Meeting report: 30th International Conference on Antiviral Research, in Atlanta, GA, USA

Vere Hodge, R Anthony

To identify E inhibitors, phenotypic screens were used to find inhibitors of dengue virus entry and these were then screened for compounds likely to affect this process through interactions with E. For the primary antiviral assay, cells were infected with dengue virus serotype 2, the compound added 1 h after infection and the assay read at three days, to allow inhibitors of every step in the viral life cycle, including viral spread, to be identified. In secondary screens, time-of-addition experiments were performed, including experiments in which compound treatment was restricted to a preincubation of the viral inoculum and the initial 1 h infection period. This enabled identification of three chemical series, 2,4-diaminopyrimidines, 4,6-disubstituted pyrimidines and cyanohydrazones that all appeared to inhibit dengue virus entry via binding to a target present in the viral inoculum. Medicinal chemistry efforts were used to improve the initial lead compounds, leading to inhibitors including 1-100-1 (4,6-disubstituted pyrimidine), 7-148-6, 2-12-2 (2,4-diaminopyrimidine) and 3-110-22 (cyanohydrazone) that had 90% effective concentration (EC 90 ) values in cell culture between 2 and 10 mM. Medicinal chemistry was also used to generate derivatives containing biotin and fluorophores, which were used to demonstrate binding to pre-fusion, E 2, rather than to post-fusion, E 3 . An in vitro fusion assay monitoring pH-triggered fusion of virions to synthetic liposomes was used to demonstrate that all three compound series inhibit E-mediated fusion. To establish where these compounds bind on E 2 , virus was serially passaged in the presence of inhibitors to select for resistance mutations. Up to passage 9, no resistance was detected with three of the compounds but resistance to 7-148-6 was obtained at passage 4, due to a single mutation, E-M196V, located in the detergentbinding pocket. This mutation reduces the affinity of recombinant E for all three series of inhibitors. The mutation T171A located in domain I away from the pocket also confers resistance to compound 3-110-22, but does so without affecting the affinity of the E-3-110-22 interaction. Additional point substitutions in the pocket were chosen from naturally occurring E sequences identified in public databases. These were introduced to the pocket (Q52A, F193L, M272S, L277M and F279S) by site-directed mutagenesis and the impact of each of these substitutions was assessed by measurement of Kd values against each of four compounds (3-110-22, 7-148-6, 2-12-2, GNF-2 and 1-100-1). The compounds were all affected by mutations in the pocket but 'footprints' of inhibitors in the same structural class were more similar to each other than to compounds in the other structural classes. Collectively, these results provide strong evidence that all three inhibitor series bind in pocket between domains I and II.