RFP Labelled Human Respiratory Syncytial Virus

Human respiratory syncytial virus (RSV) is a negative-sense RNA virus that is an important cause of respiratory tract infections in infants and the elderly. Lumitabine (ALS-8176) is an antiviral drug developed for the treatment of RSV and hMPV. It is an RNA polymerase inhibitor. The parent molecule of ALS-8176, ALS-8112, undergoes intracellular phosphorylation to produce an active 5'-triphosphate metabolite. Here, researchers have identified the activation pathway of ALS-8112 and show that it is distinct from other antiviral cytidine analogs. The first step is driven by deoxycytidine kinase (dCK) with great efficiency, while the second step limits the formation of the active 5'-triphosphate species. ALS-8112 is a 2'- and 4'-modified nucleoside analog, which prompted us to investigate the recognition of other 2'- and 4'-modified nucleosides by dCK. These results highlight the great potential of optimizing nucleoside analogs based on the second activation step and draw more attention to nucleoside diphosphate and triphosphate prodrugs in drug discovery.

Here, the researchers analyzed the effects of dCK inhibitors on RSV replication in the presence of ALS-8112 at a nontoxic concentration of 10 μM. RSV-RFP (red fluorescent protein)-infected HEp-2 cells were treated with DMSO, ALS-8112 alone, ALS-8112 + inhibitor 1, or ALS-8112 + inhibitor 2. Fluorescence micrographs of infected cells (Figure 1A) showed an 86% reduction in red fluorescence in ALS-8112-treated cells compared to drug-naive RSV-RFP-infected cells, indicating potent antiviral activity. Administration of ALS-8112 with dCK inhibitors 1 or 2 showed 123% and 90% rescue of RSV-RFP infection, respectively (Figure 1B). Collectively, these biochemical and cellular results indicate that dCK inhibitors antagonize the phosphorylation and antiviral activity of ALS-8112, confirming the essential role of dCK as the major kinase involved in the first step of ALS-8112 phosphorylation.

Figure 1. dCK inhibitors 1 and 2 rescue RSV-RFP infected HEp-2 cells. (Jordan P C, et al. 2017)