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.
The COVID-19 pandemic, caused by the SARS-CoV-2 virus, has a huge impact on the world. Although several vaccines have recently reached the market, the development of specific antiviral drugs against SARS-CoV-2 is an important additional strategy in fighting the pandemic. One of the most promising pharmacological targets is the viral main protease (Mpro). Here, we present an optimized biochemical assay procedure for SARS-CoV-2 Mpro. We have comprehensively investigated the influence of different buffer components and conditions on the assay performance and characterized Förster resonance energy transfer (FRET) substrates with a preference for 2-Abz/Tyr(3-NO2) FRET pairs. The substrates 2-AbzSAVLQSGTyr(3-NO2)R-OH, a truncated version of the established DABCYL/EDANS FRET substrate, and 2-AbzVVTLQSGTyr(3-NO2)R-OH are promising candidates for screening and inhibitor characterization. In the latter substrate, the incorporation of Val at position P5 improved the catalytic efficiency. Based on the obtained results, we present here a reproducible, reliable assay protocol using highly affordable buffer components.
The viral serine protease NS2B-NS3
is one of the promising targets
for drug discovery against dengue virus and other flaviviruses. The
molecular recognition preferences of the protease favor basic, positively
charged moieties as substrates and inhibitors, which leads to pharmacokinetic
liabilities and off-target interactions with host proteases such as
thrombin. We here present the results of efforts that were aimed specifically
at the discovery and development of noncharged, small-molecular inhibitors
of the flaviviral proteases. A key factor in the discovery of these
compounds was a cellular reporter gene assay for the dengue protease,
the DENV2proHeLa system. Extensive structure–activity relationship
explorations resulted in novel benzamide derivatives with submicromolar
activities in viral replication assays (EC50 0.24 μM),
selectivity against off-target proteases, and negligible cytotoxicity.
This structural class has increased drug-likeness compared to most
of the previously published active-site-directed flaviviral protease
inhibitors and includes promising candidates for further preclinical
development.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.