Inhibition of human cytomegalovirus signaling and replication by the immunosuppressant FK778

FK778 (Fujisawa Healthcare Inc.) is an immunosuppressant structurally similar to A771726, the active metabolite of leflunomide (Aventis Pharmaceuticals), but with a clinically relevant shorter serum half-life. Leflunomide, a tolerated and efficacious immunosuppressive agent in patients receiving allograft transplantations, was reported to be active against HCMV and HSV-1. Here we report that FK778 is a potent and effective inhibitor of HCMV, and that its mode of antiviral action appears to mirror the biochemical mechanisms elsewhere described to be responsible for its immunosuppressive properties: inhibition of protein tyrosine phosphorylation and inhibition of cellular de novo pyrimidine biosynthesis. Initial HCMV-mediated activation of the EGF receptor/phosphatidylinositol 3-kinase (PI3-K) pathways and Sp1 and NF-κB were partially inhibited by FK778. The second tier (phase) of PI3-K, Sp1, and NF-κB induction by HCMV was more sensitive to FK778. Treatment of HCMV-infected cells with FK778 prevented the appearance of HCMV proteins some 12-24 h post infection, and inhibited viral DNA synthesis. In our assays, leflunomide also reduced HCMV DNA levels. The antiviral activity of FK778 was reversed in cell culture by treatment with uridine, consistent with specific inhibition of dihydroorotate dehydrogenase (DHODH), a required enzyme in the de novo biosynthesis of pyrimidines. This report substantiates the clinical possibility of a single drug treatment to achieve immunosuppression and inhibit opportunistic herpesvirus infections. Our results differ from descriptions of leflunomide acting as an inhibitor of HCMV cytoplasmic capsid formation. Additionally, this study indicates that DHODH may be an effective cellular antiviral target.