Figure 1.
HTS of 348K compounds and identification of the hit compound.
A flow diagram of various assays used in the screen. The number of hits remaining after each run is indicated in bold.
Figure 2.
Selected small molecules with potent antiviral activity.
* Percent inhibition at a concentration of 20 µM with the primary screening.
Table 1.
Spectrum of antiviral activity of CID15997213.
Figure 3.
Anti-VEEV activity of CID15997213.
(A) Dose response curve of CID15997213 in the CPE-based anti-VEEV TC-83 assay using Vero 76 cells from a representative experiment. (B) Titer reduction assay results for CID15997213. Vero 76 cells grown in 6-well plates were infected with 0.05 MOI of TC-83 or TrD and then incubated in the presence of CID15997213 at the denoted concentrations. Forty hours later the supernatant was harvested and the titer of the progeny virus was determined. Each point represents the mean from two independent experiments. The horizontal line in the figure indicates the detection limit of the assay. (C, D) Viral RNA and protein analysis. Vero 76 cells were infected with VEEV TC-83 at MOI of 5 and then incubated in the presence of DMSO or CID15997213 for 16 hours. In C, viral RNA was quantified using a quantitative real-time RT-PCR method with the total RNA from the cells. In D, proteins from VEEV TC83-infected cell extracts were analyzed by western blot assay. Closed triangles indicate bands corresponding to actin (internal control) and open arrows indicate bands that are specific to the viral proteins.
Figure 4.
CID15997213 targets viral nsP2.
(A) Time of addition study. Test compound, CID15997213, was added to the designated wells by replenishing the culture media with fresh culture media containing 5 µM of the compound at the time points denoted on the x axis. The graph denotes the virus titers at 16 hours post-infection from various time of addition points. Each data point is the mean from two independent replicates with duplication in titration. (B) Location of the mutations in the CID15997213 resistant viruses. The mutations mapped within the N-terminus of nsP2 protein (pink). There were no missense mutations in either nsP1, nsP3 or nsP4 genes. * Methyl-transferase like domain. (C) Sequence alignment of nsP2 alphaviruses. Amino acid sequences of nsP2 of following alphaviruses were aligned with Clustal W (www.clustal.org): VEEV (L01442.2, GenBank Access No. hereafter), EEEV (NC_003899), WEEV (NC_003908), Fort Morgan virus (FMV, NC_013528), Ross River virus (RRV, NC_001544), Semliki Forest virus (SFV, NC_003215), O'nyong-nyong virus (ONYV, NC_001512.1), CHIKV (L37661.3), Sindbis virus (SINV, NP_740671.1). Y102 position is highlighted in red.
Table 2.
Antiviral activity of CID15997213 with VEEV and VEEV mutants.
Figure 5.
Effect of CID15997213 on survival of VEEV TC-83 infected mice.
Six groups of ten C3H/HeN mice were used to assess antiviral activity in vivo; Group 1-Vehicle control; Group 2-VEEV only; Group 3- VEEV and 2 mg/kg/day CID15997213; Group 4- VEEV and 10 mg/kg/day CID15997213; Group 5- VEEV and 50 mg/kg/day CID15997213; Group 6- VEEV and 200 mg/kg/day CID15997213. Vehicle or CID15997213 were administered by i.p. four hours prior to mock or i.n. infection of VEEV TC-83. Treatments continued for from D0 though D5. Survival of mice in each group in plotted by time. Each group was compared to Group 3 for measurement of the p-value using the Mantel-Cox test. Analyses of each P value generated by the Mantel-Cox test were evaluated by comparison to a Bonferroni corrected threshold of 0.0125 (p = 0.05) to measure any potential significant differences between groups.