M102 activates both NRF2 and HSF1 transcription factor pathways and is neuroprotective in cell and animal models of amyotrophic lateral sclerosis

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Keerie, Raquel Rua Martins, Chloe F. Allen, Sufana Al Mashhadi, and 17 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6964528/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 04 Nov, 2025 Read the published version in Molecular Neurodegeneration → Version 1 posted 11 You are reading this latest preprint version Abstract M102 is a central nervous system (CNS) penetrant small molecule electrophile which activates in vivo the NF-E2 p45-related factor 2 - antioxidant response element (NRF2-ARE) pathway, as well as transcription of heat-shock element (HSE) associated genes. In the TDP-43 Q331K transgenic mouse model of ALS dosed subcutaneously at 5mg/kg OD or 2.5mg/kg BD with M102, significant improvements in compound muscle action potential (CMAP) amplitude of hind limb muscles and gait parameters were observed at 6 months of age, with associated target engagement. An oral dose response study of M102 in SOD1 G93A transgenic mice showed a dose-dependent improvement in CMAP of hindlimb muscles which correlated with preservation of lumbar spinal motor neurons at the same time point. These data enabled prediction of human efficacious exposures and doses, which were well within the safety margin predicted from Good Laboratory Practice (GLP) toxicology studies. A parallel program of work in vitro showed that M102 rescued motor neuron survival in co-culture with patient-derived astrocytes from sporadic, C9orf72 and SOD1 ALS cases. Markers of oxidative stress, as well as indices of TDP-43 proteinopathy were also reduced by exposure to M102 in these in vitro models. This comprehensive package of preclinical efficacy data across two mouse models as well as patient-derived astrocyte toxicity assays, provides a strong rationale for clinical evaluation of M102 in ALS patients. Combined with the development of target engagement biomarkers and the completed preclinical toxicology package, a clear translational pathway to testing in ALS patients has been developed. S-apomorphine M102 NRF-2 activation HSF1 activation neuroprotection amyotrophic lateral sclerosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 INTRODUCTION Amyotrophic lateral sclerosis (ALS) is a rapidly progressive, fatal neurodegenerative disorder in which motor neuron injury and cell death causes muscle weakness and wasting, leading to progressive loss of motor control of the upper and lower limbs as well as bulbar and respiratory functions. The lifetime risk is approximately 1 in 300-350 (1) and the average course of the disease is 2.5-3 years from symptom onset (2). Currently, there are no effective treatments to halt or reverse the progression of ALS and approved drugs only marginally increase survival (riluzole) or disease progression (edaravone) (3-5). The anti-sense oligonucleotide (ASO) treatment tofersen (QALSODY) has beneficial clinical effects and biomarker readouts, but applies only to the ~2 percent of ALS patients who harbor a SOD1 mutation (6). A key feature of ALS is the speed of progression. This poses huge problems of adjustment for affected individuals, an escalating burden on carers and families, and a challenge to those purchasers and providers of healthcare who are involved in meeting the variable, rapidly changing and complex care needs (7, 8). The pathophysiology of ALS is complex. More than 30 genes are known to cause or contribute to motor neuron degeneration in ALS (9). Even in the presence of a mutation in a gene such as SOD1, the structure and function of which are well understood, it is recognised that a cascade of multiple pathophysiological mechanisms within both motor neurons and neighbouring glial cells contribute to neurodegeneration (9- 11). Data from disease model systems and human biosamples provide strong evidence for a role of redox imbalance, inflammation, mitochondrial dysfunction and altered proteostasis, as key drivers of the pathobiology of ALS (12-16). Therapies targeting individual pathways have failed in the clinic or have shown marginal efficacy. In addition, few studies in ALS have shown target engagement for the proposed therapeutic agent in the clinic (17) with the exception of tofersen (6). There is clearly a huge unmet need for effective neuroprotective therapies to slow disease progression in ALS. Transcription factor NF-E2 p45-related factor 2 (NRF2) is a master regulator of the antioxidant response and activates the expression of over 1000 genes with cytoprotective properties (18-20). NRF2 protein levels are highly regulated through several different mechanisms including negative regulation at the protein level by Kelch-like ECH-associated protein 1 (KEAP1) (21). There is a body of evidence from both cellular and animal models and human biosamples that this cytoprotective NRF2 system is dysregulated in ALS (22-27). We previously identified M102 (chemical name: S-(+)-10,11-dihydroxyaporphine) in a compound screen to discover blood brain barrier penetrant NRF2-ARE pathway activators (28). We reported that M102 enhanced glutathione (GSH) secretion from astrocytes in co-culture; protected neuromuscular junctions from denervation in SOD1 G93A mice; and slowed the decline in motor function when dosed at 5 mg/kg subcutaneously daily. M102 also reduced the elevated basal oxidative stress seen in fibroblasts from ALS cases (28). M102 (S[+]-apomorphine) is an enantiomer of R-apomorphine which is used as a dopamine agonist in Parkinson’s disease. M102 itself is a very weak dopamine antagonist (29) and its structure is consistent with known NRF2 activators predicted to function through modification of cysteine residues on KEAP1, thereby reducing the degradation of NRF2, increasing NRF2 translocation to the nucleus and upregulating the expression of multiple cytoprotective genes (18, 20). M102, which we now demonstrate activates both the NRF2 and HSF1 transcription factor pathways, has the potential to modulate all four of the key drivers of neurodegeneration highlighted above. By targeting multiple pathways, we expect to significantly increase the probability of neuroprotection of motor neurons and clinical success. Lack of translation between mouse models and clinical trials has hampered ALS treatment discovery over the last 20 years, with many compounds showing potential in mouse models, but failing to show efficacy in clinical trials (17, 30, 31). Here, we have generated an encouraging pre-clinical data set demonstrating significant positive effects on the same behavioural outputs in two robust and reproducible ALS mouse models, as well as a preclinical toxicology package that predicts safe efficacious doses in humans. In addition, we provide evidence that M102 increases motor neuron survival in an in vitro model of patient-derived astrocyte toxicity (10) across multiple subtypes of ALS. M102 reduces toxicity from astrocytes derived from C9orf72, SOD1 and sporadic ALS patients and results in neuroprotection of co-cultured motor neurons. Overall, to our knowledge, this is one of the most comprehensive packages of pre-clinical efficacy for therapy development in ALS. RESULTS M102 activates NRF2 and HSF1 transcription factor pathways in vivo We previously identified M102 in a screen for CNS penetrant NRF2 activating molecules. We demonstrated that M102 activated NRF2-directed transcription in vitro and in the CNS and was able to rescue motor deficits in the SOD1 G93A mouse model of ALS at a daily subcutaneous dose of 5 mg/kg (28). In vitro evidence showed that M102 was able to increase production of glutathione from astrocytes, thereby mediating neuroprotection to co-cultured motor neurons. In order to expand the preclinical validation to other genetic subtypes of ALS we set out to establish a dose response for NRF2 activation in the CNS as a precursor to investigating efficacy in a second, TARDBP mutant ALS mouse model for which we have validated readouts (32). Electrophilic compounds such as M102 also have the potential to activate HSF1 (heat shock factor 1) signalling pathways (33, 34) and since HSF1 activation would address additional pathogenic mechanisms in ALS (35), we also sought to establish whether M102 was able to transcriptionally activate canonical HSF1 targets in vitro and in vivo as well as NRF2-related targets. We treated HeLa-HSE-Luciferase cells expressing luciferase under the transcriptional control of the HSP70.1 promoter with varying concentrations of M102, or M102 in the presence of CuCl 2 to mimic oxidation in vivo , and showed that M102 was a robust activator of the HSP70.1 promoter ( Suppl. Fig. 1 A, B ). Western blotting of lysates of SHSY5Y cells, showed that M102 increased expression of both the NRF2 target haem oxygenase 1 (HO-1) and the HSF1 target HSP70 (Hspa1a), and that siRNA treatment reducing HSF1 or NRF2 was able to ablate the response for their respective targets ( Suppl. Fig. 1 C, D ) To confirm activation of both pathways in vivo , we dosed wild-type (WT) mice with M102 at 0.5, 1.5, 5 and 10 mg/kg SC, once daily for 7 days, and measured the transcriptional response in cerebral cortex tissue using RT-qPCR. Fig. 1 shows the transcriptional response of NRF2 ( Fig. 1A ) and HSF1 ( Fig. 1B ) regulated genes. Gene targets of both transcription factors showed a clear dose response, with 5mg/kg being optimal in most cases. This represents the first indication that M102 is also able to activate HSF1 directed transcription in vivo , with HSF1 regulated genes including Hspa1a (HSP70), Hspa8, and Syn1 showing elevated transcriptional responses. M102 rescues motor, weight and, neurophysiological phenotypes in the TDP-43 Q331K transgenic mouse model of ALS The study design for evaluating the effect of M102 in huTDP-43 Q331K transgenic mice is shown in Fig. 1C . Female TDP-43 Q331K mice were block randomised into three dosing groups and dosed subcutaneously with 5mg/kg M102 as a split dose twice daily at 2.5mg/kg twice daily (BD) or a single 5mg/kg once daily (OD) dose from 25 days until 6 months of age. A variety of behavioural tests were performed at time points previously shown to correlate with significant milestones of disease progression, and CNS tissue was collected at 3 and 6 months. Transgenic TDP-43 Q331K mice gain significantly more weight compared to their non-transgenic littermates (32). This is due to the increased amount of food they consume and the reduction in activity seen with this mutation in TDP-43 that is linked to an apathy fronto-temporal dementia (FTD) phenotype (36). Mouse weights were recorded daily before dosing to determine dose volume. Fig. 1D shows that weight gain is significantly reduced from 161 days in the 2.5mg/kg BD group (two-way ANOVA with Dunnett’s post-test). At the end of the study there was a significant decrease in weight of the M102 2.5mg/kg BD dosed mice when compared to vehicle controls. At 177 days of age, M102 2.5mg/kg BD dosed animals weighed 23.2 +/- 1.9g compared to vehicle animals that weighed 25.4 +/- 2.3g (p = 0.03). When analysed as area under the curve, weight is significantly reduced in both 5mg/kg and 2.5 mg/kg BD dosed groups ( Fig. 1E ; one-way ANOVA with Dunnett’s post-test). Rotarod performance was also improved in the 5mg/kg dose group at 19 weeks of age ( Fig. 1F -two- way ANOVA with Dunnett’s post-testing). Gait analysis was carried out at 3 and 6 months of age. An improvement in gait ( Fig. 1 G,H ) was observed in the 2.5mg/kg BD dosed group when compared to vehicle controls, using Catwalk (NoldusXT) gait analysis. This was shown as an increase in the amount of time spent using diagonal paws, a marker of normal mouse gait, versus a decrease in the amount of time spent on three paws, suggesting a reduction of gait unsteadiness (one-way ANOVA, with Dunnett’s post-test). Muscle electrophysiology was performed at 6 weeks and 6 months of age. At 6 weeks of age there was no significant difference in compound muscle action potential (CMAP) amplitude between either of the M102 dose groups and the vehicle dosed group. However, at 6 months of age there was a significant improvement in CMAP amplitude in both M102 treated groups when compared to the vehicle group ( Fig. 2A , two-way ANOVA with Dunnett’s post-test). The percentage change in CMAP amplitude between 6 weeks and 6 months of age also showed significant improvement of the M102 2.5mg/kg BD dosed group when compared to the vehicle dosed group ( Fig. 2B , one-way ANOVA with Dunnet’s post-test). At 6 months of age there was also a significant improvement in response to repetitive stimulation in the 2.5mg/kg BD dosed animals when compared to vehicle dosed animals, suggesting improved maintenance of the neuromuscular junctions (NMJs) ( Fig. 2C , one-way ANOVA with Dunnett's post-test). Motor neuron counts and size distribution from the ventral horns of the lumbar spinal cord showed no significant difference between M102 dosed groups and vehicle dosed groups ( Fig. 2D , two-way ANOVA with Dunnett’s post-test). However, in this model, we were unable to detect any significant difference between WT and TDP-43 Q331K transgenic mice in lumbar spinal cord motor neuron counts at a similar timepoint ( Suppl. Fig. 2 ). Analysis of RNA levels in the cerebral cortex by RT-qPCR showed an increase in downstream targets of NRF2 and HSF1 targets at 3 months of age in M102 dosed mice when compared to vehicle dosed mice, demonstrating activation of these pathways over time in the target tissue of the CNS ( Fig. 2E , two-way ANOVA with Dunnett’s post-test). Pharmacokinetic data in wild-type mice (Suppl. Fig. 3) Following a single subcutaneous dose of 5 mg/kg in mice, M102 exhibited a mean Cmax (maximum plasma concentration) and AUC last (area under the plasma concentration-time curve to the last measurable plasma concentration) of 989 ng/mL and 380 ng/mL*h, respectively. Pharmacokinetic (PK) evaluation of M102 post single dose oral administration was also performed. After a single oral dose of 10 mg/kg in mice, M102 showed a mean C max and AUC last of 168 ng/mL and 187 ng/mL*h, respectively. In the preclinical pharmacology studies in transgenic mouse models of ALS, M102 has demonstrated significant efficacy at a daily subcutaneous dose of 5 mg/kg (28). Assuming a correlation of M102 efficacy to its systemic exposure and dose proportionality, the efficacious oral dose of M102 was estimated to be 25 mg/kg or lower. This estimate was further confirmed in an additional in vivo pharmacology study in SOD1 G93A mice, where efficacy was observed at 12.5 and 25 mg/kg of M102 administered orally. Oral administration of M102 in SOD1 G93A mice improves body weight, neurophysiology, and spinal cord motor neuron counts We have previously shown disease modifying effects of M102 in the SOD1 G93A mouse model when M102 was dosed subcutaneously at 5 mg/kg daily (28). In that study there was an upregulation of Hmox1 and Nqo1 in the spinal cord, a significant preservation of innervation at the neuromuscular junctions, a significant decrease in oxidised glutathione with an increase in the GSH/GSSG ratio in CNS tissue, and an improvement in rotarod performance and gait analysis parameters in the M102 exposed mice compared to the control group. We wanted to further expand on these data, by exploring the effect of M102 dosed orally at an equivalent dose to the subcutaneous dose, as this is the preferred route of administration in humans. Transgenic female SOD1 G93A animals were block randomised into different groups and dosed orally with either vehicle, 5 mg/kg, 12. 5mg/kg or 25 mg/kg M102 daily from 25 days until 90 days of age (n=8 per group). The doses were chosen as 5 mg/kg subcutaneous dosing has an equivalent exposure as 25 mg/kg oral dosing ( Suppl. Fig. 3 ). Behavioral tests were carried out at various time points to determine motor function and tissue was collected at 90 days for histological and mRNA analysis ( Fig. 3A ). Animals were weighed daily before dosing to determine dose volume ( Fig. 3B , two-way ANOVA followed by Dunnett’s post-test). SOD1 G93A transgenic mice are hypermetabolic and lose weight in the later stages of disease when compared to non-transgenic mice due to muscle and fat loss (37, 38). Area under the curve analysis showed a significant increase in body weight, and thus improvement of disease progression, of the 5 mg/kg and 25 mg/kg M102 groups when compared to vehicle dosed animals ( Fig. 3C ; p <0.05 at 5 mg/kg and <0.001 at 25 mg/kg; one-way ANOVA followed by Dunnett’s post-test). The number of motor neurons in the lumbar ventral horns was counted and a significant dose-dependent increase in the number of surviving motor neurons was observed in the M102 groups when compared to vehicle treated animals ( Fig. 3D , one-way ANOVA with Dunnett’s post-test). At 90 days of age there was a significant improvement in the CMAP amplitude of the M102 25 mg/kg dose group when compared to vehicle controls ( Fig. 3E, F , one-way ANOVA with Dunnett’s post-test). The percentage change in CMAP amplitude between 60 and 90 days of age showed a dose-dependent increase in the M102 dosed groups when compared to the vehicle dosed group with 1/7 mice showing an increase in in CMAP in the vehicle group versus 6/8 in the 25mg/kg M102 treated group ( Fig. 3G , one-way ANOVA with Dunnett’s post-test). Dose prediction of M102 to humans and safety margin Efficacy was observed in the mouse SOD1 G93A model at an oral dose of 12.5 mg/kg which resulted in a AUC last of 419 ng*h/mL. Adjusting for the difference in human vs. mouse free fraction yields an estimated human equivalent exposure of 541 ng*h/mL. This value was used as the target efficacy exposure in the human dose projections as well as in the safety margin calculations. To confirm brain penetration of M102, additional plasma and brain PK studies were conducted in rats ( Suppl. Table 1 ). After a single dose oral administration of 12 mg/kg M102, the brain-to-plasma ratios of M102 were determined to be 0.70 and 0.36 at 30 and 60 min post-dose, respectively. Subsequently, M102 was evaluated in non-Good Laboratory Practice (GLP) and GLP general toxicology studies in rats and non-human primates (NHPs) and the findings are summarised in Supplementary tables 2 (rats) and 3 (NHPs). In the non-GLP toxicology studies, M102-related mild liver toxicity findings were observed at 250 mg/kg and 100 mg/kg in rats and NHPs, respectively. In 28-day GLP toxicology studies, 75 mg/kg was declared as the no observed adverse effect level (NOAEL) in both rats and non-human primates (NHPs). The free AUC on Day 28 at the NOAEL in both species, as well as the margin to the projected human free AUC necessary for efficacy, is 9.3-fold for rats, 10.1-fold for female NHPs, and 15.1-fold for male NHPs. Human clearance, volume of distribution and half-life were estimated using PK data from rat and monkey studies. Various allometric scaling techniques were applied following the rule of exponents guidelines. Human clearance was also estimated from in vitro hepatocyte incubations. Correcting for brain weight, free fraction and using the multi-exponential method (39) resulted in estimates ranging from 55-111.7 mL/min/kg, while scaling intrinsic clearance from human hepatocytes resulted in an estimate of 84 mL/min/kg. Human volume of distribution was estimated by allometric scaling of preclinical species values and using an exponent of 1 and was approximately 81 L/kg. M102 has a bioavailability range of 10-30% in preclinical species. As a predictive model of human bioavailability does not exist, this range of potential values were modelled. Using the projected human efficacious exposure target of 302 ng*h/mL that was based on mouse efficacy at an oral dose of 12.5 mg/kg PO, a daily dose range in humans of 352-1417 mg is predicted. Increased oxidised RNA in affected CNS areas, and in the CSF and i-Astrocytes derived from ALS cases Encouraged by the compelling data obtained in two in vivo models of ALS, we decided to evaluate the levels of oxidative stress detectable in patient biosamples and in a patient-derived cellular model of ALS, as potential biomarkers of target engagement and efficacy. Oxidative stress is one of the hallmarks of ALS, with oxidised lipids and proteins being identified in post- mortem tissues (40). Due to the recently identified involvement of RNA dysregulation in ALS (41) and the known role for oxidised RNA in neurodegeneration (42), we aimed to map the presence of oxidised RNA in the areas of the CNS that are affected by the disease. As guanine is the base that is most susceptible to oxidation, we used an antibody against 8-oxo-2-oxyguanosine (8-OHG), as a marker of direct nucleic acid oxidation. Staining of frontal and motor cortex with adjacent white matter, as well as the cervical spinal cord, from 6 sALS patients, 4 patients carrying C9orf72 repeat expansion mutations and 3 healthy controls (subject information in Suppl. Table 4 ) revealed that ALS patients display much higher levels of oxidised RNA compared to age-matched, neurologically unaffected controls ( Fig. 4 A-C ). Of note, patients carrying C9orf72 mutations with both ALS and fronto-temporal dementia (FTD), displayed significantly higher levels of oxidised RNA staining in the frontal cortex compared to both controls and ALS patients who displayed only a motor phenotype ( Fig. 4B ). Interestingly, although ALS patients displayed higher levels of oxidised RNA in the spinal cord compared to controls (Fig. 4C) , amongst all the CNS areas analysed, the spinal cord was the tissue with the highest oxidative stress burden ( Fig. 4A ). Given the positive correlation between 8-OHG staining and pathology, we proceeded to examine whether oxidised RNA levels in the CSF could be used as a biomarker of oxidative stress. Using an ELISA kit specific for oxidised RNA, we detected higher levels of 8-OHG in the CSF of ALS patients (n=13; 9 males and 4 females; average age= 56.2 ± 10 years) compared to controls (n= 12; 5 males and 7 females; average age= 44.2 ± 12 years ( Fig. 4 D , unpaired t-test). However, there did not seem to be any correlation between clinical/genetic characteristics and levels of oxidised RNA (clinical information in Suppl. Table 5 ). Furthermore, the levels of oxidised RNA did not correlate with age in healthy controls, suggesting the higher levels of oxidised RNA identified in the ALS cases are not due to a potential age difference (Suppl. Figure 4). To determine whether increased levels of oxidised RNA were also present in ALS patient-derived astrocytes, we stained iAstrocytes differentiated from induced neural progenitor cells (iNPCs) directly reprogrammed from fibroblasts collected via skin biopsy from ALS cases and age-matched healthy controls (patient information in Suppl. Table 6 ). These iAstrocytes are known to retain the features of ageing that are likely to contribute to patient-specific phenotypes (43), and therefore constitute a robust in vitro model of the astrocyte contribution to ALS. iAstrocytes derived from SOD1 , C9orf72 and sporadic ALS patients displayed higher levels of oxidised RNA compared to healthy controls ( Fig. 4E,F , one-way ANOVA followed by Dunnett's multiple comparisons test), recapitulating the findings in post-mortem tissue. M102 treatment is a dual activator of the NRF2 and HSF1 pathways in ALS patient-derived iAstrocytes, and is sufficient to reduce oxidative stress, misfolded SOD1, and TDP-43 proteinopathy in vitro . As ALS patient-derived iAstrocytes recapitulate the high levels of oxidative stress observed in post-mortem tissue, we set out to evaluate the ability of M102 treatment to activate the antioxidant NRF2 pathway and therefore contribute to decreased oxidative stress in these cells. It is known that the NRF2 pathway is downregulated in astrocytes from models of ALS (27). Consistently, iAstrocytes derived from SOD1 (n=1), C9orf72 (n=3) and sporadic (n=2) ALS cases displayed lower levels of NQO1, a downstream target of NRF2 (18, 20), compared to age-matched healthy controls, under baseline conditions ( Fig. 5A,B , one-way ANOVA followed by Dunnett's multiple comparisons test; full blots in Suppl. Figure 5 ). Importantly, when treated with 10 μM of M102 for 48h, the levels of NQO1 increase significantly in iA astrocytes from healthy controls and ALS patients, including SOD1 , C9orf72 and sporadic patient lines ( Fig. 5A,C , two-way ANOVA followed by Šídák's multiple comparisons test). Interestingly, iAstrocytes from ALS cases with a C9orf72 mutation showed a higher response to M102 compared to iAstrocytes from sporadic patients or patients with a SOD1 mutation. Overall NRF2 levels in the nucleus vs cytoplasm also increased upon 24h treatment with M102 ( Figure 5D, E , two-way ANOVA), providing evidence that M102 activates the NRF2-ARE pathway in ALS patient-derived iAstrocytes. Similar to what is observed in mouse models of ALS, M102 treatment also leads to increased expression of HSF1 in patient-derived iAstrocytes ( Fig. 5F, G , two-way ANOVA followed by Šídák's multiple comparisons test), indicating that M102 is a dual activator of the NRF2-ARE and HFS1-HSE pathways in vitro . Consistent with these findings, iAstrocytes derived from SOD1 , C9orf72 and sporadic ALS patients treated with 10 μM M102 for 48h showed decreased levels of oxidised RNA, thus confirming that M102 is capable of reducing the levels of oxidative stress in ALS astrocytes ( Fig. 6A,B , two-way ANOVA followed by Šídák's multiple comparisons test). The HSF1-HSE pathway is known to play an essential role in maintaining proteostasis by facilitating protein folding and avoiding protein misfolding (35). As we showed that M102 is a strong activator of the HSF1 pathway, we set out to investigate whether M102 treatment has a beneficial effect on the reduction of misfolded SOD1 and TDP-43 proteinopathy, two hallmarks of ALS pathology (44, 45). By using an antibody raised against misfolded SOD1 (B8H10), we detected perinuclear staining in iAstrocytes from SOD1 cases, as well as cases carrying C9orf72 mutations and sALS patients, consistent with previous findings in post-mortem tissues. Upon treatment with M102, we observed a significant reduction of misfolded SOD1 in iAstrocytes derived from SOD1, C9orf72 , and sporadic ALS patients ( Figure 6C,D , two-way ANOVA followed by Šídák's multiple comparisons test). Induced astrocytes recapitulate one of the key hallmarks of ALS, i.e. TDP-43 proteinopathy, detected as the presence of TDP-43 fragments (observed at 35 kDa) in sporadic and C9orf72 ALS patient iAstrocytes ( Fig. 6E ), but not in SOD1 cases ( Suppl. Fig. 6). Importantly, we observed that time-dependent treatment with M102 leads to a reduction of TDP-43 proteinopathy, particularly upon 48h exposure ( Fig. 6E , F, paired t-test). M102 rescues MN survival in co-culture with ALS patient-derived iAstrocytes by targeting multiple mechanisms known to underlie ALS pathophysiology Astrocytes from ALS patients are known to be toxic to MNs, contributing to death of healthy motor neurons in co-culture (10). We have previously reported that iAstrocytes differentiated from iNPCs directly reprogrammed from fibroblasts of SOD1, C9orf72 and sporadic ALS patients are toxic to MNs (10). As M102 seems to target multiple mechanisms associated with ALS, including oxidative stress and protein misfolding and aggregation, we asked whether M102 treatment would be sufficient to rescue MN survival when in co-culture with toxic ALS astrocytes. For this, we used our previously described iAstrocyte-MN co-culture model (10). Briefly, ALS patient-derived iAstrocytes were treated with DMSO or M102, using the Echo 550 liquid dispenser, and 24h later co-cultured with healthy mouse MNs expressing GFP under a Hb9 promoter. The MNs were then scanned in an InCell Analyser 24h and 72h after treatment, and the numbers of viable MNs counted ( Fig. 7A ). To assess the optimal dose of M102 to use, we started by testing 6 different concentrations of M102, ranging from 0.03 μM to 10 μM, in 4 sALS iAstrocyte lines. As expected, under baseline conditions, MNs co-cultured with ALS iAstrocytes showed reduced survival compared to MNs co-cultured with iAstrocytes derived from healthy controls. Furthermore, our dose-curve response showed that M102 has an EC 50 of 1.33 μM and the maximum neuroprotective effect in co-culture was achieved with a dose of 10 μM ( Fig. 7B ). We therefore used 10 μM M102 to further evaluate whether M102 is sufficient to rescue MN survival when in co-culture with multiple iAstrocyte lines derived from both familial and sporadic ALS cases. Under baseline conditions, MNs co-cultured with ALS patient-derived iAstrocytes displayed reduced survival (between 30%-60% reduction in MN survival depending on the patient donor) compared to MNs co-cultured with iAstrocytes derived from healthy controls ( Fig. 7C ). Treatment with 10 μM M102 led to a significant increase in MN survival in co-cultures with 7 out of 9 different iAstrocyte patient lines, including SOD1, C9orf72 and sporadic ALS cases ( Fig. 7C , two-way ANOVA followed by Šídák's multiple comparisons test). Interestingly, the response to M102 varied between patient lines, with 4 patient lines responding more strongly by increasing MN survival by >50% compared to baseline levels. In order to further explore the effects of M102 and better understand how M102 could play a role in iAstrocyte toxicity and MN survival, we performed RNA Sequencing in patient-derived iAstrocytes before and after 10 μM M102 treatment for 48h. Our data show that there is a clear separation of the transcriptomic profile before and after M102 treatment, as shown by a principal component analysis (PCA) plot ( Fig. 7D ). This transcriptomic shift in response to M102 is observed in iAstrocytes derived from SOD1, C9orf72 , and sporadic patients ( Suppl. Fig. 7 A,B,C, respectively ). Differential expression analysis between treated and untreated iAstrocytes identified a total of 160, 283, and 267 differentially expressed genes (DEGs) in C9- ALS cases, SOD1 cases, and sALS cases, respectively ( Suppl. Fig.7 D ). We further performed gene ontology analysis to investigate the predominant pathways affected by M102 treatment. The results show that M102 treatment altered the expression of genes associated with neuroinflammation, mitochondrial dysfunction, autophagic response and cell adhesion ( Fig. 7E, Suppl. Table 7 ), all known to be important drivers of the pathophysiology of ALS (9). Taken together with our results demonstrating that M102 is a dual activator of the NRF2 and HSF1 pathways, these results indicate that M102 targets multiple pathophysiological mechanisms operating in ALS. As we observed that some iAstrocyte lines responded more strongly to M102 than others, we then set out to test whether we could discriminate high and low responders to M102 based on the individual patient transcriptomic profiles. For this, we classified the iAstrocytes in ‘high’ and ‘low’ responders to M102 based on the levels of motor neuron survival upon M102 treatment in iAstrocyte-MN co-cultures. iAstrocytes that responded to M102 with an increase in motor neuron survival of over 50% were considered ‘high responders’ (4 patients) whereas the iAstrocytes that responded with an increase in motor neuron survival less than 50% were considered 'low responders’ (4 patients). A heatmap of healthy controls and ALS patient-derived iAstrocytes before and after M102 treatment showed a set of 161 transcripts identified significant differences between “high” and “low” responders to M102. Interestingly, the low responders to M102 show a transcriptomic profile for these 161 transcripts that is closer to healthy controls compared to high responders, under baseline conditions. Upon M102 treatment, high responders displayed a shift in their transcriptomic profile for these 161 transcripts, which became more similar to the profile of healthy controls ( Fig. 7F ), thus indicating that a subgroup of these genes could be used as a biomarker of drug response. MATERIALS AND METHODS (For Full Materials and Methods, see Supplementary Material) Study design This study aims to provide a comprehensive package of preclinical efficacy data for M102, a CNS penetrant small molecule electrophile capable of activating both NRF2-ARE and HSF-1-HSE pathways, in vivo and in vitro . To do this, the pharmacokinetic profile was assessed in C57Bl/6 mice; GLP general toxicology studies were performed in rats and NHPs; and oral dose, target engagement and efficacy were assessed across two ALS mouse models: TDP-43 Q331K and SOD1 G93A . RT-qPCR was used to assess target engagement after M102 treatment. Immunohistochemistry (IHC) was used to identify and count the number of motor neurons in the spinal ventral horn after M102 treatment. Compound muscle action potential (CMAP) amplitude of hind limb muscles, rotarod performance, and gait parameters were used to assess treatment efficacy on readouts of motor function. Together, these data enabled prediction of human efficacious exposures and doses, which were established to be well within the safety margin predicted from GLP toxicology studies. Post-mortem tissue, human CSF samples, and ALS patient-derived astrocytes were used to confirm that ALS patients present higher levels of oxidative stress compared to healthy controls, and that M102 is capable of reducing the levels of oxidative stress. A combination of immunocytochemistry (ICC) and western blotting analysis was used to assess the effects of M102 on NRF2-ARE and HSF-1-HSE pathway activation, as well as to assess TDP-43 proteinopathy in patient-derived astrocytes. MN-astrocyte co-cultures were performed to assess whether M102 can rescue motor neuron survival in the presence of toxic ALS iAstrocytes, and to determine high and low responders to M102. RNA Sequencing of untreated and M102 treated patient-derived iAstrocytes was used to identify additional biological mechanisms targeted by M102. No study size calculations or randomization were carried out. All samples were quantified in a blinded manner for microscopy and mouse experiments. No cell or animal samples were excluded. Ethics statement GLP general toxicology studies in rats and NHPs to evaluate potential toxicity of M102 were conducted at WuXi AppTec (Suzhou, China). The protocol and any amendments or procedures involving the care or use of animals in this study were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) prior to the initiation of such procedures. A staff veterinarian monitored the study for animal welfare issues. All mouse studies were carried out under a UK Home Office project license by individuals that held the appropriate UK Home Office personal license and had appropriate training for procedures. All work was carried out under the terms of the UK Animals (Scientific Procedures) Act 1986 and animals were housed and maintained in line with Home Office Code of Practice for House and Care of Animals Used in Scientific Procedures. Formalin-fixed, paraffin-embedded (FFPE) human CNS post-mortem tissue was obtained from the Sheffield Brain Tissue Bank with Research Ethics Committee approval (Sheffield Brain Bank –SBB-, Ethics Committee reference 08/MRE00/103). Human CSF was obtained from the University of Sheffield Biorepository with Research Ethics Committee approval number STH16573. Fibroblasts were collected from skin biopsies donated by ALS patients and controls with informed consent (Ethical Committee approval references: 12/YH/0330; 16/LO/2136). Mice Mice were housed in same sex groups of between 2 and 5 mice per cage. Each cage consisted of a plastic house, sawdust covering the floor (Datesand) and paper wool bedding (Datesand). The mice had ad libitum access to water and food (standard rodent diet 2018, Envigo). Temperatures in the rooms were maintained at 21°C with a 12 hour light/dark cycle (7am – 7pm). Wild type animals used were C67BL/6 mice either from Envigo or non transgenic mice from the SOD1 G93A colony. The SOD1 G93A C57BL/6 transgenic mice were bred in-house. The B6SJL-Tg(SOD1-G93A)1Gur/J mice were backcrossed onto the C57BL/6J OlaHsd background for at least 20 generations. This line has been extensively characterised in-house and develops a reproducible progressive motor phenotype (46). The TDP-43 Q331K C57BL/6NJ transgenic mice were bred in-house and were obtained from Jackson laboratory (stock number 017933). These mice were originally on a C57BL/6NCrl background (47) but have been crossed onto a C57BL/6NJ background for a minimum of 4 generations and have been extensively characterised by the authors (32). Mice were ear-clipped for identification and genotyping. Genotyping for the two colonies was carried out as previously described (32, 46). Differentiation of patient-derived neurons, motor neurons and astrocytes Tissue culture to generate induced neural progenitor cells (iNPCs) and iAstrocytes from donated fibroblasts. Fibroblasts collected from skin biopsy material donated by ALS patients and controls were directly reprogrammed into iNPCs, using a combination of retroviral vectors (Oct3/4, Sox2, Klf4, and c-Myc), as previously described (10). Briefly, the fibroblasts were treated with 700 μL medium/viral vector overnight, then washed 2× with PBS, and fed once per day with fibroblast medium (DMEM plus 10% FBS) for 3 days. At day 4 the cells were switched to NPC conversion medium consisting of DMEM/F12, 1% N2, 1% B27, 20 ng/mL FGF2, 20 ng/mL EGF, and heparin (5 μg/mL; Sigma-Aldrich) and fed every day thereafter. When the cells changed shape and presented sphere-like structures, they were lifted with accutase, centrifuged, resuspended in NPC conversion medium, and replated for expansion. Once the NPC culture was established, the medium was switched to NPC medium consisting of DMEM/F12, 1% N2, 1% B27, and FGF2 (40 ng/mL). To differentiate iNPCs into iAstrocytes, iNPCs were seeded in NPC medium at low density in a fibronectin-coated 10-cm dish. The day after, the medium was changed to DMEM containing 10% FBS and 0.3% N2 and the cells were allowed to mature for at least 7 days. Motor neuron differentiation from embryonic stem cells. Mouse embryonic stem cells expressing GFP under the MN-specific promoter HB9 (HBG3 cells; kind gift from Tom Jessell, Columbia University, New York) were cultured on primary mouse embryonic fibroblasts (Millipore). For differentiation into MNs, cells were lifted with trypsin and resuspended in DFK10 culture medium consisting of knockout DMEM/F12, 10% knockout serum replacement, 1% N2, 0.5% L-glutamine, 0.5% glucose (30% in water), and 0.0016% 2-mercaptoethanol. The cells were plated on nonadherent Petri dishes to allow formation of embryoid bodies. After 1 d of recovery, 2 μM retinoic acid (Sigma) and 1 μM smoothened antagonist (SAG, Merk) were added freshly every day with fresh medium. After 5 days of differentiation, the embryoid bodies were dissociated and sorted for GFP on a BD FACSVantage/DiVa sorter. Human iAstrocyte-murine motor neuron co-culture assay. Human plasma fibronectin (Merck Millipore) was diluted 1:400 in PBS, and 5 µL was added per well on 384-well plates (Greiner Bio-one, 781091. Plates were coated for at least 5 min at RT. A total of 2,000 human iAstrocytes were seeded in 35 μL iAstrocyte media per well on fibronectin-coated 384-well plates. Plates were centrifuged at 1,760 x g for 60 s, and cells were incubated for 24 h. Drugs were then delivered to iAstrocytes in 100 % anhydrous DMSO (Sigma, 276855) using an Echo550 liquid handler (Labcyte). The final concentration of DMSO was 0.1 % (v/v) in the media in all wells. Plates were centrifuged at 1,760 x g for 60 s, and cells were incubated for a further 24 h. A total of 2,500 murine Hb9-GFP+ motor neurons were seeded per well in motor neuron media (KnockOut DMEM (45% v/v), F12 medium (45% v/v), KO Serum Replacement (10% v/v), 50 units/ml penicillin/streptomycin (Lonza), 1 mM L-glutamine, 1X N-2 supplement (Thermo-Fisher Scientific), 0.15% filtered glucose, 0.0008% (v/v) 2-mercaptoethanol, 20 ng/ml GDNF, 20 ng/ml BDNF, 20 ng/ml CNTF) on top of the pre-treated iAstrocytes. Plates were centrifuged at 1,760 x g for 60 s. Hb9-GFP+ motor neurons were imaged after 24 and 72 hours using an INCELL analyser 2000 (GE Healthcare), and the number of viable motor neurons was counted using the Columbus™ analysis software (Perkin Elmer). The number of viable motor neurons (defined as GFP+ motor neurons with at least 1 process) that survived after 72 hours in co-culture was calculated as a percentage of the number of viable motor neurons after 24 hours in co-culture. The percentage survival of motor neurons was then normalised to the DMSO control for each individual iAstrocyte line. GLP general toxicological study in rats A total of 142 rats (71/sex) were randomly assigned to 4 groups, i.e., Groups 1-4 with M102 dose levels by oral gavage at 0, 25, 50, and 75 mg/kg, respectively. The main study (toxicity study) animals were 10/sex/group, while Groups 1 and 4 had an additional 5/sex/group to assess recovery of any observed effects. Toxicokinetics (TK) animals were 3/sex in the control group and 6/sex/group in the treated groups. Animals were approximately 6-7 weeks of age with body weights ranging from 178.46 to 211.76 g in females and 252.21 to 295.14 g in males at dosing initiation. The dosage volume was 10 mL/kg. The control group (Group 1) was administered 10% (w/v) HP-β-CD in purified water (HP-β-CD) by oral gavage. Criteria for evaluation included viability (morbidity/mortality), clinical observations, body weight, food consumption, ophthalmology, clinical pathology (hematology, coagulation, serum chemistry, and urinalysis), TK, gross pathology, organ weights, and histopathology. The concentration and homogeneity results of M102 in the dosing formulations met acceptance criteria, which demonstrated the formulations were accurately prepared and homogenous. GLP general toxicological study in non-human primates (NHPs) A total of 32 (16/sex) NHPs were randomly assigned to 4 groups including 5/sex/group in control and high dose groups, and 3/sex/group in low and middle dose groups. Dose groups were vehicle control [10% (w/v) HP-β-CD in purified water (HP-β-CD)] or M102 in vehicle at doses of 25, 50, or 75 mg/kg/day. At the end of the dosing phase, the last 2 surviving NHPs/sex/group in control and high dose groups were held for an additional 14 days without administration of the test article. Animals were approximately 2.4 to 2.9 months of age and with body weights ranging from 2.1 to 3.8 kg in males and 2.1 to 2.9 kg in females at dosing initiation. Criteria for evaluation included viability (morbidity/mortality), clinical observations, body weight, food consumption, ophthalmic examinations, electrocardiograms, clinical pathology (hematology, serum chemistry, coagulation, urinalyses), gross (necropsy) evaluation, organ weight, histopathological evaluation and toxicokinetics. Statistical analysis Statistical analysis was performed using GraphPad Prism v11.00. Normality was assessed by the Shapiro-Wilk test. Unpaired t-test or the Mann Whitney test were used to compare 2 data points, depending on whether the samples presented a Gaussian distribution or not, respectively. Paired t-test was used to compare the same samples before and after treatment. One-way ANOVA followed by Dunnett's multiple comparisons tests was used to compare more than 2 data points in 1 group. Two-way ANOVA followed by Šídák's multiple comparisons tests was used to compare more than 2 data points in 2 groups. DISCUSSION Our understanding of the genetic underpinnings and pathophysiological mechanisms in ALS has substantially increased in recent years (9). However, apart from the recent promising emergence of tofersen as a disease modifying therapy for the two percent of ALS patients who harbor mutations in the SOD1 gene, other approved drugs have only marginal effects on life expectancy (riluzole) or indices of disease progression (edaravone) (48). Therapies targeting individual pathways have failed to generate significant benefit during several decades of clinical trials. Previous attempts at identifying effective neuroprotective therapies for ALS have failed, as they do not account for disease heterogeneity and the multiple mechanisms driving motor neuron injury (17, 49). Data from disease model systems and from human biosamples provide strong evidence for a role of redox imbalance (40), inflammation (50), mitochondrial dysfunction (51) and altered proteostasis, including autophagy and mitophagy (51, 52), as four key drivers in the pathobiology of ALS (9, 49). Few, if any, studies in ALS have shown multi-target engagement for the proposed therapeutic agent in the clinic (17). We previously identified M102, a CNS-penetrant, small molecule, activator of the NRF2-ARE pathway. Here, we have also shown that it also activates the HSF-1-HSE transcription factor pathway, which targets multiple genetically validated pathophysiological mechanisms in ALS. We identified M102 in a screen to discover CNS penetrant NRF2-ARE pathway activators (28). M102 (S-apomorphine hydrochloride hemihydrate) is a proprietary new chemical entity (NCE) and the S-enantiomer of the marketed R-apomorphine (Apokyn®; pure R-enantiomer). The R-enantiomer is a dopamine agonist administered subcutaneously for the management of advanced Parkinson's disease. M102 is a very weak dopamine antagonist and does not show the adverse effects associated with dopamine agonism (29). Initial work showed beneficial effects in the SOD1 G93A mouse model and in indices of oxidative stress in ALS patient fibroblasts (28). Here, we demonstrate that M102 is a dual activator of NRF2 and HSF1 transcription factor pathways, two upstream master regulators of neuroprotective mechanisms, with the potential to modulate all four of these key drivers of neurodegeneration and with excellent penetration across the blood brain barrier (34, 39). NRF2 is a stress-responsive transcription factor and a master regulator of anti-oxidant and anti-inflammatory genes, activating the transcription of >1000 cytoprotective genes and with a multi-modal contribution to healthy mitochondrial function and regulation of key autophagy genes (53, 54). These pleiotropic effects enable the cell to maintain homeostasis under stress conditions. NRF2 activity is tightly regulated via a complex set of transcriptional and post-translational controls, principally by KEAP1 in the cytoplasm and BTB domain and CNC homolog 1 ( BACH1) in the nucleus (55). Under physiological conditions, KEAP1 promotes constitutive proteasomal degradation of Nrf2 (56) while BACH1 acts as a transcriptional antagonist of NRF2 target genes (57). Under oxidative stress conditions, KEAP1 is inactivated by modification of its reactive cysteine residues allowing NRF2 to escape degradation and newly synthesized NRF2 is able to translocate to the nucleus and bind to the anti-oxidant response elements (AREs) of multiple cytoprotective genes. This cytoprotective gene expression response provides protection against several key pathophysiological mechanisms operating in ALS including oxidative stress, mitochondrial dysfunction, inflammation and dysregulated proteostasis (18). The stress response of the KEAP1-Nrf2-ARE system is stronger in astrocytes compared to neurons (58). A body of evidence from in vitro and in vivo model systems and from post-mortem CNS tissue from ALS patients has indicated that the NRF2 response is impaired in ALS and has also been shown to decline with age (59-61). HSF1 is a stress-inducible transcription factor that is the key driver for the expression of multiple heat shock proteins which act as chaperones responsible for the correct folding of newly synthesized proteins, the refolding of denatured proteins and the prevention of aggregation of misfolded proteins. This heat shock response is a pro-survival pathway activated under conditions of cellular stress. This cellular stress response declines with age, with decreased ability of HSF1 to bind to the promoters of genes encoding heat shock proteins (35). Motor neurons have been reported to display a high threshold for the induction of the heat shock stress response (62). Nevertheless, activation of the HSF1/HSP pathway has shown beneficial effects in cellular and animal models of ALS, including clearance of TDP-43 aggregates (63). Activation of HSF1 is an attractive pharmacological target for ALS and other neurodegenerative conditions characterized by proteotoxic stress. However, to date, many small molecule activators of HSF-1 have shown undesirable properties e.g. by acting as Hsp90 inhibitors or by exerting direct proteotoxic effects (35, 64). In vitro and in vivo data presented here show that M102 is a multi-target drug with predicted neuroprotective benefits for the treatment of ALS. As a dual activator of NRF2 and HSF1 pathways, M102 is able to activate anti-oxidative and anti-inflammatory pathways, as well as beneficial modulating effects on proteostasis and mitochondrial function. In vitro data also show that M102 can reduce TDP-43 proteinopathy and biomarkers of oxidative stress in patient-derived astrocytes. Moreover, M102 is capable of rescuing MN survival in co-cultures with iAstrocytes derived from C9orf72, SOD1, and sporadic ALS patients, as well as spinal cord MNs in the SOD1 G93A mouse model. The efficacy of M102 was shown in vivo in both SOD1 G93A and TDP-43 Q331K transgenic mouse models of ALS, with beneficial effects on a translatable neurophysiological biomarker (CMAP) which correlates with motor neuron survival in the spinal cord. The transcriptome of patient-derived astrocytes exposed in vitro to M102 confirm the activation of multiple key neuroprotective pathways in astrocyte lines from several subgroups of ALS. There is a degree of heterogeneity in the level of the in vitro response to M102, with the transcriptome of high responders shifting substantially towards the profile observed in the astrocytes of healthy controls. This comprehensive package of pre-clinical efficacy data for therapy development in ALS is accompanied by strong safety data. We have completed the process of Good Manufacturing Practice (GMP) manufacturing for M102 and a series of GLP toxicology and safety pharmacology studies in rats and non-human primates, which have demonstrated that M102 will have an acceptable safety margin (9.3-15 for free drug) in clinical studies. Pharmacokinetic studies show strong penetration of M102 across the blood-brain-barrier, with a brain-plasma ratio of 0.7 at 30 min post-dosing. Other NRF2 activators have been investigated in clinical trials or have been approved for medical use. These include dimethylfumarate (DMF) (Tecfidera®, Biogen) and omaveloxolone (Reata, Biogen). DMF was originally approved for the treatment of psoriasis (Fumaderm®) and was later repurposed for the treatment of relapsing-remitting multiple sclerosis (Tecfidera®). A phase 2 trial of DMF in ALS provided Class 1 evidence of safety at a dose of 480mg/day and lack of disease-modifying efficacy (65). DMF treatment is associated with dose-limiting lymphopenia and flushing (Tecfidera® Prescribing Information). Omaveloxolone (Skyclarys®) is a potent NRF2 activator that has been approved by the FDA and EMA for the treatment of Friedreich's ataxia. By activating the NRF2 pathway, omaveloxolone ameliorates oxidative stress and improves mitochondrial function. As a potent NRF2 activator, omaveloxolone exhibited significant liver toxicity with elevated AST/ALT levels in 37% of patients exposed to a dose of 150 mg (66). Toxicity has also been reported with other potent NRF2 activators, such as bardoxolone methyl (EC50: 53 nM) which showed significant heart, liver, and renal toxicity in humans (67). In contrast, our preclinical toxicological studies indicate that M102 has a much higher safety margin in relation to liver toxicity. Arimoclomol is a co-activator of HSF1 and has been ineffective in two clinical trials targeting SOD1-ALS initially and then ALS more broadly (68,69). There is a huge unmet need for more effective neuroprotective therapies to slow disease progression in ALS. Here, we have demonstrated that M102, a combined activator of NRF2 and HSF1 signalling pathways, has positive therapeutic effects in two different ALS transgenic mouse models and improves motor neuron survival and multiple pathological markers (i.e. oxidative stress, misfolded SOD1, TDP-43 proteinopathy) in a range of human ALS cellular model systems. Importantly, these neuroprotective effects are seen across multiple subtypes of ALS, including the two most common genetic subtypes caused by mutations in the C9orf72 and SOD1 genes, as well as sporadic ALS cases. Taken together, M102 has the potential to modulate multiple key drivers of neurodegeneration, increasing the chances of achieving impactful neuroprotection and disease modifying effects in ALS. Its positive effect on TDP-43 proteinopathy and on rescuing motor neuron survival both in vivo and in co-culture with iAstrocytes derived from both familial and sporadic ALS patients also suggest that M102 may be beneficial for a wide range of ALS subtypes as ~97% of ALS cases display TDP-43 proteinopathy and ~90% of cases are sporadic. Abbreviations ALS – amyotrophic lateral sclerosis ARE - anti-oxidant response element ASO – anti-sense oligonucleotide AUC last - area under the plasma concentration-time curve to the last measurable plasma concentration BACH1 – BTB domain and CNC homolog 1 BCA - bicinchoninic acid C9orf72 - chromosome 9 open reading frame 72 CMAP – compound muscle action potential CMAX – maximum plasma concentration CNS – central nervous system CSF – cerebrospinal fluid DEG – differentially expressed gene DMF – dimethylfumarate DTT – dithiothreitol ELISA – enzyme -linked immunosorbent assay EMA – European Medicines Agency FDA – Food and Drug Admonistration FFPE - formalin-fixed, paraffin-embedded FTD – fronto-temporal dementia GAPDH - glyceraldehyde-3-phosphate dehydrogenase GLP – Good Laboratory Practice GMP – Good Manufacturing Practice GRASPS - Genome-wide RNA analysis of stalled protein synthesis GSH – glutathione HSF1 – heat shock factor 1 HSE - heat-shock element HSP70 – heat shock protein 70 IACUC - Institutional Animal Care and Use Committee ICC - immunocytochemistry IHC - immunohistochemistry iNPC – induced neural progenitor cell KEAP1 - Kelch-like ECH-associated protein 1 M102 - S-(+)-10,11-dihydroxyaporphine NCE – new chemical entity NFR2-ARE - NF-E2 p45-related factor 2 - antioxidant response element NHP – non-human primate NMJ – neuromuscular junction NOAEL - no observed adverse effect level NQ01 - NAD(P)H dehydrogenase [quinone]1 8-OHG - 8-oxo-2-oxyguanosine PCA – principal component analysis PK – pharmacokinetic sALS – sporadic amyotrophic lateral sclerosis SOD1 – Cu-Zn superoxide dismutase 1 TDP-43 – transactive response DNA binding protein 43 TK – toxicokinetics WT – wild type Declarations Ethical approvals GLP general toxicology studies in rats and NHPs were conducted at WuXi AppTec (Suzhou, China). The protocol and any amendments or procedures involving the care or use of animals in this study were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) prior to the initiation of such procedures. A staff veterinarian monitored the study for animal welfare issues. All mouse studies were carried out under a UK Home Office project license (PP3890603) ethically reviewed and approved by the local ethics committee (University of Sheffield Animal Welfare and Ethical Review Body). All individuals held the appropriate UK Home Office personal license and had appropriate training for procedures. All work was carried out under the terms of the UK Animals (Scientific Procedures) Act 1986 and animals were housed and maintained in line with Home Office Code of Practice for House and Care of Animals Used in Scientific Procedures. All procedures were reviewed by the University of Sheffield. Human CNS post-mortem tissue was obtained from the Sheffield Brain Tissue Bank with Research Ethics Committee approval (Sheffield Brain Bank –SBB-, Ethics Committee reference 08/MRE00/103). Human CSF was obtained from the University of Sheffield Biorepository with Research Ethics Committee approval number STH16573. Fibroblasts were collected from skin biopsies donated by ALS patients and controls with informed consent (Ethical Committee approval references: 12/YH/0330; 16/LO/2136). Acknowledgements We are very grateful to the ALS patients and healthy control subjects who generously donated biosamples to support this work. Funding Medical Research Council Developmental Pathway Funding Scheme: MR/V027735/1 (RJM, PJS, LF, NS) FightMND Australia grant: 03_DDG_2020_Shan (NS, PJS, RJM, LF). Amyotrophic Lateral Sclerosis Research Program supported by the Assistant Secretary of Defense for Health Affairs endorsed by the Department of Defense: W81XWH2210175 (NS, PJS, RJM, LF). Motor Neuron Disease Association: A Multi-Centre Biomarker Resource Strategy in ALS (AMBRoSIA). MNDA 972-797(PJS). NIHR Sheffield Biomedical Research Centre: NIHR 203321 (PJS). Author contributions Conceptualization: PJS, RJM, LF, NS. Methodology : AFK, RRM, CFA, KB, SM, TM, MM, NT, SNB, AS, SS, SNM, AD, TW, RJM, LF, PJS, MH, ATD-K. Investigation : AFK, RRM, CFA, KB, SM, TM, MM, NT, SNB, AS, SS, SNM, AD, TW, RJM, LF, PJS, MH, ATD-K. Visualization : AFK, RRM, CFA, KB, SM, TM, MM, NT, SNB, AS, SS, SNM, AD, TW, RJM, LF, PJS. Funding acquisition : PJS, RJM, LF, NS. Project administration : PJS, RJM, LF, NS. Supervision : PJS, RJM, LF, NS. Writing – original draft: PJS, RJM, LF, NS, AFK, RRM, CFA Writing – review & editing: All authors Competing interests PJS is a member of the Scientific Advisory Board for Aclipse Therapeutics and PJS, RJM and LF are share-holders of Aclipse Therapeutics. NS and INK are employees of Aclipse Therapeutics. Patents relevant to M102 PCT/US2019/056996 - Treatment of Neurodegenerative diseases. Inventors: Ning Shan, Richard Mead, Laura Ferraiuolo, Pamela J Shaw. This covers the mechanism of action of a drug identified at SITraN for the treatment of neurodegenerative diseases, including ALS. Submitted in 2019. PCT/US2019/056998 - Treatment of Neurodegenerative diseases. Inventors: Laura Ferraiuolo, Ning Shan, Pamela J Shaw. This covers the specific properties of a neuroprotective compound identified at SITraN. Submitted in 2019. PCT/US2020/45321 - Pharmaceutical Composition For Use In The Treatment Of Neurological Diseases. Inventor: Ning Shan. This covers the pharmaceutical compositions and associated pharmacokinetics of a drug identified at SITraN. Submitted in 2019. Availability of Data and Materials Materials used in this study can be made available subject to Materials Transfer agreements (MTAs). 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RNA Dysregulation in Amyotrophic Lateral Sclerosis. Front Genet. 2018;9:712. Li Z, Chen X, Liu Z, Ye W, Li L, Qian L, et al. Recent Advances: Molecular Mechanism of RNA Oxidation and Its Role in Various Diseases. Front Mol Biosci. 2020;7:184. Gatto N, Dos Santos Souza C, Shaw AC, Bell SM, Myszczynska MA, Powers S, et al. Directly converted astrocytes retain the ageing features of the donor fibroblasts and elucidate the astrocytic contribution to human CNS health and disease. Aging Cell. 2021;20(1):e13281. Mackenzie IR, Bigio EH, Ince PG, Geser F, Neumann M, Cairns NJ, et al. Pathological TDP-43 distinguishes sporadic amyotrophic lateral sclerosis from amyotrophic lateral sclerosis with SOD1 mutations. Ann Neurol. 2007;61(5):427-34. Pare B, Lehmann M, Beaudin M, Nordstrom U, Saikali S, Julien JP, et al. Misfolded SOD1 pathology in sporadic Amyotrophic Lateral Sclerosis. Sci Rep. 2018;8(1):14223. Mead RJ, Bennett EJ, Kennerley AJ, Sharp P, Sunyach C, Kasher P, et al. Optimised and rapid pre-clinical screening in the SOD1(G93A) transgenic mouse model of amyotrophic lateral sclerosis (ALS). PLoS One. 2011;6(8):e23244. Arnold ES, Ling SC, Huelga SC, Lagier-Tourenne C, Polymenidou M, Ditsworth D, et al. ALS-linked TDP-43 mutations produce aberrant RNA splicing and adult-onset motor neuron disease without aggregation or loss of nuclear TDP-43. Proc Natl Acad Sci U S A. 2013;110(8):E736-45. Evans LJ, O'Brien D, Shaw PJ. Current neuroprotective therapies and future prospects for motor neuron disease. Int Rev Neurobiol. 2024;176:327-84. Hardiman O, Al-Chalabi A, Chio A, Corr EM, Logroscino G, Robberecht W, et al. Amyotrophic lateral sclerosis. Nat Rev Dis Primers. 2017;3:17071. McCauley ME, Baloh RH. Inflammation in ALS/FTD pathogenesis. Acta Neuropathol. 2019;137(5):715-30. Smith EF, Shaw PJ, De Vos KJ. The role of mitochondria in amyotrophic lateral Evans CS, Holzbaur ELF. Autophagy and mitophagy in ALS. Neurobiol Dis. 2019;122:35-40. Bonay M. Molecular Targets of Oxidative Stress: Focus on the Nrf2 Signaling Pathway in Health and Disease. Antioxidants (Basel). 2024;13(3). Murakami S, Kusano Y, Okazaki K, Akaike T, Motohashi H. NRF2 signalling in cytoprotection and metabolism. Br J Pharmacol. 2023. Mayer C, Riera-Ponsati L, Kauppinen S, Klitgaard H, Erler JT, Hansen SN. Targeting the NRF2 pathway for disease modification in neurodegenerative diseases: mechanisms and therapeutic implications. Front Pharmacol. 2024;15:1437939. McMahon M, Itoh K, Yamamoto M, Hayes JD. Keap1-dependent proteasomal degradation of transcription factor Nrf2 contributes to the negative regulation of antioxidant response element-driven gene expression. J Biol Chem. 2003;278(24):21592-600. Dhakshinamoorthy S, Jain AK, Bloom DA, Jaiswal AK. Bach1 competes with Nrf2 leading to negative regulation of the antioxidant response element (ARE)-mediated NAD(P)H:quinone oxidoreductase 1 gene expression and induction in response to antioxidants. J Biol Chem. 2005;280(17):16891-900. Vargas MR, Johnson JA. The Nrf2-ARE cytoprotective pathway in astrocytes. Expert Rev Mol Med. 2009;11:e17. Bono S, Feligioni M, Corbo M. Impaired antioxidant KEAP1-NRF2 system in amyotrophic lateral sclerosis: NRF2 activation as a potential therapeutic strategy. Mol Neurodegener. 2021;16(1):71. Shilovsky GA. Lability of the Nrf2/Keap/ARE Cell Defense System in Different Models of Cell Aging and Age-Related Pathologies. Biochemistry (Mosc). 2022;87(1):70-85. Shilovsky GA, Ashapkin VV. Transcription Factor Nrf2 and Mitochondria - Friends or Foes in the Regulation of Aging Rate. Biochemistry (Mosc). 2022;87(12):1477-86. Batulan Z, Shinder GA, Minotti S, He BP, Doroudchi MM, Nalbantoglu J, et al. High threshold for induction of the stress response in motor neurons is associated with failure to activate HSF1. J Neurosci. 2003;23(13):5789-98. Chen HJ, Mitchell JC, Novoselov S, Miller J, Nishimura AL, Scotter EL, et al. The heat shock response plays an important role in TDP-43 clearance: evidence for dysfunction in amyotrophic lateral sclerosis. Brain. 2016;139(Pt 5):1417-32. Neef DW, Jaeger AM, Thiele DJ. Heat shock transcription factor 1 as a therapeutic target in neurodegenerative diseases. Nat Rev Drug Discov. 2011;10(12):930-44. Vucic S, Henderson RD, Mathers S, Needham M, Schultz D, Kiernan MC, et al. Safety and efficacy of dimethyl fumarate in ALS: randomised controlled study. Ann Clin Transl Neurol. 2021;8(10):1991-9. Lynch DR, Chin MP, Delatycki MB, Subramony SH, Corti M, Hoyle JC, et al. Safety and Efficacy of Omaveloxolone in Friedreich Ataxia (MOXIe Study). Ann Neurol. 2021;89(2):212-25. Ruggenenti P. The CARDINAL Trial of Bardoxolone Methyl in Alport Syndrome: When Marketing Interests Prevail over Patients Clinical Needs. Nephron. 2023;147(8):465-9. Benatar M, Hansen T, Rom D, Geist MA, Blaettler T, Camu W, et al. Safety and efficacy of arimoclomol in patients with early amyotrophic lateral sclerosis (ORARIALS-01): a randomised, double-blind, placebo-controlled, multicentre, phase 3 trial. Lancet Neurol. 2024;23(7):687-99. Benatar M, Wuu J, Andersen PM, Atassi N, David W, Cudkowicz M, et al. Randomized, double-blind, placebo-controlled trial of arimoclomol in rapidly progressive SOD1 ALS. Neurology. 2018;90(7):e565-e74. Stopford MJ, Allen SP, Ferraiuolo L. A High-throughput and Pathophysiologically Relevant Astrocyte-motor Neuron Co-culture Assay for Amyotrophic Lateral Sclerosis Therapeutic Discovery. Bio Protoc. 2019;9(17). Westerheide SD, Bosman JD, Mbadugha BNA, Kawahara TLS, Matsumoto G, Kim S, et al. Celastrols as inducers of the heat shock response and cytoprotection. J Biol Chem 2004;279:56053-56060. Lin YH, Dodd JE, Cutillo L, Castelli LM, Mihaylov SR, Norris K, et al. GRASPS: a simple-to-operate translatome technology reveals omics-hidden disease-associated pathways in TDP-43-related amyotrophic lateral sclerosis. bioRxiv 2024;2003:2004.583294. Additional Declarations Competing interest reported. PJS is a member of the Scientific Advisory Board for Aclipse Therapeutics and PJS, RJM and LF are share-holders of Aclipse Therapeutics. NS and INK are employees of Aclipse Therapeutics. Supplementary Files SUPPLEMENTARYMATERIALA.Keerieetal.docx Cite Share Download PDF Status: Published Journal Publication published 04 Nov, 2025 Read the published version in Molecular Neurodegeneration → Version 1 posted Editorial decision: Revision requested 11 Aug, 2025 Reviews received at journal 07 Aug, 2025 Reviews received at journal 05 Aug, 2025 Reviewers agreed at journal 24 Jul, 2025 Reviewers agreed at journal 23 Jul, 2025 Reviewers agreed at journal 23 Jul, 2025 Reviewers agreed at journal 22 Jul, 2025 Reviewers invited by journal 14 Jul, 2025 Editor assigned by journal 10 Jul, 2025 Submission checks completed at journal 27 Jun, 2025 First submitted to journal 24 Jun, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6964528","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":485421221,"identity":"7336aaf0-4d61-40c7-b68a-bcef30473ac2","order_by":0,"name":"Amy F. 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TDP-43\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eQ331K\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e mice.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6964528/v1/ceb2380de91b129100d9962a.png"},{"id":87045407,"identity":"fb7ad726-86b2-4b8a-9b8f-fcc52206eba7","added_by":"auto","created_at":"2025-07-18 14:30:56","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":333838,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eM102 treatment improves\u003c/strong\u003e \u003cstrong\u003e\u0026nbsp;electrophysiological parameters in TDP-43\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eQ331K\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e mice\u003c/strong\u003e\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6964528/v1/069ee7c52d3778caa67f729d.png"},{"id":87048294,"identity":"2b9b6f14-f196-4b96-8c9c-b9f850a7a6c5","added_by":"auto","created_at":"2025-07-18 14:46:56","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":499970,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOral M102 treatment in SOD1\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eG93A\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e mice shows improvement in multiple parameters of disease progression.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6964528/v1/9be15d971ecf900aea548ad8.png"},{"id":87050070,"identity":"a7114b7e-596e-4fd8-bd73-995088fa7915","added_by":"auto","created_at":"2025-07-18 14:54:56","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1338015,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eALS cases display high levels of oxidised RNA compared to healthy controls.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6964528/v1/f8971859133d52650d182557.png"},{"id":87047082,"identity":"40398d75-e3fc-47ec-a9a9-33c9f2c3a6ed","added_by":"auto","created_at":"2025-07-18 14:38:56","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":589155,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eM102 treatment activates both NRF2 and HSF1 pathways in sporadic, \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eC9orf72\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e, and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eSOD1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e patient-derived iAstrocytes.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6964528/v1/44eb5ddff83e7855d8a54280.png"},{"id":87045408,"identity":"98736950-b63d-4d25-ba62-37267852978f","added_by":"auto","created_at":"2025-07-18 14:30:56","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":901795,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eM102 treatment reduces oxidised RNA, misfolded SOD1 and TDP-43 proteinopathy in ALS patient-derived iAstrocytes.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6964528/v1/e0f417190acc779ff27564c6.png"},{"id":87047086,"identity":"1976eaff-d25b-4093-b5c9-ee2971e2d7b8","added_by":"auto","created_at":"2025-07-18 14:38:57","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":587746,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eM102 treatment rescues MN survival in co-cultures with \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eC9, SOD1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and sporadic ALS patient-derived astrocytes.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-6964528/v1/d3b30e7467804cdf8a42355a.png"},{"id":95564725,"identity":"e435c873-6189-4683-b39b-e539cd705e5a","added_by":"auto","created_at":"2025-11-10 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PJS is a member of the Scientific Advisory Board for Aclipse Therapeutics and PJS, RJM and LF are share-holders of Aclipse Therapeutics.\nNS and INK are employees of Aclipse Therapeutics.","formattedTitle":"M102 activates both NRF2 and HSF1 transcription factor pathways and is neuroprotective in cell and animal models of amyotrophic lateral sclerosis","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eAmyotrophic lateral sclerosis (ALS) is a rapidly progressive, fatal neurodegenerative disorder in which motor neuron injury and cell death causes muscle weakness and wasting, leading to progressive loss of motor control of the upper and lower limbs as well as bulbar and respiratory functions. The lifetime risk is approximately 1 in 300-350 (1) and the average course of the disease is 2.5-3 years from symptom onset (2). Currently, there are no effective treatments to halt or reverse the progression of ALS and approved drugs only marginally increase survival (riluzole) or disease progression (edaravone) (3-5). The anti-sense oligonucleotide (ASO) treatment tofersen (QALSODY) has beneficial clinical effects and biomarker readouts, but applies only to the ~2 percent of ALS patients who harbor a SOD1 mutation (6). A key feature of ALS is the speed of progression. This poses huge problems of adjustment for affected individuals, an escalating burden on carers and families, and a challenge to those purchasers and providers of healthcare who are involved in meeting the variable, rapidly changing and complex care needs (7, 8).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe pathophysiology of ALS is complex. More than 30 genes are known to cause or contribute to motor neuron degeneration in ALS (9). Even in the presence of a mutation in a gene such as SOD1, the structure and function of which are well understood, it is recognised that a cascade of multiple pathophysiological mechanisms within both motor neurons and neighbouring glial cells contribute to neurodegeneration (9- 11). Data from disease model systems and human biosamples provide strong evidence for a role of redox imbalance, inflammation, mitochondrial dysfunction and altered proteostasis, as key drivers of the pathobiology of ALS (12-16). Therapies targeting individual pathways have failed in the clinic or have shown marginal efficacy. In addition, few studies in ALS have shown target engagement for the proposed therapeutic agent in the clinic (17) with the exception of tofersen (6). There is clearly a huge unmet need for effective neuroprotective therapies to slow disease progression in ALS.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTranscription factor NF-E2 p45-related factor 2\u0026nbsp;(NRF2) is a master regulator of the antioxidant response and activates the expression of over 1000 genes with cytoprotective properties (18-20). NRF2 protein levels are highly regulated through several different mechanisms including negative regulation at the protein level by Kelch-like ECH-associated protein 1 (KEAP1) (21). There is a body of evidence from both cellular and animal models and human biosamples that this cytoprotective NRF2 system is dysregulated in ALS (22-27).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe previously identified M102 (chemical name: S-(+)-10,11-dihydroxyaporphine) in a compound screen to discover blood brain barrier penetrant NRF2-ARE pathway activators (28). We reported that M102 enhanced glutathione (GSH) secretion from astrocytes in co-culture; protected neuromuscular junctions from denervation in SOD1\u003csup\u003eG93A\u003c/sup\u003e mice; and slowed the decline in motor function when dosed at 5 mg/kg subcutaneously daily. M102 also reduced the elevated basal oxidative stress seen in fibroblasts from ALS cases (28). M102 (S[+]-apomorphine) is an enantiomer of R-apomorphine which is used as a dopamine agonist in Parkinson\u0026rsquo;s disease. M102 itself is a very weak dopamine antagonist (29) and its structure is consistent with known NRF2 activators predicted to function through modification of cysteine residues on KEAP1, thereby reducing the degradation of NRF2, increasing NRF2 translocation to the nucleus and upregulating the expression of multiple cytoprotective genes (18, 20). M102, which we now demonstrate activates both the NRF2 and HSF1 transcription factor pathways, has the potential to modulate all four of the key drivers of neurodegeneration highlighted above. By targeting multiple pathways, we expect to significantly increase the probability of neuroprotection of motor neurons and clinical success.\u003c/p\u003e\n\u003cp\u003eLack of translation between mouse models and clinical trials has hampered ALS treatment discovery over the last 20 years, with many compounds showing potential in mouse models, but failing to show efficacy in clinical trials (17, 30, 31). Here, we have generated an encouraging pre-clinical data set demonstrating significant positive effects on the same behavioural outputs in two robust and reproducible ALS mouse models, as well as a preclinical toxicology package that predicts safe efficacious doses in humans. In addition, we provide evidence that M102 increases motor neuron survival in an \u003cem\u003ein vitro\u003c/em\u003e model of patient-derived astrocyte toxicity (10) across multiple subtypes of ALS. M102 reduces toxicity from astrocytes derived from \u003cem\u003eC9orf72, SOD1\u003c/em\u003e and sporadic ALS patients and results in neuroprotection of co-cultured motor neurons. Overall, to our knowledge, this is one of the most comprehensive packages of pre-clinical efficacy for therapy development in ALS.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e\u003cstrong\u003eM102 activates NRF2 and HSF1 transcription factor pathways \u003cem\u003ein vivo\u003c/em\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe previously identified M102 in a screen for CNS penetrant NRF2 activating molecules. We demonstrated that M102 activated NRF2-directed transcription \u003cem\u003ein vitro\u003c/em\u003e and in the CNS and was able to rescue motor deficits in the SOD1\u003csup\u003eG93A\u003c/sup\u003e mouse model of ALS at a daily subcutaneous dose of 5 mg/kg (28). \u0026nbsp;\u003cem\u003eIn vitro\u003c/em\u003e evidence showed that M102 was able to increase production of glutathione from astrocytes, thereby mediating neuroprotection to co-cultured motor neurons. In order to expand the preclinical validation to other genetic subtypes of ALS we set out to establish a dose response for NRF2 activation in the CNS as a precursor to investigating efficacy in a second, \u003cem\u003eTARDBP\u003c/em\u003e mutant ALS mouse model for which we have validated readouts (32).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eElectrophilic compounds such as M102 also have the potential to activate HSF1 (heat shock factor 1) signalling pathways (33, 34) and since HSF1 activation would address additional pathogenic mechanisms in ALS (35), we also sought to establish whether M102 was able to transcriptionally activate canonical HSF1 targets \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e as well as NRF2-related targets.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe treated HeLa-HSE-Luciferase cells expressing luciferase under the transcriptional control of the HSP70.1 promoter with varying concentrations of M102, or M102 in the presence of CuCl\u003csub\u003e2\u003c/sub\u003e to mimic oxidation \u003cem\u003ein vivo\u003c/em\u003e, and showed that M102 was a robust activator of the HSP70.1 promoter (\u003cstrong\u003eSuppl. Fig. 1 A, B\u003c/strong\u003e). Western blotting of lysates of SHSY5Y cells, showed that M102 increased expression of both the NRF2 target haem oxygenase 1 (HO-1) and the HSF1 target HSP70 (Hspa1a), and that siRNA treatment reducing HSF1 or NRF2 was able to ablate the response for their respective targets (\u003cstrong\u003eSuppl. Fig. 1 C, D\u003c/strong\u003e)\u003c/p\u003e\n\u003cp\u003eTo confirm activation of both pathways \u003cem\u003ein vivo\u003c/em\u003e, we dosed wild-type (WT) mice with M102 at 0.5, 1.5, 5 and 10 mg/kg SC, once daily for 7 days, and measured the transcriptional response in cerebral cortex tissue using RT-qPCR. \u003cstrong\u003eFig. 1\u003c/strong\u003e shows the transcriptional response of NRF2 (\u003cstrong\u003eFig. 1A\u003c/strong\u003e) and HSF1 (\u003cstrong\u003eFig. 1B\u003c/strong\u003e) regulated genes. Gene targets of both transcription factors showed a clear dose response, with 5mg/kg being optimal in most cases. This represents the first indication that M102 is also able to activate HSF1 directed transcription \u003cem\u003ein vivo\u003c/em\u003e, with HSF1 regulated genes including Hspa1a (HSP70), Hspa8, and Syn1 showing elevated transcriptional responses.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eM102 rescues motor, weight and, neurophysiological phenotypes in the TDP-43\u003csup\u003eQ331K\u003c/sup\u003e transgenic mouse model of ALS\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study design for evaluating the effect of M102 in huTDP-43\u003csup\u003eQ331K\u003c/sup\u003e transgenic mice is shown in \u003cstrong\u003eFig. 1C\u003c/strong\u003e. Female TDP-43\u003csup\u003eQ331K\u003c/sup\u003e mice were block randomised into three dosing groups and dosed subcutaneously with 5mg/kg M102 as a split dose twice daily at 2.5mg/kg twice daily (BD) or a single 5mg/kg once daily (OD) dose from 25 days until 6 months of age. A variety of behavioural tests were performed at time points previously shown to correlate with significant milestones of disease progression, and CNS tissue was collected at 3 and 6 months.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTransgenic TDP-43\u003csup\u003eQ331K\u003c/sup\u003e mice gain significantly more weight compared to their non-transgenic littermates (32). This is due to the increased amount of food they consume and the reduction in activity seen with this mutation in TDP-43 that is linked to an apathy fronto-temporal dementia (FTD) phenotype (36). Mouse weights were recorded daily before dosing to determine dose volume. \u003cstrong\u003eFig. 1D\u003c/strong\u003e shows that weight gain is significantly reduced from 161 days in the 2.5mg/kg BD group (two-way ANOVA with Dunnett\u0026rsquo;s post-test). \u0026nbsp; At the end of the study there was a significant decrease in weight of the M102 2.5mg/kg BD dosed mice when compared to vehicle controls. At 177 days of age, M102 2.5mg/kg BD dosed animals weighed 23.2 +/- 1.9g compared to vehicle animals that weighed 25.4 +/- 2.3g (p = 0.03). \u0026nbsp;When analysed as area under the curve, weight is significantly reduced in both 5mg/kg and 2.5 mg/kg BD dosed groups (\u003cstrong\u003eFig. 1E\u003c/strong\u003e; one-way ANOVA with Dunnett\u0026rsquo;s post-test). Rotarod performance was also improved in the 5mg/kg dose group at 19 weeks of age (\u003cstrong\u003eFig. 1F\u003c/strong\u003e -two- way ANOVA with Dunnett\u0026rsquo;s post-testing).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eGait analysis was carried out at 3 and 6 months of age. \u0026nbsp;An improvement in gait (\u003cstrong\u003eFig. 1 G,H\u003c/strong\u003e) was observed in the 2.5mg/kg BD dosed group when compared to vehicle controls, using Catwalk (NoldusXT) gait analysis. This was shown as an increase in the amount of time spent using diagonal paws, a marker of normal mouse gait, versus a decrease in the amount of time spent on three paws, suggesting a reduction of gait unsteadiness (one-way ANOVA, with Dunnett\u0026rsquo;s post-test).\u003c/p\u003e\n\u003cp\u003eMuscle electrophysiology was performed at 6 weeks and 6 months of age. At 6 weeks of age there was no significant difference in compound muscle action potential (CMAP) amplitude between either of the M102 dose groups and the vehicle dosed group. However, at 6 months of age there was a significant improvement in CMAP amplitude in both M102 treated groups when compared to the vehicle group (\u003cstrong\u003eFig. 2A\u003c/strong\u003e, two-way ANOVA with Dunnett\u0026rsquo;s post-test). The percentage change in CMAP amplitude between 6 weeks and 6 months of age also showed significant improvement of the M102 2.5mg/kg BD dosed group when compared to the vehicle dosed group (\u003cstrong\u003eFig. 2B\u003c/strong\u003e, one-way ANOVA with Dunnet\u0026rsquo;s post-test). At 6 months of age there was also a significant improvement in response to repetitive stimulation in the 2.5mg/kg BD dosed animals when compared to vehicle dosed animals, suggesting improved maintenance of the neuromuscular junctions (NMJs) (\u003cstrong\u003eFig. 2C\u003c/strong\u003e, one-way ANOVA with Dunnett\u0026apos;s post-test).\u003c/p\u003e\n\u003cp\u003eMotor neuron counts and size distribution from the ventral horns of the lumbar spinal cord showed no significant difference between M102 dosed groups and vehicle dosed groups (\u003cstrong\u003eFig. 2D\u003c/strong\u003e, two-way ANOVA with Dunnett\u0026rsquo;s post-test). However, in this model, we were unable to detect any significant difference between WT and TDP-43\u003csup\u003eQ331K\u003c/sup\u003e transgenic mice in lumbar spinal cord motor neuron counts at a similar timepoint (\u003cstrong\u003eSuppl. Fig. 2\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eAnalysis of RNA levels in the cerebral cortex by RT-qPCR showed an increase in downstream targets of NRF2 and HSF1 targets at 3 months of age in M102 dosed mice when compared to vehicle dosed mice, demonstrating activation of these pathways over time in the target tissue of the CNS (\u003cstrong\u003eFig. 2E\u003c/strong\u003e, two-way ANOVA with Dunnett\u0026rsquo;s post-test).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePharmacokinetic data in wild-type mice (Suppl. Fig. 3)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFollowing a single subcutaneous dose of 5 mg/kg in mice, M102 exhibited a mean Cmax (maximum plasma concentration) and AUC\u003csub\u003elast\u003c/sub\u003e (area under the plasma concentration-time curve to the last measurable plasma concentration) of 989 ng/mL and 380 ng/mL*h, respectively. Pharmacokinetic (PK) evaluation of M102 post single dose oral administration was also performed. After a single oral dose of 10 mg/kg in mice, M102 showed a mean C\u003csub\u003emax\u003c/sub\u003e and AUC\u003csub\u003elast\u003c/sub\u003e of 168 ng/mL and 187 ng/mL*h, respectively. In the preclinical pharmacology studies in transgenic mouse models of ALS, M102 has demonstrated significant efficacy at a daily subcutaneous dose of 5 mg/kg (28). Assuming a correlation of M102 efficacy to its systemic exposure and dose proportionality, the efficacious oral dose of M102 was estimated to be 25 mg/kg or lower. This estimate was further confirmed in an additional \u003cem\u003ein vivo\u003c/em\u003e pharmacology study in SOD1\u003csup\u003eG93A\u003c/sup\u003e mice, where efficacy was observed at 12.5 and 25 mg/kg of M102 administered orally.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOral administration of M102 in \u003cem\u003eSOD1\u003c/em\u003e\u003csup\u003eG93A\u003c/sup\u003e mice improves body weight, neurophysiology, and spinal cord motor neuron counts\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe have previously shown disease modifying effects of M102 in the SOD1\u003csup\u003eG93A\u003c/sup\u003e mouse model when M102 was dosed subcutaneously at 5 mg/kg daily (28). In that study there was an upregulation of \u003cem\u003eHmox1\u003c/em\u003e and \u003cem\u003eNqo1\u003c/em\u003e in the spinal cord, a significant preservation of innervation at the neuromuscular junctions, a significant decrease in oxidised glutathione with an increase in the GSH/GSSG ratio in CNS tissue, and an improvement in rotarod performance and gait analysis parameters in the M102 exposed mice compared to the control group. We wanted to further expand on these data, by exploring the effect of M102 dosed orally at an equivalent dose to the subcutaneous dose, as this is the preferred route of administration in humans.\u003c/p\u003e\n\u003cp\u003eTransgenic female SOD1\u003csup\u003eG93A\u003c/sup\u003e animals were block randomised into different groups and dosed orally with either vehicle, 5 mg/kg, 12. 5mg/kg or 25 mg/kg M102 daily from 25 days until 90 days of age (n=8 per group). The doses were chosen as 5 mg/kg subcutaneous dosing has an equivalent exposure as 25 mg/kg oral dosing (\u003cstrong\u003eSuppl. Fig. 3\u003c/strong\u003e). Behavioral tests were carried out at various time points to determine motor function and tissue was collected at 90 days for histological and mRNA analysis (\u003cstrong\u003eFig. 3A\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAnimals were weighed daily before dosing to determine dose volume (\u003cstrong\u003eFig. 3B\u003c/strong\u003e, two-way ANOVA followed by Dunnett\u0026rsquo;s post-test). SOD1\u003csup\u003eG93A\u003c/sup\u003e transgenic mice are hypermetabolic and lose weight in the later stages of disease when compared to non-transgenic mice due to muscle and fat loss (37, 38). Area under the curve analysis showed a significant increase in body weight, and thus improvement of disease progression, of the 5 mg/kg and 25 mg/kg M102 groups when compared to vehicle dosed animals (\u003cstrong\u003eFig. 3C\u003c/strong\u003e; p \u0026lt;0.05 at 5 mg/kg and \u0026lt;0.001 at 25 mg/kg; one-way ANOVA followed by Dunnett\u0026rsquo;s post-test).\u003c/p\u003e\n\u003cp\u003eThe number of motor neurons in the lumbar ventral horns was counted and a significant dose-dependent increase in the number of surviving motor neurons was observed in the M102 groups when compared to vehicle treated animals (\u003cstrong\u003eFig. 3D\u003c/strong\u003e, one-way ANOVA with Dunnett\u0026rsquo;s post-test). At 90 days of age there was a significant improvement in the CMAP amplitude of the M102 25 mg/kg dose group when compared to vehicle controls (\u003cstrong\u003eFig. 3E, F\u003c/strong\u003e, one-way ANOVA with Dunnett\u0026rsquo;s post-test). The percentage change in CMAP amplitude between 60 and 90 days of age showed a dose-dependent increase in the M102 dosed groups when compared to the vehicle dosed group with 1/7 mice showing an increase in in CMAP in the vehicle group versus 6/8 in the 25mg/kg M102 treated group (\u003cstrong\u003eFig. 3G\u003c/strong\u003e, one-way ANOVA with Dunnett\u0026rsquo;s post-test).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDose prediction of M102 to humans and safety margin \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEfficacy was observed in the mouse \u003cem\u003eSOD1\u003c/em\u003e\u003csup\u003eG93A\u003c/sup\u003e model at an oral \u0026nbsp;dose of 12.5 mg/kg which resulted in a AUC\u003csub\u003elast\u003c/sub\u003e of 419 ng*h/mL. Adjusting for the difference in human vs. mouse free fraction yields an estimated human equivalent exposure of 541 ng*h/mL. This value was used as the target efficacy exposure in the human dose projections as well as in the safety margin calculations. To confirm brain penetration of M102, additional plasma and brain PK studies were conducted in rats (\u003cstrong\u003eSuppl. Table 1\u003c/strong\u003e). After a single dose oral administration of 12 mg/kg M102, the brain-to-plasma ratios of M102 were determined to be 0.70 and 0.36 at 30 and 60 min post-dose, respectively. Subsequently, M102 was evaluated in non-Good Laboratory Practice (GLP) and GLP general toxicology studies in rats and non-human primates (NHPs) and the findings are summarised in \u003cstrong\u003eSupplementary tables 2\u003c/strong\u003e (rats) \u003cstrong\u003eand 3\u0026nbsp;\u003c/strong\u003e(NHPs). In the non-GLP toxicology studies, M102-related mild liver toxicity findings were observed at 250 mg/kg and 100 mg/kg in rats and NHPs, respectively. In 28-day GLP toxicology studies, 75 mg/kg was declared as the no observed adverse effect level (NOAEL) in both rats and non-human primates (NHPs). \u0026nbsp;The free AUC on Day 28 at the NOAEL in both species, as well as the margin to the projected human free AUC necessary for efficacy, is 9.3-fold for rats, 10.1-fold for female NHPs, and 15.1-fold for male NHPs.\u003c/p\u003e\n\u003cp\u003eHuman clearance, volume of distribution and half-life were estimated using PK data from rat and monkey studies. Various allometric scaling techniques were applied following the rule of exponents guidelines. Human clearance was also estimated from \u003cem\u003ein vitro\u003c/em\u003e hepatocyte incubations. \u0026nbsp; Correcting for brain weight, free fraction and using the multi-exponential method (39) resulted in estimates ranging from 55-111.7 mL/min/kg, while scaling intrinsic clearance from human hepatocytes resulted in an estimate of 84 mL/min/kg. Human volume of distribution was estimated by allometric scaling of preclinical species values and using an exponent of 1 and was approximately 81 L/kg. M102 has a bioavailability range of 10-30% in preclinical species. As a predictive model of human bioavailability does not exist, this range of potential values were modelled. Using the projected human efficacious exposure target of 302 ng*h/mL that was based on mouse efficacy at an oral dose of 12.5 mg/kg PO, a daily dose range in humans of 352-1417 mg is predicted. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIncreased oxidised RNA in affected CNS areas, and in the CSF and i-Astrocytes derived from ALS cases\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEncouraged by the compelling data obtained in two \u003cem\u003ein vivo\u003c/em\u003e models of ALS, we decided to evaluate the levels of oxidative stress detectable in patient biosamples and in a patient-derived cellular model of ALS, as potential biomarkers of target engagement and efficacy.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOxidative stress is one of the hallmarks of ALS, with oxidised lipids and proteins being identified in post- mortem tissues (40). Due to the recently identified involvement of RNA dysregulation in ALS (41) and the known role for oxidised RNA in neurodegeneration (42), we aimed to map the presence of oxidised RNA in the areas of the \u0026nbsp;CNS that are affected by the disease. As guanine is the base that is most susceptible to oxidation, we used an antibody against 8-oxo-2-oxyguanosine (8-OHG), as a marker of direct nucleic acid oxidation. Staining of frontal and motor cortex with adjacent white matter, as well as the cervical spinal cord, from 6 sALS patients, 4 patients carrying \u003cem\u003eC9orf72\u0026nbsp;\u003c/em\u003erepeat expansion mutations and 3 healthy controls (subject information in \u003cstrong\u003eSuppl. Table 4\u003c/strong\u003e) revealed that ALS patients display much higher levels of oxidised RNA compared to age-matched, neurologically unaffected controls (\u003cstrong\u003eFig. 4 A-C\u003c/strong\u003e). Of note, patients carrying \u003cem\u003eC9orf72\u003c/em\u003e mutations with both ALS and fronto-temporal dementia (FTD), displayed significantly higher levels of oxidised RNA staining in the frontal cortex compared to both controls and ALS patients who displayed only a motor phenotype (\u003cstrong\u003eFig. 4B\u003c/strong\u003e). Interestingly, although ALS patients displayed higher levels of oxidised RNA in the spinal cord compared to controls \u003cstrong\u003e(Fig. 4C)\u003c/strong\u003e, amongst all the CNS areas analysed, the spinal cord was the tissue with the highest oxidative stress burden (\u003cstrong\u003eFig. 4A\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eGiven the positive correlation between 8-OHG staining and pathology, we proceeded to examine whether oxidised RNA levels in the CSF could be used as a biomarker of oxidative stress. Using an ELISA kit specific for oxidised RNA, we detected higher levels of 8-OHG in the CSF of ALS patients (n=13; 9 males and 4 females; average age= 56.2 \u0026plusmn; 10 years) compared to controls (n= 12; \u0026nbsp;5 males and 7 females; average age= 44.2 \u0026plusmn; 12 years (\u003cstrong\u003eFig. 4\u003c/strong\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp;\u003cstrong\u003eD\u003c/strong\u003e, unpaired t-test). However, there did not seem to be any correlation between clinical/genetic characteristics and levels of oxidised RNA (clinical \u0026nbsp;information in \u003cstrong\u003eSuppl. Table 5\u003c/strong\u003e). Furthermore, the levels of oxidised RNA did not correlate with age in healthy controls, suggesting the higher levels of oxidised RNA identified in the ALS cases are not due to a potential age difference \u003cstrong\u003e(Suppl. Figure 4).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo determine whether increased levels of oxidised RNA were also present in ALS patient-derived astrocytes, we stained iAstrocytes differentiated from induced neural progenitor cells (iNPCs) directly reprogrammed from fibroblasts collected via skin biopsy from ALS cases and age-matched healthy controls (patient information in \u003cstrong\u003eSuppl. Table 6\u003c/strong\u003e). These iAstrocytes are known to retain the features of ageing that are likely to contribute to patient-specific phenotypes (43), and therefore constitute a robust \u003cem\u003ein vitro\u003c/em\u003e model of the astrocyte contribution to ALS. iAstrocytes derived from \u003cem\u003eSOD1\u003c/em\u003e, \u003cem\u003eC9orf72\u003c/em\u003e and sporadic ALS patients displayed higher levels of oxidised RNA compared to healthy controls (\u003cstrong\u003eFig. 4E,F\u003c/strong\u003e, one-way ANOVA followed by Dunnett\u0026apos;s multiple comparisons test), recapitulating the findings in post-mortem tissue.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eM102 treatment is a dual activator of the NRF2 and HSF1 pathways in ALS patient-derived iAstrocytes, and is sufficient to reduce oxidative stress, misfolded SOD1, and TDP-43 proteinopathy \u003cem\u003ein vitro\u003c/em\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs ALS patient-derived iAstrocytes recapitulate the high levels of oxidative stress observed in post-mortem tissue, we set out to evaluate the ability of M102 treatment to activate the antioxidant NRF2 pathway and therefore contribute to decreased oxidative stress in these cells.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIt is known that the NRF2 pathway is downregulated in astrocytes from models of ALS (27). Consistently, iAstrocytes derived from SOD1 (n=1), C9orf72 (n=3) and sporadic (n=2) ALS \u0026nbsp; \u0026nbsp; \u0026nbsp; cases displayed lower levels of NQO1, a downstream target of NRF2 (18, 20), compared to age-matched healthy controls, under baseline conditions (\u003cstrong\u003eFig. 5A,B\u003c/strong\u003e, one-way ANOVA followed by Dunnett\u0026apos;s multiple comparisons test; full blots in \u003cstrong\u003eSuppl. Figure 5\u003c/strong\u003e ). Importantly, when treated with 10 \u0026mu;M of M102 for 48h, the levels of NQO1 increase significantly in iA astrocytes from healthy controls and ALS patients, including \u003cem\u003eSOD1\u003c/em\u003e, \u003cem\u003eC9orf72\u003c/em\u003e and sporadic patient lines (\u003cstrong\u003eFig. 5A,C\u003c/strong\u003e, two-way ANOVA followed by \u0026Scaron;\u0026iacute;d\u0026aacute;k\u0026apos;s multiple comparisons test). Interestingly, iAstrocytes from ALS cases with a \u003cem\u003eC9orf72\u003c/em\u003e mutation showed a higher response to M102 compared to iAstrocytes from sporadic patients or patients with a \u003cem\u003eSOD1\u0026nbsp;\u003c/em\u003emutation. Overall NRF2 levels in the nucleus vs cytoplasm also increased upon 24h treatment with M102 (\u003cstrong\u003eFigure 5D, E\u003c/strong\u003e, two-way ANOVA), providing evidence that M102 activates the NRF2-ARE pathway in ALS patient-derived iAstrocytes. Similar to what is observed in mouse models of ALS, M102 treatment also leads to increased expression of HSF1 in patient-derived iAstrocytes (\u003cstrong\u003eFig. 5F, G\u003c/strong\u003e, two-way ANOVA followed by \u0026Scaron;\u0026iacute;d\u0026aacute;k\u0026apos;s multiple comparisons test), indicating that M102 is a dual activator of the NRF2-ARE and HFS1-HSE pathways \u003cem\u003ein vitro\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eConsistent with these findings, iAstrocytes derived from \u003cem\u003eSOD1\u003c/em\u003e, \u003cem\u003eC9orf72\u003c/em\u003e and sporadic ALS patients treated with 10 \u0026mu;M M102 for 48h showed decreased levels of oxidised RNA, thus confirming that M102 is capable of reducing the levels of oxidative stress in ALS astrocytes (\u003cstrong\u003eFig. 6A,B\u003c/strong\u003e, two-way ANOVA followed by \u0026Scaron;\u0026iacute;d\u0026aacute;k\u0026apos;s multiple comparisons test).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe HSF1-HSE pathway is known to play an essential role in maintaining proteostasis by facilitating protein folding and avoiding protein misfolding (35). As we showed that M102 is a strong activator of the HSF1 pathway, we set out to investigate whether M102 treatment has a beneficial effect on the reduction of misfolded SOD1 and TDP-43 proteinopathy, two hallmarks of ALS pathology (44, 45). By using an antibody raised against misfolded SOD1 (B8H10), we detected perinuclear staining in iAstrocytes from \u003cem\u003eSOD1\u003c/em\u003e cases, as well as cases carrying \u003cem\u003eC9orf72\u0026nbsp;\u003c/em\u003emutations and sALS patients, consistent with previous findings in post-mortem tissues. \u0026nbsp;Upon treatment with M102, we observed a significant reduction of misfolded SOD1 in iAstrocytes derived from \u003cem\u003eSOD1, C9orf72\u003c/em\u003e, and sporadic ALS patients (\u003cstrong\u003eFigure\u003c/strong\u003e \u003cstrong\u003e6C,D\u003c/strong\u003e, two-way ANOVA followed by \u0026Scaron;\u0026iacute;d\u0026aacute;k\u0026apos;s multiple comparisons test). Induced astrocytes recapitulate one of the key hallmarks of ALS, i.e. TDP-43 proteinopathy, detected as the presence of TDP-43 fragments (observed at 35 kDa) in sporadic and \u003cem\u003eC9orf72\u003c/em\u003e ALS patient iAstrocytes (\u003cstrong\u003eFig. 6E\u003c/strong\u003e), but not in SOD1 cases (\u003cstrong\u003eSuppl. Fig.\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e6).\u003c/strong\u003e Importantly, we observed that time-dependent treatment with M102 leads to a reduction of TDP-43 proteinopathy, particularly upon 48h exposure (\u003cstrong\u003eFig. 6E\u003c/strong\u003e, F, paired t-test).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eM102 rescues MN survival in co-culture with ALS patient-derived iAstrocytes by targeting multiple mechanisms known to underlie ALS pathophysiology\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAstrocytes from ALS patients are known to be toxic to MNs, contributing to death of healthy motor neurons in co-culture (10). We have previously reported that iAstrocytes differentiated from iNPCs directly reprogrammed from fibroblasts of \u003cem\u003eSOD1,\u003c/em\u003e \u003cem\u003eC9orf72\u003c/em\u003e and sporadic ALS patients are toxic to MNs (10). As M102 seems to target multiple mechanisms associated with ALS, including oxidative stress and protein misfolding and aggregation, we asked whether M102 treatment would be sufficient to rescue MN survival when in co-culture with toxic ALS astrocytes. For this, we used our previously described iAstrocyte-MN co-culture model (10). Briefly, ALS patient-derived iAstrocytes were treated with DMSO or M102, using the Echo 550 liquid dispenser, and 24h later co-cultured with healthy mouse MNs expressing GFP under a Hb9 promoter. The MNs were then scanned in an InCell Analyser 24h and 72h after treatment, and the numbers of viable MNs counted (\u003cstrong\u003eFig. 7A\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo assess the optimal dose of M102 to use, we started by testing 6 different concentrations of M102, ranging from 0.03 \u0026mu;M to 10 \u0026mu;M, in 4 sALS iAstrocyte lines. As expected, under baseline conditions, MNs co-cultured with ALS iAstrocytes showed reduced survival compared to MNs co-cultured with iAstrocytes derived from healthy controls. Furthermore, our dose-curve response showed that M102 has an EC\u003csub\u003e50\u003c/sub\u003e of 1.33 \u0026mu;M and the maximum neuroprotective effect in co-culture was achieved with a dose of 10 \u0026mu;M (\u003cstrong\u003eFig. 7B\u003c/strong\u003e). We therefore used 10 \u0026mu;M M102 to further \u0026nbsp;evaluate whether M102 is sufficient to rescue MN survival when in co-culture with multiple iAstrocyte lines derived from both familial and sporadic ALS cases. Under baseline conditions, MNs co-cultured with ALS patient-derived iAstrocytes displayed reduced survival (between 30%-60% reduction in MN survival depending on the patient donor) compared to MNs co-cultured with iAstrocytes derived from healthy controls (\u003cstrong\u003eFig. 7C\u003c/strong\u003e). Treatment with 10 \u0026mu;M M102 led to a significant increase in MN survival in co-cultures with 7 out of 9 different iAstrocyte patient lines, including \u003cem\u003eSOD1,\u003c/em\u003e \u003cem\u003eC9orf72\u003c/em\u003e and sporadic ALS cases (\u003cstrong\u003eFig. 7C\u003c/strong\u003e, two-way ANOVA followed by \u0026Scaron;\u0026iacute;d\u0026aacute;k\u0026apos;s multiple comparisons test). Interestingly, the response to M102 varied between patient lines, with 4 patient lines responding more strongly by increasing MN survival by \u0026gt;50% compared to baseline levels.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn order to further explore the effects of M102 and better understand how M102 could play a role in iAstrocyte toxicity and MN survival, we performed RNA Sequencing in patient-derived iAstrocytes before and after 10 \u0026mu;M M102 treatment for 48h. Our data show that there is a clear separation of the transcriptomic profile before and after M102 treatment, as shown by a principal component analysis (PCA) plot (\u003cstrong\u003eFig. 7D\u003c/strong\u003e). This transcriptomic shift in response to M102 is observed in iAstrocytes derived from \u003cem\u003eSOD1, C9orf72\u003c/em\u003e, and sporadic patients (\u003cstrong\u003eSuppl. Fig. 7\u003c/strong\u003e \u003cstrong\u003eA,B,C, respectively\u003c/strong\u003e). Differential expression analysis between treated and untreated iAstrocytes identified a total of 160, 283, and 267 differentially expressed genes (DEGs) in \u003cem\u003eC9-\u003c/em\u003eALS cases, \u003cem\u003eSOD1\u0026nbsp;\u003c/em\u003ecases, and sALS cases, respectively (\u003cstrong\u003eSuppl. Fig.7 D\u003c/strong\u003e). We further performed gene ontology analysis to investigate the predominant pathways affected by M102 treatment. The results show that M102 treatment altered the expression of genes associated with neuroinflammation, mitochondrial dysfunction, autophagic response and cell adhesion (\u003cstrong\u003eFig. 7E, Suppl. Table 7\u003c/strong\u003e), all known to be important drivers of the pathophysiology of ALS (9). Taken together with our results demonstrating that M102 is a dual activator of the NRF2 and HSF1 pathways, these results indicate that M102 targets multiple pathophysiological mechanisms operating in ALS.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAs we observed that some iAstrocyte lines responded more strongly to M102 than others, we then set out to test whether we could discriminate high and low responders to M102 based on the individual patient transcriptomic profiles. For this, we classified the iAstrocytes in \u0026lsquo;high\u0026rsquo; and \u0026lsquo;low\u0026rsquo; responders to M102 based on the levels of motor neuron survival upon M102 treatment in iAstrocyte-MN co-cultures. iAstrocytes that responded to M102 with an increase in motor neuron survival of over 50% were considered \u0026lsquo;high responders\u0026rsquo; (4 patients) whereas the iAstrocytes that responded with an increase in motor neuron survival less than 50% were considered \u0026apos;low responders\u0026rsquo; (4 patients). A heatmap of healthy controls and ALS patient-derived iAstrocytes before and after M102 treatment showed a set of 161 transcripts identified significant differences between \u0026ldquo;high\u0026rdquo; and \u0026ldquo;low\u0026rdquo; responders to M102. Interestingly, the low responders to M102 show a transcriptomic profile for these 161 transcripts that is closer to healthy controls compared to high responders, under baseline conditions. Upon M102 treatment, high responders displayed a shift in their transcriptomic profile for these 161 transcripts, which became more similar to the profile of healthy controls (\u003cstrong\u003eFig. 7F\u003c/strong\u003e), thus indicating that a subgroup of these genes could be used as a biomarker of drug response.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS (For Full Materials and Methods, see Supplementary Material) ","content":"\u003cp\u003e\u003cstrong\u003eStudy design\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study aims to provide a comprehensive package of preclinical efficacy data for M102, a CNS penetrant small molecule electrophile capable of activating both NRF2-ARE and HSF-1-HSE pathways, \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e. To do this, the pharmacokinetic profile was assessed in C57Bl/6 mice; GLP general toxicology studies were performed in rats and NHPs; and oral dose, target engagement and efficacy were assessed across two ALS mouse models: TDP-43\u003csup\u003eQ331K\u003c/sup\u003e and SOD1\u003csup\u003eG93A\u003c/sup\u003e. RT-qPCR was used to assess target engagement after M102 treatment. Immunohistochemistry (IHC) was used to identify and count the number of motor neurons in the spinal ventral horn after M102 treatment. Compound muscle action potential (CMAP) amplitude of hind limb muscles, rotarod performance, and gait parameters were used to assess treatment efficacy on readouts of motor function. Together, these data enabled prediction of human efficacious exposures and doses, which were established to be well within the safety margin predicted from GLP toxicology studies.\u003c/p\u003e\n\u003cp\u003ePost-mortem tissue, human CSF samples, and ALS patient-derived astrocytes were used to confirm that ALS patients present higher levels of oxidative stress compared to healthy controls, and that M102 is capable of reducing the levels of oxidative stress. A combination of immunocytochemistry (ICC) and western blotting analysis was used to assess the effects of M102 on NRF2-ARE and HSF-1-HSE pathway activation, as well as to assess TDP-43 proteinopathy in patient-derived astrocytes. MN-astrocyte co-cultures were performed to assess whether M102 can rescue motor neuron survival in the presence of toxic ALS iAstrocytes, and to determine high and low responders to M102. RNA Sequencing of untreated and M102 treated patient-derived iAstrocytes was used to identify additional biological mechanisms targeted by M102.\u003c/p\u003e\n\u003cp\u003eNo study size calculations or randomization were carried out. All samples were quantified in a blinded manner for microscopy and mouse experiments. No cell or animal samples were excluded.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics statement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGLP general toxicology studies in rats and NHPs to evaluate potential toxicity of M102 were conducted at WuXi AppTec (Suzhou, China). The protocol and any amendments or procedures involving the care or use of animals in this study were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) prior to the initiation of such procedures. A staff veterinarian monitored the study for animal welfare issues.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll mouse studies were carried out under a UK Home Office project license by individuals that held the appropriate UK Home Office personal license and had appropriate training for procedures. All work was carried out under the terms of the UK Animals (Scientific Procedures) Act 1986 and animals were housed and maintained in line with Home Office Code of Practice for House and Care of Animals Used in Scientific Procedures.\u003c/p\u003e\n\u003cp\u003eFormalin-fixed, paraffin-embedded (FFPE) human CNS post-mortem tissue was obtained from the Sheffield Brain Tissue Bank with Research Ethics Committee approval (Sheffield Brain Bank \u0026ndash;SBB-, Ethics Committee reference 08/MRE00/103). Human CSF was obtained from the University of Sheffield Biorepository with Research Ethics Committee approval number STH16573. Fibroblasts were collected from skin biopsies donated by ALS patients and controls with informed consent (Ethical Committee approval references: 12/YH/0330; 16/LO/2136).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMice were housed in same sex groups of between 2 and 5 mice per cage. Each cage consisted of a plastic house, sawdust covering the floor (Datesand) and paper wool bedding (Datesand). The mice had ad libitum access to water and food (standard rodent diet 2018, Envigo). Temperatures in the rooms were maintained at 21\u0026deg;C with a 12 hour light/dark cycle (7am \u0026ndash; 7pm). Wild type animals used were C67BL/6 mice either from Envigo or non transgenic mice from the SOD1\u003csup\u003eG93A\u003c/sup\u003e colony.\u003c/p\u003e\n\u003cp\u003eThe SOD1\u003csup\u003eG93A\u003c/sup\u003e C57BL/6 transgenic mice were bred in-house. The B6SJL-Tg(SOD1-G93A)1Gur/J mice were backcrossed onto the C57BL/6J OlaHsd background for at least 20 generations. This line has been extensively characterised in-house and develops a reproducible progressive motor phenotype (46). The TDP-43\u003csup\u003eQ331K\u003c/sup\u003e C57BL/6NJ transgenic mice were bred in-house and were obtained from Jackson laboratory (stock number 017933). These mice were originally on a C57BL/6NCrl background (47) but have been crossed onto a C57BL/6NJ background for a minimum of 4 generations and have been extensively characterised by the authors (32). Mice were ear-clipped for identification and genotyping. Genotyping for the two colonies was carried out as previously described (32, 46).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDifferentiation of patient-derived neurons, motor neurons and astrocytes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTissue culture to generate induced neural progenitor cells (iNPCs) and iAstrocytes from donated fibroblasts.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFibroblasts collected from skin biopsy material donated by ALS patients and controls were directly reprogrammed into iNPCs, using a combination of retroviral vectors (Oct3/4, Sox2, Klf4, and c-Myc), as previously described (10). Briefly, the fibroblasts were treated with 700 \u0026mu;L medium/viral vector overnight, then washed 2\u0026times; with PBS, and fed once per day with fibroblast medium (DMEM plus 10% FBS) for 3 days. At day 4 the cells were switched to NPC conversion medium consisting of DMEM/F12, 1% N2, 1% B27, 20 ng/mL FGF2, 20 ng/mL EGF, and heparin (5 \u0026mu;g/mL; Sigma-Aldrich) and fed every day thereafter. When the cells changed shape and presented sphere-like structures, they were lifted with accutase, centrifuged, resuspended in NPC conversion medium, and replated for expansion. Once the NPC culture was established, the medium was switched to NPC medium consisting of DMEM/F12, 1% N2, 1% B27, and FGF2 (40 ng/mL). To differentiate iNPCs into iAstrocytes, iNPCs were seeded in NPC medium at low density in a fibronectin-coated 10-cm dish. The day after, the medium was changed to DMEM containing 10% FBS and 0.3% N2 and the cells were allowed to mature for at least 7 days.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMotor neuron differentiation from embryonic stem cells.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMouse embryonic stem cells expressing GFP under the MN-specific promoter HB9 (HBG3 cells; kind gift from Tom Jessell, Columbia University, New York) were cultured on primary mouse embryonic fibroblasts (Millipore). For differentiation into MNs, cells were lifted with trypsin and resuspended in DFK10 culture medium consisting of knockout DMEM/F12, 10% knockout serum replacement, 1% N2, 0.5% L-glutamine, 0.5% glucose (30% in water), and 0.0016% 2-mercaptoethanol. The cells were plated on nonadherent Petri dishes to allow formation of embryoid bodies. After 1 d of recovery, 2 \u0026mu;M retinoic acid (Sigma) and 1 \u0026mu;M smoothened antagonist (SAG, Merk) were added freshly every day with fresh medium. After 5 days of differentiation, the embryoid bodies were dissociated and sorted for GFP on a BD FACSVantage/DiVa sorter.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHuman iAstrocyte-murine motor neuron co-culture assay.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHuman plasma fibronectin (Merck Millipore) was diluted 1:400 in PBS, and \u0026nbsp;5 \u0026micro;L was added per well on 384-well plates (Greiner Bio-one, 781091. Plates were coated for at least 5 min at RT. A total of 2,000 human iAstrocytes were seeded in 35 \u0026mu;L iAstrocyte media per well on fibronectin-coated 384-well plates. Plates were centrifuged at 1,760 x g for 60 s, and cells were incubated for 24 h. Drugs were then delivered to iAstrocytes in 100 % anhydrous DMSO (Sigma, 276855) using an Echo550 liquid handler (Labcyte). The final concentration of DMSO was 0.1 % (v/v) in the media in all wells. Plates were centrifuged at 1,760 x g for 60 s, and cells were incubated for a further 24 h. A total of 2,500 murine Hb9-GFP+ motor neurons were seeded per well in motor neuron media (KnockOut DMEM (45% v/v), F12 medium (45% v/v), KO Serum Replacement (10% v/v), 50 units/ml penicillin/streptomycin (Lonza), 1 mM L-glutamine, 1X N-2 supplement (Thermo-Fisher Scientific), 0.15% filtered glucose, 0.0008% (v/v) 2-mercaptoethanol, 20 ng/ml GDNF, 20 ng/ml BDNF, 20 ng/ml CNTF) on top of the pre-treated iAstrocytes. Plates were centrifuged at 1,760 x g for 60 s. Hb9-GFP+ motor neurons were imaged after 24 and 72 hours using an INCELL analyser 2000 (GE Healthcare), and the number of viable motor neurons was counted using the Columbus\u0026trade; analysis software (Perkin Elmer). The number of viable motor neurons (defined as GFP+ motor neurons with at least 1 process) that survived after 72 hours in co-culture was calculated as a percentage of the number of viable motor neurons after 24 hours in co-culture. The percentage survival of motor neurons was then normalised to the DMSO control for each individual iAstrocyte line.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGLP general toxicological study in rats\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of 142 rats (71/sex) were randomly assigned to 4 groups, i.e., Groups 1-4 with M102 dose levels by oral gavage at 0, 25, 50, and 75 mg/kg, respectively. The main study (toxicity study) animals were 10/sex/group, while Groups 1 and 4 had an additional 5/sex/group to assess recovery of any observed effects. Toxicokinetics (TK) animals were 3/sex in the control group and 6/sex/group in the treated groups. Animals were approximately 6-7 weeks of age with body weights ranging from 178.46 to 211.76 g in females and 252.21 to 295.14 g in males at dosing initiation. The dosage volume was 10 mL/kg. The control group (Group 1) was administered 10% (w/v) HP-\u0026beta;-CD in purified water (HP-\u0026beta;-CD) by oral gavage.\u003c/p\u003e\n\u003cp\u003eCriteria for evaluation included viability (morbidity/mortality), clinical observations, body weight, food consumption, ophthalmology, clinical pathology (hematology, coagulation, serum chemistry, and urinalysis), TK, gross pathology, organ weights, and histopathology. The concentration and homogeneity results of M102 in the dosing formulations met acceptance criteria, which demonstrated the formulations were accurately prepared and homogenous.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGLP general toxicological study in non-human primates (NHPs)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of 32 (16/sex) NHPs were randomly assigned to 4 groups including 5/sex/group in control and high dose groups, and 3/sex/group in low and middle dose groups. Dose groups were vehicle control [10% (w/v) HP-\u0026beta;-CD in purified water (HP-\u0026beta;-CD)] or M102 in vehicle at doses of 25, 50, or 75 mg/kg/day. At the end of the dosing phase, the last 2 surviving NHPs/sex/group in control and high dose groups were held for an additional 14 days without administration of the test article. Animals were approximately 2.4 to 2.9 months of age and with body weights ranging from 2.1 to 3.8 kg in males and 2.1 to 2.9 kg in females at dosing initiation.\u003c/p\u003e\n\u003cp\u003eCriteria for evaluation included viability (morbidity/mortality), clinical observations, body weight, food consumption, ophthalmic examinations, electrocardiograms, clinical pathology (hematology, serum chemistry, coagulation, urinalyses), gross (necropsy) evaluation, organ weight, histopathological evaluation and toxicokinetics.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatistical analysis was performed using GraphPad Prism v11.00. Normality was assessed by the Shapiro-Wilk test. Unpaired t-test or the Mann Whitney test were used to compare 2 data points, depending on whether the samples presented a Gaussian distribution or not, respectively. Paired t-test was used to compare the same samples before and after treatment. One-way ANOVA \u0026nbsp;followed by Dunnett\u0026apos;s multiple comparisons tests \u0026nbsp;was \u0026nbsp;used \u0026nbsp; to compare more than 2 data points in 1 group. \u0026nbsp;Two-way ANOVA followed by \u0026Scaron;\u0026iacute;d\u0026aacute;k\u0026apos;s multiple comparisons tests was used to compare more than 2 data points in 2 groups. \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eOur understanding of the genetic underpinnings and pathophysiological mechanisms in ALS has substantially increased in recent years (9). \u0026nbsp;However, apart from the recent promising emergence of tofersen as a disease modifying therapy for the two percent of ALS patients who harbor mutations in the SOD1 gene, other approved drugs have only marginal effects on life expectancy (riluzole) or indices of disease progression (edaravone) (48). Therapies targeting individual pathways have failed to generate significant benefit during several decades of clinical trials. Previous attempts at identifying effective neuroprotective therapies for ALS have failed, as they do not account for disease heterogeneity and the multiple mechanisms driving motor neuron injury (17, 49). Data from disease model systems and from human biosamples provide strong evidence for a role of redox imbalance (40), inflammation (50), mitochondrial dysfunction (51) \u0026nbsp;and altered proteostasis, including autophagy and mitophagy (51, 52), as four key drivers in the pathobiology of ALS (9, 49). Few, if any, studies in ALS have shown multi-target engagement for the proposed therapeutic agent in the clinic (17). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe previously identified M102, a CNS-penetrant, small molecule, \u0026nbsp;activator of the NRF2-ARE pathway. Here, we have also shown that it also activates the HSF-1-HSE transcription factor pathway, which targets multiple genetically validated pathophysiological mechanisms in ALS. We identified M102 in a screen to discover CNS penetrant NRF2-ARE pathway activators (28). M102 (S-apomorphine hydrochloride hemihydrate) is a proprietary new chemical entity (NCE) and the S-enantiomer of the marketed R-apomorphine (Apokyn\u0026reg;; pure R-enantiomer). The R-enantiomer is a dopamine agonist administered subcutaneously for the management of advanced Parkinson\u0026apos;s disease. M102 is a very weak dopamine antagonist and does not show the adverse effects associated with dopamine agonism (29). Initial work showed beneficial effects in the SOD1\u003csup\u003eG93A\u003c/sup\u003e mouse model and in indices of oxidative stress in ALS patient fibroblasts (28). Here, we demonstrate that M102 is a dual activator of NRF2 and HSF1 transcription factor pathways, two upstream master regulators of neuroprotective mechanisms, with the potential to modulate all four of these key drivers of neurodegeneration and with excellent penetration across the blood brain barrier (34, 39).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNRF2 is a stress-responsive transcription factor and a master regulator of anti-oxidant and anti-inflammatory genes, activating the transcription of \u0026gt;1000 cytoprotective genes and with a multi-modal contribution to healthy mitochondrial function and regulation of key autophagy genes (53, 54). These pleiotropic effects enable the cell to maintain homeostasis under stress conditions. \u0026nbsp;NRF2 activity is tightly regulated via a complex set of transcriptional and post-translational controls, principally by KEAP1 in the cytoplasm and \u0026nbsp;BTB domain and CNC homolog 1 ( BACH1) in the nucleus (55). Under physiological conditions, KEAP1 promotes constitutive proteasomal degradation of Nrf2 (56) while BACH1 acts as a transcriptional antagonist of NRF2 target genes (57). Under oxidative stress conditions, KEAP1 is inactivated by modification of its reactive cysteine residues allowing NRF2 to escape degradation and newly synthesized NRF2 is able to translocate to the nucleus and bind to the anti-oxidant response elements (AREs) of multiple cytoprotective genes. This cytoprotective gene expression response provides protection against several key pathophysiological mechanisms operating in ALS including oxidative stress, mitochondrial dysfunction, inflammation and dysregulated proteostasis (18). The stress response of the KEAP1-Nrf2-ARE system is stronger in astrocytes compared to neurons (58). \u0026nbsp;A body of evidence from \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e model systems and from post-mortem CNS tissue from ALS patients has indicated that the NRF2 response is impaired in ALS and has also been shown to decline with age \u0026nbsp;(59-61).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHSF1 is a stress-inducible transcription factor that is the key driver for the expression of multiple heat shock proteins which act as chaperones responsible for the correct folding of newly synthesized proteins, the refolding of denatured proteins and the prevention of aggregation of misfolded proteins. This heat shock response is a pro-survival pathway activated under conditions of cellular stress. This cellular stress response declines with age, with decreased ability of HSF1 to bind to the promoters of genes encoding heat shock proteins (35). Motor neurons have been reported to display a high threshold for the induction of the heat shock stress response (62). Nevertheless, activation of the HSF1/HSP pathway has shown beneficial effects in cellular and animal models of ALS, including clearance of TDP-43 aggregates (63). Activation of HSF1 is an attractive pharmacological target for ALS and other neurodegenerative conditions characterized by proteotoxic stress. However, to date, many small molecule activators of HSF-1 have shown undesirable properties e.g. by acting as Hsp90 inhibitors or by exerting direct proteotoxic effects (35, 64).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn vitro and \u003cem\u003ein vivo\u003c/em\u003e data presented here show that M102 is a multi-target drug with predicted neuroprotective benefits for the treatment of ALS. As a dual activator of NRF2 and HSF1 pathways, M102 is able to activate anti-oxidative and anti-inflammatory pathways, as well as beneficial modulating effects on proteostasis and mitochondrial function. \u003cem\u003eIn vitro\u003c/em\u003e data also show that M102 can reduce TDP-43 proteinopathy and biomarkers of oxidative stress in patient-derived astrocytes. Moreover, M102 is capable of rescuing MN survival in co-cultures with iAstrocytes derived from \u003cem\u003eC9orf72, SOD1,\u003c/em\u003e and sporadic ALS patients, as well as spinal cord MNs in the SOD1\u003csup\u003eG93A\u003c/sup\u003e mouse model. \u0026nbsp;The efficacy of M102 was shown \u003cem\u003ein vivo\u003c/em\u003e in both SOD1\u003csup\u003eG93A\u003c/sup\u003e and TDP-43\u003csup\u003eQ331K\u003c/sup\u003e transgenic mouse models of ALS, with beneficial effects on a translatable neurophysiological biomarker (CMAP) which correlates with motor neuron survival in the spinal cord. \u0026nbsp; The transcriptome of patient-derived astrocytes exposed \u003cem\u003ein vitro\u003c/em\u003e to M102 confirm the activation of multiple key neuroprotective pathways in astrocyte lines from several subgroups of ALS. There is a degree of heterogeneity in the level of the \u003cem\u003ein vitro\u003c/em\u003e response to M102, with the transcriptome of high responders shifting substantially towards \u0026nbsp;the profile observed in the astrocytes of healthy controls. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis comprehensive package of pre-clinical efficacy data for therapy development in ALS is accompanied by strong safety data. We have completed the process of Good Manufacturing Practice (GMP) manufacturing for M102 and a series of GLP toxicology and safety pharmacology studies in rats and non-human primates, which have demonstrated that M102 will have an acceptable safety margin (9.3-15 for free drug) in clinical studies. Pharmacokinetic studies show strong penetration of M102 across the blood-brain-barrier, with a brain-plasma ratio of 0.7 at 30 min post-dosing. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOther NRF2 activators have been investigated in clinical trials or have been approved for medical use. These include dimethylfumarate (DMF) (Tecfidera\u0026reg;, Biogen) and omaveloxolone (Reata, Biogen). DMF was originally approved for the treatment of psoriasis (Fumaderm\u0026reg;) and was later repurposed for the treatment of relapsing-remitting multiple sclerosis (Tecfidera\u0026reg;). \u0026nbsp;A phase 2 trial of DMF in ALS provided Class 1 evidence of safety at a dose of 480mg/day and lack of disease-modifying efficacy (65). DMF treatment is associated with dose-limiting lymphopenia and flushing (Tecfidera\u0026reg; Prescribing Information). Omaveloxolone (Skyclarys\u0026reg;) is a potent NRF2 activator that has been approved by the FDA and EMA for the treatment of Friedreich\u0026apos;s ataxia. By activating the NRF2 pathway, omaveloxolone ameliorates oxidative stress and improves mitochondrial function. As a potent NRF2 activator, omaveloxolone exhibited significant liver toxicity with elevated AST/ALT levels in 37% of patients exposed to a dose of 150 mg (66). Toxicity has also been reported with other potent NRF2 activators, such as bardoxolone methyl (EC50: 53 nM) which showed significant heart, liver, and renal toxicity in humans (67). In contrast, our preclinical toxicological studies indicate that M102 has a much higher safety margin in relation to liver toxicity. Arimoclomol is a co-activator of HSF1 and has been ineffective in two clinical trials targeting SOD1-ALS initially and then ALS more broadly (68,69).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThere is a huge unmet need for more effective neuroprotective therapies to slow disease progression in ALS. \u0026nbsp; Here, we have demonstrated that M102, a combined activator of NRF2 and HSF1 signalling pathways, has positive therapeutic effects in two different ALS transgenic mouse models and improves motor neuron survival and multiple pathological markers (i.e. oxidative stress, misfolded SOD1, TDP-43 proteinopathy) in a range of human ALS cellular model systems. \u0026nbsp;Importantly, these neuroprotective effects are seen across multiple subtypes of ALS, including the two most common genetic subtypes caused by mutations in the \u003cem\u003eC9orf72\u003c/em\u003e and \u003cem\u003eSOD1\u003c/em\u003e genes, as well as sporadic ALS cases. Taken together, M102 has the potential to modulate multiple key drivers of neurodegeneration, increasing the chances of achieving impactful neuroprotection and disease modifying effects in ALS. \u0026nbsp;Its positive effect on TDP-43 proteinopathy and on rescuing motor neuron \u0026nbsp;survival both \u003cem\u003ein vivo\u003c/em\u003e and in co-culture with iAstrocytes derived from both familial and sporadic ALS patients also suggest that M102 may be beneficial for a wide range of ALS subtypes as ~97% of ALS cases display TDP-43 proteinopathy and ~90% of cases are sporadic.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eALS \u0026ndash; amyotrophic lateral sclerosis\u003c/p\u003e\n\u003cp\u003eARE - anti-oxidant response element\u003c/p\u003e\n\u003cp\u003eASO \u0026ndash; anti-sense oligonucleotide\u003c/p\u003e\n\u003cp\u003eAUC\u003csub\u003elast -\u0026nbsp;\u003c/sub\u003earea under the plasma concentration-time curve to the last measurable plasma concentration\u003c/p\u003e\n\u003cp\u003eBACH1 \u0026ndash; BTB domain and CNC homolog 1\u003c/p\u003e\n\u003cp\u003eBCA - bicinchoninic acid\u003c/p\u003e\n\u003cp\u003eC9orf72 -\u0026nbsp;\u0026nbsp;chromosome 9 open reading frame 72\u003c/p\u003e\n\u003cp\u003eCMAP \u0026ndash;\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003ecompound muscle action potential\u003c/p\u003e\n\u003cp\u003eCMAX \u0026ndash; maximum plasma concentration\u003c/p\u003e\n\u003cp\u003eCNS \u0026ndash; central nervous system\u003c/p\u003e\n\u003cp\u003eCSF \u0026ndash; cerebrospinal fluid\u003c/p\u003e\n\u003cp\u003eDEG \u0026ndash; differentially expressed gene\u003c/p\u003e\n\u003cp\u003eDMF \u0026ndash; dimethylfumarate\u003c/p\u003e\n\u003cp\u003eDTT \u0026ndash; dithiothreitol\u003c/p\u003e\n\u003cp\u003eELISA \u0026ndash; enzyme -linked immunosorbent assay\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eEMA \u0026ndash; European Medicines Agency\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFDA \u0026ndash; Food and Drug Admonistration\u003c/p\u003e\n\u003cp\u003eFFPE - formalin-fixed, paraffin-embedded\u003c/p\u003e\n\u003cp\u003eFTD \u0026ndash; fronto-temporal dementia\u003c/p\u003e\n\u003cp\u003eGAPDH -\u0026nbsp;glyceraldehyde-3-phosphate dehydrogenase\u003c/p\u003e\n\u003cp\u003eGLP \u0026ndash; Good Laboratory Practice\u003c/p\u003e\n\u003cp\u003eGMP \u0026ndash; Good Manufacturing Practice\u003c/p\u003e\n\u003cp\u003eGRASPS - Genome-wide RNA analysis of stalled protein synthesis\u003c/p\u003e\n\u003cp\u003eGSH \u0026ndash; glutathione\u003c/p\u003e\n\u003cp\u003eHSF1 \u0026ndash; heat shock factor 1\u003c/p\u003e\n\u003cp\u003eHSE \u0026nbsp; - heat-shock element\u003c/p\u003e\n\u003cp\u003eHSP70 \u0026ndash; heat shock protein 70\u003c/p\u003e\n\u003cp\u003eIACUC - Institutional Animal Care and Use Committee\u003c/p\u003e\n\u003cp\u003eICC - immunocytochemistry\u003c/p\u003e\n\u003cp\u003eIHC - immunohistochemistry\u003c/p\u003e\n\u003cp\u003eiNPC \u0026ndash; induced neural progenitor cell\u003c/p\u003e\n\u003cp\u003eKEAP1 - Kelch-like ECH-associated protein 1\u003c/p\u003e\n\u003cp\u003eM102 - S-(+)-10,11-dihydroxyaporphine\u003c/p\u003e\n\u003cp\u003eNCE \u0026ndash; new chemical entity\u003c/p\u003e\n\u003cp\u003eNFR2-ARE \u0026nbsp; - \u0026nbsp;NF-E2 p45-related factor 2 -\u0026nbsp;antioxidant response element\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNHP \u0026ndash; non-human primate\u003c/p\u003e\n\u003cp\u003eNMJ \u0026ndash; neuromuscular junction\u003c/p\u003e\n\u003cp\u003eNOAEL - no observed adverse effect level\u003c/p\u003e\n\u003cp\u003eNQ01 - \u0026nbsp;NAD(P)H dehydrogenase [quinone]1\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e8-OHG - 8-oxo-2-oxyguanosine\u003c/p\u003e\n\u003cp\u003ePCA \u0026ndash; principal component analysis\u003c/p\u003e\n\u003cp\u003ePK \u0026ndash; pharmacokinetic\u003c/p\u003e\n\u003cp\u003esALS \u0026ndash; sporadic amyotrophic lateral sclerosis\u003c/p\u003e\n\u003cp\u003eSOD1 \u0026ndash; Cu-Zn superoxide dismutase 1\u003c/p\u003e\n\u003cp\u003eTDP-43 \u0026ndash;\u0026nbsp;\u0026nbsp;transactive response DNA binding protein 43\u003c/p\u003e\n\u003cp\u003eTK \u0026ndash; toxicokinetics\u003c/p\u003e\n\u003cp\u003eWT \u0026ndash; wild type\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthical approvals\u0026nbsp;\u003c/strong\u003eGLP general toxicology studies in rats and NHPs were conducted at WuXi AppTec (Suzhou, China). The protocol and any amendments or procedures involving the care or use of animals in this study were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) prior to the initiation of such procedures. A staff veterinarian monitored the study for animal welfare issues.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll mouse studies were carried out under a UK Home Office project license (PP3890603) ethically reviewed and approved by the local ethics committee (University of Sheffield Animal Welfare and Ethical Review Body).\u003c/p\u003e\n\u003cp\u003eAll \u0026nbsp; \u0026nbsp; \u0026nbsp;individuals held the appropriate UK Home Office personal license and had appropriate training for procedures. \u0026nbsp;All work was carried out under the terms of the UK Animals (Scientific Procedures) Act 1986 and animals were housed and maintained in line with Home Office Code of Practice for House and Care of Animals Used in Scientific Procedures. All procedures were reviewed by the University of Sheffield.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHuman CNS post-mortem tissue was obtained from the Sheffield Brain Tissue Bank with Research Ethics Committee approval (Sheffield Brain Bank \u0026ndash;SBB-, Ethics Committee reference 08/MRE00/103). Human CSF was obtained from the University of Sheffield Biorepository with Research Ethics Committee approval number STH16573. Fibroblasts were collected from skin biopsies donated by ALS patients and controls with informed consent (Ethical Committee approval references: 12/YH/0330; 16/LO/2136).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe are very grateful to the ALS patients and healthy control subjects who generously donated biosamples to support this work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMedical Research Council Developmental Pathway Funding Scheme: MR/V027735/1 (RJM, PJS, LF, NS)\u003c/p\u003e\n\u003cp\u003eFightMND Australia grant: 03_DDG_2020_Shan (NS, PJS, RJM, LF).\u003c/p\u003e\n\u003cp\u003eAmyotrophic Lateral Sclerosis Research Program supported by the Assistant Secretary of Defense for Health Affairs endorsed by the Department of Defense: W81XWH2210175 (NS, PJS, RJM, LF).\u003c/p\u003e\n\u003cp\u003eMotor Neuron Disease Association: A Multi-Centre Biomarker Resource Strategy in ALS (AMBRoSIA). MNDA 972-797(PJS).\u003c/p\u003e\n\u003cp\u003eNIHR Sheffield Biomedical Research Centre: NIHR 203321 (PJS).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: PJS, RJM, LF, NS.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethodology\u003c/strong\u003e: AFK, RRM, CFA, KB, SM, TM, MM, NT, SNB, AS, SS, SNM, AD, TW, RJM, LF, PJS, MH, ATD-K.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInvestigation\u003c/strong\u003e: AFK, RRM, CFA, KB, SM, TM, MM, NT, SNB, AS, SS, SNM, AD, TW, RJM, LF, PJS, MH, ATD-K.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVisualization\u003c/strong\u003e: AFK, RRM, CFA, KB, SM, TM, MM, NT, SNB, AS, SS, SNM, AD, TW, RJM, LF, PJS.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding acquisition\u003c/strong\u003e: PJS, RJM, LF, NS.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProject administration\u003c/strong\u003e: PJS, RJM, LF, NS.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupervision\u003c/strong\u003e: PJS, RJM, LF, NS.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWriting\u003c/strong\u003e \u0026ndash; original draft: PJS, RJM, LF, NS, AFK, RRM, CFA\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWriting\u003c/strong\u003e \u0026ndash; review \u0026amp; editing: All authors\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePJS is a member of the Scientific Advisory Board for Aclipse Therapeutics and PJS, \u0026nbsp; RJM and LF are share-holders of Aclipse Therapeutics.\u003c/p\u003e\n\u003cp\u003eNS and INK are employees of Aclipse Therapeutics.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePatents relevant to M102\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePCT/US2019/056996\u003c/strong\u003e - Treatment of Neurodegenerative diseases. Inventors: Ning Shan, Richard Mead, Laura Ferraiuolo, Pamela J Shaw. \u0026nbsp;This covers the mechanism of action of a drug identified at SITraN for the treatment of neurodegenerative diseases, including ALS. \u0026nbsp; Submitted in 2019.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePCT/US2019/056998\u003c/strong\u003e - Treatment of Neurodegenerative diseases. Inventors: Laura Ferraiuolo, Ning Shan, Pamela J Shaw. \u0026nbsp;This covers the specific properties of a neuroprotective compound identified at SITraN. Submitted in 2019. \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePCT/US2020/45321\u003c/strong\u003e - Pharmaceutical Composition For Use In The Treatment Of Neurological Diseases. Inventor: Ning Shan. This covers the pharmaceutical compositions and associated pharmacokinetics of a drug identified at SITraN. Submitted in 2019.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of Data and Materials\u0026nbsp;\u003c/strong\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMaterials used in this study can be made available subject to Materials Transfer agreements (MTAs). \u0026nbsp;RNA Sequencing data will be made available on an appropriate public database following publication of the manuscript. \u0026nbsp;All other data are available in the manuscript main text and supplementary materials. \u0026nbsp;\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eRyan M, Heverin M, McLaughlin RL, Hardiman O. Lifetime Risk and Heritability of Amyotrophic Lateral Sclerosis. JAMA Neurol. 2019;76(11):1367-74.\u003c/li\u003e\n \u003cli\u003eMasrori P, Van Damme P. Amyotrophic lateral sclerosis: a clinical review. Eur J Neurol. 2020;27(10):1918-29.\u003c/li\u003e\n \u003cli\u003eBensimon G, Lacomblez L, Meininger V. 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Recent Advances: Molecular Mechanism of RNA Oxidation and Its Role in Various Diseases. Front Mol Biosci. 2020;7:184.\u003c/li\u003e\n \u003cli\u003eGatto N, Dos Santos Souza C, Shaw AC, Bell SM, Myszczynska MA, Powers S, et al. Directly converted astrocytes retain the ageing features of the donor fibroblasts and elucidate the astrocytic contribution to human CNS health and disease. Aging Cell. 2021;20(1):e13281.\u003c/li\u003e\n \u003cli\u003eMackenzie IR, Bigio EH, Ince PG, Geser F, Neumann M, Cairns NJ, et al. Pathological TDP-43 distinguishes sporadic amyotrophic lateral sclerosis from amyotrophic lateral sclerosis with SOD1 mutations. Ann Neurol. 2007;61(5):427-34.\u003c/li\u003e\n \u003cli\u003ePare B, Lehmann M, Beaudin M, Nordstrom U, Saikali S, Julien JP, et al. Misfolded SOD1 pathology in sporadic Amyotrophic Lateral Sclerosis. Sci Rep. 2018;8(1):14223.\u003c/li\u003e\n \u003cli\u003eMead RJ, Bennett EJ, Kennerley AJ, Sharp P, Sunyach C, Kasher P, et al. Optimised and rapid pre-clinical screening in the SOD1(G93A) transgenic mouse model of amyotrophic lateral sclerosis (ALS). PLoS One. 2011;6(8):e23244.\u003c/li\u003e\n \u003cli\u003eArnold ES, Ling SC, Huelga SC, Lagier-Tourenne C, Polymenidou M, Ditsworth D, et al. ALS-linked TDP-43 mutations produce aberrant RNA splicing and adult-onset motor neuron disease without aggregation or loss of nuclear TDP-43. Proc Natl Acad Sci U S A. 2013;110(8):E736-45.\u003c/li\u003e\n \u003cli\u003eEvans LJ, O\u0026apos;Brien D, Shaw PJ. Current neuroprotective therapies and future prospects for motor neuron disease. Int Rev Neurobiol. 2024;176:327-84.\u003c/li\u003e\n \u003cli\u003eHardiman O, Al-Chalabi A, Chio A, Corr EM, Logroscino G, Robberecht W, et al. Amyotrophic lateral sclerosis. Nat Rev Dis Primers. 2017;3:17071.\u003c/li\u003e\n \u003cli\u003eMcCauley ME, Baloh RH. Inflammation in ALS/FTD pathogenesis. 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Heat shock transcription factor 1 as a therapeutic target in neurodegenerative diseases. Nat Rev Drug Discov. 2011;10(12):930-44.\u003c/li\u003e\n \u003cli\u003eVucic S, Henderson RD, Mathers S, Needham M, Schultz D, Kiernan MC, et al. Safety and efficacy of dimethyl fumarate in ALS: randomised controlled study. Ann Clin Transl Neurol. 2021;8(10):1991-9.\u003c/li\u003e\n \u003cli\u003eLynch DR, Chin MP, Delatycki MB, Subramony SH, Corti M, Hoyle JC, et al. Safety and Efficacy of Omaveloxolone in Friedreich Ataxia (MOXIe Study). Ann Neurol. 2021;89(2):212-25.\u003c/li\u003e\n \u003cli\u003eRuggenenti P. The CARDINAL Trial of Bardoxolone Methyl in Alport Syndrome: When Marketing Interests Prevail over Patients Clinical Needs. Nephron. 2023;147(8):465-9.\u003c/li\u003e\n \u003cli\u003eBenatar M, Hansen T, Rom D, Geist MA, Blaettler T, Camu W, et al. Safety and efficacy of arimoclomol in patients with early amyotrophic lateral sclerosis (ORARIALS-01): a randomised, double-blind, placebo-controlled, multicentre, phase 3 trial. Lancet Neurol. 2024;23(7):687-99.\u003c/li\u003e\n \u003cli\u003eBenatar M, Wuu J, Andersen PM, Atassi N, David W, Cudkowicz M, et al. Randomized, double-blind, placebo-controlled trial of arimoclomol in rapidly progressive SOD1 ALS. Neurology. 2018;90(7):e565-e74.\u003c/li\u003e\n \u003cli\u003eStopford MJ, Allen SP, Ferraiuolo L. A High-throughput and Pathophysiologically Relevant Astrocyte-motor Neuron Co-culture Assay for Amyotrophic Lateral Sclerosis Therapeutic Discovery. Bio Protoc. 2019;9(17).\u003c/li\u003e\n \u003cli\u003eWesterheide SD, Bosman JD, Mbadugha BNA, Kawahara TLS, Matsumoto G, Kim S, et al. Celastrols as inducers of the heat shock response and cytoprotection. J Biol Chem 2004;279:56053-56060.\u003c/li\u003e\n \u003cli\u003eLin YH, Dodd JE, Cutillo L, Castelli LM, Mihaylov SR, Norris K, et al. GRASPS: a simple-to-operate translatome technology reveals omics-hidden disease-associated pathways in TDP-43-related amyotrophic lateral sclerosis. bioRxiv 2024;2003:2004.583294.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"molecular-neurodegeneration","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mond","sideBox":"Learn more about [Molecular Neurodegeneration](http://molecularneurodegeneration.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mond/default.aspx","title":"Molecular Neurodegeneration","twitterHandle":"@MolNeuro","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"S-apomorphine, M102, NRF-2 activation, HSF1 activation, neuroprotection, amyotrophic lateral sclerosis","lastPublishedDoi":"10.21203/rs.3.rs-6964528/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6964528/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eM102 is a central nervous system (CNS) penetrant small molecule electrophile which activates \u003cem\u003ein vivo\u003c/em\u003e the NF-E2 p45-related factor 2 - antioxidant response element (NRF2-ARE) pathway, as well as transcription of heat-shock element (HSE) associated genes. In the TDP-43\u003csup\u003eQ331K\u003c/sup\u003e transgenic mouse model of ALS dosed subcutaneously at 5mg/kg OD or 2.5mg/kg BD with M102, significant improvements in compound muscle action potential (CMAP) amplitude of hind limb muscles and gait parameters were observed at 6 months of age, with associated target engagement.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAn oral dose response study of M102 in SOD1\u003csup\u003eG93A\u003c/sup\u003e transgenic mice showed a dose-dependent improvement in CMAP of hindlimb muscles which correlated with preservation of lumbar spinal motor neurons at the same time point. These data enabled prediction of human efficacious exposures and doses, which were well within the safety margin predicted from Good Laboratory Practice (GLP) toxicology studies.\u003c/p\u003e\n\u003cp\u003eA parallel program of work \u003cem\u003ein vitro\u003c/em\u003e showed that M102 rescued motor neuron survival in co-culture with patient-derived astrocytes from sporadic, \u003cem\u003eC9orf72\u003c/em\u003e and \u003cem\u003eSOD1\u003c/em\u003e ALS cases. Markers of oxidative stress, as well as indices of TDP-43 proteinopathy were also reduced by exposure to M102 in these \u003cem\u003ein vitro\u003c/em\u003e models.\u003c/p\u003e\n\u003cp\u003eThis comprehensive package of preclinical efficacy data across two mouse models as well as patient-derived astrocyte toxicity assays, provides a strong rationale for clinical evaluation of M102 in ALS patients.\u0026nbsp; Combined with the development of target engagement biomarkers and the completed preclinical toxicology package, a clear translational pathway to testing in ALS patients has been developed.\u0026nbsp;\u003c/p\u003e","manuscriptTitle":"M102 activates both NRF2 and HSF1 transcription factor pathways and is neuroprotective in cell and animal models of amyotrophic lateral sclerosis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-18 14:30:51","doi":"10.21203/rs.3.rs-6964528/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-08-11T12:21:22+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-07T14:26:48+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-05T15:48:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"52772832818217557177906877243719852464","date":"2025-07-24T07:30:07+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"303258009078134565105361516891432515266","date":"2025-07-23T22:04:06+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"45972999884084931163992864737095123814","date":"2025-07-23T12:17:47+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"147849352899345206499035936520927008796","date":"2025-07-22T18:21:54+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-14T22:34:51+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-10T17:14:12+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-06-27T08:39:12+00:00","index":"","fulltext":""},{"type":"submitted","content":"Molecular Neurodegeneration","date":"2025-06-24T10:02:24+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"molecular-neurodegeneration","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mond","sideBox":"Learn more about [Molecular Neurodegeneration](http://molecularneurodegeneration.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mond/default.aspx","title":"Molecular Neurodegeneration","twitterHandle":"@MolNeuro","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"0201c5ad-99dd-46f4-a817-3f14f651530e","owner":[],"postedDate":"July 18th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-11-10T16:08:54+00:00","versionOfRecord":{"articleIdentity":"rs-6964528","link":"https://doi.org/10.1186/s13024-025-00908-y","journal":{"identity":"molecular-neurodegeneration","isVorOnly":false,"title":"Molecular Neurodegeneration"},"publishedOn":"2025-11-04 15:57:52","publishedOnDateReadable":"November 4th, 2025"},"versionCreatedAt":"2025-07-18 14:30:51","video":"","vorDoi":"10.1186/s13024-025-00908-y","vorDoiUrl":"https://doi.org/10.1186/s13024-025-00908-y","workflowStages":[]},"version":"v1","identity":"rs-6964528","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6964528","identity":"rs-6964528","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}