Research Article: Solution conformations of Zika NS2B-NS3pro and its inhibition by natural products from edible plants

Date Published: July 10, 2017

Publisher: Public Library of Science

Author(s): Amrita Roy, Liangzhong Lim, Shagun Srivastava, Yimei Lu, Jianxing Song, Eugene A. Permyakov.


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.

Partial Text

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 Forest of Uganda in 1947 [3], and it was later found ZIKV is transmitted to humans by Aedes mosquitoes. However since 2007, large epidemics of Asian genotype ZIKV have been reported around the world [4,5]. Recently 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]. Recent studies have associated ZIKV infection with other diseases: Guillain-Barré syndrome and microcephaly in newborn infants of mothers infected with ZIKV during pregnancy [7,9–11], thrombocytopenia [12], multiple-organ failures [13], and possibly male infertility [14]. Consequently 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 unfortunately there is no available effective vaccine or therapy so far.

Knowledge of catalysis, structures and dynamics of all structural states is beneficial for design of inhibitors with high affinity and specificity towards enzymes including viral proteases [49–53]. This knowledge is particularly relevant to the flaviviral NS2B-NS3pro complexes as it is proposed that their catalytic activities require a transition from the open (inactive) to closed (active) conformation [21,28,29,40]. Interestingly, regardless of being in the open or closed conformations, NS3pro domains of different flaviviral NS2B-NS3pro complexes universally adopt the same chymotrypsin fold. By contrast, while the N-half of NS2B assumes a similar β-strand packed to the NS3pro domain in both open and closed conformations, the C-half of NS2B shows a significant structural diversity in different structures determined so far. In the closed conformation, NS2B structures of flaviviral NS2B-NS3pro complexes show a similar a short β-hairpin formation over the C-half of NS2B and is tightly bound to the NS3pro chymotrypsin fold (Fig 4A). However in the open conformation, the structural properties of the NS2B C-half have been shown to be quite diverse. For the well-studied Dengue-2 NS2B-NS3pro in the open conformation, most NS2B residues are tightly packed with the NS3pro domain as revealed by the crystal structure [27], and evident from its well-dispersed HSQC spectrum (S2C Fig) reconstructed from a previous report [30].




0 0 vote
Article Rating
Notify of
Inline Feedbacks
View all comments