Small-molecule screening using a whole-cell viral replication reporter gene assay identifies 2-{[2-(benzoylamino)benzoyl]amino}-benzoic acid as a novel antiadenoviral compound

Abstract

Adenovirus infections are widespread in society and are occasionally associated with severe, but rarely with life-threatening, disease in otherwise healthy individuals. In contrast, adenovirus infections present a real threat to immunocompromised individuals and can result in disseminated and fatal disease. The number of patients undergoing immunosuppressive therapy for solid organ or hematopoietic stem cell transplantation is steadily increasing, as is the number of AIDS patients, and this makes the problem of adenovirus infections even more urgent to solve. There is no formally approved treatment of adenovirus infections today, and existing antiviral agents evaluated for their antiadenoviral effect give inconsistent results. We have developed a whole cell-based assay for high-throughput screening of potential antiadenoviral compounds. The assay is unique in that it is based on a replication-competent adenovirus type 11p green fluorescent protein (GFP)-expressing vector (RCAd11pGFP). This allows measurement of fluorescence changes as a direct result of RCAd11pGFP genome expression. Using this assay, we have screened 9,800 commercially available small organic compounds. Initially, we observed approximately 400 compounds that inhibited adenovirus expression in vitro by > or = 80%, but only 24 were later confirmed as dose-dependent inhibitors of adenovirus. One compound in particular, 2-{[2-(benzoylamino)benzoyl]amino}-benzoic acid, turned out to be a potent inhibitor of adenovirus replication.

FIG. 1.

(a) Dose response for A02 inhibition of GFP expression from the RCAd11pGFP vector in K562 cells. The fluorescence intensity was measured after 24 h of incubation with compound A02 and vector. (b) Inhibition of GFP expression from the RCAd11pGFP vector in A549 cells. The fluorescence intensity assayed by FACS analysis after 24 h of incubation with compound A02 and vector.

FIG. 2.

Chemical structures of compound A02 and its analogues separated from the purchased sample. A02, 2-{[2-(benzoylamino)benzoyl]amino}-benzoic acid; A01, 2-(benzoylamino)-benzoic acid; A03, 2-{[2-[[2-(benzoylamino)benzoyl]amino]benzoyl]amino}-benzoic acid.

FIG. 3.

Effect of A02, A01, and A03 on Ad replication. The separated compounds were tested for inhibitory effect on Ad replication by QPCR. Ad5 was allowed to infect A549 cells with or without compound. After 24 h of incubation DNA was prepared from cells and virus and analyzed by QPCR. As an internal control, the cellular gene RNase P was included in the assay. All values are normalized to RNase P. Error bars represent the standard deviation of the means from three independent experiments run in duplicates. The statistical significance was determined by unpaired t test, and a P value of < 0.05 was considered significant. Statistical analyses were performed by using GraphPad Prism.

FIG. 4.

Titration of the effect of A02 on Ad5, Ad11p, and the toxic effect in A549 cells after 24 h of incubation with virus and/or compound A02. The EC₅₀s for Ad5 and Ad11p were 3.7 and 2.9 μM, respectively. The CC₅₀ for compound A02 in A549 cells was 199 μM. The EC₅₀ is the concentration at which the Ad replication is inhibited by 50% as determined by QPCR, and CC₅₀ is the concentration at which the cytotoxicity is 50%, i.e., 50% of the cells are viable, as determined by the XTT assay. Error bars represent the standard deviation of the means from three independent duplicate experiments.

FIG. 5.

(a) Binding of ³⁵S-labeled Ad5 or Ad11p to A549 cells in the presence of A02. Error bars represent the standard deviation of the means from three independent duplicate experiments. (b) Flow cytometry assay detecting Ad hexon protein after 24 h of incubation with virus and compound A02. Error bars represent the standard deviation of the means from two independent duplicate experiments. (c) Flow cytometry assay detecting dead cells where propidium iodide has intercalated the DNA. FACS analysis was performed after 24 h of incubation with a 15 μM concentration of compound A02. K562 cells were used mainly in the screening assay; A549 cells were used for most verification assays. Error bars represent the standard deviation of the means from two independent duplicate experiments.