A New Class of Dengue and West Nile Virus Protease Inhibitors with Submicromolar Activity in Reporter Gene DENV-2 Protease and Viral Replication Assays

Abstract

Dengue and West Nile virus are rapidly spreading global pathogens for which no specific therapeutic treatments are available. One of the promising targets for drug discovery against dengue and other flaviviruses is the viral serine protease NS2B-NS3. We present the design, synthesis, and in vitro and cellular characterization of a novel chemotype of potent small-molecule non-peptidic dengue protease inhibitors derived from 4-benzyloxyphenylglycine. A newly developed luciferase-based DENV-2 protease reporter system in HeLa cells (DENV2proHeLa) was employed to determine the activity of the compounds in a cellular environment. Specificity and selectivity of the DENV2proHeLa system were confirmed by viral titer reduction assays. The compounds reach low micromolar to upper nanomolar inhibitory potency in cell-based assays, are selective against other serine proteases, and do not show relevant cytotoxicity. An extensive structure–activity relationship study provides a perspective for further drug development against flaviviral infections.

Efficiency Improvements and Discovery of New Substrates for a SARS-CoV-2 Main Protease FRET Assay

“…Substrates 7 and 8 are substrates developed and routinely used in our group for the dengue virus and West Nile virus NS2B/NS3 protease assays, and they were used here as negative controls. 39 , 40 …”

“…Substrates 7 and 8 are substrates developed and routinely used in our group for the dengue virus and West Nile virus NS2B/NS3 protease assays, and they were used here as negative controls. 39,40 We first screened the activity of the protease at a substrate concentration of 50 µM. The highest cleavage was observed for substrates 3 and 4 (Fig.…”

Efficiency Improvements and Discovery of New Substrates for a SARS-CoV-2 Main Protease FRET Assay

“…Substrates 7 and 8 are substrates developed and routinely used in our group for the dengue and West Nile vims NS2B/NS3 protease assays, and were used here as negative controls. 36–37…”

Viral Proteases

“…These compounds are decarboxylated peptides containing a C-terminal 4-guanidinobenzylamino group which is responsible for interacting with S1 pocket of the protease. The most potent inhibitor obtained in these studies (5) showed a EC 50 value of 0.5 mM in cell-based assays with no relevant cytotoxicity (Kühl et al 2020).…”