Assessment of Dengue virus helicase and methyltransferase as targets for fragment-based drug discovery.
Seasonal and pandemic flaviviruses continue to be leading global health concerns. With the view to help drug discovery against Dengue virus (DENV), a fragment-based experimental approach was applied to identify small molecule ligands targeting two main components of the flavivirus replication complex: the NS3 helicase (Hel) and the NS5 mRNA methyltransferase (MTase) domains. A library of 500 drug-like fragments was first screened by thermal-shift assay (TSA) leading to the identification of 36 and 32 fragment hits binding Hel and MTase from DENV, respectively. In a second stage, we set up a fragment-based X-ray crystallographic screening (FBS-X) in order to provide both validated fragment hits and structural binding information. No fragment hit was confirmed for DENV Hel. In contrast, a total of seven fragments were identified as DENV MTase binders and structures of MTase-fragment hit complexes were solved at resolution at least 2.0Å or better. All fragment hits identified contain either a five- or six-membered aromatic ring or both, and three novel binding sites were located on the MTase. To further characterize the fragment hits identified by TSA and FBS-X, we performed enzymatic assays to assess their inhibition effect on the N7- and 2'-O-MTase enzymatic activities: five of these fragment hits inhibit at least one of the two activities with IC50 ranging from 180μM to 9mM. This work validates the FBS-X strategy for identifying new anti-flaviviral hits targeting MTase, while Hel might not be an amenable target for fragment-based drug discovery (FBDD). This approach proved to be a fast and efficient screening method for FBDD target validation and discovery of starting hits for the development of higher affinity molecules that bind to novel allosteric sites.
Aix-Marseille Université, AFMB UMR 7257, 163 Avenue de Luminy, Case 932, 13288 Marseille Cedex 09, France; CNRS, AFMB UMR 7257, 163 Avenue de Luminy, Case 932, 13288 Marseille Cedex 09, France.
Bruno
B
Coutard
Aix-Marseille Université, AFMB UMR 7257, 163 Avenue de Luminy, Case 932, 13288 Marseille Cedex 09, France; CNRS, AFMB UMR 7257, 163 Avenue de Luminy, Case 932, 13288 Marseille Cedex 09, France.
Etienne
E
Decroly
Aix-Marseille Université, AFMB UMR 7257, 163 Avenue de Luminy, Case 932, 13288 Marseille Cedex 09, France; CNRS, AFMB UMR 7257, 163 Avenue de Luminy, Case 932, 13288 Marseille Cedex 09, France.
Changqing
C
Li
Global Phasing Ltd, Sheraton House, Castle Park, Cambridge CB3 0AX, England, United Kingdom.
Andrew
A
Sharff
Aix-Marseille Université, AFMB UMR 7257, 163 Avenue de Luminy, Case 932, 13288 Marseille Cedex 09, France; CNRS, AFMB UMR 7257, 163 Avenue de Luminy, Case 932, 13288 Marseille Cedex 09, France.
Julien
J
Lescar
Global Phasing Ltd, Sheraton House, Castle Park, Cambridge CB3 0AX, England, United Kingdom.
Gérard
G
Bricogne
Aix-Marseille Université, AFMB UMR 7257, 163 Avenue de Luminy, Case 932, 13288 Marseille Cedex 09, France; CNRS, AFMB UMR 7257, 163 Avenue de Luminy, Case 932, 13288 Marseille Cedex 09, France. Electronic address: karine.barral@afmb.univ-mrs.fr.
Karine
K
Barral
D000998:Antiviral Agents / Q000302:isolation & purification* / Q000494:pharmacology; D018360:Crystallography, X-Ray; D003716:Dengue Virus / Q000187:drug effects / Q000201:enzymology*; D055808:Drug Discovery / Q000379:methods*; D004791:Enzyme Inhibitors / Q000302:isolation & purification* / Q000494:pharmacology; D006801:Humans; D020128:Inhibitory Concentration 50; D008780:Methyltransferases / Q000037:antagonists & inhibitors*; D011485:Protein Binding; D020365:RNA Helicases / Q000037:antagonists & inhibitors*
D000998:Antiviral Agents; D004791:Enzyme Inhibitors; D008780:Methyltransferases; D020365:RNA Helicases
Antiviral screening; Dengue virus; Fragment-based drug discovery; NS3 helicase; NS5 methyltransferase