Damfo, Shymaa Abdullah M; (2022) Toward the Discovery of Inhibitors Targeting Dengue and Zika virus Methyltransferase Using Fragment-based Screening. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

BACKGROUND: The Zika and dengue viruses, members of the family Flaviviridae, present threats to global health, yet no effective therapy is currently available against these human pathogens. The Dengvaxia vaccine has recently become available to prevent dengue, but its application is limited to certain age groups and depends on the recipients’ pre-vaccination infection status. The highly conserved non-structural protein 5 (NS5), encompassing the RNA-dependent RNA polymerase and the N7 and 2’-O methyltransferase (MTase) domains, offers a potential antiviral target. AIM: This study aimed to identify and develop compounds targeting the MTase domain of NS5 through fragment-based drug discovery. METHODOLOGY: To identify fragment hits as starting points, a fragment library was screened against dengue virus serotype 3 (DENV3) MTase using X-ray crystallography. This project presents the results of the fragment-based screening, including the optimisation of crystallisation conditions to yield the high-quality crystals required for screening, diffraction data collection, structure determination and refinement. The crystallographic fragment screening was followed by orthogonal biophysical assays, such as microscale thermophoresis and thermal shift assays, to identify novel ligand binding pockets on the DENV3 MTase. Finally, the structureactivity relationship by catalogue methodology was employed in an effort to improve the affinity of the identified hits. RESULTS AND CONCLUSION: Almost 600 crystals of DENV3 MTase with an average resolution of 2.5 Å were produced for fragment-based screening. The primary crystallographic screening identified 20 fragment hits that showed clear electron densities. These fragments bind to 10 binding sites in total, eight of which are unpublished and two of which were previously discovered. A structure-activity relationship by catalogue study with 66 analogues improved the affinity of six of the fragment hits. Further investigation and optimisation of these analogues will significantly expand our options for developing small molecules with favourable druglike properties targeting the DENV3 MTase domain.

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