Functional Loop Dynamics and Characterization of the Inactive State of the NS2B-NS3 Dengue Protease due to Allosteric Inhibitor Binding

Dengue virus, a flavivirus that causes dengue shock syndrome
and
dengue hemorrhagic fever, is currently prevalent worldwide. A two-component
protease (NS2B-NS3) is essential for maturation, representing an important
target for designing anti-flavivirus drugs. Previously, consideration
has been centered on developing active-site inhibitors of NS2B-NS3pro.
However, the flat and charged nature of its active site renders difficulties
in developing inhibitors, suggesting an alternative strategy for identifying
allosteric inhibitors. The allosterically sensitive site of the dengue
protease is located near Ala125, between the 120s loop and 150s loop.
Using atomistic molecular dynamics simulations, we have explored the
protease’s conformational dynamics upon binding of an allosteric
inhibitor. Furthermore, characterization of the inherent flexible
loops (71–75s loop, 120s loop, and 150s loop) is carried out
for allosteric-inhibitor-bound wild-type and mutant A125C variants
and a comparison is performed with its unbound state to extract the
structural changes describing the inactive state of the protease.
Our study reveals that compared to the unliganded system, the inhibitor-bound
system shows large structural changes in the 120s loop and 150s loop
in contrast to the rigid 71–75s loop. The unliganded system
shows a closed-state pocket in contrast to the open state for the
wild-type complex that locks the protease into the open and inactive-state
conformations. However, the mutant complex fluctuates between open
and closed states. Also, we tried to see how mutation and binding
of an allosteric inhibitor perturb the connectivity in a protein structure
network (PSN) at contact levels. Altogether, our study reveals the
mechanism of conformational rearrangements of loops at the molecular
level, locking the protein in an inactive conformation, which may
be useful for developing allosteric inhibitors.