DNL343 is an investigational CNS penetrant eIF2B activator that prevents and reverses the effects of neurodegeneration caused by the Integrated Stress Response

Reviewer #1 (Public Review):

Summary:
In this study, the authors evaluated a novel eIF2B activator, DNL343, in two mouse models representing different forms of the integrated stress response (ISR). They first assessed the pharmacokinetics of DNL343, demonstrating its ability to cross the blood-brain barrier and exhibit good bioavailability. In an acute ISR model induced by optic nerve crush (ONC) injury, DNL343 treatment reduced ISR-induced transcriptional changes and neuronal loss, demonstrating neuroprotective effects. Next, the authors generated an eIF2B loss-of-function mice model by knocking in disease-causing Eif2b5 variants. The model presents a chronic ISR and mimics vanishing white matter disease (VWMD). DNL343 treatment from the pre-symptomatic stage improved body weight and motor functions corrected transcriptional changes, and reversed proteomic and metabolomic alterations in the brain and cerebrospinal fluid. DNL343 treatment initiated at an advanced disease stage also showed positive effects, restoring body weight gain, suppressing ISR, reducing neurodegeneration biomarkers, and extending lifespan. These findings highlight DNL343 as an effective ISR inhibitor with potential applications in treating VWMD and other neurodegenerative disorders involving ISR.

Strengths:
The study's findings regarding the novel compound DNL343 offer significant promise in addressing VWMD, a condition currently lacking disease-modifying treatment. DNL343 directly targets eIF2B, the disease-causing complex in VWMD, and demonstrates notable efficacy in reversing the integrated stress response (ISR) and mitigating neurodegeneration in a VWMD mouse model. These results raise hope for the potential application of DNL343 in VWMD treatment, a development eagerly anticipated by patients and the VWMD research community. Moreover, the study hints at the broader potential of DNL343 in treating other ISR-related neurodegenerative disorders, such as amyotrophic lateral sclerosis, a prospect that holds broader interest. Additionally, the study's identification of potential biomarkers for VWMD represents a notable strength, potentially leading to improved disease progression assessment pending further confirmation in future research.

Weaknesses:
There are a couple of notable concerns in this study. Firstly, while the in vivo evidence strongly supports the efficacy of DNL343 in mitigating ISR and neurodegeneration, there is a lack of direct biochemical evidence to confirm its activity in eIF2B activation. Secondly, the potential for cardiovascular toxicity, which has been reported for a related eIF2B activator in a canine model (as mentioned in the manuscript), has not been evaluated for DNL343 in this study. This data gap regarding toxicity could be crucial for informing the future development of DNL343 for potential human use. Further investigation into these areas would be valuable for a comprehensive understanding of the compound's mechanisms and safety profile.

Reviewer #2 (Public Review):

Summary:
The authors developed DNL343, a CNS-penetrant small molecule integrated stress response (ISR) inhibitor, to treat neurodegenerative diseases caused by ISR.

Strengths:
DNL343 is an investigational CNS-penetrant small molecule integrated stress response (ISR) inhibitor designed to activate the eukaryotic initiation factor 2B (eIF2B) and suppress aberrant ISR activation. The therapeutic efficacy of DNL343 has been extensively characterized in two animal models. Importantly, plasma biomarkers of neuroinflammation and neurodegeneration can be reversed with DNL343 treatment. Remarkably, several of these biomarkers show differential levels in CSF and plasma from patients with vanishing white matter disease (VWMD) upon DNL343 treatment. Overall, this is a very exciting study to target ISR for therapeutic interventions.

Weaknesses:
My main questions center around the characterization of DNL343.

1. Is there any biochemical evidence showing DNL343 activates eIF2B, such as binding assays or in vitro biochemical activity assays? A conference presentation was cited - "Osipov, M. (2022). Discovery of DNL343: a Potent Selective and Brain-penetrant eIF2B Activator Designed for the Treatment of Neurodegenerative Diseases. Medicinal Chemistry Gordon Research Conference. New London, NH." However, there needs to be public information about this presentation.

2. How was the selectivity of DNL343 demonstrated? What are the off-targets of DNL343, in particular when DNL343 is administered at a high dose? Thermal-proteasome profiling or photoaffinity labeling experiments could be considered.

3. What are the total drug concentrations in the brain and plasma? What are the unbound ratios?

4. If DNL343 is given intravenously, what are the concentrations in the brain and plasma after 5 minutes and 1 hour or longer time points? In other words, does DNL343 cross BBB through passive diffusion or an active process?

5. What is the complete PK profile of DNL343 for intravenous and oral dosing?

6. Are there any major drug metabolites that could be of concern?

Reviewer #3 (Public Review):

Summary:
ISR contributes to the pathogenesis of multiple neurodegenerative diseases, such as ALS, FTD, VWMD, etc. Targeting ISR is a promising avenue for potential therapeutics. However, previously identified ways to target ISR present some challenges. PERK inhibitors suppress ISR by inhibiting eIF2alpha phosphorylation and cause pancreatic toxicity in mice. In order to bypass eIF2alpha, previous studies have identified ISR suppressors that target eIF2B, such as ISRIB and 2BAct. These molecules suppress neurodegeneration but do not cause detrimental effects in mouse models. However, ISRIB is water-insoluble, and 2BAct causes cardiovascular complications in dogs, preventing their use in clinics. Here, the authors showed that DNL343, a new ISR inhibitor targeting eIF2B, suppresses neurodegeneration in mouse models. Combined with their previous results of a clinical phase I trial showing the safety of DNL343, these findings suggest the promise of DNL343 as a potential drug for neurodegenerative diseases in which ISR contributes to pathogenesis.

Strengths:
The finding is important and has disease implications, and the conclusion is not surprising.

Weaknesses:
The experimental design and data are hard to comprehend for an audience with a basic research background. This reviewer suggests that the authors use the same way that previous studies on ISRIB and 2BAct (e.g., Wong et al; eLife, 2019) designed experiments and interpret data.