Multiple sclerosis (MS) is a multifaceted demyelinating disease, involving deleterious interactions between the immune system and CNS neuronal/glial cells, resulting in axonal and neuronal degeneration. Currently, the approved therapies in general for MS are not as effective as thought previously. This may have been due to lack of understanding the disease manifestation and more importantly, MS is now recognized as a heterogenous disease. However, some agents have shown promise but have severe negative side effects following treatments (Wipfler et al., 2011). Unfortunately, many patients continue to progress in disease severity while receiving treatment. Although the pro-inflammatory actions of autoreactive T-cells and other immune cells in MS are well established (Ahmad et al., 2023a; Aldossari et al., 2023), axonal degeneration is an important contributing factor to disability in progressive cases of MS (Silber and Sharief, 1999; Bjartmar et al., 2003). However, the mechanisms of myelin breakdown and damage to neurons, axons, and glial cells in MS are not as clearly understood. Thus, it is important to investigate the activation, migration, and production of mediators by autoreactive T-cells and other immune cells (immune component) vs. neuronal/glial cell death, damage of axonal myelin (neurodegenerative component), in a rodent MS model, experimental allergic encephalomyelitis (EAE). Recent studies have shown that inhibition of STAT3, histamine H4 receptor (H4R), chemokine receptors (CCR1, CCR4), and mitogen-activated protein kinase can suppress EAE via reduction of pro-inflammatory cells and expansion of anti-inflammatory cells/factors in mice (Ansari et al., 2022; Ahmad et al., 2023a; Aldossari et al., 2023; Alomar et al., 2023). While current therapies for MS primarily focus on attenuating the pro-inflammatory immunologic aspect of the disease, it does not consistently prevent disease relapse or progression. Therefore, new therapeutic strategies must be developed to block both inflammatory and neurodegenerative components of MS in order to attenuate disease progression and improve function.

The immune arm of the disease is largely mediated by helper T (Th) cells. Activation of naïve CD4+ Th cell receptors (and co-receptors) leads to differential signal transducer and activator of transcription proteins (STAT) signaling that drives lineage development through selective cytokine production (Jee et al., 2001; Rodriguez et al., 2006; Glatigny and Bettelli, 2018). STAT 1 and 4 drive the development of inflammatory Th1 cells while STAT3 drives development of inflammatory Th17 cells. Anti-inflammatory Th2 cells depend on STAT6 for development. A shift from inflammatory associated STAT proteins (STAT1, -4, -3) to non-inflammatory associated STAT proteins (STAT6) is correlated with a reduction of disease severity in EAE (Jee et al., 2001). Along with STAT proteins, other transcription factors are highly associated with the development, commitment, and survival of Th subtypes. While T-bet and ROR-γt/RORC are associated with Th1 and Th17 inflammatory cells respectively, GATA3 and Foxp3 are non-inflammatory associated transcription factors for Th2 and regulatory (Treg) T-cells, respectively (Trager et al., 2014). Low levels of ROR-γt/RORC and T-bet, and higher levels of STAT6, are associated with lower disease states in EAE (Li et al., 2010). Therefore, therapies which decrease Th1/Th17 inflammatory cells while increasing Th2 and Treg cells may lead to reduced disease severity in EAE.

Several strategies have been explored for treating EAE and MS. One of the approaches is to target proteases that degrade myelin and axonal proteins, contributing to disability via myelin and axonal degeneration (Aktas et al,. 2010). While the exact etiology of MS and EAE is incompletely understood, both acidic and neutral proteases are thought to play a role in disease development. One such protease with increased activity in EAE/MS, is the Ca2+-activated neutral protease, calpain (Sato et al., 1982, 1984; Banik et al., 1987; Berlet, 1987). The degeneration of axons and neuronal death mediated by calpain has also been demonstrated in EAE (Shields et al., 1999; Schaecher et al., 2001). Previous studies demonstrated increased calpain activity and cell-specific overexpression in glial cells (astrocytes, microglia) and other cell types (macrophages, T-cells) in EAE and MS, suggesting a pivotal role for calpain in myelin breakdown in these diseases (Shields and Banik, 1998a; Shields et al., 1998, 1999). Calpain inhibitors have been shown to decrease EAE disease signs in rats and mice by reducing inflammatory and neurodegenerative events (Guyton et al., 2005, 2009, 2010). Our recent studies have shown that treatment of EAE animals with calpain inhibitors protect cells and preserve axons with reduction of disease severity (Guyton et al., 2006; Smith et al., 2011). Treatment with the water soluble calpain inhibitor SNJ1945 has also been found to have neuroprotective effects (Ryu et al., 2012), but the mechanisms of axonal damage and the complexity of the EAE disease remain to be investigated. STAT6 (another calpain substrate) is involved in Th2 cell survival and cytokine production. Thus, one proposed mechanism of calpain inhibitor action in EAE treatment is via a STAT-mediated increase in the relative population of anti-inflammatory Th2-cells.

Some strategies are aimed to bias the T-cell repertoire away from inflammation, thus preventing further damage by addressing the immune arm of the disease process. One such treatment is the use of altered peptide ligands (APLs). APLs are analogs of immunogenic peptides in which the T-cell receptor (TCR) contact residues have been altered at one or more positions (Tate et al., 1995; Rabinowitz et al., 1996, 1997; Collins and Frelinger, 1998; Deraos et al., 2015). Activation of CD4+ T-cells follows the engagement of the TCR by a peptide bound to a class II MHC, together with co-stimulatory signals from the antigen presenting cells (APCs) (Kappler et al., 1987; Tate et al., 1995; Lee et al., 1998; Donermeyer et al., 2006). Optimal TCR engagement causes proliferation, cytokine induction, and effector function. Presentation of APL in the presence of antigen stimulation perturbs the effector functions of T-cells (Ryan et al., 2004). APLs have also been shown to produce partial or even full deactivation of T-cells (Smilek et al., 1991; Emmanouil et al., 2018). This leads to anergy and unresponsiveness of the T-cell to further stimulation by the antigen (Sloan-Lancaster et al., 1993). Some APLs have also been shown to induce Tregs which suppress inflammatory immune responses (Kroemer et al., 1996; Nicholson et al., 1997). In a rat model of EAE, this type of therapy showed promise, and was moved into clinical trials (Karin et al., 1994). Although APLs were thought to be a promising therapeutic for MS, their shortcomings were evident in clinical trials where higher doses of APLs caused hypersensitivity reactions which resulted in its withdrawal from the market. Peptides in the body, either native signaling peptides or peptides used as drugs, are subject to degradation by non-specific proteases on cell surfaces, and thus require high doses for efficacy as therapeutics (Boonen et al., 2009). One way to address changes in the peptide sequence is to convert the APL to an Aza-APL; this substitution of an alpha carbon in the peptide sequence to a nitrogen, has been demonstrated to show resistance to amino- and carboxypeptidases (Wipf et al., 1996). Thus, we synthesized a small peptide and created an Aza-APL (3aza-APL) that is resistant to protease degradation and may overcome the shortcomings of APL therapy (Hart and Beeson, 2001a; Trager et al., 2018).

Since the pathophysiology of MS is complex and multifaceted, clinicians often consider combination therapies for MS treatment (Milo and Panitch, 2011). Thus, the goal of our study is to design a novel combination approach to target both the immune-mediated attack and neurodegenerative aspects of the disease using novel compounds. APLs are immunomodulatory, while calpain inhibitors have both immunomodulatory and neuroprotective effects. Therefore, an approach of combination of 3aza-APL and calpain inhibitor SNJ1945 treatment may create a unique treatment plan for EAE that will address both immune and neurodegenerative components of the disease.