Page 131 - GPD-4-2

Gene & Protein in Disease Binding of 11q to DENV and WNV proteases

primarily transmitted through mosquito bites, but WNV Despite several efforts, there are currently no clinically
infection can also be contracted through alternative routes, approved small-molecule inhibitors that effectively target
such as blood transfusion, organ transplantation, and the NS2B–NS3 protease of DENV and WNV. Although
vertical transmission. Although most WNV infections are several promising compounds, such as BP2109,
28
asymptomatic, a small proportion (~1%) can lead to severe glycyrrhizin acid derivatives, 29 8-hydroxyquinoline
neuroinvasive diseases, such as encephalitis, meningitis, or derivatives, and myricetin, have demonstrated
30
31
acute flaccid paralysis. Therefore, there is a critical need to inhibitory activities in vitro, these compounds possess
6
design and develop effective antiviral compounds to treat severe limitations, including a lack of specificity, low
these infections. However, no approved medications are potency, poor pharmacokinetic properties, and a failure to
currently available for these diseases. target the dynamic active site of the protease. Therefore,
32
It is known that the NS2B–NS3 protease of the DENV there is a need to identify novel small molecules that can
plays a prominent role in disease progression by cleaving tightly bind to the active site of the protease to inhibit its
the viral polyprotein chain to generate virulent proteins, activity.
7-9
thereby facilitating viral genome replication in host In a recent study, several derivatives of aryl benzoyl
cells. 10,11 The crystal structure of the DENV–NS2B–NS3 hydrazide were synthesized to target different influenza
protease (Protein Data Bank [PDB] ID 3U1I) provides viruses. Among these derivatives, 10b, 10c, 10g, 11p, and
12
33
detailed structural insights that can aid in the design 11q showed encouraging activities against the avian H5N1
of antiviral compounds to inhibit protease activity. The flu strain. Among them, N-(((2,6-dibromophenyl) amino)
DENV protease contains two subunits (chains A and B), methyl)-4-morpholinobenzamide (11q) (Figure 2A)
7
which are NS2B (chain A) and NS3 (chain B). NS3 is a showed nanomolar antiviral activities against H1N1 and
large protease domain (182 residues), bound to its cofactor flu B viruses. It also showed excellent bioavailability
33
NS2B (45 residues) that wraps around NS3. The NS2B against the influenza A virus. Remarkably, the binding
33
cofactor inserts hydrophobic patches into the protease of 11q to the ZIKV NS2B–NS3 protease was found to
core to stabilize its active conformation. 13,14 NS3 adopts a be about 5 kcal/mol more stable than that of SYC-1307,
chymotrypsin-like fold, composed of two β-barrel domains, a known protease inhibitor (Table 1). Since SYC–1307
34
and contains a catalytic triad – H51, D75, and S135 – inhibits the ZIKV protease with a half-maximal inhibitory
essential for enzymatic cleavage of the viral polyprotein. In concentration value of 0.2 ± 0.01 µM, the stronger
35
addition to its catalytic role, NS3 contributes significantly binding affinity of 11q suggests that it may inhibit the ZIKV
to substrate recognition and binding. Residues, such as protease more efficiently than SYC–1307. Interestingly, due
D129, F130, A132, G151, G153, and Y161, are involved to the structural similarities between ZIKV, DENV, and
in substrate binding. Similarly, several residues from the WNV, SYC–1307 was also reported to inhibit the proteases
NS2B cofactor, such as D81, G82, and T83, contribute to of DENV (serotype-3) and WNV, with half-maximal
stabilizing substrate binding. 7-9,15 inhibitory concentration values of 0.52 ± 0.06 and 0.78 ±
The NS2B–NS3 protease of the WNV plays a similar 0.02 µM, respectively. 35
role in disease progression. It also contains NS2B (chain Given that ZIKV, DENV, and WNV proteases share
A) (47 residues) and NS3 (B chain) (186 residues) (PDB identical sequences (Figure 1) and structures, 36,37 it
ID 2FP7). Remarkably, both the DENV and WNV was necessary to examine whether 11q can bind to the
13
proteases share significant similarities at the genomic, NS2B–NS3 proteases of DENV and WNV with similar
structural, and functional levels. 3,16-18 For example, both affinity as observed for ZIKV protease. We have recently
viruses possess a positive-sense single-stranded RNA shown that paritaprevir binds tightly to the proteases of ZIKV,
genome of approximately 11 kilobases, which encodes a DENV, and WNV, with binding free energies of −11.51 ± 2.82
single polyprotein. This polyprotein is post-translationally – −17.3 ± 2.55 kcal/mol, suggesting its potential as a pan-
22
cleaved into three structural proteins – envelope, pre- antiviral agent against ZIKV, DENV, and WNV infections.
cursor membrane, and capsid – and seven non-structural Hence, it was necessary to evaluate whether 11q could act as
proteins, including NS1, NS2A, NS2B, NS3, NS4A, NS4B, a pan-antiviral agent against these infections.
and NS5. 19-27 Structural alignments of NS2B–NS3 proteases
of DENV and WNV (Figure 1) suggest that their catalytic 2. Materials and methods
residues (S135, H51, and D75) are conserved. These
residues are also conserved for the Zika virus (ZIKV) 2.1. System preparation
protease (Figure 1). Other than these catalytic residues, The X-ray crystal structures of the NS2B–NS3 protease
D130, T134, and S136 are also conserved in ZIKV, DENV, of the DENV (PDB ID 3U1I) and the WNV (PDB ID
12
and WNV proteases (Figure 1). 2FP7) were retrieved from the PDB (www.rcsb.org/)
13
Volume 4 Issue 2 (2025) 2 doi: 10.36922/gpd.8293