N-arachidonoyletanolamine (AEA) and 2-arachidonoylglycerol (2-AG) are the most representative members of a class of endogenous ligands named endocannabinoid, which are able to activate cannabinoids receptors. The endocannabinoid neuromodulation plays important roles in various aspects of neural functions including learning and memory, anxiety, depression, appetite and feeding behavior, nociception, neuroprotection and movement control. Since 2-AG has been considered the true on-demand released endocannabinoid ligand for CB1 receptor, a promising way to explore and promote its therapeutic potential could be the selective inhibition of the monoacylglycerol lipase (MGL), the enzyme responsible of 2-AG degradation. Three critical residues of cysteine have been identified as regulatory gates for the activity of the enzyme: Cys242, within the active site close to the catalytic triad, Cys201 and Cys208 located on the lid domain, a flexible solvent-exposed region that regulate the access of the substrate to the active site. Cys201 and Cys208 have been recently shown to be redox switches that regulate the activity of MGL by undergoing oxidation after oxidative stimulus induced by hydrogen peroxide.
Despite the potential of MGL as target for 2-AG signaling modulation, few potent and selective MGL inhibitors are available until now. Recently two class of partially-reversible MGL inhibitors has been reported: one is populated by covalent disulfide-bond forming compounds such as isothiazol-3(2H)-one (ITZ) and benzo[d]isothiazol-3(2H)-one (BTZ) derivatives, the second one is populated by triterpenes such as euphol and pristimerin. Both these classes of compounds interact with critical residues Cys201 and Cys208 of MGL thus preserving the catalytic activity of the enzyme. The modulation of MGL activity by interaction with these critical cysteine residues is a crucial point to fine tune the 2-AG signaling avoiding behavioral side-effects associated to the total disruption of the catalytic activity of MGL exerted by inhibitors addressing the catalytic site
The research work reported in this PhD dissertation has been focused on the exploration of the structure-activity relationships (SAR) around the BTZ warhead in order to explore the pharmacophoric space around the targeted cysteines of MGL.
The structure of the driver portion of compounds sharing an identical warhead has been broadly changed. I investigated the effect of substituent groups with different steric demand and electronic inductive effects on the nitrogen atom and the substituents on the phenyl ring of BTZ.
I performed the design and the synthesis of several compounds, applying different synthetic strategies.
The inhibitory potency of synthesized BTZs on MGL and was evaluated and SAR of this class were described. We identified promising compounds with inhibitory potency on MGL in the low nanomolar range. Moreover, the lead compound emerged from this screening was subjected to mechanistic studies that revealed a behavior similar to that of hydrogen peroxide in inactivating MGL supporting the theory that BTZs mimic the effect of an endogenous oxidation stimulus. To further elucidate the role of critical Cys201 and Cys208 in MGL activity, site-directed mutagenesis studies were performed identifying both cysteines as possible counterpart in the interaction with BTZs.
Moreover, in order to evaluate the stability and reactivity of these compounds towards biological thiols we investigated, trough analytical methods the effect of substituent groups with different steric hindrance and electronic effects on the sulfenamidic reactive core of BTZ. I investigated by means of NMR experiments the reactivity of selected BTZs with a model biological thiol, N-acetyl cysteine (NAC). These studies allowed us to define a structure-reactivity relationship landscape that further validated our biological results.
To increse the knolewdge in the field of the terpene-based inhibitors of MGL, I synthesized 3 new compounds starting form commercially available celastrol: these scaffold modifications led to less active compounds than reference compound pristimerin thus discouraging the development of triperpene-based MGL inhibitors.
Fibroblast growth factor-2 (FGF2) is a member of a large family of proteins that bind heparin and heparan sulfate and modulate the function of a wide range of cell types. FGF2 stimulates the growth and development of new blood vessels (angiogenesis) that contribute to the pathogenesis of several diseases (i.e. cancer, atherosclerosis), normal wound healing and tissue development. FGF2 exerts its activity on endothelial cells by interacting with high affinity tyrosine-kinase FGF receptors (FGFRs) and low affinity heparan sulphate proteoglycans (HSPGs), leading to the formation of productive HSPG/FGF2/FGFR ternary complexes. Presta and coworkers identified, from a virtual screening study of an NCI library, a promising molecule NSC172288 able to inhibit FGF2-FGFR interactions. During my PhD research I designed and performed the synthesis of this compound not described in literature. The major challenge associated was the necessity of planning the synthesis on the base of a poorly described structure.
A synthetic pathway for NSC172285 was setup and an efficient method for using HFA trihydrate as electrophilic partner in an aldol reaction, with standard laboratory equipment was developed
