I. Strategies to treat ALS caused by SOD1 pathogenetic variants
Tg-SOD1G93A mice represent the first animal model for ALS and remains the most widely used rodent model of human ALS (Gurney et al., 1994). Chemicals that act on various biological pathways have been tested in these mice to investigate their putative effect on disease progression and overall survival. It is plausible though that the biological pathways in the ALS form recapitulated by Tg-SOD1G93A mice are not implicated in all ALS subtypes. The chemical compounds used to treat SOD1 -ALS can be classified based on their mechanism of action, as outlined below.
Reduction of excitotoxicity. Excitotoxicity describes the neuronal damage caused by excessive stimulation due to glutamate accumulation in the synaptic cleft. ALS patients and mouse models show decreased levels of the excitatory amino acid transporter 2 (EAAT2) that is responsible for synaptic glutamate clearance. Overexpression ofEAAT2 in Tg-SOD1G93A mice significantly delays grip strength decline but does not alter the onset of disease symptoms or the lifespan of Tg-EAAT2 /Tg-SOD1G93A mice compared to Tg-SOD1G93A (Guo et al., 2003) indicating that excitotoxicity may not represent an ideal target for pharmacological intervention in ALS. This is corroborated by clinical and preclinical data. For example, riluzole that inhibits the release of glutamate, extended the lifespan of ALS patients by only 2-3 months, while it had a modest effect in delaying disease progression (Contestabile, 2011). When administered at the onset of symptoms, it does not have any effect on lifespan or motor function of Tg-SOD1G93A , Tg-TDP43A315T and Tg-FUS(1-359) mouse models (Hogg et al., 2018). The efficacy of riluzole in patients may also be compromised by the rapid metabolism of the drug by CYP1A2. Thus, prodrugs that withstand CYP1A2 metabolism and have increased in vivo stability were designed (Limbert et al., 2013). Since serum levels of administered riluzole in patients are determined by the expression of CYP1A2 (Limbert et al., 2013), another way to increase the likelihood of response to riluzole could be to select ALS patients with low CYP1A2 levels.
Ceftriaxone (IV ), a cephalosporine antibiotic, that increases the activity of the EAAT2 gene promoter resulting in elevated EAAT2 expression that in turn reduces glutamate excitotoxicity (Cudkowicz et al., 2015), significantly improved the ALS phenotype of Tg-SOD1G93A mice and extended their lifespan by 10 days (Rothsein et al., 2005). Consistently, earlier case studies had reported improvement of symptoms in some ALS patients after administration of ceftriaxone (Smith, 1992). A later clinical trial (NCT00349622) showed no beneficial effect of ceftriaxone in non-stratified ALS patients. Whether SOD1 -ALS patients were included in this trial is unknown as no genetic data are available for retrospect analysis, to validate whether ceftiaxome was beneficial in this subgroup (Cudkowicz et al., 2015).
Targeting inflammation. TNFα is a major pro-inflammatory cytokine with a wide variety of biological responses including the apoptosis of neuronal cells. Increased levels of TNFα and FasL have been found in biopsy sections from lumbar spinal cord of ALS patients (FALS-SOD1I113T and SALS) and Tg-SOD1G93A mice (Kiaei et al., 2006). Thus, thalidomide (V ) and lenalidomide (VI ), which inhibit TNFα production, were tested in Tg-SOD1G93Amice. When administered pre-symptomatically, both compounds improved motor performance, attenuated weight loss and extended lifespan by approximately 3 weeks (16% and 18.5% increase in mean survival, respectively) (Kiaei et al., 2006). When administered at the onset of symptoms, lenalidomide improved motor performance, weight loss and extended the lifespan of Tg-SOD1G93A mice by approximately 19 days (Neymotin et al., 2009). A clinical trial with non-stratified ALS patients found that thalidomide does not improve the ALSFRS-R (ALS F unctional R atingS ystem-R evised) score or the forced vital capacity (FVC) (Stommel et al., 2009). Thus, these compounds may only show effect inSOD1 -ALS given their beneficial effect in Tg-SOD1G93A mice. To this end, it is interesting to note that deletion of Tnfα in Tg-SOD1G93A and Tg-SOD1G37R mice did not increase lifespan and did not inhibit the extent of neuronal loss (Gowing et al., 2006) indicating that thalidomide and lenalidomide may have other than TNFα-inhibiting (off-target) functions in vivo probably linked to the extended lifespan of Tg-SOD1G93A mice. Mapping the off-target effects in mice may reveal novel targets for pharmacological intervention. This endeavor could be accelerated or advanced by use of drug-based activity-based probes (ABPs) as will be described below.
Mitochondria targeting. Mitochondria dysfunction is a characteristic feature of ALS. Tg-SOD1G93A mice show decreased respiratory capacity in astrocytes. Dichloroacetate (DCA) (VII ) is a pyruvate dehydrogenase kinase inhibitor that stimulates mitochondrial metabolism and, when administered in drinking water of Tg-SOD1G93A mice, it extended their survival by 2 weeks in males and 10 days in females while it improved grip strength (Miquel et al., 2012).
Olesoxime (VIII ) is a mitochondrial pore modulator that reduces neuronal cell death in Tg-SOD1G93A mice (Sunyach et al., 2012) but a phase II/III clinical trial did not validate these positive effects in non-stratified ALS patients (Lenglet et al., 2014).
Coenzyme Q10 slightly increases the lifespan of Tg-SOD1G93A mice (Matthews et al., 1998), but a clinical trial with non-stratified ALS patients (NCT00243932) did not show phenotype improvement for a high dose of coenzyme Q10 (Kaufmann et al., 2009). Other dietary approaches to alleviate ALS symptoms have also been taken. The Deana Protocol Supplement involves arginine α-ketoglutarate, γ-aminobutyric acid (GABA), coenzyme Q10 and medium chain triglycerides. When administered to ten-week-old Tg-SOD1G93A mice, this supplement improved survival and motor functions (Ari et al., 2014). Similarly, caprylic triglyceride which was used in a recent clinical trial (NCT02716662), enhanced motor performance in different tests, increased mitochondrial respiration compared to controls, but did not extend survival. Also, vitamin E delayed onset of disease symptoms in Tg-SOD1G93A mice but did not increase lifespan (Gurney et al., 1996). In a large clinical trial, high doses (5 g per day) of vitamin E did not have significant beneficial effects on survival or alleviation of symptoms (Graf et al., 2005). The SS31 antioxidant peptide improved survival and motor performance inTg-SOD1G93A mice and reduced cell apoptosis due to hydrogen peroxide in vitro (Petri et al., 2006) but it has not yet been validated in clinical trials. Since all these studies showed effectiveness of the aforementioned supplements in Tg-SOD1G93A their therapeutic effect may be limited to SOD1 -ALS patients.
Rasagiline (IX ), a MAO-B inhibitor that has antioxidant and anti-apoptotic functions extends the lifespan and improves the running wheel performance of Tg-SOD1G93A mice. Co-administered with riluzole, it showed additive effects in Tg-SOD1G93A mice (Waibel et al., 2004). Currently, rasagiline is being tested in ALS patients of various disease genotypes (NCT01879241, NCT01786603), with positive results in reducing oxidative stress in mitochondria, and increasing the mitochondrial membrane potential (Macchi et al., 2015).Dexpramipexole (X ), a dopamine receptor antagonist, had protective effects in vitro manifested by improved mitochondrial function, prevented apoptosis, and reduced ROS (Cheah et al., 2010) but failed to exhibit positive results in Tg-SOD1G93A mice and, in a phase III clinical trial in ALS patients (Wilkins et al., 2017).
Targeting SOD1 aggregation. Formation of SOD1 aggregates can be suppressed by either inhibiting SOD1 aggregation directly or by reducing the expression of SOD1 (Limpert et al., 2013), as for example with pyrimethamine. In SOD1 -ALS patients, pyrimethamine lowers the levels of SOD1 in CSF (Lange et al., 2017). This clinical trial was designed to specifically include SOD1 -ALS patients and to monitor the levels of SOD1 in CSF (NCT01083667).
Another strategy involves the stabilization of SOD1 dimers to diminish the formation of aggregates through chemical crosslinking between adjacent Cys111 of two SOD1 molecules with maleimide derivatives and thiol-disulfide exchange approaches, e.g., with 1,4-bismaleimidobutane (Auclair et al., 2010).
A recent strategy involves targeting the interaction between SOD1 and derlin-1, which plays a role in endoplasmic reticulum (ER) machinery and disrupting this interaction has a positive role in alleviating ALS symptoms (Tsuburaya et al., 2018). High-throughput screening (HTS) identified compound XI that prevents the interaction of mutant SOD1 with derlin-1. A series of analogues were synthesized andXII with better physicochemical properties was tested as a candidate drug. XII alleviated pathology of Tg-SOD1G93A  mice and of motor-neurons derived from iPSC of patients with SOD1 -ALS (Tsuburaya et al., 2018).
Screening of 640 FDA approved drugs found that statins (simvastatin, lovastatin, mevastatin) and vitamin D3 derivatives (alfacalcidol, calcidiol, calcitriol) inhibited aggregation of apo-SOD1G37R (Anzai et al., 2016). Unexpectedly, statins accelerate disease progression, decrease the lifespan ofTg-SOD1G93A mice (Su et al., 2016) and worsen the phenotype of ALS patients manifested by increased rates of ALSFRS-R decline and higher frequency of muscle cramps (Zinman et al., 2008). These observations uncover obstacles of HTS against pure components like the SOD1 aggregates to identify new drugs, but vitamin D3 supplementation in Tg-SOD1G93A mice improves motor function (Gianforcaro and Hamadeh, 2014). Nevertheless, although earlier studies showed that patient intake of vitamin D3 slowed the rate of ALSFRS-R decline (Karam et al., 2013), later studies did not replicate these findings (Trojsi et al., 2019; Libonati et al., 2017). These contradictory clinical evidences may be due to the quite varying genetic background of patients.
Ebselen (XIII ) is an organoselenium cysteine reactive compound that assists in the formation of the intramolecular disulfide bond of SOD1 and its correct folding. Thus, it facilitates the production of the functional SOD1 dimer instead of the toxic aggregates. In addition, ebselen has antioxidant activity (Capper et al., 2018). Although it only marginally extended the lifespan of Tg-SOD1G93A mice it significantly delayed the onset of disease symptoms (Amporndanai et al., 2020). Thus, it may alleviate the symptoms in SOD1 -ALS patients.
In analogous manner, treatment with Cu(II)ASTM (XIV ) improves symptoms and extends survival in Tg-SOD1G93A(Hilton et al., 2017) and Tg-SOD1G37R mice (Roberts et al., 2014). A mechanism accounting for its function involves copper transfer to SOD1. Thus, the loading of SOD1 with metal ions may provide a new type for SOD1 -ALS specific therapy. A phase I clinical trial to assess the pharmacokinetics of Cu(II)ASTM in ALS patients has been completed (NCT02870634) and another study (NCT04082832) is ongoing to assess its efficacy without, however, selecting for SOD1 -ALS patients in which drug efficacy has been proven.
Finally, pyrazolone derivatives have been identified as SOD1 aggregation inhibitors (Zhang et al., 2015; Chen et al., 2011) and the pyrazolone XV increased the lifespan of Tg-SOD1G93A by 13% (Chen et al., 2012). In this case, an activity-based probe (ABP) was designed to map its complete interactome (i.e. biological targets and off-targets). For this, a biotin moiety with a spacer was introduced to one side of the molecule and the new derivative (XVI ) was immobilized on streptavidin beads. The beads were incubated with cellular lysates and the precipitants were analyzed by mass spectrometry to identify the biological targets (Trippier et al., 2014). This assay revealed an unexpected function of pyrazolones, i.e ., proteosomal activation without heat shock response.
Targeting proteolysis. Biochemical pathways involving proteases may provide novel targets for ALS treatment. In this respect, the widely studied metalloprotease MMP9 mediates the degeneration of fast motor neurons in Tg-SOD1G93A mice through enhancement of ER stress (Kaplan et al., 2014) and/or regulation of Tnfα and FasL expression (Kiaei et al. 2007). Deletion of one Mmp9 allele in Tg-SOD1G93A increased the lifespan by 14% while deletion of both alleles (Tg-SOD1G93AMmp9-/- ) by 25%, which is the longest prolongation of lifespan ever reported for an intervention in any of ALS mouse models. Intracerebroventricular (icv) administration of an MMP9 inhibitor in Tg-SOD1G93A mice delayed denervation and reduced ER stress (Kaplan et al., 2014). Knockdown or knockout of MMP9 attenuates the neuromuscular defects in rNLS8 (Tg-TDP-43ΔNLS ) mice. Intriguingly though, Tg-TDP-43ΔNLSMmp9-/-mice have significantly shortened survival and run slower than the rNLS8, despite their attenuated neuromuscular defects (Spiller et al., 2019). Thus, targeting MMP9 for inhibition may represent a beneficial therapeutic strategy only for SOD1 -ALS patients.
Maintenance of proteostasis. The dynamic regulation of a balanced, functional proteome (proteostasis) orchestrates multiple cellular systems and functions like the ubiquitin proteasome system, autophagy, ER stress/unfolded protein response, stress granules and heat shock proteins, to provide “quality control” for proteome maintenance. The proteostasis drug methylene blue (XVII ) that induces autophagy (Congdon et al., 2012) could rescue motor defects in Tg-TDP-43A315T and Tg-FUSS57Δ C. elegans models and in Tg-TDP-43G348C and Tg-FUS521H D. rerio (Vaccaro et al., 2012) but not in Tg-TDP-43G348C (Audet et al., 2012) and Tg-SOD1G93A mice (Lougheed and Turnbull, 2011). These results indicate that selection of the appropriate animal model is essential for extrapolating animal data to putative clinical significance.
Sephin 1 (XVIII ) is a selective inhibitor of the stress-induced phosphatase PPP1R15A that prolongs eIF2 phosphorylation upon stress and protects cells from ER stress. In Tg-SOD1G93A mice, sephin 1 prevents weight loss and improves motor function (Das et al., 2015).
Another approach to target SOD1 -ALS involves the activation of heat shock family chaperones that protect cells from various stresses. Arimoclomol (XIX ), a heat shock protein inducer, delays onset of symptoms, extends the lifespan, promotes muscle function, and prevents the aggregation of SOD1 in Tg-SOD1 G93Amice (Kieran et al., 2004; Kalmar et al., 2012). In the clinical trial NCT00706147, rapidly progressive SOD1 -mutant ALS patients were treated with up to 200 mg arimoclomol and this phase II trial showed that arimoclomol is safe and well-tolerated (Benatar et al., 2018). An ongoing phase III clinical trial (NCT03836716) with 231 participants aims to define the long-term safety and efficacy of arimoclomol inSOD1 -ALS patients.