title: A Sensitive and Robust High-Throughput Screening Assay for Inhibitors of the Chikungunya Virus nsP1 Capping Enzyme.
abstract: Chikungunya virus (CHIKV) is a mosquito-borne Alphavirus that causes severe and debilitating disease symptoms. Alarmingly, transmission rates of CHIKV have increased dramatically over the last decade resulting in 1.7 million suspected cases in the Western hemisphere alone. There are currently no antivirals for treatment of CHIKV infection and novel anti-alphaviral compounds are badly needed. nsP1 is the alphavirus protein responsible for the methyltransferase and guanylyltransferase activities necessary for formation of the 5' type 0 cap structure added to newly formed viral RNA. Formation of this cap depends on nsP1 binding GTP and transferring a methylated GMP to nascent viral RNA. We have developed a fluorescence polarization-based assay that monitors displacement of a fluorescently-labeled GTP analog in real time. Determining the relative affinities of 15 GTP analogs for nsP1 GTP revealed important structural aspects of GTP that will inform identification of inhibitors able to outcompete GTP for the nsP1 binding site. Validation of the assay for HTS was completed and a secondary orthogonal assay that measures guanylation activity was developed in order to evaluate hits from future drug screens. This platform provides an avenue for identification of potent nsP1 inhibitors, which would potentially provide compounds capable of treating disease caused by CHIKV infection.
authors.affiliation: Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, Colorado, United States of America.
authors.forename: Kristen M
authors.initials: KM
authors.lastname: Bullard-Feibelman
authors.affiliation: Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, Colorado, United States of America.
authors.forename: Benjamin P
authors.initials: BP
authors.lastname: Fuller
authors.affiliation: Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, Colorado, United States of America.|Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, Colorado, United States of America.|School of Biomedical Engineering, Colorado State University, Fort Collins, Colorado, United States of America.
authors.forename: Brian J
authors.initials: BJ
authors.lastname: Geiss
mesh_terms: D000998:Antiviral Agents / Q000737:chemistry / Q000494:pharmacology; D065632:Chikungunya Fever / Q000188:drug therapy / Q000821:virology*; D002646:Chikungunya virus / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000494:pharmacology*; D006160:Guanosine Triphosphate / Q000378:metabolism; D057166:High-Throughput Screening Assays / Q000379:methods; D006801:Humans; D008780:Methyltransferases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D009713:Nucleotidyltransferases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D012315:RNA Caps / Q000378:metabolism
chemical_list: D000998:Antiviral Agents; D004791:Enzyme Inhibitors; D012315:RNA Caps; D006160:Guanosine Triphosphate; D008780:Methyltransferases; D009713:Nucleotidyltransferases; C020842:guanylyltransferase