title: Stabilization of dengue virus polymerase in de novo initiation assay provides advantages for compound screening.
abstract: Dengue virus (DENV) NS5 protein comprises an N-terminal methyltransferase domain and a C-terminal RNA-dependent RNA polymerase domain (RdRp). DENV RdRp is responsible for viral RNA synthesis via a de novo initiation mechanism and represents an attractive target for anti-viral therapy. Herein we describe the characterization of its de novo initiation activities by PAGE analyses and the knowledge gained was used to develop a fluorescent-based assay. A highly processive and robust assay was achieved by addition of cysteine in the assay buffer. This stabilized the apo-enzyme, and rendered optimal de novo initiation activity while balancing its intrinsic terminal transferase activity. Steady-state kinetic parameters of the NTP and RNA substrates under these optimal conditions were determined for DENV1-4 FL NS5. Heavy metal ions such as Zn(++) and Co(++) as well as high levels of monovalent salts, suppressed DENV polymerase de novo initiation activities. This assay was validated with nucleotide chain terminators and used to screen two diverse small library sets. The screen data obtained was further compared with concurrent screens performed with a DENV polymerase elongation fluorescent assay utilizing pre-complexed enzyme-RNA. A higher hit-rate was obtained for the de novo initiation assay compared to the elongation assay (∼2% versus ∼0.1%). All the hits from the latter assay are also identified in the de novo initiation assay, indicating that the de novo initiation assay performed with the stabilized apo-enzyme has the advantage of providing additional chemical starting entities for inhibiting this enzyme.
authors.affiliation: Novartis Institute for Tropical Diseases, Singapore; Maveta Company Limited, 26/522 Paholyothin 62/1 S, Saimai, Bangkok 10220, Thailand(1).
authors.forename: Pornwaratt
authors.initials: P
authors.lastname: Niyomrattanakit
authors.affiliation: Novartis Institute for Tropical Diseases, Singapore. Electronic address: kah_fei.wan@novartis.com.
authors.forename: Kah Fei
authors.initials: KF
authors.lastname: Wan
authors.affiliation: Novartis Institute for Tropical Diseases, Singapore; Division of Structural Biology and Biochemistry, School of Biological Sciences, Nanyang Technological University, Singapore.
authors.forename: Ka Yan
authors.initials: KY
authors.lastname: Chung
authors.affiliation: Novartis Institute for Tropical Diseases, Singapore.
authors.forename: Siti Nurdiana
authors.initials: SN
authors.lastname: Abas
authors.affiliation: Novartis Institute for Tropical Diseases, Singapore.
authors.forename: Cheah Chen
authors.initials: CC
authors.lastname: Seh
authors.affiliation: Novartis Institute for Tropical Diseases, Singapore.
authors.forename: Hongping
authors.initials: H
authors.lastname: Dong
authors.affiliation: Novartis Institute for Tropical Diseases, Singapore.
authors.forename: Chin Chin
authors.initials: CC
authors.lastname: Lim
authors.affiliation: Novartis Institute for Tropical Diseases, Singapore.
authors.forename: Alexander Theodore
authors.initials: AT
authors.lastname: Chao
authors.affiliation: Novartis Institute for Tropical Diseases, Singapore.
authors.forename: Chang Bok
authors.initials: CB
authors.lastname: Lee
authors.affiliation: Novartis Institute for Tropical Diseases, Singapore.
authors.forename: Shahul
authors.initials: S
authors.lastname: Nilar
authors.affiliation: Division of Structural Biology and Biochemistry, School of Biological Sciences, Nanyang Technological University, Singapore; INSERM UMRS 945 "Immunité et Infection", Centre Hospitalier Universitaire Pitié-Salpêtrière, Faculté de Médecine et Université Pierre et Marie Curie, 91 Bd de l'Hôpital, 75013 Paris, France.
authors.forename: Julien
authors.initials: J
authors.lastname: Lescar
authors.affiliation: Novartis Institute for Tropical Diseases, Singapore.
authors.forename: Pei-Yong
authors.initials: PY
authors.lastname: Shi
authors.affiliation: Novartis Institute for Tropical Diseases, Singapore.
authors.forename: David
authors.initials: D
authors.lastname: Beer
authors.affiliation: Novartis Institute for Tropical Diseases, Singapore. Electronic address: siew_pheng.lim@novartis.com.
authors.forename: Siew Pheng
authors.initials: SP
authors.lastname: Lim
mesh_terms: D000998:Antiviral Agents / Q000494:pharmacology*; D001051:Apoenzymes / Q000378:metabolism; D003545:Cysteine / Q000378:metabolism; D003716:Dengue Virus / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004795:Enzyme Stability; D006801:Humans; D007700:Kinetics; D008826:Microbial Sensitivity Tests; D012367:RNA, Viral / Q000235:genetics; D012324:RNA-Dependent RNA Polymerase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D014158:Transcription, Genetic; D017361:Viral Nonstructural Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D000998:Antiviral Agents; D001051:Apoenzymes; D004791:Enzyme Inhibitors; C582223:NS5 protein, dengue virus; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; D012324:RNA-Dependent RNA Polymerase; D003545:Cysteine
keywords: Activity; Assay; Compounds; De novo initiation; Dengue virus NS5 protein; Elongation; Flavivirus; Inhibition; Kinetics; RNA dependent RNA polymerase