DNL343

eIF2B, integrated stress response (ISR)[1]

使用荧光显微镜在ALS体外模型中评估DNL343对ISR的抑制作用。将共表达可诱导的GFP标记的TDP43细胞质形式和应激颗粒标记物G3BP1-mCherry的H4细胞用亚砷酸钠应激2小时以诱导应激颗粒的形成。在2小时的时间过程中，DNL343以剂量依赖的方式抑制TDP43（86–414）阳性应激颗粒的形成，IC50值为13 nM。该数据支持DNL343在疾病模型中有效抑制细胞应激后的ISR途径[1]。

为了证明DNL343在体内具有抑制中枢神经系统ISR抑制的活性，我们利用了Eif2b5 R191H小鼠，该小鼠模拟了由中枢神经系统慢性ISR激活驱动的神经退行性疾病。DNL343（50mg/kg）在纯合突变动物及其野生型同窝仔中通过灌胃每天给药一次，持续2天。两组的未结合脑浓度与未结合血浆水平相当，与在大鼠和cyno中观察到的高中枢神经系统外显率一致（见上文）。如我们自己的模型表征以及其他人的模型表征所示，在2.9–3.8个月大时，与野生型对照相比，纯合突变体的大脑中ISR转录标记物的表达水平更高。DNL343的急性治疗降低了这些ISR标记物，表明DNL343在抑制大脑中的ISR过度激活方面是有效的[1]。

萤光素酶测定[1]
ATF4报告子克隆17细胞以2500个细胞/孔的速度在384孔板中以15μL进行铺板。第二天，通过使用Echo Acoustic液体处理器（10μM最终1/2 log稀释度）在750 nL中点样化合物，制备DNL343板。然后将培养基添加到稀释板（50μL/孔）中，以产生适当的稀释液添加到细胞中。然后使用Apricot液体处理器将含有DNL343（10μL）的培养基转移到细胞培养板中。将细胞与DNL343在37°C和5%CO2下孵育45分钟，然后加入5μL亚砷酸钠至最终浓度为30μM。然后将板在药物和压力的存在下再孵育6小时。对照孔包括在每个平板上，并被指定为“低”（无亚砷酸钠）和“高”（30μM亚砷酸盐，无DNL343）
将NanoGlo测定缓冲液和底物解冻并在室温下放置1小时（NanoGlo-萤光素酶试剂盒（Promega N1150））。为了制备工作测定试剂，在测定前直接将底物（200μL/板）加入缓冲液（10mL/板。最后6小时孵育后，向每个孔中加入30μL的测定工作试剂（体积与样品体积相等）。然后将板在600rpm下振荡3分钟以混合，然后在EnVision板读取器上读取发光。NLuc报告子定量后，使用以下公式计算DNL343实现的抑制百分比：100-[（样本-低）/（高-低）×100]。“高”（亚砷酸钠，无化合物）和“低”（无亚砷酸盐）由每个平板上运行的对照定义。

H4诱导的TDP43细胞的应激颗粒IC50[1]
H4细胞分别被表达G3BP1 mCherry和多西环素诱导的GFP-TDP43（86–414）的慢病毒感染，以产生稳定的细胞系。细胞在DMEM中培养，DMEM补充有1%丙酮酸钠和10%无tet胎牛血清，温度为37°C，含5%CO2。[1]
对于应力颗粒测定，将细胞接种在PDL涂布的96w Cell Carrier Ultra板中。在接种时用1μg/mL多西环素诱导转基因表达。诱导后约24小时，使用液体处理器（Tecan D300e数字分配器）用DNL343预处理细胞，从10mM终浓度和10点1/2对数稀释系列开始，在37°C、5%CO2中预处理30分钟。然后，在37°C、5%CO2中向细胞中加入200μM亚砷酸钠2小时。然后将细胞固定在4%多聚甲醛中，并在室温下孵育10分钟。固定后，用1×PBS洗涤细胞两次，并在0.1%Triton X-100中在室温下透化20分钟。将细胞封闭在3%BSA+0.1%Triton X-100中1小时。在室温下用DAPI（1:5000，Thermo Fisher）对细胞核染色1小时。然后将细胞在1×PBS中洗涤4次。然后使用40×物镜在Opera Phenix高含量显微镜上对板进行成像。 为了量化应激颗粒和TDP43点状，通过Harmony HCS软件在每个通道中分割并自动识别斑点，并计算每个细胞的总斑点面积之和。该值用于通过使用以下方程计算DNL343的抑制百分比：100-[（样品-低）/（高-低）×100]，其中高（亚砷酸钠，无DNL343）和低（无亚砷酸盐）定义为对照。

DNL343 Preparation for Oral Gavage Dosing [1]
DNL343 was prepared as a suspension in a 0.5% methylcellulose solution. Briefly, DNL343 was placed in individual glass vials to which purified water (half of the final volume) was added. The mixture was gently mixed using a probe sonicator at 40% amplitude 10 times, each lasting for 20 s on pulse sonication. The result was a suspension with dispersed particles that was uniform throughout the product. Equal volume of a 1% methylcellulose (2× final target concentration, in half of the final target volume) solution in purified water was added to the above suspension and vortexed until uniform. Suspension was visually inspected to be uniformly distributed. Dosing solutions were prepared on the first dosing day and were maintained at 4 °C, on a laboratory rotator, until use. DNL343 suspension was remixed on dosing instances and administered via oral gavage at a volume of 10 mL/kg body weight.[1]

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Mouse Tissue Collection[1]
Animals were humanely anesthetized with 2.5% tribromoethanol. Whole blood was collected via cardiac puncture into EDTA-coated tubes and spun down at 12,700 rpm for 7 min at 4 °C before collecting the top plasma layer for analysis. Animals were transcardially perfused with ice-cold PBS. Tissues were then collected, weighed, frozen on dry ice, and stored at 80 °C for subsequent analysis.[1]

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DNL343 Pharmacokinetics[1]
DNL343 in the mouse brain and plasma were measured using LC–MS/MS by Quintara Discovery. Brain samples were homogenized in 3 volumes of ice-cold water and then further diluted 2-fold with blank mouse plasma. An aliquot of 20 μL of plasma sample or plasma diluted brain homogenate was extracted with 100 μL of acetonitrile containing internal standard (warfarin). The mixture was vortexed on a shaker for 15 min and subsequently centrifuged at 4000 rpm for 15 min. An aliquot of 70 μL of the supernatant was mixed with 70 μL of 35% acetonitrile in water with 0.1% formic acid for injection to the LC–MS/MS. Calibration standards and quality control samples were prepared by dilutions of the 2 mg/mL DNL343 stock solution with 70% acetonitrile added to blank mouse plasma. These standards and QC samples were extracted alongside unknown samples in each bioanalytical batch. The final extracts were analyzed by reversed-phase liquid chromatography and negative electrospray ionizations (LC–MS/MS) with multiple reaction-monitoring (MRM) of the test article and the internal standard. The standard curve was fitted by linear regression to quantify DNL343 in the matrix using Analyst 1.6.3 software (AB Sciex). Total DNL343 concentrations were adjusted for brain or plasma protein binding (fraction unbound, fu) that were determined in vitro via an ultracentrifugation method and expressed as unbound (free DNL343) exposures.

[1]. Discovery of DNL343: A Potent, Selective, and Brain-Penetrant eIF2B Activator Designed for the Treatment of Neurodegenerative Diseases. J Med Chem. 2024 Apr 11;67(7):5758-5782.
[2]. Yulyaningsih E, et al. DNL343 is an investigational CNS penetrant eIF2B activator that prevents and reverses the effects of neurodegeneration caused by the Integrated Stress Response[J]. bioRxiv, 2023: 2023.8.21.554203

DNL343 is under investigation in clinical trial NCT05842941 (HEALEY ALS Platform Trial - Regimen G DNL343).

C20H19CLF3N3O4

457.83077454567

457.10

C, 52.47; H, 4.18; Cl, 7.74; F, 12.45; N, 9.18; O, 13.98

2278265-85-1

137491382

Light yellow to green yellow solid powder

3.7

86.5Ų

1

9

7

31

678

0

ClC1C=CC(=CC=1)OCC(NC12CC(C3=NN=C(C4CC(C4)OC(F)(F)F)O3)(C1)C2)=O

IKPNRMYDOSNVAU-QEJBIBHGSA-N

InChI=1S/C20H19ClF3N3O4/c21-12-1-3-13(4-2-12)29-7-15(28)25-19-8-18(9-19,10-19)17-27-26-16(30-17)11-5-14(6-11)31-20(22,23)24/h1-4,11,14H,5-10H2,(H,25,28)/t11-,14+,18?,19?

2-(4-chlorophenoxy)-N-(3-(5-((1s,3s)-3-(trifluoromethoxy)cyclobutyl)-1,3,4-oxadiazol-2-yl)bicyclo[1.1.1]pentan-1-yl)acetamide

DNL343; DNL 343; DNL-343

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

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注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

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注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

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口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

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口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

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注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

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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。