Effects of NS2B-NS3 protease and furin inhibition on West Nile and Dengue virus replication

Abstract

West Nile virus (WNV) and Dengue virus (DENV) replication depends on the viral NS2B-NS3 protease and the host enzyme furin, which emerged as potential drug targets. Modification of our previously described WNV protease inhibitors by basic phenylalanine analogs provided compounds with reduced potency against the WNV and DENV protease. In a second series, their decarboxylated P1-trans-(4-guanidino)cyclohexylamide was replaced by an arginyl-amide moiety. Compound 4-(guanidinomethyl)-phenylacetyl-Lys-Lys-Arg-NH inhibits the NS2B-NS3 protease of WNV with an inhibition constant of 0.11 µM. Due to the similarity in substrate specificity, we have also tested the potency of our previously described multibasic furin inhibitors. Their further modification provided chimeric inhibitors with additional potency against the WNV and DENV proteases. A strong inhibition of WNV and DENV replication in cell culture was observed for the specific furin inhibitors, which reduced virus titers up to 10,000-fold. These studies reveal that potent inhibitors of furin can block the replication of DENV and WNV.

Optimization of Substrate‐Analogue Furin Inhibitors

“…The hemagglutinin precursor HA0 is cleaved by furin into HA1 and HA2. Results obtained with this screening assay usually correlate well with the antiviral activity of inhibitors in cell‐based infection studies as shown previously for H7N1 and H5N1 HPAIV, canine distemper virus, Semliki Forest virus, chikungunya virus, West Nile virus, and Dengue‐2 virus . In the current study, strong differences in potency were found in this assay, as shown by the Western blot in Figure .…”

“…A slight concentration‐dependent inhibition was observed for the aminopyridine analogue 20 , which provided ≈10‐fold reduced DENV‐2 titers at 50 μ m , whereas a negligible inhibition was found for compounds 14 , 17 , 19 , and 23 (data not shown). Therefore, only the most potent inhibitors 24 and 27 were selected for a further dose‐response assay, using the recently investigated compound 4 and the nucleotide analogue ribavirin as reference inhibitors (Figure ) . Also in this assay, the strongest antiviral efficacy was found for the reference compound 4 , which is more potent than ribavirin at similar concentrations.…”

“…Cell culture supernatants were harvested 48 h post‐infection and virus titers ± standard deviation ( n =3) were determined by plaque assay in VeroE6 cells. The effects of the reference compounds 4 and ribavirin, as well as details of the assay methods used, were described recently …”

“…The 4‐Amba group binds in a deep, well‐defined S1 pocket and is involved in numerous contacts with furin, thereby contributing to the picomolar potency of these analogues. Thus far, the strongest potency against anthrax or diphtheria toxin cytotoxicity and the inhibition of virus replication after infection of cells by HPAIV of the type H5N1 and H7N1, canine distemper virus, chikungunya virus, Semliki Forest virus, Dengue or West Nile virus was found for compound 4 (MI‐1148) …”

Design, Synthesis, and Characterization of Macrocyclic Inhibitors of the Proprotein Convertase Furin

“…Our previously described linear inhibitors revealed as ignificant activity against numerousf urin-dependent pathogenic viruses in cell culture experiments at lower micromolar concentrations. [20,24,29,[36][37][38][39] Due to limited tolerability of the most potent compounds in mice, [29] we prepared different types of new macrocyclic analogues to find alternative inhibitor structures and determined their potencya gainst furin. The development of these compounds began with the crystal structure of the hexapeptided erivative 1 in complex with furin.…”