Discovery of novel probes for a multi-target approach to Multiple Sclerosis treatment

Abstract Multiple Sclerosis (MS) is a life-limiting, multifactorial disease of the Central Nervous System, characterized by inflammation, demyelination, and neurodegeneration, which still represents an unmet medical need. In the search for innovative therapeutic agents, we envisaged that a multi-target (MT) approach might be a valid drug-discovery strategy. My Ph.D. project aimed to discover dual ligands able to simultaneously tackle two selected targets, N-Acylethanolamine-hydrolyzing Acid Amidase (NAAA) and Glycogen-Synthase Kinase 3β (GSK-3β), two enzymes involved in the inflammation and neurodegeneration components of MS, respectively. Supported by preliminary in vivo studies in an MS mouse model, which demonstrated the efficacious concurrent administration of an NAAA and a GSK-3β inhibitor in reducing the severity of the symptoms, novel dual-ligands were designed and synthesized. The combination of the structural features of the two individual compounds, 1 and 2 (as the NAAA and GSK-3β inhibitor, respectively), led initially to the conjugated derivatives 3 and 4, which showed encouraging in vitro dual inhibition but suboptimal drug-like properties. Structure-Activity Relationship (SAR) investigations led to identifying the merged hybrid compound 74, which guided the rational design of the more potent analog 73. X-ray cocrystal structure of 73 with GSK-3β guided further structural modifications, which ultimately led to 114 and its close analogs. In testing these novel dual inhibitors, strange results were obtained in the NAAA assay. After extensive investigations, we discovered that compound 114 emits fluorescence at a wavelength that interferes with the assay readout. NAAA assay uses N-(4-methyl-2-oxo-chromen-7-yl)-hexadecanamide (PAMCA) as the substrate, which is cleaved by the enzyme releasing the fluorophore 7-amino 4-methyl coumarin (AMC). In order to obviate the fluorescence interference, a novel fluorogenic substrate, N-(4-trifluoromethyl-2-oxo-chromen-7-yl)-hexadecanamide (PAMFA) was synthesized, which allows shifting the fluorescence emission of the released coumarin to longer wavelengths. The new substrate strongly reduced the fluorescence interference. Under these new assay conditions, compound 114, and its close analogs, turned out to be inactive. Despite this negative result, the new NAAA assay format, based on PAMFA as the substrate, will allow testing new analogs of our dual inhibitors without fluorescence interference, possibly leading to the discovery of novel potent dual NAAA/GSK-3β ligands. During my third year of Ph.D., I spent 6 months in Prof. Caflisch’s lab (University of Zurich) where I learned a few computational approaches to be applied in structure-based drug design. Computational tools, developed by Caflisch’s group, allowed identifying promising small molecules, which could be able to inhibit the protein-protein interaction (PPI) of the METTL3-METTL14 methyltransferase complex.