A 1H‐isoindol‐3‐amine was identified as suitable P1 group for the proprotein convertase furin using a crystallographic screening with a set of 20 fragments known to occupy the S1 pocket of trypsin‐like serine proteases. Its binding mode is very similar to that observed for the P1 group of benzamidine‐derived peptidic furin inhibitors suggesting an aminomethyl substitution of this fragment to obtain a couplable P1 residue for the synthesis of substrate‐analogue furin inhibitors. The obtained inhibitors possess a slightly improved picomolar inhibitory potency compared to their benzamidine‐derived analogues. The crystal structures of two inhibitors in complex with furin revealed that the new P1 group is perfectly suited for incorporation in peptidic furin inhibitors. Selected inhibitors were tested for antiviral activity against respiratory syncytial virus (RSV) and a furin‐dependent influenza A virus (SC35M/H7N7) in A549 human lung cells and demonstrated an efficient inhibition of virus activation and replication at low micromolar or even submicromolar concentrations. First results suggest that the Mas‐related G‐protein coupled receptor GPCR‐X2 could be a potential off‐target for certain benzamidine‐derived furin inhibitors.

Inhibitors of the serine protease furin have been widely studied as antimicrobial agents due to their ability to block the cleavage and activation of certain viral surface proteins and bacterial toxins. In this study, the antipseudomonal effects and safety profiles of the furin inhibitors MI-1851 and MI-2415 were assessed. Fluorescence quenching studies suggested no relevant binding of the compounds to human serum albumin and α₁-acid glycoprotein. Both inhibitors demonstrated significant antipseudomonal activity in Madin–Darby canine kidney cells, especially compound MI-1851 at very low concentrations (0.5 µM). Using non-tumorigenic porcine IPEC-J2 cells, neither of the two furin inhibitors induced cytotoxicity (CCK-8 assay) or altered significantly the intracellular (Amplex Red assay) or extracellular (DCFH-DA assay) redox status even at a concentration of 100 µM. The same assays with MI-2415 conducted on primary human hepatocytes also resulted in no changes in cell viability and oxidative stress at up to 100 µM. Microsomal and hepatocyte-based CYP3A4 activity assays showed that both inhibitors exhibited a concentration-dependent inhibition of the isoenzyme at high concentrations. In conclusion, this study indicates a good safety profile of the furin inhibitors MI-1851 and MI-2415, suggesting their applicability as antimicrobials for further in vivo investigations, despite some inhibitory effects on CYP3A4.

The proprotein convertase (PC) furin emerged as promising drug target for the treatment of numerous infectious diseases, cancer and cystic fibrosis. A recently described nonpeptidic lead structure served as template to develop a new series of PC inhibitors containing a dichlorobiphenyl-derived core segment decorated with a left and right inhibitor arm. The compounds were tested for their inhibitory potency against furin and the structurally related PC7. The most potent compounds inhibited furin with Ki values <5 nM, whereas most of them were significantly weaker inhibitors of PC7. Only for one compound, a significant potency with a Ki value of 7.3 nM against PC7 was found. Furthermore, crystal structures of six inhibitors in complex with furin were determined. Selected inhibitors were additionally tested for their antiviral potency against the furin-dependent H7N7 influenza A strain SC35M; a significant antiviral potency was found for compound 9.

Background/Objectives: Enterobacteriaceae, including pathogenic Shigella (S.) flexneri and Escherichia (E.) coli, cause severe gastrointestinal infections through toxins like Shiga and Shiga-like toxins. Antibiotic use is often discouraged due to its potential to increase toxin effects or contribute to the development of resistance. The host protease furin is capable of activating several viral glycoproteins and bacterial toxins, thus enhancing pathogen infectivity. Methods: To assess the therapeutic potential of furin inhibitors, cultured epithelial cell models (IPEC-J2 and MDCK) were used. The effects of MI-1851 and MI-2415 were evaluated after short-term (2 h) and long-term (6 h) exposure to S. flexneri, enterohemorrhagic E. coli (EHEC), and enteropathogenic E. coli (EPEC). Cytotoxicity was determined using the CCK-8 assay, and the inflammatory response was assessed by measuring interleukin (IL)-6 and IL-8 levels. Additionally, extracellular hydrogen peroxide production was monitored in IPEC-J2 cells to evaluate the potential alterations in redox status. Results: Infections with EHEC, EPEC, and S. flexneri significantly reduced the viability of epithelial cells after 6 h of incubation. Furin inhibitors MI-1851 and MI-2415 decreased cytotoxicity and compensated for IL-6 and IL-8 overproduction in cells during infection with EHEC and S. flexneri, but not in cells exposed to EPEC. In addition, they alleviated oxidative stress, particularly during S. flexneri addition. Conclusions: The development of new antimicrobial drugs that act via alternative mechanisms and effectively manage life-threatening enterobacterial infections is of key importance. Targeting furin with inhibitors MI-1851 and MI-2415, thus blocking toxin activation, could prevent the development of antimicrobial resistance, reduce the need for antibiotics and enhance overall treatment outcomes.

The Front Cover shows the crystal structure of a highly potent inhibitor of the proprotein convertase furin containing a C-terminal aminoisoindol group superimposed with a previously developed benzamidine analogue. This new P1 anchor was identified by crystallographic fragment screening with furin and enabled the design of peptidic inhibitors with inhibition constants < 10 pM. The inhibitors showed significant antiviral activity against respiratory syncytial virus and a furin-dependent influenza A virus. More information can be found in the Research Article 202400057 by Roman W. Lange, Sven O. Dahms, Torsten Steinmetzer, and co-workers.