Compound ID IC50 (µM) Docking Results Mg2+ preincubation No Mg2+ preincubation % solutions in preferred cluster C-Score Chemgauss 3 Number of Hydrophobic Interactions Number of Hydrophilic Interactions Amiloride 139 ± 18 20 ± 1 100 7.13 -57.04 4 7 DMA 424 ± 31 82 ± 29 82 7.12 -53.55 6 7 EIPA 79 ± 10 11 ± 2 50 7.79 -59.66 7 5 HMA >500a 136 ± 22 70 6.96 -61.22 9 3 Benzamil 255 ± 20 52 ± 11 55 7.8 -62.41 8 5 DCB 45 ± 1 15 ± 3 77 7.53 -58.98 11 7 HMB >75c 22 ± 4 70 7.59 -58.48 10 6 NMB inactiveb ND NA NA NA NA NA CHG inactiveb ND NA NA NA NA NA CHMG inactiveb ND NA NA NA NA NA CMG inactiveb ND NA NA NA NA NA aLess than 50% inhibition at 500 μM bNo effect up to 500 μM concentration. cLess than 50% inhibition at highest possible concentration in solution (75 μM) ND–not determined NA–not applicable, i.e. no consistent binding poses were observed The compounds were pre-incubated with 3Dpol and RNA primer/template at different concentrations (0 to 500 μM), either in the presence (left column) or absence (right column) of Mg2+, and then reactions were started by addition of ATP or ATP and Mg2+, respectively. The IC50 values are mean ± standard error of the mean for 2-4 independent experiments and were determined as described in Materials and Methods. C-score is the consensus scoring function within Surflex (higher scores denote better docking). Chemgauss3 is the atypical scoring function within FRED and is based upon energy (lower values denote better docking).

Table 1: Inhibitory potencies of amiloride analogues in an assay of enzymatic activity of CVB3 3Dpol and their scoring functions in Surflex-Dock and FRED algorithms.