OFF-State-Specific Inhibition of the Proprotein Convertase Furin

Abstract

The pro-protein convertase furin is a highly specific serine protease involved in the proteolytic maturation of many proteins in the secretory pathway. It also activates surface proteins of many viruses including the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Furin inhibitors effectively suppress viral replication and thus are promising antiviral therapeutics with broad application potential. Polybasic substrate-like ligands typically trigger conformational changes shifting furin’s active site cleft from the OFF-state to the ON-state. Here, we solved the X-ray structures of furin in complex with four different arginine mimetic compounds with reduced basicity. These guanylhydrazone-based inhibitor complexes showed for the first time an active site-directed binding mode to furin’s OFF-state conformation. The compounds undergo unique interactions within the S1 pocket, largely different compared to substrate-like ligands. A second binding site was identified at the S4/S5 pocket of furin. Crystallography-based titration experiments confirmed the S1 site as the primary binding pocket. We also tested the proprotein convertases PC5/6 and PC7 for inhibition by guanylhydrazones and found an up to 7-fold lower potency for PC7. Interestingly, the observed differences in the K i values correlated with the sequence conservation of the PCs at the allosteric sodium binding site. Therefore, OFF-state-specific targeting of furin can serve as a valuable strategy for structure-based development of PC-selective small-molecule inhibitors.

Dichlorophenylpyridine-Based Molecules Inhibit Furin through an Induced-Fit Mechanism

“…Compounds 1−5 were synthesized as described previously 16 Furin was expressed, purified, and crystallized as described previously. 5,25,15 On the basis of the fit to the electron density map, the R and S enantiomers of 2 and 5, respectively, were modeled in the structures.…”

“… 12 , 13 Structural studies revealed different interaction patterns of these compound classes compared with canonical furin inhibitors. 14 , 15 Thus, noncanonical small-molecule furin inhibitors might be a promising opportunity to identify more drug-like compounds with improved bioavailability.…”

“…Beyond substrate-like inhibitors, several types of small-molecule furin inhibitors have been described, including 2,5-dideoxystreptamine- and guanylhydrazone-derived compounds. , Structural studies revealed different interaction patterns of these compound classes compared with canonical furin inhibitors. , Thus, noncanonical small-molecule furin inhibitors might be a promising opportunity to identify more drug-like compounds with improved bioavailability.…”

A perspective on the applications of furin inhibitors for the treatment of SARS-CoV-2

“…Of note, these compounds are not required to be positively charged to establish electrostatic interaction with the active site of furin, which overcomes the limitation of poor cell permeability [ 44 ]. In another study, guanyl hydrazone inhibitors evidenced an active site-directed binding mode to the furin OFF-state conformation [ 45 ]. The compounds were found to interact with the S1 pocket and with a second binding site at the S4 / S5 pocket of furin.…”

COVID-19 Therapies: Protease Inhibitions and Novel Degrader Strategies

“…SARS-CoV-2 bears a polybasic sequence PRRAR at the S1/S2 cleavage site that can be cleaved by furin (Hoffmann et al, 2020a;Peacock et al, 2021). Several peptide-based and small-molecule inhibitors of furin have been developed (Dahms et al, 2021;Osman et al, 2022). A combination of the furin inhibitor MI-1851 (Figure 1Bc) with various TMPRSS2 inhibitors enhances the antiviral potency (Bestle et al, 2020).…”