LOC14

PDI (Kd = 62 nM)

LOC-14（0.01-100μM；24小时）显示出抑制重组（r）PDIA3的能力，IC50约为5μM。经PDI抑制剂处理后，LOC14抑制了肺上皮细胞中的PDIA3活性，减少了分子内二硫键，随后在H1N1（a/PR8/34）和H3N2（X31，a/Aichi/68）感染的肺上皮细胞中HA的寡聚化（成熟）[2]。

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LOC14是通过高通量筛选约10000种铅优化化合物来鉴定的，这些化合物可有效挽救表达突变亨廷顿蛋白的PC12细胞的存活率，然后在体外筛选中评估化合物对PDI还原酶活性的影响。等温滴定量热法和荧光实验表明，与PDI的结合是可逆的，Kd为62 nM，表明LOC14是迄今为止报道的最有效的PDI抑制剂。使用2D异核单量子相关NMR实验，我们能够将LOC14的结合位点定位为与活性位点相邻，并观察到LOC14的连接迫使PDI采用氧化构象。此外，我们发现LOC14诱导的PDI氧化不仅在细胞培养中，而且在皮质纹状体脑切片培养中都具有神经保护作用[1]。

LOC-14（灌胃口服；20mg/kg；每日一次；12-28周）延长了N171-82Q HD小鼠的存活时间，减轻了脑萎缩，并显著改善了运动功能。雄性N171-82Q HD小鼠是动物模型。每公斤20毫克。20mg/kg，每日口服一次，持续12至28周。因此，HD小鼠的运动性能得到了改善。LOC14是新制备的，首先将其与1-甲基-2-吡咯烷酮（NMP）混合，形成80mg/ml的储备溶液，然后用0.5%的甲基纤维素稀释至所需浓度（40倍稀释）[3]。

酶促胰岛素还原试验。[1]
该测定在384孔黑色透明底板中进行。每个孔含有80μL缓冲液A（10 mM Tris⋅HCl，pH 8，150 mM NaCl和2 mM EDTA）中的反应混合物，其中含有5μM PDIa、100μM牛胰岛素、350μM DTT和75μM测试化合物。所有实验均一式两份。将测定板在25°C下孵育1小时，然后在Tecan Infinite 200酶标仪上连续读取每个样品在650 nm处的吸光度，间隔5分钟，持续1小时。吸光度的增加表明胰岛素的β链聚集并从溶液中沉淀出来[1]。

细胞MTEC浓度：0.01μM；0.1μM；0.5μM；1μM；5μM；10μM；100μM 24小时潜伏期；结果：重组PDIA3活性降低。
如前所述，从年龄和性别匹配的野生型（WT）C57BL/6NJ小鼠中分离和培养原代MTEC。在DMEM/F12（Gibco）无生长因子培养基中，细胞以2×106个细胞/皿的密度铺板，当融合率大于90%时，感染小鼠适应的H1N1甲型流感病毒波多黎各8/34（PR8）或H3N2 A X-31，A/Aichi/68（X31），每细胞2.5个卵感染单位（EIU）。使用紫外线（UV）照射的复制缺陷病毒（模拟）作为对照。感染后，将细胞在37°C下孵育1小时，然后用2 mL PBS洗涤平板两次以去除未结合的病毒，并补充无生长因子的培养基。在病毒感染期间，MTEC用10μMLOC14预处理2小时，在病毒感染后1小时，DMSO用作对照。所有治疗均在无生长因子的培养基中进行[2]。

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LOC图书馆的高通量屏幕。在使用前，将含有4 mg/mL化合物的LOC母板解冻并旋转（215×g，20°C，1分钟）。Biomek FX机器人液体分配器用于处理所有液体转移和混合。复制子板（D1）是通过将2μL化合物从母板转移到384个深井透明圆底聚丙烯板中制备的，该板含有98μL不含选择性试剂遗传霉素的PC12培养基，在2%（体积/体积）DMSO中获得80μL/mL的化合物浓度。通过将50μL化合物（80μL/mL）从D1板转移到子板D2中的50μL PC12培养基中，混合，然后对剩余的三个板重复该过程，在五个子板上进行两倍连续稀释。然后，将化合物浓度为80μL/mL的子板D1、化合物浓度为20μL/mL的子板D3和化合物浓度为5μL/mL时的子板D5用于筛选。通过将虫酰肼诱导的PC12 mHTQ103细胞接种到384孔的黑色透明底板中，在不含遗传霉素的57μL PC12培养基中以每孔7500个细胞的密度建立检测板。将三微升来自子平板（D1、D3和D5）的化合物加入到测定平板中，使最终化合物浓度分别为4、1和0.25μL/mL。每个平板上还包括四个含有未诱导的PC12 mHTQ103细胞的孔和四个仅含有培养基的孔作为对照。将测定板在37°C、9.5%CO2下孵育48小时。向每个孔中加入20微升40%（vol/vol）Alamar blue（目录号DAL1100；Life Technologies）PC12培养基溶液（1:10最终稀释），并在37°℃、9.5%CO2条件下再孵育12-24小时。在具有530nm激发滤光片和590nm发射滤光片的荧光板阅读器上读取Alamar蓝色荧光。每种化合物浓度都进行了三次测试。[1]

Chronic administration of a reversible, brain penetrable small molecule PDI modulator, LOC14 (20 mg/kg/day), significantly improved motor function, attenuated brain atrophy and extended survival in the N171–82Q HD mice. Moreover, LOC14 preserved medium spiny neuronal marker dopamine- and cyclic-AMP-regulated phosphoprotein of molecular weight 32 000 (DARPP32) levels in the striatum of HD mice. Mechanistic study revealed that LOC14 suppressed mHtt-induced ER stress, indicated by repressing the abnormally upregulated ER stress proteins in HD models. These findings suggest that LOC14 is promising to be further optimized for clinical trials of HD, and modulation of signaling pathways coping with ER stress may constitute an attractive approach to reduce mHtt toxicity and identify new therapeutic targets for treatment of HD.[3]

[1]. Small molecule-induced oxidation of protein disulfide isomerase is neuroprotective. Proc Natl Acad Sci U S A. 2015 Apr 28;112(17):E2245-52.

[2]. Lung epithelial protein disulfide isomerase A3 (PDIA3) plays an important role in influenza infection, inflammation, and airway mechanics. Redox Biol. 2019 Apr;22:101129.

[3]. Small molecule modulator of protein disulfide isomerase attenuates mutant huntingtin toxicity and inhibits endoplasmic reticulum stress in a mouse model of Huntington's disease. Hum Mol Genet. 2018 May 1;27(9):1545-1555

Protein disulfide isomerase (PDI) is a chaperone protein in the endoplasmic reticulum that is up-regulated in mouse models of, and brains of patients with, neurodegenerative diseases involving protein misfolding. PDI's role in these diseases, however, is not fully understood. Here, we report the discovery of a reversible, neuroprotective lead optimized compound (LOC)14, that acts as a modulator of PDI. LOC14 was identified using a high-throughput screen of ∼10,000 lead-optimized compounds for potent rescue of viability of PC12 cells expressing mutant huntingtin protein, followed by an evaluation of compounds on PDI reductase activity in an in vitro screen. Isothermal titration calorimetry and fluorescence experiments revealed that binding to PDI was reversible with a Kd of 62 nM, suggesting LOC14 to be the most potent PDI inhibitor reported to date. Using 2D heteronuclear single quantum correlation NMR experiments, we were able to map the binding site of LOC14 as being adjacent to the active site and to observe that binding of LOC14 forces PDI to adopt an oxidized conformation. Furthermore, we found that LOC14-induced oxidation of PDI has a neuroprotective effect not only in cell culture, but also in corticostriatal brain slice cultures. LOC14 exhibited high stability in mouse liver microsomes and blood plasma, low intrinsic microsome clearance, and low plasma-protein binding. These results suggest that LOC14 is a promising lead compound to evaluate the potential therapeutic effects of modulating PDI in animal models of disease.[1]

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Protein disulfide isomerases (PDI) are a family of redox chaperones that catalyze formation or isomerization of disulfide bonds in proteins. Previous studies have shown that one member, PDIA3, interacts with influenza A virus (IAV) hemagglutinin (HA), and this interaction is required for efficient oxidative folding of HA in vitro. However, it is unknown whether these host-viral protein interactions occur during active infection and whether such interactions represent a putative target for the treatment of influenza infection. Here we show that PDIA3 is specifically upregulated in IAV-infected mouse or human lung epithelial cells and PDIA3 directly interacts with IAV-HA. Treatment with a PDI inhibitor, LOC14 inhibited PDIA3 activity in lung epithelial cells, decreased intramolecular disulfide bonds and subsequent oligomerization (maturation) of HA in both H1N1 (A/PR8/34) and H3N2 (X31, A/Aichi/68) infected lung epithelial cells. These reduced disulfide bond formation significantly decreased viral burden, and also pro-inflammatory responses from lung epithelial cells. Lung epithelial-specific deletion of PDIA3 in mice resulted in a significant decrease in viral burden and lung inflammatory-immune markers upon IAV infection, as well as significantly improved airway mechanics. Taken together, these results indicate that PDIA3 is required for effective influenza pathogenesis in vivo, and pharmacological inhibition of PDIs represents a promising new anti-influenza therapeutic strategy during pandemic and severe influenza seasons.[2]

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Huntington's disease (HD) is caused by a cytosine-adenine-guanine (CAG) trinucleotide repeat expansion in the huntingtin (HTT) gene encoding an elongated polyglutamine tract within the N-terminal of the huntingtin protein (Htt) and leads to Htt misfolding, aberrant protein aggregation, and progressive appearance of disease symptoms. Chronic activation of endoplasmic reticulum (ER) stress by mutant Htt (mHtt) results in cellular dysfunction and ultimately cell death. Protein disulfide isomerase (PDI) is a chaperone protein located in the ER. Our previous studies demonstrated that mHtt caused PDI to accumulate at mitochondria-associated ER membranes and triggered cell death, and that modulating PDI activity using small molecules protected cells again mHtt toxicity in cell and brain slice models of HD. In this study, we demonstrated that PDI is upregulated in the HD human brain, in cell and mouse models. Chronic administration of a reversible, brain penetrable small molecule PDI modulator, LOC14 (20 mg/kg/day), significantly improved motor function, attenuated brain atrophy and extended survival in the N171-82Q HD mice. Moreover, LOC14 preserved medium spiny neuronal marker dopamine- and cyclic-AMP-regulated phosphoprotein of molecular weight 32 000 (DARPP32) levels in the striatum of HD mice. Mechanistic study revealed that LOC14 suppressed mHtt-induced ER stress, indicated by repressing the abnormally upregulated ER stress proteins in HD models. These findings suggest that LOC14 is promising to be further optimized for clinical trials of HD, and modulation of signaling pathways coping with ER stress may constitute an attractive approach to reduce mHtt toxicity and identify new therapeutic targets for treatment of HD.[3]

C16H19N3O2S

317.405962228775

317.12

C, 60.55; H, 6.03; N, 13.24; O, 10.08; S, 10.10

877963-94-5

877963-94-5

9117962

White to off-white solid powder

1.3

69.2Ų

0

4

3

22

460

0

YVBSNHLFRIVWFQ-UHFFFAOYSA-N

InChI=1S/C16H19N3O2S/c20-15(12-5-6-12)18-9-7-17(8-10-18)11-19-16(21)13-3-1-2-4-14(13)22-19/h1-4,12H,5-11H2

2-[[4-(cyclopropanecarbonyl)piperazin-1-yl]methyl]-1,2-benzothiazol-3-one

LOC14; LOC 14; LOC-14; 2-[(4-cyclopropanecarbonylpiperazin-1-yl)methyl]-2,3-dihydro-1,2-benzothiazol-3-one; 2-[[4-(Cyclopropylcarbonyl)-1-piperazinyl]methyl]-1,2-benzisothiazol-3(2H)-one; LOC-14; CHEMBL4637290; 2-((4-(Cyclopropanecarbonyl)piperazin-1-yl)methyl)benzo[d]isothiazol-3(2H)-one; 2-[[4-(cyclopropanecarbonyl)piperazin-1-yl]methyl]-1,2-benzothiazol-3-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： (1). 本产品在运输和储存过程中需避光。  (2). 该产品在溶液状态不稳定，请现配现用。

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

DMSO: ~50 mg/mL (~157.5 mM)
Water: ˂1 mg/mL
Ethanol: ~19 mg/mL (~60 mM)

配方 1 中的溶解度: ≥ 2.08 mg/mL (6.55 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 20.8 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: ≥ 2.08 mg/mL (6.55 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 20.8 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: ≥ 2.08 mg/mL (6.55 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 20.8 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。