Multiple Sclerosis (MS) is a chronic autoimmune disease that affects the central nervous system (CNS), including the brain and spinal cord. Symptoms include weakness, tingling, numbness, visual disturbances, muscle stiffness, and memory loss. There are currently treatments to dampen the symptoms and delay disease progression, but no known cure for multiple sclerosis has been found to date (1).
Causes of multiple sclerosis
The disease is provoked by auto aggressive immune cells that cause devastating symptoms in the central nervous system. Myelin sheath damage on nerve cell axons is known to be the primary cause of MS, slowing the nerve system’s ability to transmit messages between brain and body (Figure 1).
Figure 1: Normal healthy neuron and demyelinated neuron in multiple sclerosis.
MS exacerbation is instigated when circulating T cells become mis-programmed to target and attack myelin. The T cells release cytokines to recruit similar T cells, B cells, and macrophages to mount the attack. The inflammatory cells in MS can cross the blood brain barrier which normally separates the immune cells in the periphery from the central nervous system. Once inside the brain, the more aggressive T cells release more cytokines (for example interleukins) to activate B cells, macrophages, and microglia to propagate a cycle of inflammation which will attack the myelin sheath, axons, and oligodendrocytes, leading to cell death.
Approaches to MS drug discovery
Reducing IL-6 secretion by dendritic cells, protecting and promoting oligodendrocyte growth (myelin producing cells), and inhibiting the differentiation of T naive cells to Th17 cells are commonly used methods to discover drugs that treat the progression of MS.
Dendritic cells (DCs) are known to stimulate naive T cells by pro-inflammatory cytokine IL-6. These cells modulate the immune responses and play a critical role in the pathogenesis of autoimmune diseases, including multiple sclerosis. Targeting DC pro-inflammatory IL-6 production is an important strategy for the treatment of autoimmune diseases. Chen et al. (2) identified a potential new drug (BVDU) that dampens the production of IL-6 by dendritic cells. The team ran a high throughput screen using mature dendritic cells stimulated with LPS (Figure 2). IL-6 secretions in dendritic cell culture supernatants were determined using the HTRF™ Mouse IL-6 assay kit, and the assay signal intensities were measured using an EnVision multimode plate reader (Revvity). The HTRF mouse IL-6 assay kit was chosen for the compound screen due to its low cost, no-wash, simple assay steps, and stable signal.
Figure 2: LPS-stimulated mature dendritic cells (mDC) to screen drug candidates for MS. Chen, S., Zhou, J., Cai, Y. et al. Discovery of BVDU as a promising Drug for autoimmune disease Therapy by Dendritic-cell-based functional screening. Sci Rep 7, 43820 (2017)
BVDU inhibited IL-6 secretion and reduced the severity of disease symptoms in the EAE mouse model (a well-known animal model of MS), as shown by decreased neuroinflammation and demyelination. A detailed description can be found in the associated literature review “Three Drug Discovery Approaches for Multiple Sclerosis”.
Oligodendrocytes are cells that produce proteins for myelin formation along neuron axons. Promoting oligodendrocyte differentiation is another approach for MS treatment. Researchers are actively searching for drugs that enhance oligodendrocyte growth to repair damaged myelin. Merten et al. (3) studied the effects of an existing drug candidate HAMI3379, a cysteinyl-leukotriene CysLT2 receptor (GPR17) antagonist, which was originally developed to address cardiovascular and inflammatory disorders. The study showed that inhibition of GPR17 receptor signaling by HAMI3379 promoted oligodendrocyte growth (Figure 3).
The orphan GPR17 receptor was interesting, since GPR17 knockout is associated with precocious myelination at the neonatal stage. MDL29.951, a specific GPR17 ligand, was used in the study to show the antagonist activity of HAMI3379 in human oligodendrocytes. The activity of HAMI3379 was determined by measuring the intracellular cAMP accumulation assay. To measure intracellular cAMP, the HTRF-based cAMP accumulation assay kits(Revvity) were used, and the signals of the assays were measured with a Revvity EnVision multilabel plate reader.
The study revealed that HAMI3379, an experimental drug that stimulates differentiation of both rodent and human oligodendrocytes, efficiently blocks GPR17 signaling and may qualify as a pharmacological tool to promote oligodendrocyte differentiation and the myelination process in MS by interrogating the GPR17 function.
RORγ (Retinoic acid-related Orphan Receptor γ) is a nuclear receptor belonging to the family of RORα and RORβ. RORγt, an isoform of RORγ that is highly expressed in immune cells such as Th17 and γδT cells, is a key player in the IL-17 pathway and in the differentiation of naive T cells into Th17 cells. Amaudrut et al. (4) identified a potent and selective RORγ inverse agonist (compound 29). The selectivity of the agonist vs the other ROR isoforms was evaluated in a coactivator recruitment assay using AlphaScreen™ technology (Figure 4), in which the binding of N-biotin co-activator PGC1g peptide and His-RORγt (amino acid T259-K518) was inhibited by the ROR isoforms (RORα, RORβ, or ROYγ).
Figure 4: The principle of AlphaScreen™ Assay Technology. AlphaScreen™ Technology (Amplified Luminescent Proximity Homogeneous Assay) was used to determine the compound dependent interaction of the RORγt ligand-binding domain (His-RORγ (amino acid T259-K518)) with the co-activator PGC1g (LXD1) peptide (N-biotin-QEAEEPSLLKKLLLAPANTQL-COOH). Following the binding of biotinylated co-activator peptide to His-Tag RORγt protein, the biotin on the co-activator binds to the streptavidin-coated donor beads and His-Tag of RORγt protein interacts with Ni-chelate alpha acceptor beads, bringing the beads into close proximity. The excitation of the donor beads provokes the release of singlet oxygen molecules which triggers a cascade of energy transfer in the Acceptor beads, resulting in a light emission at 520-615 nm that can be detected by an Envision plate reader.
Conclusion
Three small molecule drug candidates (BVDU, HAMI3379, and Compound 29) investigated by three separate laboratories were identified and validated for the treatment of multiple sclerosis (MS). All three molecules hold promise for pharmacological exploitation either to reduce immune cell attacks on neuron myelin or to enhance remyelination in patients. HTRF based IL-6 and cAMP assays and AlphaScreen™ Assay Technology have played critical roles in MS drug discovery.
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- Dobson R. and Giovannoni G.Multiple Sclerosis – A Review.Eur J Neurol 2019, 26:27-40.
- Chen S., Zhou J., Cai Y. et al.Discovery of BVDU as a promising Drug for autoimmune disease Therapy by Dendritic-cell-based functional screening.Sci Rep 7, 43820 (2017).
- Merten N., Fischer J., Simon K. et al.Repurposing HAMI3379 to Block GPR17 and Promote Rodent and Human Oligodendrocyte Differentiation.Cell Chem Biol. 2018;25(6):775‐786.e5.
- Amaudrut J., Argiriadi MA., Barth M. et al.Discovery of novel quinoline sulphonamide derivatives as potent, selective and orally active RORγ inverse agonists.Bioorg. Med. Chem. Lett. 29, 1799–1806 (2019).
