**1. Introduction**

Flaviviruses are arthropod-borne pathogens (arboviruses) that pose a serious threat to global health and cause millions of infections annually. Regardless of extensive research and public health

issues, currently there are no specific antiviral treatments in clinical use for flavivirus infections and, despite licensed vaccines, outbreaks still occur, highlighting challenges in executing effective control measures [1]. Some flaviviruses, like Japanese encephalitis virus (JEV), West Nile virus (WNV), and tick-borne encephalitis virus (TBEV) are associated with severe encephalitis; dengue virus (DENV) and yellow fever (YFV), cause hemorrhagic fever; and the newly emerging Zika virus may cause microcephaly in neonates and Guillain–Barré syndrome in adults [2,3]. Flaviviruses are enveloped, icosahedral with positive sense single-stranded RNA viruses that enter host cells by receptor-mediated endocytosis and transport to endosomes, where an acidic condition triggers conformational changes in the envelope (E) protein that prompt virus and host cell membrane fusion [4]. The released genomic RNA is translated to polyprotein precursor of about 3.4 k amino acids in length. This polypeptide gives rise to three structural (core (C), precursor membrane (prM), and envelope (E)) and seven nonstructural (NS) (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) proteins by host cell signalases and virus-encoded proteases [5]. NS proteins are mainly involved in virus replication and evasion from host immune response while structural proteins are responsible for virus assembly and successful viral entrance into and exit from host cells [6,7].

Immature virions assemble in the endoplasmic reticulum (ER). These non-infectious, immature viral particles contain heterodimers of E and prM proteins [8,9]. Maturation of virions occurs after the exocytic pathway in the trans-Golgi network (TGN) through the host cell endoprotease, called furin [10,11]. This calcium-dependent endoprotease cleaves prM to M protein in an acidic environment of the TGN after recognition of the sequence R-X-R/K-R in all flaviviruses. Mature particles then become infectious and are released in the extracellular environment by exocytosis [12]. This furin-mediated cleavage of prM is a critical step for flavivirus assembly and maturation [13]. Inhibition of furin functioning during the viral life cycle may debilitate flavivirus infectivity and pathogenicity.

Decanoyl-Arg-Val-Lys-Arg-chloromethylketone (dec-RVKR-cmk) and hexa-D-arginine (D6R) are small synthetic furin inhibitors that are suitable for clinical purposes. CMK has now been used by many groups as a reference inhibitor to study the effect of furin and related proprotein convertases. It also significantly inhibits viral infection because of its capacity to irreversibly block furin [14,15]. As reported previously, CMK is more effective than D6R in the reduction of HBV replication by inhibiting furin-mediated processing of the hepatitis B e antigen (HBeAg) precursor into mature HBeAg [16]. Dec-RVKR-cmk is a small, synthetic, irreversible, and cell-permeable competitive inhibitor of all proprotein convertases (PC1, PC2, PC4, PACE 4, PC5, PC7, and furin). This peptidyl chloromethylketone is reported to inhibit furin-mediated cleavage and fusion activity of viral glycoproteins, and acts as an antiviral agent against different viruses, including human immunodeficiency virus [17], Chikungunya virus [18], chronic hepatitis B virus, influenza A, Ebola virus infection [16], duck hepatitis B virus [19], and papilloma virus [20]. The structural-activity relationship of dec-RVKR-cmk was studied by Becker and colleagues by replacing the P1-arginine group with 4-amidinobenzylamide and the N-terminal deconyl group with phenyl acetyl group, and they derived a new compound, named phenylacetyl-Arg-Val-Arg-4-amidinobenzylamide, that exhibited more potency to inhibit the cleavage of hemagglutinin of fowl plague virus compared to dec-RVKR-cmk [15]. Smith et al. and Steinmetzer et al. also patented a peptidomimetic furin inhibitor by modifying the C-terminal of dec-RVKR-cmk with decarboxylated arginine mimetics, resulting in highly potent furin inhibitors [21].

There has been skepticism about whether a wide-range inhibitor of furin and other proprotein convertases (PCs) would interfere with normal cellular processes [22,23]. In the case of the PCs family, the concept of redundancy was observed, which constitutes an advantage over the other protease families. According to this concept, it is assumed that the inhibitory effect of a PC inhibitor in normal cells would be minimized by the redundant actions of other co-expressed PCs. Although the mechanism of the redundancy concept is still not well defined [23], this concept has been extensively validated in vitro, and recently has been explored in an animal study. It was observed that liver-specific

furin knockout mice showed no obvious adverse effects, thus suggesting that the redundancy effects of other PCs can compensate for the molecular ablation of furin in normal cellular process [24]. Recent developments in medicinal chemistry have explored whether a peptide-based inhibitor has overcome the particular issues associated to their use, bioavailability, and toxicity [23].

The furin-mediated flavivirus maturation encouraged us to evaluate the therapeutic potential of dec-RVKR-cmk against flaviviruses. Our results highlight the efficacy of dec-RVKR-cmk as an interesting anti-flavivirus agent with significant antiviral activities at a non-cytotoxic concentration, suggesting dec-RVKR-cmk as a potential candidate for the treatment of flavivirus.
