The hCoV 229E antiviral potency is more than 50-fold greater than that for SARS CoV-1 in the Vero 76 line, even though 4 possesses nearly equivalent 3CLpro biochemical potency for the hCoV 229E and CoV-1 viruses. While undefined differences between hCoV 229E and SARS CoV-1 could account for these large differences in antiviral EC50/enzyme IC50 ratios, different active transporter expression levels between MRC-5 and Vero 76 cell lines may also be a contributing factor. Consistent with the previously stated goal to characterize the impact of efflux on potency measured in the SARS CoV-1 antiviral assay, we co-dosed a known P-glycoprotein transport inhibitor at a fixed concentration (0.5 μM CP-100356)48 in a full dose–response of inhibitor 4. This resulted in a pronounced potency shift of SARS CoV-1 antiviral activity (EC50 = 0.11 μM) that now more closely matched the antiviral potency seen for hCoV 229E. As a control, the same co-dosing of efflux inhibitor led to no such shift in the hCoV 229E antiviral potency of 4, demonstrating that the human lung-derived MRC-5 cells show less P-glycoprotein-based transporter under similar assay conditions. These results suggest that the high antiviral EC50/enzyme IC50 ratio observed in Vero 76 cells is an artifact of the high efflux potential of that assay cell line and may underestimate the antiviral potency in human lung cells, the relevant tissue for SARS and COVID-19. Assays to directly measure the antiviral potency of 4 against SARS CoV-2 in clinically relevant human lung cells are currently under investigation.