mTORC1; mTORC2; Natural product; secondary metabolite

(+)-松萝酸 (1) 是一种常见的生物活性地衣衍生次级代谢物，具有特征性的二苯并呋喃骨架。它表现出低微摩尔抗增殖活性水平，并且值得注意的是，在一组不同的乳腺癌细胞系中诱导自噬，表明雷帕霉素的机械（以前称为“哺乳动物”）靶标 (mTOR) 是潜在的大分子靶标。由于 mTOR 下游效应子的抑制，细胞自噬标志物显著上调。此外，1 在 mTOR 激酶口袋中表现出最佳结合姿势，并借助与关键氨基酸的多种相互作用。合理设计的 1 的苯亚甲基类似物通过与 Tyr2225 残基的酚侧链堆叠，显示出与激酶裂隙核心的目标深疏水口袋完美契合。我们生成了几种有效的类似物，包括 52，它们对来自多种乳腺癌克隆系的细胞表现出强效（nM 浓度）的抗增殖、抗迁移和抗侵袭活性，而不会影响非致瘤性 MCF-10A 乳腺上皮细胞。类似物 52 还表现出强效的 mTOR 抑制和自噬诱导。此外，52 在两种无胸腺裸鼠乳腺癌异种移植模型中表现出强效的体内抗肿瘤活性。总的来说，松萝酸和类似物是潜在的领先 mTOR 抑制剂，适合未来用于控制乳腺恶性肿瘤。[1]
我们将 (+)-松萝酸装入改性聚氨酯中，并使用图像分析定量评估了 (+)-松萝酸在层流条件下控制金黄色葡萄球菌或铜绿假单胞菌生物膜形成的能力。 （+）-松萝酸负载聚合物不会抑制金黄色葡萄球菌细胞的初始附着，但杀死附着的细胞会导致生物膜的抑制。有趣的是，虽然铜绿假单胞菌生物膜确实在（+）-松萝酸负载聚合物的表面形成，但生物膜的形态发生了改变，这可能表明（+）-松萝酸干扰了信号通路。[2]

(+)-松萝酸 类似物52在裸小鼠异种移植模型中抗乳腺癌症的体内活性[1]
使用mTOR-失调的原位异种移植物癌症模型评估52的抗肿瘤功效，所述模型通过用MCF-7或MDA-MB-231/GFP细胞接种裸鼠产生。在研究结束时，以10mg/kg、3×/周的剂量方案腹腔注射52，显著减轻了两种模型的肿瘤生长（图14）。在MDA-MB-231/GFP模型中，载体处理的对照组动物在牺牲时间（接种后34天）的平均肿瘤体积为1835±251 mm3（±SE）。同时，治疗组小鼠的平均肿瘤体积达到692±146 mm3，肿瘤生长抑制率接近62.3%（图14A）。在MCF-7模型中，到研究结束时（接种后75天），载体治疗的对照组小鼠的平均肿瘤体积为1093±185 mm3。另一方面，接受52种肿瘤治疗的小鼠，其平均体积仅为385±96 mm3（图14），表明平均肿瘤生长抑制率为64.8%。总的来说，这些结果清楚地证明了52在抑制MCF-7和MDA-MB-231/GFP乳腺肿瘤生长方面的强大功效，这两种细胞系的功能失调，会聚集在mTOR调控的途径上。此外，在研究期间监测了动物的体重，并将结果作为毒性的一般指标。与赋形剂处理的对照组相比，类似物52没有引起任何明显的毒性症状或治疗组动物平均体重的显著降低）。

聚合物中抗菌剂的负载。[2]
通过制备含有（+）-松脂酸（2%[wt/vol]）或PEUADED（5%[wt/val]）的丙酮溶液，在PEUADED中加载（+）-usnic acid（图1B）。（+）-通过将上述溶液浇铸在特氟纶板上，然后在30°C的真空下蒸发溶剂，获得负载Usnic酸的PEUADED盘（处理过的聚合物）。通过将侧链中带有碱性叔氨基的聚氨酯与显示酸性基团的抗菌剂（+）-松萝酸结合，建立了高亲和力抗生素-聚合物相互作用。1H核磁共振分析和酰胺化反应效力的酸碱滴定显示，聚合物侧链中存在75%的氨基（数据未显示）。

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（+）-usnic acid从聚合物中释放。[2]
通过光谱法测量浸入圆盘的水中（+）-松萝酸的浓度，每24小时观察270 nm处的吸光度，持续6天（计划的实验期），从而确定抗生素从水中聚合物圆盘释放的动力学。由于松萝酸在水中的溶解度有限，标准溶液是在95%水和5%丙酮的溶液中制备的。在此期间未检测到（+）-松萝酸浸出（数据未显示）。

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（+）-usnic acid/（+）-松萝酸最低抑菌浓度的测定。[2]
采用微量稀释法测定（+）-松萝酸对铜绿假单胞菌和金黄色葡萄球菌的MIC（26）。由于（+）-松萝酸在水中的溶解度有限，在通过镀层活性排除丙酮的任何固有活性后，使用丙酮作为抗菌剂的溶剂介体。制备0.2%（重量/体积）的（+）-松萝酸溶液，然后用LB肉汤稀释铜绿假单胞菌，用胰蛋白酶大豆肉汤稀释金黄色葡萄球菌至所需浓度。两种物种都使用了5×105 CFU/ml的接种物。金黄色葡萄球菌1945GFPuvr和铜绿假单胞菌pMF230的（+）-松萝酸MIC分别为32μg/ml和256μg/ml。

在没有剪切力和营养物质存在的情况下，经过 30 分钟和 24 小时的暴露后，金黄色葡萄球菌附着在 (+)-松萝酸处理的聚合物表面（图 6），但未生长形成成熟的生物膜。活力染色显示，附着活细胞的相对比例从 30 分钟后的约 80% 下降到 24 小时后的不到 1%。[2]
(+)-松萝酸可用于开发抗菌导管，以抵抗金黄色葡萄球菌和可能的其他革兰氏阳性菌的生物膜形成。在高浓度细菌的多重挑战下，改性聚合物在流动条件下成功抑制了金黄色葡萄球菌生物膜的形成，持续时间长达 6 天。[2]

In Vivo Nude Mice Xenograft Models [1]
Female athymic nude mice (5–6 weeks old) were housed at the Animal Facility and maintained under clean conditions in sterile filter-top cages, at a temperature of 24 ± 2 °C, 50 ± 10% relative humidity, and 12:12 h artificial light–dark cycle. Mice received mouse chow and water ad libitum.
MDA-MB-231 green florescent protein-tagged (MDA-MB-231/GFP) cells were harvested, washed with PBS, and resuspended in RPMI-1640 medium. Cells (2 × 106 cells/25 μL) were injected into the mammary fat pad of each nude mouse, using a 29G hypodermic needle. Animals were observed daily for the growth of palpable tumors at the site of injection. Ten days postimplantation, tumors became visible with an approximate average volume of 50 mm3. Mice were randomized and allocated to control and treatment groups (5 mice/group). In the MCF-7 xenografted animals, a 17β-estradiol pellet delivering controlled release over 90 days was implanted beneath the skin of each nude mouse. After recovery from surgery (5 days), animals were injected with 2 × 106 cells suspended in 25 μL of serum-free media into the mammary fat pad of each nude mouse. It took approximately 20 days for generated tumors to reach the average volume of 50 mm3. Mice were randomized and allocated to control and treatment groups (5 mice/group). (+)-usnic acid analogue 52 was prepared as a stock solution in sterile DMSO (1 mg/20 μL), diluted with sterile PBS containing 0.1% Tween 80, and injected intraperitoneally at a dose regimen of 10 mg/kg body weight, three times per week, for the indicated times in each set of experiments. Animals in the control groups received the same volume of vehicle, following the same treatment protocol. Tumor dimensions were measured using a digital caliper. Tumor volume was calculated using the well-established formula: tumor volume (mm3) = [(length × width2)/2]. Animals were monitored daily for any signs of treatment- or vehicle-associated toxicity. Animals were sacrificed at the indicated times, unless they appeared to be moribund or a tumor showed signs of necrosis. At termination, tumors were excised from the connective tissues and snap-frozen for subsequent analysis.

Hepatotoxicity
Several cases of clinically apparent acute liver injury have been attributed to commercial dietary supplements that contain usnic acid. “LipoKinetix” was one such supplement advertised as a weight loss and body building supplement. Each tablet contained sodium usniate (100 mg), norephedrine (25 mg), diiodothyronine (100 mg), yohimbine (3 mg) and caffeine (100 mg). The product has been linked to multiple instances of acute liver injury. The time to onset was 2 to 12 weeks and the clinical presentation resembled acute viral hepatitis with onset of fatigue and nausea, followed by jaundice. The pattern of serum enzyme elevations was hepatocellular, with marked elevations in serum ALT and minimal increases in alkaline phosphatase levels. Liver biopsy demonstrated acute hepatocellular necrosis and inflammation. Immunoallergic features (fever, rash and eosinophilia) were not common and autoantibodies were usually not present. Recovery was rapid with stopping the dietary supplement, but some cases were severe and led to acute liver failure and either death or need for emergency liver transplantation. Instances of acute hepatitis have also been reported with other multi-ingredient dietary supplements that contain usnic acid, but much more rarely than with LipoKinetix which was withdrawn from distribution after a FDA warning letter. Rare instances of hepatotoxicity have also been reported with use of lichen based teas known as Kombucha tea, but whether these were due to usnic acid or another contaminant of the tea was not shown.
Likelihood score (usnic acid): B (highly likely cause of clinically apparent liver injury).
Likelihood score (Kombucha tea): C (probable cause of clinically apparent liver injury).

[1]. Usnic Acid Benzylidene Analogues as Potent Mechanistic Target of Rapamycin Inhibitors for the Control of Breast Malignancies. J Nat Prod. 2017 Apr 28;80(4):932-952.

[2]. Usnic acid, a natural antimicrobial agent able to inhibit bacterial biofilm formation on polymer surfaces. Antimicrob Agents Chemother. 2004;48(11):4360-4365

(-)-usnic acid is the (-)-enantiomer of usnic acid. It has a role as an EC 1.13.11.27 (4-hydroxyphenylpyruvate dioxygenase) inhibitor. It is a conjugate acid of a (-)-usnic acid(2-). It is an enantiomer of a (+)-usnic acid.
Usnic acid is a furandione found uniquely in lichen that is used widely in cosmetics, deodorants, toothpaste and medicinal creams as well as some herbal products. Taken orally, usnic acid can be toxic and has been linked to instances of clinically apparent, acute liver injury.
(-)-Usnic acid has been reported in Ramalina hierrensis, Stereocaulon alpinum, and other organisms with data available.

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(+)-Usnic acid (1) is a common bioactive lichen-derived secondary metabolite with a characteristic dibenzofuran scaffold. It displayed low micromolar antiproliferative activity levels and, notably, induced autophagy in a panel of diverse breast cancer cell lines, suggesting the mechanistic (formerly "mammalian") target of rapamycin (mTOR) as a potential macromolecular target. The cellular autophagic markers were significantly upregulated due to the inhibition of mTOR downstream effectors. Additionally, 1 showed an optimal binding pose at the mTOR kinase pocket aided by multiple interactions to critical amino acids. Rationally designed benzylidene analogues of 1 displayed excellent fitting into a targeted deep hydrophobic pocket at the core of the kinase cleft, through stacking with the phenolic side chain of the Tyr2225 residue. Several potent analogues were generated, including 52, that exhibited potent (nM concentrations) antiproliferative, antimigratory, and anti-invasive activities against cells from multiple breast cancer clonal lines, without affecting the nontumorigenic MCF-10A mammary epithelial cells. Analogue 52 also exhibited potent mTOR inhibition and autophagy induction. Furthermore, 52 showed potent in vivo antitumor activity in two athymic nude mice breast cancer xenograft models. Collectively, usnic acid and analogues are potential lead mTOR inhibitors appropriate for future use to control breast malignancies.[1]

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In modern medicine, artificial devices are used for repair or replacement of damaged parts of the body, delivery of drugs, and monitoring the status of critically ill patients. However, artificial surfaces are often susceptible to colonization by bacteria and fungi. Once microorganisms have adhered to the surface, they can form biofilms, resulting in highly resistant local or systemic infections. At this time, the evidence suggests that (+)-usnic acid, a secondary lichen metabolite, possesses antimicrobial activity against a number of planktonic gram-positive bacteria, including Staphylococcus aureus, Enterococcus faecalis, and Enterococcus faecium. Since lichens are surface-attached communities that produce antibiotics, including usnic acid, to protect themselves from colonization by other bacteria, we hypothesized that the mode of action of usnic acid may be utilized in the control of medical biofilms. We loaded (+)-usnic acid into modified polyurethane and quantitatively assessed the capacity of (+)-usnic acid to control biofilm formation by either S. aureus or Pseudomonas aeruginosa under laminar flow conditions by using image analysis. (+)-Usnic acid-loaded polymers did not inhibit the initial attachment of S. aureus cells, but killing the attached cells resulted in the inhibition of biofilm. Interestingly, although P. aeruginosa biofilms did form on the surface of (+)-usnic acid-loaded polymer, the morphology of the biofilm was altered, possibly indicating that (+)-usnic acid interfered with signaling pathways.[2]

C18H16O7

344.32

344.089

C, 62.79; H, 4.68; O, 32.53

7562-61-0

Usnic acid;125-46-2

442614

Light yellow to yellow solid

1.5±0.1 g/cm3

638.2±55.0 °C at 760 mmHg

201-203 °C(lit.)

236.0±25.0 °C

0.0±2.0 mmHg at 25°C

1.679

2.3

121.13

3

7

2

25

734

1

O1C2=C(C(C([H])([H])[H])=O)C(=C(C([H])([H])[H])C(=C2[C@]2(C([H])([H])[H])C(C(C(C([H])([H])[H])=O)=C(C([H])=C12)O[H])=O)O[H])O[H]

WEYVVCKOOFYHRW-GOSISDBHSA-N

InChI=1S/C18H16O7/c1-6-14(22)12(8(3)20)16-13(15(6)23)18(4)10(25-16)5-9(21)11(7(2)19)17(18)24/h5,21-23H,1-4H3/t18-/m1/s1

(9bS)-2,6-diacetyl-3,7,9-trihydroxy-8,9b-dimethyldibenzofuran-1-one

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

注意： 本产品在运输和储存过程中需避光。

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: ~4 mg/mL warmed (~11.6 mM)
Water: <1 mg/mL
Ethanol: <1 mg/mL

配方 1 中的溶解度: ≥ 0.63 mg/mL (1.83 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 6.3 mg/mL 澄清 DMSO 储备液加入900 μL 玉米油中，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。