ML 254

rat mGluR5 ( EC50 = 9.3 nM ); rat mGluR5 ( EC50 = 8.03 nM )

正变构调节剂(Positive allosteric modulator, PAM)，化合物38t (ML254)是一种低谷氨酸折叠位移的变构配体(最大折叠位移~ 3.0)，在体外化合物表征系统和两种天然电生理系统中均无激动作用。PAM 38t (ML254)将有助于探讨协同性和变构激动作用对这类mGlu5 PAM的不良反应和神经毒性的相对贡献。[1]

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为了验证38t(ML254)是在MPEP结合位点与mGlu5相互作用，我们与[3H]methoxyPEPy进行了放射性配体结合研究。38t浓度的增加导致[3H]甲氧基pepy结合完全抑制，支持两个配体之间的竞争性相互作用(图9)。38t的Ki值为90 nM，与结合相比，其功能活性高10倍(EC50 = 9.3 nM)。为了在体内使用化合物，确定它们与其他mGlu亚型相比是否对mGlu5具有选择性是很重要的。当使用表达任何其他mGlu亚型(mglu1 - 4,6 - 8，见补充材料部分I)的细胞进行评估时，10µM浓度的PAM 38t没有改变谷氨酸(或L-AP4)浓度响应曲线，表明对mGlu5具有高选择性。此外，对68个gpcr、离子通道和转运体的10µM 38t进行筛选，没有发现明显的脱靶活性(Eurofins Inc.)。最后，在渐进折叠移位实验(50 nM至30µM)中评估氧乙烷38t。调制剂浓度增加时谷氨酸浓度响应曲线的变化如图10所示。随着调制剂浓度的增加，在5µM处的fold shift达到最大值约3.0倍，预测亲和力为- 6.81 (154 nM)，谷氨酸和指示变构调制剂之间的功效协同因子(logβ)为0.34(协同性~2.2)。[1]

采用大鼠脑匀浆结合法测定38t(ML254)脑内未结合部分;这些研究表明，fu脑值为1.6%。为了评估药物-药物相互作用，在人肝微粒体中检测了主要的人细胞色素P450 (CYP)酶(2C9, 2D6, 3A4, 1A2)的抑制作用，发现38t (ML254)在1A2处显示抑制活性(IC50 = 5.30µM)，而对其他CYP没有活性(IC50 >30µM)。38t的溶解度适中，空腹模拟肠液溶解度为10-23µg/mL [1]。

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为了验证其在天然系统中的PAM药理学特征，我们检测了38t (ML254)在海马区Schaffer侧支-CA1 (SC-CA1)突触上诱导长期抑郁(LTD)的作用。已知该突触中的LTD受mGlu5激活的调节，并且正位mGlu5激动剂如(S)-3,5- dhpg已被证明可引发LTD.48。类似地，ago-PAM 19可诱导LTD. 38，然而，38t本身不会诱导LTD(图11;复合洗脱后55min基线(100.3±3.7%)。这提供了进一步的证据，证明38t本身不会在原生系统中引起反应。此外，先前涉及19的研究表明，海马制剂中的CA3锥体神经元可诱导癫痫样活动。我们用38t进行了类似的研究，以评估这种天然中枢神经系统制剂的激动剂活性。PAM 38t对自发性放电的事件间间隔(基线的127.9±7.7%)或幅度(基线的101.2±5.0%)均无显著影响，支持38t无激动作用的特征(数据未显示)。这些数据表明，38t在两个海马原生系统中作为纯PAM起作用。[1]

选择性筛选[1]
为了评估测试化合物对mGlu1的影响，按照前面的描述进行了Ca2+动员试验(Hammond等人，2010;Noetzel et al.， 2012)。将稳定表达大鼠mGlu1的HEK293细胞以2万个细胞/孔的密度，简单地镀于黑壁、透明底、聚d -赖氨酸包被的384孔板中。随着时间的推移，钙通量随着Ca2+指示剂Fluo-4AM荧光的增加而测量，使用FDSS 6000。加入载体或固定浓度的测试化合物(10µM，最终浓度)，140秒后加入谷氨酸CRC。数据分析方法如上所述。[1]

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如前所述，通过测量通过GIRK通道的铊通量，在大鼠II组和III组mGlu受体上评估了感兴趣化合物的功能活性(Niswender et al.， 2008)。简单地说，将表达mGlu亚型2、3、4、6、7或8的HEK293-GIRK细胞以15000个细胞/孔的密度，在检测培养基中被镀于384孔、黑壁、清底聚d -赖氨酸包被板中。加入单一浓度的测试化合物(10µM)或对照物，140秒后加入用铊缓冲液(125 mM NaHCO3, 1 mM MgSO4, 1.8 mM CaSO4, 5 mM葡萄糖，12 mM硫酸铊，10 mM HEPES)稀释的谷氨酸CRC(或L-AP4 for mGlu7)，并使用FDSS 6000测量荧光。数据分析如前所述(Niswender et al.， 2008)。

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放射性配体结合[1]
从表达大鼠mGlu5的HEK293A细胞制备膜。收集细胞，离心成球，再悬浮于冷冻匀浆缓冲液(50 mM Tris-HCl, 10 mM EDTA, 0.9% NaCl, pH7.4)中，以3 × 10秒的间歇匀浆。离心分离细胞组分，将所得颗粒重悬于冷冻缓冲液(50 mM Tris-HCl, 0.9% NaCl, pH7.4)中。为了进行抑制结合实验，将膜(50 Yg/孔)与7 nM [3H]甲氧基pepy和一定浓度的测试配体在室温下孵育1小时，并在实验缓冲液中摇晃。10µM MPEP检测非特异性结合。使用Brandel 96孔板收收机快速过滤终止实验，并用冰冷的实验缓冲液洗涤三次。第二天加入MicroScint20并计算放射性。

Fluorescence-Based Calcium Flux Assay (Concentration-response curve (potency) and glutamate fold shift (efficacy)[1]/基于荧光的钙通量测定(浓度-反应曲线(效价)和谷氨酸折叠位移(效价))[1]
为了测量化合物引起的细胞内钙的增加，将稳定表达大鼠mGlu5的HEK293细胞在20µL的检测培养基(DMEM中添加10%的透析胎牛血清，20 mM HEPES和1 mM丙酮酸钠)中，以15000个细胞/孔的密度，在384孔，44个聚d-赖氨酸包被的黑壁清底板中镀上。细胞在37°C/5% CO2下培养过夜。第二天，从细胞中取出培养基，用20µl/孔的1 00B5;M Fluo-4AM配制为2.3 mM的基质，在二甲亚砜(DMSO)中孵育，与10% (w/v) pluronic acid F-127按1:1的比例混合，并在钙测定缓冲液(Hank 's Balanced Salt Solution，添加20 mM HEPES和2.5 mM probenecid, pH 7.4)中稀释，37°C下孵育50分钟。去除染料上样液，用20µl/孔的实验缓冲液代替。对于PAM效价曲线，mGlu5化合物在钙测定缓冲液中稀释后加入细胞，140秒后加入EC20浓度的谷氨酸，60秒后加入EC80浓度的谷氨酸。在折叠移位实验中，加入单一浓度(10µM)或多个固定浓度(50 nM - 30µM)的mGlu5化合物或载体，并在140秒后加入谷氨酸的浓度响应曲线(CRC)。使用功能药物筛选系统6000 (FDSS 6000)测量钙通量随时间的荧光增加。在对谷氨酸的最大反应归一化之前，计算相对荧光在基底上的变化。

Electrophysiology (LTD and epileptiform studies)[1]
All animals used in these studies were cared for in accordance with the NIH Guide for the Care and Use of Laboratory Animals. 30–40 (LTD experiments) or 24–30 (epileptiform experiments) day old male Sprague–Dawley rats were used. The brains were quickly removed and submerged into ice-cold cutting solution (in mM: 110 sucrose, 60 NaCl, 3 KCl, 1.25 NaH2PO4, 28 NaHCO3, 5 glucose, 0.6 (+)-sodium-L-ascorbate, 0.5 CaCl2, 7 MgCl2). All solutions were continuously bubbled with 95% O2/5% CO2. Transverse slices (400 µm) were made using a vibratome. For LTD experiments, individual hippocampi were microdissected out and transferred to a room temperature mixture containing equal volumes of cutting solution and artificial cerebrospinal fluid (ACSF; in mM: 125 NaCl, 2.5 KCl, 1.25 NaH2PO4, 25 NaHCO3, 25 glucose, 2 CaCl2, 1 MgCl2) and equilibrated for 30 min, followed by room temperature ACSF for 1 h. For epileptiform experiments, individual hippocampi were transferred directly into room temperature ACSF (in mM: 124 NaCl, 5 KCl, 1.25 NaH2PO4, 26 NaHCO3, 10 glucose, 2 CaCl2, 1.2 MgSO4) and equilibrated for 1 h. Slices were transferred to a submersion recording chamber and equilibrated for 5–10 min at 30–32°C. A bipolar-stimulating electrode was placed in the stratum radiatum near the CA3-CA1 border in order to stimulate the Schaffer collaterals. Recording electrodes were filled with ACSF and placed in the stratum radiatum of area CA1 (LTD experiments) or in the pyramidal cell body layer of CA3 (epileptiform experiments). Field potential recordings were acquired using a Multiclamp 700B amplifier and pClamp 9.2 software. For stimulation based experiments an intensity that produced 50–60% of the maximum was used as the baseline stimulation. mGlu5 compounds were diluted to the appropriate concentrations in DMSO and applied to the bath using a perfusion system. Sampled data was analyzed by averaging three sequential field excitatory postsynaptic potentials (fEPSPs) slopes, followed by normalizing to the average slope calculated during the predrug period (percent of baseline). For epileptiform experiments, spontaneous events were measured using MiniAnalysis and inter-event interval (IEI) was normalized to the baseline response.

[1]. Exploration of allosteric agonism structure-activity relationships within an acetylene series of metabotropic glutamate receptor 5 (mGlu5) positive allosteric modulators (PAMs): discovery of 5-((3-fluorophenyl)ethynyl)-N-(3-methyloxetan-3-yl)picolinamide (ML254). J Med Chem. 2013;56(20):7976-7996.

Positive allosteric modulators (PAMs) of metabotropic glutamate receptor 5 (mGlu5) represent a promising therapeutic strategy for the treatment of schizophrenia. Both allosteric agonism and high glutamate fold-shift have been implicated in the neurotoxic profile of some mGlu5 PAMs; however, these hypotheses remain to be adequately addressed. To develop tool compounds to probe these hypotheses, the structure-activity relationship of allosteric agonism was examined within an acetylenic series of mGlu5 PAMs exhibiting allosteric agonism in addition to positive allosteric modulation (ago-PAMs). PAM 38t, a low glutamate fold-shift allosteric ligand (maximum fold-shift ~ 3.0), was selected as a potent PAM with no agonism in the in vitro system used for compound characterization and in two native electrophysiological systems using rat hippocampal slices. PAM 38t (ML254) will be useful to probe the relative contribution of cooperativity and allosteric agonism to the adverse effect liability and neurotoxicity associated with this class of mGlu5 PAMs.[1]

C18H15FN2O2

310.322307825089

310.33

C, 69.67; H, 4.87; F, 6.12; N, 9.03; O, 10.31

1428630-86-7

1428630-86-7

53382545

Off-white to light yellow solid powder

2.5

51.2Ų

1

4

4

23

506

0

YMYCVXPMSMNWEP-UHFFFAOYSA-N

InChI=1S/C18H15FN2O2/c1-18(11-23-12-18)21-17(22)16-8-7-14(10-20-16)6-5-13-3-2-4-15(19)9-13/h2-4,7-10H,11-12H2,1H3,(H,21,22)

5-[2-(3-fluorophenyl)ethynyl]-N-(3-methyloxetan-3-yl)pyridine-2-carboxamide

ML 254; VU 0430644; VU0430644-2; VU0430644; 1428630-86-7; 5-[2-(3-fluorophenyl)ethynyl]-N-(3-methyloxetan-3-yl)pyridine-2-carboxamide; CHEMBL2431173; 5-[2-(3-fluorophenyl)ethynyl]-1-(3-methyloxetan-3-yl)-1,6-dihydropyridine-2-carboxamide; 5-[2-(3-fluorophenyl)ethynyl]-N-(3- methyloxetan-3-yl)pyridine- 2-carboxamide; MLS003871695; EX-A4809;VU-0430644; ML-254; ML254

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 (~322.3 mM)

配方 1 中的溶解度: ≥ 2.5 mg/mL (8.06 mM) (饱和度未知) in 10% DMSO + 40% PEG300 +5% Tween-80 + 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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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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7、 以上所有助溶剂都可在 Invivochem.cn网站购买。