Potent Cationic Inhibitors of West Nile Virus NS2B/NS3 Protease With Serum Stability, Cell Permeability and Antiviral Activity

Abstract

West Nile virus (WNV) has spread rapidly around the globe, efficiently crossing species from migrating birds into humans and other mammals. The viral protease NS2B-NS3 is important for WNV replication and recognizes dibasic substrate sequences common to other flaviviral proteases but different from most mammalian proteases. Potent inhibitors of WNV protease with antiviral activity have been elusive to date. We report the smallest and most potent inhibitors known for this enzyme, cationic tripeptides with nonpeptidic caps at the N-terminus and aldehyde at the C-terminus. One of these, compound 3 ( Ki = 9 nM) is stable in serum (>90% intact after 3 h, 37 degrees C), cell permeable, and shows antiviral activity (IC 50 1.6 microM) without cytotoxicity (IC 50 >400 microM), thereby validating the approach of inhibiting WNV protease to suppress WNV replication.

Development and Characterization of New Peptidomimetic Inhibitors of the West Nile Virus NS2B–NS3 Protease

“…This tendency was also observed in the previously described arginal series. 66 A similar activity was also found for most other analogues, whereby the lowest K i values were determined for the dichloro-substituted inhibitors 73-75. Derivatives 54-75 were prepared as described for inhibitor 26 in Scheme 3.1 using commercially available P4 acyl residues.…”

“…Later, additional arginal derivatives have been reported by Stoermer et al. 66 The use of the sequence P4-Lys-Lys-Arginal provided strongly improved inhibitors, selected analogues are shown in Table 1.4. Various agmatine and agmatine-like peptidomimetic inhibitors of the WNV were first reported in 2011.…”

“…66 In most cases ring-substituted phenylacetyl moieties were incorporated, and in two inhibitors an N-terminal Fmoc-or p-hyroxyphenyl-propionyl group was used (Table 3.4). Compared with the reference inhibitor 26 (phenylacetyl-Lys-Lys-agmatine,…”

NMR Analysis of a Novel Enzymatically Active Unlinked Dengue NS2B-NS3 Protease Complex

“…Much effort has been invested into screening for DENV protease inhibitors as potential drug targets (33)(34)(35)(36)(37)(38)(39)(40)(41). The linked DENV protease construct has been widely utilized for in vitro biochemical assays and in silico compound screening.…”

Mechanisms of activation and inhibition of Zika virus NS2B-NS3 protease

“…The NS2B-NS3pro fusion protein was purified from E. coli, and gel filtration analysis revealed that majority of this complex existed in a monomeric form at various protein concentrations (Supplementary information, Figure S1C). NS2B-NS3pro can be activated readily by adding a widely used synthetic substrate AC-LKKR-AMC [10]. Using Michaelis-Menten kinetics we determined that the K m and k cat are 85.3 ± 2.1 μM and 1.149 ± 0.069 s -1 , respectively (Supplementary information, Figure S1D), which are comparable to the dengue virus (DENV) and the West Nile virus (WNV) NS2B-NS3pros [11,12], but different from ZIKV NS2B-NS3 (R95A/ R29G), which has an increased activity [8].…”