Γ-HYDROXY LACTONE AS A VERSATILE SCAFFOLD FOR THE DEVELOPMENT OF NOVEL BIOACTIVE COMPOUNDS

Drug resistance in mycobacteria is a rapidly increasing phenomenon requiring the identification of new drugs. The inhibition of Protein Tyrosine Phosphatase B (MptpB), which interferes with host immune responses, may provide a new strategy to fight tuberculosis (TB), while preventing cross-resistance issues. On this basis, starting from a virtual screening (VS) campaign and subsequent structure elucidation studies guided by X-ray analysis, an unexpected γ-hydroxy lactone derivative (compound VS1) with significant enzymatic activity against MptpB was identified. Structure-activity relationship (SAR) studies led to several simplified derivatives that maintained the inhibitory activity of VS1 against MptpB, albeit without significantly improving it. Interestingly, VS1 shares its characteristic core lactone moiety with a natural product (BLI) isolated from the marine sponge-derived fungus Aspergillus terreus SCSIO 4100. Given that BLI was recently reported as an innovative inhibitor of the phosphorylation of peroxisome proliferator-activated receptor γ (PPARγ), we applied a repurposing strategy to find a new pharmacological application of our in-house library of γ-hydroxy lactone compounds in the field of insulin resistance. Indeed, PPARγ represents a key target for the treatment of type 2 diabetes and metabolic syndrome. To avoid the serious adverse effects related to the PPARγ agonism profile of traditional antidiabetic drugs, a new opportunity is represented by the development of molecules acting as inhibitors of PPARγ phosphorylation by the cyclin-dependent kinase 5 (Cdk5). Their mechanism of action is mediated by the stabilization of the PPARγ β-sheet containing Ser245. We approached this study by performing a biological screening, through Surface Plasmon Resonance (SPR) technology-based experiments, of the in-house library of synthetic γ-hydroxy-lactone derivatives. These studies, together with transactivation assay, and phosphorylation inhibition assay led to the identification of compound 8 as a promising PPARγ non-agonist (PPARγ KD = 3.75 μM). Crystallographic experiments allowed us to deeply investigate the interaction of 8 with the enzyme, and the co-crystal data were used as starting point for computational studies, leading to optimized derivatives of 8, endowed with higher affinity for PPARγ. These new γ-hydroxy lactone-based PPARγ non-agonists represent promising anti-diabetic candidates, effective for the treatment of insulin resistance, but without adverse effects. Furthermore, during my period abroad, at the Institute for Bioengineering of Catalonia in Spain, I focused my attention on the field of nanomedicine to improve the selectivity and efficacy of anti-TB candidates. Two pro-drugs of the first-in-class Salicylate Synthase I (MbtI) inhibitor (MIC = 32 μM), previously developed in our laboratory, were successfully encapsulated in poly(2-(methacryloyloxy) ethyl-phosphorylcholine)-co-poly(2-(diisopropylamino)ethyl methacrylate) (PMPC-PDPA) polymersomes (POs). Such a carrier allows us to (i) solubilize the hydrophobic drugs, (ii) target infected phagocytes via phenotypic association to scavenger receptors class B, (iii) efficiently deliver the cargo within the cell cytosol where most bacilli harbour, and (iv) effectively kill intracellular pathogens selectively. The POs were fully characterized by DLS, HPLC, and TEM; their cytotoxicity was assessed against macrophage cell line (THP-1) and Primary Human Lung Fibroblasts (HLF). Future work includes evaluating the antimycobacterial activity of the drug-containing POs in Mtb-infected primary cells. These multidisciplinary projects were developed thanks to the cooperation of scientists from the University of Milan (Italy), the University of Pavia (Italy), the University of Pisa (Italy), the University of Bari (Italy), the CNR of Rome (Italy), and the Institute for Bioengineering of Catalonia (Barcelona, Spain).