Figure 2.

Incorporation of GS-646939 under competitive conditions by HRV-16 and EV-71 RdRp. A, RNA primer/template (top) supporting a single incorporation of ATP (“i₁”) or GS-646939 (“i₂”) at position 6. G indicates incorporation of [α-³²P]-GTP at position 5. Migration pattern of RNA synthesis products catalyzed by HRV-16 (middle) and EV-71 RdRp (bottom). Product formation resulting in AMP- or GS-646939-terminated primers was compared across increasing GS-646939 concentrations at ATP concentrations of 10, 100, and 1000 μM. A 5′-³²P-labeled 4-nt primer serves as a size marker. B, graphical representation of AMP-terminated primers (%) at increasing GS-646939 concentrations as shown in (A). Independent 8-data point experiments were performed at least three times (n = 3) and error bars represent standard error associated with the fit. C, the MC₅₀ value is defined as the concentration at which GS-646939 matches ATP for incorporation at position 6. The competition index (CI) is the ratio of the MC₅₀ value to the ATP concentration present in the reaction. D, CI values determined for HRV-16 and EV-71 RdRp and the CI average (CI_avg) and standard deviation (±) across all ATP concentrations. RdRp, RNA-dependent RNA polymerase; HRV, human rhinovirus; EV-71, enterovirus 71; MC₅₀, matching concentration.