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[Preprint]. 2024 Apr 29:2024.04.27.591279.
doi: 10.1101/2024.04.27.591279.

High-throughput crystallographic fragment screening of Zika virus NS3 Helicase

Andre S Godoy  1   2 , Nathalya C M R Mesquita  1 , Gabriela Dias Noske  1 , Victor Oliveira Gawriljuk  1 , Ryan M Lithgo  3   4   2 , Blake H Balcomb  3   4   2 , Jasmin Cara Aschenbrenner  3   4   2 , Charles W E Tomlinson  3   4   2 , Max Winokan  3   4   2 , Jenke Scheen  5   2 , Peter George Marples  3   4   2 , Anu V Chandran  3   4   2 , Xiaomin Ni  3   4   2 , Warren Thompson  3   4   2 , Michael Fairhead  6   2 , Daren Fearon  3   4   2 , Lizbé Koekemoer  6   2 , Mary-Ann Elvina Xavier  3   4   6   2 , Martin Walsh  3   4   2 , Glaucius Oliva  1 , Frank von Delft  3   4   6   2
Affiliations

High-throughput crystallographic fragment screening of Zika virus NS3 Helicase

Andre S Godoy et al. bioRxiv. .

Abstract

The Zika virus (ZIKV), discovered in Africa in 1947, swiftly spread across continents, causing significant concern due to its recent association with microcephaly in newborns and Guillain-Barré syndrome in adults. Despite a decrease in prevalence, the potential for a resurgence remains, necessitating urgent therapeutic interventions. Like other flaviviruses, ZIKV presents promising drug targets within its replication machinery, notably the NS3 helicase (NS3Hel) protein, which plays critical roles in viral replication. However, a lack of structural information impedes the development of specific inhibitors targeting NS3Hel. Here we applied high-throughput crystallographic fragment screening on ZIKV NS3Hel, which yielded structures that reveal 3D binding poses of 46 fragments at multiple sites of the protein, including 11 unique fragments in the RNA-cleft site. These fragment structures provide templates for direct design of hit compounds and should thus assist the development of novel direct-acting antivirals against ZIKV and related flaviviruses, thus opening a promising avenue for combating future outbreaks.

Keywords: NS3; Zika virus; flavivirus; fragment-screening; helicase.

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Figures

Fig. 1.
Fig. 1.. Fragment screening reveal multiple binding sites.
(a) Surface view of ZIKV NS3Hel fragment screening results in two orientations. Surface is colored in grey, and fragments are depicted as sticks with yellow carbons. Selected cluster of fragments are marked with orange (1), blue (2) and green (3) boxes. (b) Overlaid structures of 111 PBDs containing superfamily 2 helicases from Flaviviridae family. Small molecules are depicted as sticks.
Fig. 2.
Fig. 2.. Overview of RNA cleft fragments obtained.
(a) Surface view of the ZIKV NS3Hel (depicted in grey) reveals distinct features: the NTP and RNA clefts are highlighted with green and brown boxes, respectively. Additionally, the RNA and phosphate surfaces are depicted in orange to accentuate the RNA binding cleft. (b) Detailed view of ZIKV Helicase from PDB 5GJB in complex with RNA. RNA is depicted as sticks. (c) ZIKV Helicase in complex with multiple fragments obtained for the RNA-cleft binding site. Fragments are depicted as sticks. Bellow, detailed view of structures with electron densities and structural details of fragments Z235341991 (d), Z1262246195 (e), Z44584886 (f), Z18618496 (g), Z2678251369 (h), POB0008 (i), Z3241250482 (j), POB0128 (k), Z56772132 (l) and Z729726784 (m). Fragments are show as sticks with yellow carbons, while protein interacting residues are depicted as grey sticks carbon. 2Fo-Fc map is depicted as blue mesh, showed here with σ = 1.0.
Fig. 3.
Fig. 3.. Details of covalent and ATP-binding fragments.
(a) Structure of ZIKV NS3Hel in complex with Z203039992. C262 covalently bound is show is depicted with gray carbon, while native apo C262 from PDB 6MH3 is showed in purple (b) Structure of ZIKV NS3Hel in complex with Z751811134 in two overlapping forms. Fragments are shown as sticks with yellow carbons, while protein interacting residues are depicted as gray sticks carbon. Cartoon is colored according to conservancy score calculated with Consurf, where purple indicated more conserved regions and white the less conserved regions. 2Fo-Fc map is depicted as blue mesh, showed here with σ = 1.0. (c) Flaviviridae sequence alignment showing selected regions of helicase that are discussed above. Sequence alignment was generated with ESPript and colored with default parameters.
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