DISEASE-MODIFYING APPROACHES TO MULTIPLE SCLEROSIS: DESIGN AND SYNTHESIS OF NOVEL POTENTIAL THERAPEUTIC AGENTS

Multiple sclerosis (MS) is a chronic demyelinating and inflammatory disease of the central nervous system (CNS). Inflammation of the white and grey matter tissues in the CNS due to immune cell infiltration and their cytokines are the incipient cause of damage. To date, MS remains a disease with many unmet clinical needs. In the development of disease-modifying therapies (DMTs), two main challenges have been recognized: 1) The complex network of crosstalk between immune system cells is based on mediators which are also expressed physiologically. 2) The hallmark of the disease is the death of oligodendrocytes (OLs). They can be regenerated, but in the context of the lesion the remyelination process fails, eventually causing axonal degeneration. In my PhD project, these challenges were addressed by facing them separately and by applying different strategies. 1. Inhibition of Neuroinflammation through astrocyte manipulation The crucial role of the glycolipid Lactosylceramide (LacCer) has been outlined in the progression of MS. LacCer is synthetized by the enzyme B4GALT6, which is mostly expressed by reactive astrocytes. The blockage of B4GALT6 caused a halt to the progression of the disease in MS models, and this effect was reverted by an external administration of LacCer. However, currently only one B4GALT6 inhibitor is known, and it is non-selective and characterized by poor potency. Therefore, we aimed at identifying new B4GALT6 inhibitors as therapeutic agents for the treatment of MS. A computational approach was applied for rational drug design. A homology model of B4GALT6 was created, refined and validated. Subsequently, new potential ligands were identified using a large virtual screening approach, using the SPECS screening database, composed of over 207’000 commercially available compounds. Among the compounds showing the best binding, two compounds were selected for the biological evaluation. The molecules were assessed for their ability to inhibit conversion toward a reactive phenotype induced by IL-1β, using the measurement of ROS production as a proxy for activation. Both ligands were active, and SPECS-5 (AO-763) demonstrated better inhibition of ROS production under inflammatory stimuli than the reference compound PDMP. 2. Enhancing Remyelination via GPR17 modulation GPR17 is a G protein-coupled receptor expressed in a stage dependent manner during the maturation of the Oligodendrocyte Progenitor Cells (OPCs) and plays a key role in remyelination. Thus, we focused on the modulation of the activity of GPR17. We planned new ligands by connecting the haptophoric fragments of potent derivatives, which are however endowed with intrinsic low specificity and unfavourable agonist profile, to the pharmacophore of a different ligand, which was shown to elicit the desired activity at the receptor. Two different series of compounds have been designed and synthesized. A first set of molecules was conceived to have an antagonist profile, by modulating the potency via modification of the haptophore (PLA-1, PLA-2, PLA-3). A second set of derivatives was designed as putative partial agonists through modification of the pharmacophoric portion (PCD-1, PCD-2, PCD-3, PCD-4, PCD-5, PCD-6). Derivatives PCD-1, PCD-2 and PCD-3 showed the desired partial agonist activity, with a potency similar to the lead compound. Derivatives PCD-4, PCD-5 and PCD-6, had a slightly higher EC50, showing instead higher efficacy at the receptor. Remarkably, derivatives PLA-1, PLA-2 and PLA-3 are able to antagonize activation with an IC50 in the low nanomolar range, exhibiting a 1000-fold increase in potency compared to known antagonists. 3. Uncovering the role of Platelets in MS and Remyelination Besides leukocytes, other blood components have historically been known to be involved in MS. In a recent publication it has been shown that following demyelination, a transient accumulation of platelets took place within the lesion. Depletion of platelets resulted in a dramatic reduction of OPC differentiation and overall remyelination, indicating a positive contribution in regeneration. Consistent with these in vivo studies, transient in vitro exposure to platelets increased OPC differentiation. Given the importance of platelets in remyelination as well as platelet abnormalities observed in MS patients, this research study was aimed to determine whether MS alters the impact of platelet-derived molecules on the modulation of OPC differentiation. To do so, blood samples were obtained from three cohorts of patients: 1- Recently Diagnosed Patients, which received no treatments (EDMS) 2-Patients with Relapsing Remitting MS (RRMS) 3- Patients with Secondary Progressive MS (SPMS). From these samples platelets were isolated and Platelet Lysate (PL) was obtained. To assess the effect on OPCs and remyelination rats’ primary OPCs and organotypic cultures, specifically cerebellar slices were used. An unexpected increase was observed in MBP+/Olig2+ (Mature) OPCs transiently exposed to PL from RRMS patients (PL-RRMS) when compared with PL from HV. However, the majority of MBP+ cells observed in PL-RRMS were organized in clusters with a low degree of differentiation, lacking primary and secondary processes. To assess whether this increase in mature cells would correspond to improved remyelination in a milieu more akin to the CNS, demyelinated cerebellar slices cultures were exposed to PL obtained from MS patients and HV. These experiments seem to confirm the observation obtained from OPCs culture, showing increased remyelination in slices exposed to PL from RRMS patients, and furthermore, they show that PL derived from SPMS patients are unable to promote remyelination upon transient exposure. These data suggest that in MS platelets activate to compensate for the loss of oligodendrocytes, but this answer slowly exhausts over time, leading to an impairment of remyelination. Innovative approaches are crucial for improving outcomes and addressing gaps in current treatments. Within the scope of the PhD project, new strategies for the treatment of MS aimed at moderating neuroinflammation (targeting reactive astrocytes through the enzyme B4GALT6) and sustaining remyelination (targeting the GPR17 receptor and studying the interaction between OPCs and PLTs) have been developed. The exciting findings warrant further studies in all the research questions that have been undertaken.