Discovery of Nanomolar Dengue and West Nile Virus Protease Inhibitors Containing a 4-Benzyloxyphenylglycine Residue

Abstract

The dengue virus (DENV) and West Nile Virus (WNV) NS2B-NS3 proteases are attractive targets for the development of dual-acting therapeutics against these arboviral pathogens. We present the synthesis and extensive biological evaluation of inhibitors that contain benzyl ethers of 4-hydroxyphenylglycine as non-natural peptidic building blocks synthesized via a copper-complex intermediate. A three-step optimization strategy, beginning with fragment growth of the C-terminal 4-hydroxyphenylglycine to the benzyloxy ether, followed by C- and N-terminal optimization, and finally fragment merging generated compounds with in vitro affinities in the low nanomolar range. The most promising derivative reached Ki values of 12 nM at the DENV-2 and 39 nM at the WNV proteases. Several of the newly discovered protease inhibitors yielded a significant reduction of dengue and West Nile virus titers in cell-based assays of virus replication, with an EC50 value of 3.4 μM at DENV-2 and 15.5 μM at WNV for the most active analogue.

Broad-spectrum agents for flaviviral infections: dengue, Zika and beyond

“…The flaviviral proteases have a strong preference for substrates with dibasic or polybasic recognition sequences [49][50][51] . Consequently, the recognition motifs in inhibitors also tend to incorporate basic or polar functionalities, which may partially explain the lower efficacy often observed in cell-based assays relative to biochemical assays, as the compounds have low passive membrane permeability [52][53][54][55][56] . The substrate-binding residues and the substrate recognition patterns of the protease are well conserved across the flaviviruses and therefore hold promise for the development of inhibitors with broad activity.…”

“…Numerous studies have focused on the development of protease inhibitors derived from a substrate-mimicking peptide. Strategies included incorporation of a carboxyterminal electrophile 52,54,[73][74][75] , optimization of the N-terminal capping moiety 53,56,76,77 , and modulation of the P 1 and P 2 basic residues through non-natural building blocks 53,78 . Aldehydic inhibitors displayed low micromolar to nanomolar affinity for DENV serotype 2 (DENV-2) 74 and WNV 52,75 proteases.…”

“…The main limitation of the boronic acid inhibitors appears to be their low selectivity against off-targets such as thrombin and trypsin 54 . This problem may be addressed by extension towards the prime site or optimization of the N-terminal cap, as shown for other peptide-based compounds 53,56,76,77 and for the aldehyde compound 3a 52 . Although the clinical potential of peptide aldehydes and peptide boronic acids remains uncertain, they are crucial for understanding the molecular recognition and structural transitions of the flaviviral protease 54,62,64,67 .…”

Dengue Virus NS2B/NS3 Protease Inhibitors Exploiting the Prime Side

“…Recently, DENV/WNV protease inhibitors targeting P site pockets with nanomolar activity in enzymatic assays have been reported (K i ϭ 12 nM and 39 nM) (15). Since these inhibitors are highly polar, possibly underlying the micromolar-level activity in cell-based assays, incorporating the critical hydrophobic interactions we identified at P3= or P4= pockets (17) is a strategy to reduce the hydrophilicity.…”

“…While most of these inhibitors bind with only micromolar affinity, a recent study found inhibitors with nanomolar K i values (15). However, the potential challenge in targeting dengue virus protease is that this enzyme has a P side substrate sequence preference similar to those of several human serine proteases (furin RXRR, thrombin P1 R, and trypsin P1 R); hence, P side-based inhibitors are not designed to be specific to the viral protease.…”