Abstract
The recent Zika viral (ZIKV) epidemic has been associated with severe neurological pathologies such as neonatal microcephaly and Guillain-Barre syndrome but unfortunately no vaccine or medication is effectively available yet. Zika NS2B-NS3pro is essential for the proteolysis of the viral polyprotein and thereby viral replication. Thus NS2B-NS3pro represents an attractive target for anti-Zika drug discovery/design. Here, we have characterized the solution conformations and catalytic parameters of both linked and unlinked Zika NS2B-NS3pro complexes and found that the unlinked complex manifested well-dispersed NMR spectra. Subsequently with selective isotope-labeling using NMR spectroscopy, we demonstrated that C-terminal residues (R73-K100) of NS2B is highly disordered without any stable tertiary and secondary structures in the Zika NS2B-NS3pro complex in the free state. Upon binding to the well-characterized serine protease inhibitor, bovine pancreatic trypsin inhibitor (BPTI), only the extreme C-terminal residues (L86-K100) remain disordered. Additionally, we have identified five flavonoids and one natural phenol rich in edible plants including fruits and vegetables, which inhibit Zika NS2B-NS3pro in a non-competitive mode, with Ki ranging from 770 nM for Myricetin to 34.02 μM for Apigenin. Molecular docking showed that they all bind to a pocket on the back of the active site and their structure-activity relationship was elucidated. Our study provides valuable insights into the solution conformation of Zika NS2B-NS3pro and further deciphers its susceptibility towards allosteric inhibition by natural products. As these natural product inhibitors fundamentally differ from the currently-known active site inhibitors in terms of both inhibitory mode and chemical scaffold, our finding might open a new avenue for development of better allosteric inhibitors to fight ZIKV infection.
Highlights
Zika virus (ZIKV) was a neglected, mosquito-borne flavivirus because of its assumed small geographical spread and mild clinical symptoms [1] such as fever, headache, rashes and etc [2]
The linked NS2B-NS3pro protein was detected in the pellet of E. coli cells with induction of 1 mM isopropyl β-D-thiogalactopyranoside (IPTG) for four hours at 37 ̊C, while a portion of recombinant protein was found to be in supernatant with induction of 0.2 mM IPTG overnight at 18 ̊C
Our results suggest that in the Zika NS2B-NS3pro complex, NS2B has a portion of residues undergo μs-ms dynamics which made their NMR peaks too broad to be detectable; while the rest of NS2B is highly disordered and lacks tight tertiary packing, which results in a narrowly-dispersed HSQC spectrum (S2B Fig)
Summary
Zika virus (ZIKV) was a neglected, mosquito-borne flavivirus because of its assumed small geographical spread and mild clinical symptoms [1] such as fever, headache, rashes and etc [2]. The first biological ZIKV sample was isolated from a sentinel rhesus monkey in the Zika. It is estimated that one-third of the world population might be at risk of infection [6]. The rapid rise in ZIKA infection is compounded by the ease of vertical [7] and sexual human-to-human transmissions [8]. WHO has declared a public health emergency for ZIKV infection [15]. ZIKV represents a significant challenge to the public health of the whole world but there is no available effective vaccine or therapy so far
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