Dengue virus is the flavivirus that causes dengue fever, dengue hemorrhagic disease, and dengue shock syndrome, which are currently increasing in incidence worldwide. Dengue virus protease (NS2B-NS3pro) is essential for dengue virus infection and is thus a target of therapeutic interest. To date, attention has focused on developing active-site inhibitors of NS2B-NS3pro. The flat and charged nature of the NS2B-NS3pro active site may contribute to difficulties in developing inhibitors and suggests that a strategy of identifying allosteric sites may be useful. We report an approach that allowed us to scan the NS2B-NS3pro surface by cysteine mutagenesis and use cysteine reactive probes to identify regions of the protein that are susceptible to allosteric inhibition. This method identified a new allosteric site utilizing a circumscribed panel of just eight cysteine variants and only five cysteine reactive probes. The allosterically sensitive site is centered at Ala125, between the 120s loop and the 150s loop. The crystal structures of WT and modified NS2B-NS3pro demonstrate that the 120s loop is flexible. Our work suggests that binding at this site prevents a conformational rearrangement of the NS2B region of the protein, which is required for activation. Preventing this movement locks the protein into the open, inactive conformation, suggesting that this site may be useful in the future development of therapeutic allosteric inhibitors.
Herein, we introduce a new strategy to estimate binding
free energies
using end-state molecular dynamics simulation trajectories. The method
is adopted from linear interaction energy (LIE) and ANI-2x neural
network potentials (machine learning) for the atomic simulation environment
(ASE). It predicts the single-point interaction energies between ligand–protein
and ligand–solvent pairs at the accuracy of the wb97x/6-31G*
level for the conformational space that is sampled by molecular dynamics
(MD) simulations. Our results on 54 protein–ligand complexes
show that the method can be accurate and have a correlation of
R
= 0.87–0.88 to the experimental binding free energies,
outperforming current end-state methods with reduced computational
cost. The method also allows us to compare BFEs of ligands with different
scaffolds. The code is available free of charge (documentation and
test files) at
.
The
dengue virus protease (NS2B-NS3pro) plays a critical role in
the dengue viral life cycle, making it an attractive drug target for
dengue-related pathologies, including dengue hemorrhagic fever. A
number of studies indicate that NS2B-NS3pro undergoes a transition
between two widely different conformational states: an “open”
(inactive) conformation and a “closed” (active) conformation.
For the past several years, the equilibrium between these states and
the resting conformation of NS2B-NS3pro have been debated, although
a strong consensus is emerging. To investigate the importance of such
conformational states, we developed versions of NS2B-NS3pro that allow
us to trap the enzyme in various distinct conformations. Our data
from these variants suggest that the enzymatic activity appears to
be dependent on the movement of NS2B and may rely on the flexibility
of the protease core. Locking the enzyme into the “closed”
conformation dramatically increased activity, strongly suggesting
that the “closed” conformation is the active conformation.
The observed resting state of the enzyme depends largely on the construct
used to express the NS2B-NS3pro complex. In an “unlinked”
construct, in which the NS2B and NS3 regions exist as independent,
co-expressed polypeptides, the enzyme rests predominantly in a “closed”,
active conformation. In contrast, in a “linked” construct,
in which NS2B and NS3 are attached by a nine-amino acid linker, NS2B-NS3pro
adopts a more relaxed, alternative conformation. Nevertheless, even
the unlinked construct samples both the “closed” and
other alternative conformations. Given our findings, and the more
realistic resemblance of NS2B-NS3pro to the native enzyme, these data
strongly suggest that studies should focus on the “unlinked”
constructs moving forward. Additionally, the results from these studies
provide a more detailed understanding of the various poses of the
dengue virus NS2B-NS3 protease and should help guide future drug discovery
efforts aimed at this enzyme.
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