Novel screening platforms for the identification of inhibitors of the human protease Furin

Furin is a cellular protease that cleaves proteins at polybasic motifs. The processing event modulates the activity of the substrates. So far, a large number of proteins are known to be cleaved by Furin, including some of viral origin. Recently, the processing of SARS-CoV-2 glycoprotein S by Furin has attracted much attention since the inhibition of this event was early shown to be an effective strategy to block infection. Despite Furin inhibitors are available, none is FDA approved thus prompting researchers towards the search of novel compounds against Furin catalytic activity in order to combat against the current pandemic. In this project, I characterized a novel in vitro assay for the identification of Furin inhibitors, using a SARS-CoV-2 S-derived fluorogenic substrate. This assay significantly outperformed the standard in vitro Furin tests and proved suitable for high throughput screenings. Based on this setting, in a proof-of-concept study, I analyzed the inhibitor potency of small compounds libraries, identifying the 3-((5-((5-bromothi- 38 ophen-2-yl) methylene)-4-oxo-4,5 dihydrothiazol-2-yl)(3-chloro-4-methylphenyl)amino)propanoic 39 acid molecule (namely, P3) as a potent new Furin inhibitor (IC50 = 35μM). Docking analysis further suggested that the mechanism of action of P3 may involve interaction with a Furin exosite rather than its catalytic pocket. In parallel, in vitro and in silico studies, were complemented by cell-based assays, using a new Furin sensor construct containing the cleavage site of the envelope glycoprotein S of SARS-CoV-2. This sensor is a chimeric protein designed to release luciferase upon cleavage, indicating Furin activity. In mammalian cells, the sensor was processed by endogenous Furin, an action that could be blocked by specific inhibitors. These preliminary studies indicate that this new system is promising for the development of a novel cell-based assays for the screening of Furin inhibitors. Overall, this thesis has contributed to the characterization of innovative platforms for identifying compounds that can modulate Furin enzymatic activity, specifically targeting a viral sequence. Of note, a promising new inhibitor (P3) was found as an original scaffold to tailor effective antivirals.