RESUMEN
We describe a set of benzisothiazolinone (BTZ) derivatives that are potent inhibitors of monoacylglycerol lipase (MGL), the primary degrading enzyme for the endocannabinoid 2-arachidonoyl-sn-glycerol (2-AG). Structure-activity relationship studies evaluated various substitutions on the nitrogen atom and the benzene ring of the BTZ nucleus. Optimized derivatives with nanomolar potency allowed us to investigate the mechanism of MGL inhibition. Site-directed mutagenesis and mass spectrometry experiments showed that BTZs interact in a covalent reversible manner with regulatory cysteines, Cys201 and Cys208, causing a reversible sulfenylation known to modulate MGL activity. Metadynamics simulations revealed that BTZ adducts favor a closed conformation of MGL that occludes substrate recruitment. The BTZ derivative 13 protected neuronal cells from oxidative stimuli and increased 2-AG levels in the mouse brain. The results identify Cys201 and Cys208 as key regulators of MGL function and point to the BTZ scaffold as a useful starting point for the discovery of allosteric MGL inhibitors.
Asunto(s)
Cisteína/química , Inhibidores Enzimáticos/farmacología , Monoacilglicerol Lipasas/antagonistas & inhibidores , Tiazoles/farmacología , Regulación Alostérica , Animales , Sitios de Unión , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/metabolismo , Células HeLa , Humanos , Ratones , Simulación del Acoplamiento Molecular , Estructura Molecular , Monoacilglicerol Lipasas/genética , Monoacilglicerol Lipasas/metabolismo , Mutagénesis Sitio-Dirigida , Mutación , Oxidación-Reducción , Unión Proteica , Ratas , Relación Estructura-Actividad , Tiazoles/síntesis química , Tiazoles/metabolismoRESUMEN
NSC12 is an orally available pan-FGF trap able to inhibit FGF2/FGFR interaction and endowed with promising antitumor activity. It was identified by virtual screening from a NCI small molecule library, but no data were available about its synthesis, stereochemistry, and physicochemical properties. We report here a synthetic route that allowed us to characterize and unambiguously identify the structure of the active compound by a combination of NMR spectroscopy and in silico conformational analysis. The synthetic protocol allowed us to sustain experiments aimed at assessing its therapeutic potential for the treatment of FGF-dependent lung cancers. A crucial step in the synthesis generated a couple of diastereoisomers, with only one able to act as a FGF trap molecule and to inhibit FGF-dependent receptor activation, cell proliferation, and tumor growth when tested in vitro and in vivo on murine and human lung cancer cells.