockout mice (Naveilhan et al., 1999) should also prove critical to demonstrate the involvement of this subtype in a given effect induced by NPY and related peptides.[1]

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Neuropeptide Y (NPY) plays an important role in the regulation of energy homeostasis in the level of central and sympathetic nervous systems (SNSs). Genetic silencing of peripheral Y2-receptors have anti-obesity effects, but it is not known whether pharmacological blocking of peripheral Y2-receptors would similarly benefit energy homeostasis. The effects of a peripherally administered Y2-receptor antagonist were studied in healthy and energy-rich conditions with or without excess NPY. Genetically obese mice overexpressing NPY in brain noradrenergic nerves and SNS (OE-NPYDβH) represented the situation of elevated NPY levels, while wildtype (WT) mice represented the normal NPY levels. Specific Y2-receptor antagonist, BIIE0246, was administered (1.3 mg/kg/day, i.p.) for 2 or 4.5 weeks to OE-NPYDβH and WT mice feeding on chow or Western diet. Treatment with Y2-receptor antagonist increased body weight gain in both genotypes on chow diet and caused metabolic disturbances (e.g., hyperinsulinemia and hypercholesterolemia), especially in WT mice. During energy surplus (i.e., on Western diet), blocking of Y2-receptors induced obesity in WT mice, whereas OE-NPYDβH mice showed reduced fat mass gain, hepatic glycogen and serum cholesterol levels relative to body adiposity. Thus, it can be concluded that with normal NPY levels, peripheral Y2-receptor antagonist has no potential for treating obesity, but oppositely may even induce metabolic disorders. However, when energy-rich diet is combined with elevated NPY levels, e.g., stress combined with an unhealthy diet, Y2-receptor antagonism has beneficial effects on metabolic status.[2]

C49H57N11O6

896.046980000001

895.449

246146-55-4

BIIE-0246 hydrochloride

9811493

White to off-white solid powder

1.7

5.48

221.68

5

8

16

66

1710

1

C1CCC(C1)(CC(=O)N[C@@H](CCCN=C(N)N)C(=O)NCCN2C(=O)N(N(C2=O)C3=CC=CC=C3)C4=CC=CC=C4)CC(=O)N5CCN(CC5)C6C7=CC=CC=C7C(=O)NC8=CC=CC=C68

RSJAXPUYVJKAAA-JPGJPTAESA-N

InChI=1S/C49H57N11O6/c50-46(51)53-25-13-22-40(45(64)52-26-27-58-47(65)59(34-14-3-1-4-15-34)60(48(58)66)35-16-5-2-6-17-35)54-41(61)32-49(23-11-12-24-49)33-42(62)56-28-30-57(31-29-56)43-36-18-7-8-19-37(36)44(63)55-39-21-10-9-20-38(39)43/h1-10,14-21,40,43H,11-13,22-33H2,(H,52,64)(H,54,61)(H,55,63)(H4,50,51,53)/t40-,43?/m0/s1

(2S)-5-(diaminomethylideneamino)-N-[2-(3,5-dioxo-1,2-diphenyl-1,2,4-triazolidin-4-yl)ethyl]-2-[[2-[1-[2-oxo-2-[4-(6-oxo-5,11-dihydrobenzo[c][1]benzazepin-11-yl)piperazin-1-yl]ethyl]cyclopentyl]acetyl]amino]pentanamide

AR-H 053591; BIIE-0246; CHEMBL540989; BIIE-0246; BIIE 0246; 246146-55-4; BIIE0246; Ar-H-053591; (2S)-5-(diaminomethylideneamino)-N-[2-(3,5-dioxo-1,2-diphenyl-1,2,4-triazolidin-4-yl)ethyl]-2-[[2-[1-[2-oxo-2-[4-(6-oxo-5,11-dihydrobenzo[c][1]benzazepin-11-yl)piperazin-1-yl]ethyl]cyclopentyl]acetyl]amino]pentanamide; N3Z657H81X; Cyclopentaneacetamide, N-((1S)-4-((aminoiminomethyl)amino)-1-(((2-(3,5-dioxo-1,2-diphenyl-1,2,4-triazolidin-4-yl)ethyl)amino)carbonyl)butyl)-1-(2-(4-(6,11-dihydro-6-oxo-5H-dibenz(b,E)azepin-11-yl)-1-piperazinyl)-2-oxoethyl)-; GTPL1547; AR H 053591; BIIE-0246; AR-H-053591; AR H053591; ARH053591; BIIE 0246; CTK8E9439; BIIE0246;

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 : ~100 mg/mL (~111.60 mM)

配方 1 中的溶解度: ≥ 2.5 mg/mL (2.79 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 25.0 mg/mL澄清DMSO储备液加入到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: ≥ 2.5 mg/mL (2.79 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 25.0 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，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: ≥ 2.5 mg/mL (2.79 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 25.0 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：
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；
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