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.
Inhibition of coronavirus (CoV)‐encoded papain‐like cysteine proteases (PLpro) represents an attractive strategy to treat infections by these important human pathogens. Herein we report on structure‐activity relationships (SAR) of the noncovalent active‐site directed inhibitor (R)‐5‐amino‐2‐methyl‐N‐(1‐(naphthalen‐1‐yl)ethyl) benzamide (2 b), which is known to bind into the S3 and S4 pockets of the SARS‐CoV PLpro. Moreover, we report the discovery of isoindolines as a new class of potent PLpro inhibitors. The studies also provide a deeper understanding of the binding modes of this inhibitor class. Importantly, the inhibitors were also confirmed to inhibit SARS‐CoV‐2 replication in cell culture suggesting that, due to the high structural similarities of the target proteases, inhibitors identified against SARS‐CoV PLpro are valuable starting points for the development of new pan‐coronaviral inhibitors.
Ligand binding to proteins often is accompanied by conformational transitions. Here, we describe a competition assay based on single molecule Förster resonance energy transfer (smFRET) to investigate the ligand-induced conformational changes of the dengue virus (DENV) NS2B-NS3 protease, which can adopt at least two different conformations. First, a competitive ligand was used to stabilize the closed conformation of the protease. Subsequent addition of the allosteric inhibitor reduced the fraction of the closed conformation and simultaneously increased the fraction of the open conformation, demonstrating that the allosteric inhibitor stabilizes the open conformation. In addition, the proportions of open and closed conformations at different concentrations of the allosteric inhibitor were used to determine its binding affinity to the protease. The K D value observed is in accordance with the IC 50 determined in the fluorometric assay. Our novel approach appears to be a valuable tool to study conformational transitions of other proteases and enzymes.
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Due to its fast international spread and substantial mortality, the coronavirus disease COVID-19 evolved to a global threat. Since currently, there is no causative drug against this viral infection available, science is striving for new drugs and approaches to treat the new disease. Studies have shown that the cell entry of coronaviruses into host cells takes place through the binding of the viral spike (S) protein to cell receptors. Priming of the S protein occurs via hydrolysis by different host proteases. The inhibition of these proteases could impair the processing of the S protein, thereby affecting the interaction with the host-cell receptors and preventing virus cell entry. Hence, inhibition of these proteases could be a promising strategy for treatment against SARS-CoV-2. In this review, we discuss the current state of the art of developing inhibitors against the entry proteases furin, the transmembrane serine protease type-II (TMPRSS2), trypsin, and cathepsin L.
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