Shiryaev, S. | Search bioRxiv

bioRxiv2022

Allosteric inhibitors of Zika virus NS2B-NS3 protease targeting protease in super-open conformation

Zika virus (ZIKV) is a member of the Flaviviridae family and is considered a major health threat causing cases of microcephaly in newborns and Guillain-Barre syndrome in adults. Here we targeted a transient deep and hydrophobic pocket of the super-open conformation of ZIKV NS2B-NS3 protease to overcome the limitations of the orthosteric inhibitors. After virtual docking screening of approximately 7 million compounds against the novel allosteric site we selected the top seven candidates and tested them in an enzymatic assay. Six out of seven top candidates selected by the docking screen inhibited ZIKV NS2B-NS3 protease proteolytic activity at low micromolar concentrations, as well as suppressing viral replication. These six compounds, targeting the selected protease pocket conserved in ZIKV as well as several other Flaviviruses, have opened an opportunity for a new kind of drug candidate that might be useful to treat several flaviviral infections.

pharmacology and toxicology↗

bioRxiv2021

DUAL FUNCTION OF ZIKA VIRUS NS2B-NS3 PROTEASE

Zika virus (ZIKV) serine protease, indispensable for viral polyprotein processing and replication, is composed of the membrane-anchored NS2B polypeptide and the N-terminal domain of the NS3 polypeptide (NS3pro). The C-terminal domain of the NS3 polypeptide (NS3hel) is necessary for helicase activity and contains an ATP-binding site. We discovered that ZIKV NS2B-NS3pro can bind single-stranded RNA with a Kd of [~]0.3 M, suggesting a novel function. We tested various structural modifications of NS2B-NS3pro and observed that constructs stabilized in the recently discovered "super-open" conformation do not bind RNA. Likewise, stabilization of NS2B-NS3pro in the "closed" (proteolytically active) conformation using substrate inhibitors abolished RNA binding. We posit that RNA binding occurs when ZIKV NS2B-NS3pro adopts the "open" conformation, which we modeled using highly homologous dengue NS2B-NS3pro crystallized in the open conformation. We identified two positively charged fork-like structures present only in the open conformation of NS3pro. These forks are conserved across Flaviviridae family and could be aligned contiguously with the positively charged grove on NS3hel that binds RNA. This led us to propose the "reverse inchworm" model for a tightly intertwined NS2B-NS3 helicase-protease machinery, which suggests that the cycles of NS2B-NS3pro binding and releasing RNA enable the unlimited processivity of NS3hel. The transition to the closed conformation, likely induced by the substrate, enables the classical protease activity of NS2B-NS3pro.

immunology↗