AZD7545

PDHK2 (IC50 = 6.4 nM); PDHK1 (IC50 = 36.8 nM); pyruvate dehydrogenase kinase 2 (PDHK2)

AZD7545（10 μM；对于 BRAF V600E 人类黑色素瘤细胞为 90 小时，对于 NRASmut 人类黑色素瘤细胞为 120 小时）特异性抑制具有 BRAF 和 NRAS 突变的细胞以及对抑制剂耐药的人类黑色素瘤细胞的生长[2]。

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PDH（丙酮酸脱氢酶）多酶复合物催化氧化糖酵解中的关键调节步骤。丝氨酸残基上复合物E1亚基的磷酸化导致酶活性失活。存在一个由四种专用PDH激酶同工酶组成的家族，每种同工酶都显示出不同的组织特异性表达谱AZD7545是为治疗2型糖尿病而开发的一系列PDH激酶抑制剂之一。本文描述了AZD7545和相关化合物的同工酶选择性谱，并讨论了其体内作用模式的后果。[1]

单剂量 AZD7545（口服；10 mg/kg，每日一次（08:00 小时）或每日两次（08:00 和 18:00 小时）；持续 7 天）可增加肝脏 PDH 活性、去磷酸化的比例在 Wistar 大鼠中以剂量相关的方式形成。在肥胖 Zucker (fa/fa) 大鼠中，单次 10 mg/kg 剂量也显着增加肌肉 PDH 活性 [2]。

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PDH（丙酮酸脱氢酶）是控制葡萄糖氧化速率和糖异生前体可用性的关键酶。骨骼肌和肝脏中PDH的激活可能会增加葡萄糖摄取并减少葡萄糖产生。本研究描述了新型PDHK（PDH激酶）小分子抑制剂AZD7545的性质。在PDHK2存在的情况下，AZD7545增加了PDH活性，EC（50）值为5.2 nM。在大鼠肝细胞中，丙酮酸氧化速率被刺激2倍（EC（50）105 nM）。Wistar大鼠单剂量服用AZD7545后，其活性去磷酸化形式的肝脏PDH比例在30mg/kg时以剂量相关的方式从24.7%增加到70.3%；骨骼肌中为21.1%至53.3%。单剂量10mg/kg也显著提高了肥胖Zucker（fa/fa）大鼠的肌肉PDH活性。肥胖、胰岛素抵抗的Zucker大鼠与瘦大鼠相比，餐后血糖水平升高（12周龄时为8.7 mM对6.1 mM）。AZD754510mg/kg）每日两次，持续7天，通过消除餐后血糖升高，显著改善了24小时血糖状况。这些结果表明，PDHK抑制剂可能是改善2型糖尿病治疗中葡萄糖控制的有益药物[2]。

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体内PDH激活[2]
通过Coore等人描述的连接分光光度酶测定法在组织提取物中离体评估PDH活性。在纯化的猪心磷酸酶完全去磷酸化前后评估活性。喂食Wistar大鼠的单次急性剂量AZD7545以剂量依赖的方式将肝脏中活性PDH的百分比从基础（赋形剂给药）水平24.7±6.2%增加到最高评估剂量（30mg/kg）的70.3±2.6%，很明显没有达到最大活化（图1）。在相似剂量的化合物下，腓肠肌中的PDH增加，但在测试的剂量下（从21.1±1.9%到53.3±4.0%）仍未达到完全激活。Aicher等人在经历了一段禁食期的大鼠身上证明了体内活性；PDH活化通过血浆乳酸间接测定或通过体外测量PDH活性测定[14]。在20µmol/kg（约8mg/kg）的剂量下，代表性化合物引起的肝脏和胫骨前肌PDH的增加相对温和，但结果并未表示为活动状态下PDH的比例，因此无法与我们自己的研究结果进行比较。

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AZD7545对Zucker（fa/fa）大鼠的影响[2]
肥胖（fa/fa）Zucker大鼠是胰岛素抵抗或糖尿病前期状态的常用模型。它表现为糖耐量受损、食欲亢进、高胰岛素血症和高脂血症。虽然没有明显的高血糖，但与瘦肉型大鼠相比，fa/fa大鼠在进食后表现出异常的葡萄糖水平（在黑暗进食阶段4小时后，血糖水平为8.7 mM，而瘦肉型动物为6.1 mM）。这与糖化血红蛋白水平小幅但持续显著升高有关（3.49%对3.26%）。在我们研究中使用的年龄（12周），与瘦Zucker或Wistar大鼠相比，fa/fa大鼠的PDH活性升高。我们测量了肥胖和瘦Zucker大鼠之间PDHK2或PDHK4的表达水平没有差异。与Wistar大鼠一样，喂食fa/fa大鼠的PDH可以被PDHK抑制剂进一步激活（例如，10mg/kgAZD7545使肌肉PDH从61.0%增加到97.0%，肝脏PDH从33.5%增加到72.8%）。肥胖的Zucker大鼠口服PDHK抑制剂AZD7545治疗7天，在此期间结束时监测24小时的葡萄糖谱（图2）。在对照组中，经赋形剂处理的大鼠血糖最高升至9.45±1.11 mM，而在每天08:00用AZD7545治疗的大鼠中，同时血糖浓度为6.55±0.58 mM。每天两次给药后，餐后血糖升高也出现了类似的消失。

实时增殖检测[3]
将5个黑色素瘤细胞系的50×103个细胞/孔接种在12孔板上，24小时后用10μM的AZD7545刺激。在IncuCyte ZOOM活细胞显微镜中监测细胞生长，每3小时拍摄一次相位对比图像，共90小时。对三个生物重复进行增殖试验，每个图显示一个代表性重复。

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长期增殖试验[3]
A375 iRFP细胞用于测试1μM PLX4032和10μM AZD7545的组合与单独使用PLX4032。每孔接种10000个细胞（6孔板），并用抑制剂处理3周。每周更换两次介质。治疗3周后，扫描细胞，并使用LI-COR Odyssey仪器测量iRFP信号的强度。使用Image Studio lite 4.0版软件对iRFP信号进行量化。

Obese male (fa/fa) Zucker rats[2];
10 mg/kg;
Oral administration; once a day (08:00 h) or Twice a day ( 08:00 and 18:00 h); for 7 days;

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Male Wistar rats (200–240 g) were dosed orally at 08:00 h with AZD7545 in suspension in 0.5% (w/w) methocel/0.1% polysorbate 80. After 2 h animals were anaesthetized with sodium pentabarbitone (60 mg/kg, intraperitoneally) and tissues excised, freeze-clamped and stored in liquid nitrogen prior to assay. Tissue extracts were prepared and PDH activity determined by the method of Coore et al. Total PDH activity (PDHt) in the extract was determined by assay following dephosphorylation by porcine heart PDP in the presence of 20 mM MgCl2/0.8 mM CaCl2. PDH activity is given as the proportion in the active (dephosphorylated) form in extracts from (
) liver and () gastrocnemius muscle. Results are compared with tissues from control.[2]

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Obese male (fa/fa) Zucker rats, housed in a 06:00 h on/18:00 h off light cycle, were dosed for 7 days with either 10 mg/kg AZD7545, given orally, at 08:00 h () or 08:00 and 18:00 h () or with vehicle (
). On day 8, glucose was measured using a hand-held glucose monitor.[2]

[1]. AZD7545 is a selective inhibitor of pyruvate dehydrogenase kinase 2. Biochem Soc Trans. 2003 Dec;31(Pt 6):1168-70.

[2]. AZD7545, a novel inhibitor of pyruvate dehydrogenase kinase 2 (PDHK2), activates pyruvate dehydrogenase in vivo and improves blood glucose control in obese (fa/fa) Zucker rats. Biochem Soc Trans. 2003 Dec;31(Pt 6):1165-7.

[3]. ROS production induced by BRAF inhibitor treatment rewires metabolic processes affecting cell growth of melanoma cells. Mol Cancer. 2017 Jun 8;16(1):102.

AZD7545 is a sulfone that is benzene substituted by [4-(dimethylcarbamoyl)phenyl]sulfonyl, chloro and [(2R)-3,3,3-trifluoro-2-hydroxy-2-methylpropanoyl]amino groups at positions 1, 3 and 4, respectively. It is a potent and non-ATP-competitive inhibitor of pyruvate dehydrogenase kinase 2 (PDHK2) with IC50 of 6.4 nM and exhibits glucose-lowering activity. Also inhibits PDHK1 at higher levels (IC50 = 36.8 nM). It has a role as a hypoglycemic agent and an EC 2.7.11.2 - [pyruvate dehydrogenase (acetyl-transferring)] kinase inhibitor. It is a member of benzamides, a sulfone, a tertiary alcohol, a tertiary carboxamide, a secondary carboxamide, a member of monochlorobenzenes and an organofluorine compound.

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This is the first report of the testing of a novel PDHK inhibitor in the obese Zucker rat and provides clear evidence that a PDHK inhibitor can improve the control of blood glucose levels in an animal model with impaired glucose homoeostasis. This is in contrast to the statement by Aicher et al. [14] that small-molecule inhibitors were ineffective in animal models of diabetes (ob/ob mice and ZDF rats). Clearly our data would suggest that an inhibitor of PDHK will be an effective novel therapy for type 2 diabetes.[2]

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Background: Most melanoma patients with BRAFV600E positive tumors respond well to a combination of BRAF kinase and MEK inhibitors. However, some patients are intrinsically resistant while the majority of patients eventually develop drug resistance to the treatment. For patients insufficiently responding to BRAF and MEK inhibitors, there is an ongoing need for new treatment targets. Cellular metabolism is such a promising new target line: mutant BRAFV600E has been shown to affect the metabolism.
Methods: Time course experiments and a series of western blots were performed in a panel of BRAFV600E and BRAFWT/NRASmut human melanoma cells, which were incubated with BRAF and MEK1 kinase inhibitors. siRNA approaches were used to investigate the metabolic players involved. Reactive oxygen species (ROS) were measured by confocal microscopy and AZD7545, an inhibitor targeting PDKs (pyruvate dehydrogenase kinase) was tested.
Results: We show that inhibition of the RAS/RAF/MEK/ERK pathway induces phosphorylation of the pyruvate dehydrogenase PDH-E1α subunit in BRAFV600E and in BRAFWT/NRASmut harboring cells. Inhibition of BRAF, MEK1 and siRNA knock-down of ERK1/2 mediated phosphorylation of PDH. siRNA-mediated knock-down of all PDKs or the use of DCA (a pan-PDK inhibitor) abolished PDH-E1α phosphorylation. BRAF inhibitor treatment also induced the upregulation of ROS, concomitantly with the induction of PDH phosphorylation. Suppression of ROS by MitoQ suppressed PDH-E1α phosphorylation, strongly suggesting that ROS mediate the activation of PDKs. Interestingly, the inhibition of PDK1 with AZD7545 specifically suppressed growth of BRAF-mutant and BRAF inhibitor resistant melanoma cells.
Conclusions: In BRAFV600E and BRAFWT/NRASmut melanoma cells, the increased production of ROS upon inhibition of the RAS/RAF/MEK/ERK pathway, is responsible for activating PDKs, which in turn phosphorylate and inactivate PDH. As part of a possible salvage pathway, the tricarboxylic acid cycle is inhibited leading to reduced oxidative metabolism and reduced ROS levels. We show that inhibition of PDKs by AZD7545 leads to growth suppression of BRAF-mutated and -inhibitor resistant melanoma cells. Thus small molecule PDK inhibitors such as AZD7545, might be promising drugs for combination treatment in melanoma patients with activating RAS/RAF/MEK/ERK pathway mutations (50% BRAF, 25% NRASmut, 11.9% NF1mut).[3]

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DCA has been tested in multiple cell culture and rodent models of cancer, and PDK1 knock-down has been described to enhance the sensitivity of BRAFV600E positive melanoma to BRAF inhibitors. We tested a pan-PDK inhibitor, AZD7545, which interferes with the lypoyl binding pocket of PDKs for its capacity to inhibit the growth of BRAFV600E and NRASmut positive cells. We observed that AZD7545 suppressed growth of BRAFV600E positive cells and kinase inhibitor-resistant cells when applied in μM concentrations. Interestingly, AZD7545 had no effect on keratinocytes (HaCaT) and normal fibroblasts, cell types which constitute the cutaneous microenvironment of melanoma tumors (data not shown), indicating selective effects on BRAF/NRAS-mutated or resistant cancer cells. Finally, we show that the combination of BRAF inhibitors with PDK inhibitors is more efficient in tumor growth suppression than the single treatment suggesting that the simultaneus targeting of metabolic pathways might indeed be beneficial for melanoma patients.[3]

C19H18CLF3N2O5S

478.87

478.057

C, 47.66; H, 3.79; Cl, 7.40; F, 11.90; N, 5.85; O, 16.71; S, 6.69

252017-04-2

16741245

White to off-white solid powder

1.5±0.1 g/cm3

683.1±55.0 °C at 760 mmHg

366.9±31.5 °C

0.0±2.2 mmHg at 25°C

1.573

2.66

67.25

2

8

5

31

778

1

ClC1C([H])=C(C([H])=C([H])C=1N([H])C([C@](C([H])([H])[H])(C(F)(F)F)O[H])=O)S(C1C([H])=C([H])C(C(N(C([H])([H])[H])C([H])([H])[H])=O)=C([H])C=1[H])(=O)=O

DTDZLJHKVNTQGZ-GOSISDBHSA-N

InChI=1S/C19H18ClF3N2O5S/c1-18(28,19(21,22)23)17(27)24-15-9-8-13(10-14(15)20)31(29,30)12-6-4-11(5-7-12)16(26)25(2)3/h4-10,28H,1-3H3,(H,24,27)/t18-/m1/s1

4-[3-chloro-4-[[(2R)-3,3,3-trifluoro-2-hydroxy-2-methylpropanoyl]amino]phenyl]sulfonyl-N,N-dimethylbenzamide

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)

配方 1 中的溶解度: ≥ 2.5 mg/mL (5.22 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 (5.22 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 (5.22 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；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。