The NS2B/NS3 serine proteases of the Zika and Dengue
flaviviruses
are attractive targets for the development of antiviral drugs. We
report the synthesis and evaluation of a new, proline-based compound
class that displays allosteric inhibition of both proteases. The structural
features relevant for protease binding and inhibition were determined
to establish them as new lead compounds for flaviviral inhibitors.
Based on our structure–activity relationship studies, the molecules
were further optimized, leading to inhibitors with submicromolar IC50 values and improved lipophilic ligand efficiency. The allosteric
binding site in the proteases was probed using mutagenesis and covalent
modification of the obtained cysteine mutants with maleimides, followed
by computational elucidation of the possible binding modes. In infected
cells, antiviral activity against Dengue virus serotype 2 using prodrugs
of the inhibitors was observed. In summary, a novel inhibitor scaffold
targeting an allosteric site shared between flaviviral NS2B/NS3 proteases
is presented whose efficacy is demonstrated in vitro and in cellulo.
The dengue virus
protease (DENV-PR) represents an attractive target
for counteracting DENV infections. It is generally assumed that DENV-PR
can exist in an open and a closed conformation and that active site
directed ligands stabilize the closed state. While crystal structures
of both the open and the closed conformation were successfully resolved,
information about the prevalence of these conformations in solution
remains elusive. Herein, we address the question of whether there
is an equilibrium between different conformations in solution which
can be influenced by addition of a competitive inhibitor. To this
end, DENV-PR was statistically labeled by two dye molecules constituting
a FRET (fluorescence resonance energy transfer) couple. Fluorescence
correlation spectroscopy and photon-burst detection were employed
to examine FRET pair labeled DENV-PRs freely diffusing in solution.
The measurements were performed with two double mutants and with two
dye couples. The data provide strong evidence that an equilibrium
of at least two conformations of DENV-PR exists in solution. The competitive
inhibitor stabilizes the closed state. Because the open and closed
conformations appear to coexist in solution, our results support the
picture of a conformational selection rather than that of an induced
fit mechanism with respect to the inhibitor-induced formation of the
closed state.
The flaviviral heterodimeric serine protease NS2B‐NS3, consisting of the NS3 protease domain and the NS2B co‐factor, is essential for ZIKA virus maturation and replication in cells. For in vitro studies a ‘linked’ construct, where a polyglycine linker connects NS2BCF and NS3pro, is often used. This construct undergoes autocatalytic cleavage. Here, we show that linked ZIKV NS2BCF‐NS3pro is cleaved in cis in the NS2BCF exclusively at position R95 and not at the previously proposed alternate cleavage site at residue R29 in the NS3pro. Cleavage neither affects protease stability nor activity, despite some observed differences in spectroscopic behavior. This minimally modified construct may thus be useful for future structural and functional studies of the flaviviral protease, for example when testing new inhibitors.
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.