AR-C155858

MCT1/2 (monocarboxylate transporter) (Ki = 2.3 nM and 10 nM)

MCT1/MCT2 C 端嵌合体被 AR-C155858 (10 nM–100 nM) 抑制 [1]。 MCT2 被 AR-C155858 抑制，在 10 nM 时抑制率为 70%，并且抑制逐渐增加，仅与远低于 10 nM 的 Ki 值一致。在非洲爪蟾卵母细胞中，AR-C155858 以浓度和时间依赖性方式抑制 MCT1 表达 [2]。

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在哺乳动物细胞中，MCT（单羧酸转运蛋白）需要与辅助蛋白结合，才能使活性转运蛋白在质膜上表达。Basigin是MCT1、MCT3和MCT4的首选结合伴侣，而embigin则是MCT2的首选结合伙伴。在大鼠和兔红细胞中，MCT1分别与embigin和basigin相关，但发现其对AR-C155858抑制的敏感性是相同的。使用RT（逆转录）-PCR，我们发现非洲爪蟾卵母细胞含有内源性basigin，但不含embigin。外源性embigin的共表达对MCT1的表达或AR-C155858对其的抑制都没有影响。相比之下，外源性embigin的共表达显著增强了卵母细胞质膜上活性MCT2的表达。这种额外的转运活性对AR-C155858的抑制不敏感，这与用内源性basigin表达的MCT2不同，后者被AR-C15858强烈抑制。在有和没有外源性胚原的情况下，卵母细胞中也表达了MCT1和MCT2的黑猩猩和C端截短。确定了这些构建体的L-乳酸Km值，并表明MCT的TM（跨膜）结构域，最有可能是TM7-TM12，但不是C末端，是L-乳酸亲和力的主要决定因素，而相关的辅助蛋白几乎没有影响。这些构建体对乳酸转运的抑制剂滴定表明，embigin通过与MCT2的细胞内C末端和TMs 3和6相互作用来调节MCT2对AR-C155858的敏感性。发现MCT2的C末端对于其与内源性basigin的表达至关重要。[1]

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在本研究中，我们描述了强效MCT1（单羧酸转运蛋白1）抑制剂AR-C155858的性质。使用大鼠红细胞中MCT1转运L-乳酸的抑制剂滴定法来确定Ki值、MCT1上AR-C155858结合位点的数量（Et）和转运蛋白的周转数（kcat）。衍生值分别为2.3+/-1.4nM、1.29+/-0.09nmol/ml包装细胞和12.2+/-1.1s-1。当在非洲爪蟾卵母细胞中表达时，MC-C155858对MCT1和MCT2有强烈的抑制作用，而MCT4则没有。对MCT1的抑制显示出时间依赖性，微量注射时该化合物也具有活性，这表明AR-C155858可能在与MCT1上的细胞内位点结合之前进入细胞。对结合MCT1和MCT4不同结构域的几种嵌合转运蛋白的抑制剂敏感性的测量表明，AR-C155858的结合位点包含在MCT1的C端半部分，涉及TM（跨膜）结构域7-10。这与之前的数据一致，即Phe360（在TM10中）和Asp302加Arg306（TM8）是MCT1底物结合和转运的关键残基。L-乳酸和丙酮酸嵌合体Km值的测量表明，分子的C端和N端半部都会影响转运动力学，这与我们提出的MCT1分子模型及其转运机制一致，该模型需要TM1中的Lys38以及TM8中的Asp302和Arg306[Wilson，Meredith，Bunnun，Sessions和Halestrap（2009）J.Biol.Chem.28420011-20021][2]。

目的：抑制单羧酸转运蛋白（MCT）是一种潜在的治疗策略，通过阻断γ-羟丁酸（GHB）在肾脏的再吸收来治疗GHB过量。本研究进一步评估了新型高效MCT抑制剂AR-C155858对GHB毒代动力学/毒代动力学（TK/TD）的影响。[2]
方法：给大鼠注射GHB（200、600或1500 mg/kg静脉注射或1500 mg/kg口服），含或不含AR-C155858。使用全身体积描记术连续监测呼吸频率。在8小时内收集血浆和尿液样本。还评估了AR-C155858对GHB脑/血浆分配的影响。[2]
结果：在本研究中使用的所有GHB剂量下，AR-C155858治疗显著提高了静脉注射GHB后GHB的肾清除率和总清除率。AR-C155858显著改善了GHB诱导的呼吸抑制，呼吸频率的改善证明了这一点。与单独使用GHB（0.25±0.02）相比，AR-C155858治疗还导致GHB的脑/血浆分配显著减少（0.1±0.03）。口服AR-C155858治疗后，口服GHB CLR和CLoral（CL/F）也显著增加（分别从1.82±0.63增加到5.74±0.86和6.52±0.88增加到10.2±0.75ml/min/kg）。[2]
结论：新型高效MCT抑制剂是治疗GHB过量的潜在选择。[2]

大鼠红细胞MCT1活性的测定[2]
如前所述，通过用pH敏感电极监测细胞外pH的变化来测量L-乳酸进入大鼠红细胞的转运。细胞在添加了5μM DIDS和100μM乙酰唑胺的轻度缓冲盐水培养基中以3.5%或7%的红细胞压积使用，以防止碳酸氢盐/CO2介导的质子运动。在测定乳酸转运之前，红细胞在室温（22-25°C）下以所需浓度与或不与AR-C155858一起预孵育1小时。这是在6°C下进行的，通过添加10 mM L-乳酸启动底物摄取。通过pH变化时间过程的一阶回归分析计算初始转运速率，并通过测定少量添加标准化NaOH引起的pH变化将其转换为每分钟nmol H+。

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爪蟾卵母细胞MCT转运活性的测定[2]
cRNA是通过体外转录从适当的线性化pGHJ质粒 制备的，并如前所述注射到X.laevis卵母细胞中[33]。对于大多数检测，注射了20 ng cRNA，但对于[14C]动力学检测，调整了注射量以确保摄取与时间呈线性关系。更多详细信息见补充表S2（http://www.BiochemJ.org/bj/425/bj4250523add.htm). 对照组收到了等量（9.2 nl）的水。然后将卵母细胞在OR3培养基中每天用新鲜培养基培养72小时。如前所述，通过使用比率pH敏感荧光染料BCECF[2′-7′-双（羧乙基）-5（6）-羧基荧光素]跟踪细胞内pH的变化，或通过测量[14C]底物（L-乳酸或丙酮酸）的摄取，确定野生型或嵌合MCT的L-乳酸转运速率。为了测量AR-C155858敏感性，将10个卵母细胞放入含有5ml摄取缓冲液[75 mM NaCl、2 mM KCl、0.82 mM MgCl2、1 mM CaCl2和20 mM Mes（pH 6.0）]的六孔板中，并根据需要在有或没有AR-C155858的情况下预孵育所需的时间（通常为45分钟）。取出五个卵母细胞，放入50μl含有l-[14C]乳酸盐（0.5 mM，7.4 MBq/ml）的摄取缓冲液中，该缓冲液含有或不含有所需浓度的AR-C155858。在室温下继续孵育一段时间，在此期间摄取与时间呈线性关系；这因所采用的构造而异（详见补充表S2）。然后用冰冷的吸收缓冲液快速洗涤卵母细胞五次，最后一次洗涤后，将每个卵转移到闪烁瓶中，通过剧烈的涡流混合在100μl 2%（w/v）SDS中均质化。然后加入闪烁液（10ml乳化剂安全），并通过闪烁计数测定[14C]。[2]
为了测定丙酮酸和L-乳酸的Km值，将卵母细胞在孵育缓冲液[75 mM NaCl、2 mM KCl、0.82 mM MgCl2、1 mM CaCl2和20 mM Tris/Hepes（pH 7.4）]中平衡5分钟，然后用40μL含有[14C]-标记的L-乳酸或丙酮酸（7.4 MBq/ml）的摄取缓冲液孵育四个卵母细胞，最终浓度为0.2、0.5、1、2、5、20、50和75 mM。继续孵育一段时间，摄取与时间呈线性关系（详见补充表S2），然后清洗卵母细胞并准备进行上述闪烁计数。通过减去同时测定的注水卵母细胞的摄取量来确定MCT1介导的L-乳酸净摄取量。[2]
如前所述，通过将卵母细胞包埋在鸡肝中切片的免疫荧光显微镜证实了质膜上的MCT表达。

红细胞MCT1活性的测定[1]
如前所述，通过用pH敏感电极监测细胞外pH的变化来测量L-乳酸进入大鼠和兔红细胞的转运。红细胞（5%红细胞压积）在室温（22°C）下以所需浓度与或不与AR-C155858一起预孵育1小时，然后在6°C下测定L-乳酸（10 mM）的转运。通过pH变化时间过程的一阶回归分析计算初始转运速率，并通过测定少量添加标准化NaOH引起的pH变化将其转换为H+/min的nmol。

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爪蟾卵母细胞MCT转运活性的测定[1]
如前所述，制备cRNA并注射到X.laevis卵母细胞中。对于所有检测，在9.2 nl水中注射10 ng MCT cRNA±10 ng大鼠胚胎cRNA，对照组仅接受水。如前所述，通过免疫荧光显微镜证实了卵母细胞质膜上MCT和embigin的表达。通过用H+敏感染料BCECF[2′，7′-双-（2-羧乙基）-5（6）-羧基荧光素]监测细胞内pH值或通过测定L-[14C]乳酸盐的摄取量（7.4 MBq/ml）进行MCT动力学测定。摄取缓冲液含有75 mM NaCl、2 mM KCl、0.82 mM MgCl2、1 mM CaCl2和20 mM Tris/Hepes（pH 7.4）AR-C155858抑制剂滴定在pH 6下进行，卵母细胞在不同的摄取缓冲液（75 mM NaCl、2 mM KCl、0.82 mM MgCl2、1 mM CaCl2和20 mM Mes，pH 6）中预孵育45分钟，在测量L-[14C]乳酸盐（0.5 mM）的摄取之前，如前所述[38]。除非另有说明，否则所有MCT构建体的摄取量都是在2.5分钟内测定的，但含有或不含有embigin的MCT2trn和含有或不含embigion的MCT2/1除外，分别使用5分钟和10分钟。我们确定这些条件代表了摄取与时间呈线性关系的最长时期（结果未显示）。

Toxicokinetic/Toxicodynamic studies [3]
Effect of AR-C155858 on toxicokinetics and respiratory depression of intravenous γ-hydroxybutyric acid (GHB) [3]
The effect of AR-C155858 on GHB-induced respiratory depression was studied using whole-body plethysmography similar to our previously published studies. Animals were allowed to acclimate to the plethysmography chambers for 45 minutes followed by collection of five baseline measurements of respiratory parameters over 15 minutes. GHB was administered intravenously as 200, 600 or 1500 mg/kg bolus with or without AR-C155858 (1 or 5 mg/kg i.v. bolus). In the GHB 600 mg/kg group, a lower dose of AR-C155858 (0.1 mg/kg i.v. bolus) was also administered. In all the treatment groups, AR-C155858 was administered 5 minutes after GHB administration. This experiment was performed at a similar time and in a similar manner to our previous study assessing respiratory effects of GHB alone; therefore, data from rats administered GHB 200, 600, and 1500 mg/kg alone were used from the previous publication for comparison purposes. The time of GHB administration was considered as time 0. Blood and urine samples were collected at intervals up to 8 hours after GHB administration. The respiratory parameters, breathing frequency, tidal volume, and minute volume (breathing frequency x tidal volume) were recorded at 2.5, 5, 7.5, 10, 15, 20, 25, and 30 minutes and every 15 minutes thereafter until 8 hours. In all groups of animals, GHB was administered as a 300 mg/ml solution in sterile water via the jugular vein cannula. The AR-C155858 bolus was administered as a 0.1, 1 or 2.5 mg/ml solution in 10 % cyclodextrin in normal saline via the jugular vein cannula. All the treatment groups included 3–6 animals and were compared with their respective GHB alone group to determine the effects of AR-C155858 on GHB-induced respiratory depression. A separate group of animals received AR-C155858 alone (1 mg/kg i.v. bolus) to study the effect of this inhibitor itself on respiration.

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Effect of AR-C155858 on GHB blood-brain partitioning at steady state [3]
To assess the effect of AR-C155858 on the transport of GHB into the brain, GHB (400 mg/kg i.v. bolus followed by 208 mg/kg/hr i.v. infusion) was administered alone or in combination with AR-C155858 (5 mg/kg i.v. bolus) (n=4). The GHB dose was selected to produce steady-state GHB plasma concentrations of 800 µg/ml, similar to the high concentrations of GHB observed in rats after 600 mg/kg GHB i.v. used in the toxicokinetic study above. In addition this GHB concentration is similar to those seen in clinical cases of GHB overdose (5). The animals were euthanized at 4 h post GHB administration under isoflurane anesthesia followed by collection of blood and brain samples at steady state. Brain samples were immediately frozen in liquid nitrogen and stored at −80°C until analysis.

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Effect of AR-C155858 on oral GHB toxicokinetics Because GHB is commonly abused orally, the effect AR-C155858 was assessed on GHB toxicokinetics after oral administration in rats. Animals were administered GHB by oral gavage with or without AR-C155858 (5 mg/kg i.v. bolus). AR-C155858 was either administered 5 minutes or 1 hour post GHB administration (n = 4–6). In another group of animals, both AR-C155858 (10 mg/kg) and GHB (1500 mg/kg) were administered at the same time by oral gavage. Rats were fasted overnight before drug administration. Blood and urine samples were collected at intervals up to 15 hours after GHB administration. GHB alone was administered as a 300 mg/ml solution in water and AR-C155858 as a 2.5 mg/ml solution in 10% cyclodextrin in normal saline.

[1]. The inhibition of monocarboxylate transporter 2 (MCT2) by AR-C155858 is modulated by the associated ancillary protein. Biochem J. 2010 Oct 15;431(2):217-25.

[2]. AR-C155858 is a potent inhibitor of monocarboxylate transporters MCT1 and MCT2 that binds to an intracellular site involving transmembrane helices 7-10. Biochem J. 2010 Jan 15;425(3):523-30.

[3]. A Novel Monocarboxylate Transporter Inhibitor as a Potential Treatment Strategy for γ-Hydroxybutyric Acid Overdose. Pharm Res. 2015 Jun;32(6):1894-906.

In order to reconcile all of the results of the present study, we propose the scheme shown in Figure 7. The key features of this proposal are as follows.
(i) MCT2 binds preferentially to embigin over endogenous basigin, with the binding to the latter requiring an interaction between the C-termini of both proteins that is not required by MCT1. Thus MCT1trn, but not MCT2trn, expresses well in the absence of co-expressed embigin.
(ii) MCT1 associates with embigin in the absence of basigin, but prefers the latter as binding partner when both are present, unless the C-terminal tail of MCT2 replaces the MCT1 C-terminus (MCT1/2c) when binding to embigin is promoted.
(iii) When bound to embigin, the binding affinity of AR-C155858 to MCT2, but not MCT1, is greatly reduced and this effect is independent of the presence of the C-terminal tail of MCT2.
Our data emphasize that the potency with which AR-C155858 inhibits MCT2 is dependent on the ancillary protein with which it associates. In mammalian cells, embigin is the preferred endogenous binding partner of MCT2 [22] and thus MCT2-mediated lactate transport is likely to be considerably less sensitive to inhibition by AR-C155858 than that mediated by MCT1. This may justify the cautious use of AR-C155858 to dissect out the different roles of MCT1 (very sensitive to AR-C155858), MCT2 (less sensitive to AR-C155858) and MCT4 (insensitive to AR-C155858) in the metabolism of tissues such as the brain, as has been reported by Bröer and colleagues [41]. However, the development of totally isoform-specific inhibitors of MCTs that are not influenced by the associated ancillary protein is clearly desirable. [1]

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Previously, a new class of specific and extremely potent inhibitors of MCT1 have been discovered by AstraZeneca that were reported to show no binding to MCT4 and exhibit lower-affinity binding to MCT2. The Kd values for these inhibitors binding to endogenous MCT1 in rat and human cells was found to be in the low nanomolar region or less, as was also found for MCT1 expressed in Ins-1 cells that contain little or no endogenous MCT1. Similar Kd values were determined for MCT1 expressed in yeast, and the Ki value of AR-C155858 for MCT1 we have determined in rat erythrocytes of 2.3±1.4 nM is entirely consistent with the Ki of 1.2 nM for binding of AR-C155858 to human erythrocyte MCT1 derived from radioligand-binding experiments. [2]

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In summary, the novel and highly potent inhibitor, AR-C155858 increases renal and total clearance of GHB following both intravenous and oral administration in rats. AR-C155858 also results in significant improvement of GHB-induced respiratory depression which may be mediated by inhibition of its renal reabsorption and brain uptake, both processes mediated by MCTs. Our studies demonstrate proof-of-concept in utilizing MCT inhibition as a potential treatment strategy by improving GHB-induced respiratory depression which leads to death in cases of GHB overdose. [3]

C21H27N5O5S

461.5346

461.173

C, 54.65; H, 5.90; N, 15.17; O, 17.33; S, 6.95

496791-37-8

10226546

Off-white to light yellow solid powder

1.4±0.1 g/cm3

763.1±70.0 °C at 760 mmHg

415.3±35.7 °C

0.0±2.7 mmHg at 25°C

1.634

0.93

150.69

2

7

5

32

769

1

CC1=C(C(=NN1)C)CC2=C(C3=C(S2)N(C(=O)N(C3=O)C)CC(C)C)C(=O)N4C[C@@H](CO4)O

ISIVOJWVBJIOFM-ZDUSSCGKSA-N

InChI=1S/C21H27N5O5S/c1-10(2)7-25-20-17(18(28)24(5)21(25)30)16(19(29)26-8-13(27)9-31-26)15(32-20)6-14-11(3)22-23-12(14)4/h10,13,27H,6-9H2,1-5H3,(H,22,23)/t13-/m0/s1

(S)-6-((3,5-dimethyl-1H-pyrazol-4-yl)methyl)-5-(4-hydroxyisoxazolidine-2-carbonyl)-1-isobutyl-3-methylthieno[2,3-d]pyrimidine-2,4(1H,3H)-dione

ARC155858; AR-C155858; 496791-37-8; (S)-6-((3,5-dimethyl-1H-pyrazol-4-yl)methyl)-5-(4-hydroxyisoxazolidine-2-carbonyl)-1-isobutyl-3-methylthieno[2,3-d]pyrimidine-2,4(1H,3H)-dione; (S)-6-[(3,5-Dimethyl-1H-pyrazol-4-yl)methyl]-5-[(4-hydroxyisoxazolidin-2-yl)carbonyl]-1-isobutyl-3-methylthieno[2,3-d]pyrimidine-2,4(1H,3H)-dione; 6-[(3,5-dimethyl-1H-pyrazol-4-yl)methyl]-5-[(4S)-4-hydroxy-1,2-oxazolidine-2-carbonyl]-3-methyl-1-(2-methylpropyl)thieno[2,3-d]pyrimidine-2,4-dione; AR-C 155858; 6-[(3,5-dimethyl-1H-pyrazol-4-yl)methyl]-5-[(4S)-4-hydroxy-1,2-oxazolidine-2-carbonyl]-3-methyl-1-(2-methylpropyl)-1H,2H,3H,4H-thieno[2,3-d]pyrimidine-2,4-dione; ARC 155858.

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 : ~70 mg/mL (~151.67 mM)

配方 1 中的溶解度: ≥ 2.75 mg/mL (5.96 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 27.5 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: ≥ 2.75 mg/mL (5.96 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 27.5mg/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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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));
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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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