eIF2B, integrated stress response (ISR)[1]

Inhibition of the ISR by DNL343 was assessed in an ALS in vitro model using fluorescence microscopy. H4 cells coexpressing an inducible GFP-tagged cytoplasmic form of TDP43 and the stress granule marker G3BP1-mCherry were stressed with sodium arsenite for 2 h to induce stress granule formation. Over the 2 h time course, DNL343 inhibited the formation of TDP43(86–414)-positive stress granules in a dose-dependent manner, with an IC50 value of 13 nM. This data supports that DNL343 potently inhibits the ISR pathway following cellular stress in a disease model [1].

To demonstrate that DNL343 is active at suppressing CNS ISR inhibition in vivo, we leveraged the Eif2b5 R191H mouse that models a neurodegenerative condition driven by chronic ISR activation in the CNS. DNL343 (50 mg/kg) was administered once daily via oral gavage for 2 days in homozygous mutant animals and their wild-type littermates. Both groups demonstrated unbound brain concentrations that were comparable to unbound plasma levels, consistent with the high CNS penetrance observed in rat and cyno (vide supra). As shown in our own model characterization as well as by others, ISR transcript markers are expressed at higher levels in the brain of homozygous mutants relative to wild-type controls at 2.9–3.8 months of age. Acute treatment with DNL343 reduced these ISR markers, suggesting that DNL343 is effective at inhibiting ISR overactivation in the brain [1].

Luciferase Assay [1]
ATF4 Reporter clone 17 cells were plated at 2500 cells/well in 15 μL in a 384-well plate. The following day, plates of DNL343 were prepared by spotting compound in 750 nL using an Echo Acoustic liquid handler (10 μM final 1/2 log dilution). Media was then added to the dilution plate (50 μL/well) to create the appropriate dilutions to add to cells. Media containing DNL343 (10 μL) was then transferred to the cell culture plate using an Apricot liquid handler. The cells were incubated with DNL343 for 45 min at 37 °C and 5% CO2, and then 5 μL of sodium arsenite was added to a final concentration of 30 μM. The plates were then incubated for a further 6 h in the presence of both drug and stress. Control wells were included on each plate and were designated as “low” (no sodium arsenite) and “high” (30 μM sodium arsenite, no DNL343).
NanoGlo Assay buffer and substrate were thawed and brought to room temperature for 1 h (NanoGlo Luciferase Kit (Promega N1150)). To prepare working assay reagent, substrate (200 μL/plate) was added to buffer (10 mL/plate) directly prior to the assay. Following the final 6 h incubation, 30 μL of assay working assay reagent was added to each well (equal volume to sample volume). The plate was then shaken for 3 min at 600 rpm to mix, and then luminescence was read on an EnVision plate reader. Following NLuc reporter quantification, the percent inhibition achieved by DNL343 was calculated using the following: 100 – [(sample – low)/(high – low) × 100]. “High” (sodium arsenite, no compound) and “low” (no sodium arsenite) are defined by the controls run on each plate.

Stress Granule IC50 in H4-Inducible TDP43 Cells [1]
H4 cells were infected by lentiviruses expressing G3BP1-mCherry and doxycycline-inducible GFP-TDP43(86–414) made from pLVX-CMV_IRES-Hygro and pLVX-TetOne_IRES-Puro, respectively, to generate a stable cell line. Cells were cultured in DMEM, supplemented with 1% sodium pyruvate, and 10% tet-free fetal bovine serum at 37 °C in 5% CO2.[1]
For stress granule assays, cells were plated in PDL-coated 96w Cell Carrier Ultra plates. Transgene expression was induced with 1 μg/mL doxycycline at the time of seeding. Approximately 24 h following induction, a liquid handler (Tecan D300e digital dispenser) was used to pretreat the cells with DNL343 starting with 10 mM final concentration and 10-point 1/2 log dilution series for 30 min at 37 °C in 5% CO2. Then, 200 μM sodium arsenite was added to cells for 2 h at 37 °C in 5% CO2. Cells were then fixed in 4% paraformaldehyde and incubated at room temperature for 10 min. After fixation, cells were washed twice with 1× PBS and permeabilized in 0.1% Triton X-100 for 20 min at room temperature. Cells were blocked in 3% BSA + 0.1% Triton X-100 for 1 h. Nuclei were stained with DAPI (1:5000, Thermo Fisher) for 1 h at room temperature. Cells were then washed 4 times in 1× PBS. Plates were then imaged on an Opera Phenix high-content microscope using a 40× objective. To quantify stress granules and TDP43 puncta, spots were segmented and automatically identified in each channel by the Harmony HCS software, and the sum of the total spot area per cell was calculated. This value was used to calculate the percent inhibition of DNL343 by using the following equation: 100 – [(sample – low)/(high – low) × 100], where high (sodium arsenite, no DNL343) and low (no sodium arsenite) are defined as the controls.

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

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

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)