We first evaluated the antiviral activity of TQ against SARS-CoV-2-Australia/VIC01/2020 [a gift from the Doherty Institute, Melbourne, Australia] in a cytopathic effect [CPE] assay. Briefly, SARS-CoV-2-infected VERO E6 cell monolayers [at multiplicity of infection [moi] of 0.05] in 96-well plates were incubated at 37°C in 5% CO2 while exposed to differing concentrations of TQ, remdesivir [RD] and HCQ in triplicate wells in minimum essential medium supplemented with 1% (w/v) L-glutamine, 2% fetal bovine serum and 0.2% DMSO [the latter for compound dissolution]. [The same medium was used for other viral assays described later]. After 4 days, cell viability was assessed via MTT and the EC50 and CC50 calculated using the method of Pauwels et al [15] . TQ, HCQ, and RD exhibited EC50s of 15.6, > 100, and 0.77 µM respectively, and TQ exhibited greater selectivity than HCQ [selectivity index of 2.4 versus < 1.0, respectively, see Table 1 ]. An EC50 could not be established for HCQ as the CC50 was lower than the highest concentration tested [see Table 1 ]. Thus, the validity of the test system used was confirmed by virtue of the observation of a sub-micromolar EC50 for RD, and TQ was found to exhibit more potent antiviral activity than HCQ.
TQ's ability to interfere with infectious virus replication and reduce the yield of progeny virus was assessed using a yield reduction [YR] assay. Briefly, SARS-CoV-2-infected VERO E6 cells [moi 0.05] in 24-well plates were incubated at 37°C in 5% CO2 while exposed to differing concentrations of TQ and HCQ. After 48h, supernatant was harvested and added to 96-well plates in triplicate, incubated for three days at 37°C in a humidified 5% CO2 atmosphere, after which virus-induced CPE scored visually by two independent operators. The TCID50 of the virus suspension was determined using the method of Reed-Muench [17] . EC50s, calculated using the method Pauwels et al [15] were 2.6 and 10.4 µM for TQ and HCQ respectively [ Table 1 ]. EC90s, estimated by multiplying the EC50 by 1.98 [19] , were 5.1 and 21 µM respectively [ Table 1 ] We note that the EC50 was reduced to 2.6 µM from 15.6 µM when virus is exposed to TQ for 48 v 96h, suggesting that the anti-viral effect of TQ is dependent on viral load.
A time-of-addition [TOA] assay was used to identify when TQ interferes with the viral replication cycle and to investigate the mechanism of action. A compound that acts early in the replication cycle should reduce the virus titers at early time points; conversely, a compound that acts later in the replication cycle should reduce the virus titers at later time points. Antiviral compounds that act early in the cycle may be interfering with the attachment of the virus into the host. Compounds that act in the middle of the cycle may be affecting one or more of the many processes involved in viral replication. Compounds that act late in the cycle may be affecting processes associated with viral release. The TOA experiment was conducted using a variation on the method of Schneider et al [19] .
Briefly, the virus was allowed to adsorb to cell monolayers [moi of 1] in 24-well plates for 1h, then parallel cultures were incubated at 37°C in 5% CO2 for 15, 30, 60 minutes, 2, 4, 6 hours prior to adding 15µM HCQ, 15µM TQ and 5µM RD, or negative control (assay media only). Concentrations were chosen to be approximately 5-fold higher than the EC50 in the 48h YR assay, except that for HCQ this was not possible due to cytotoxicity at concentrations ≥ 50 µM, so TQ and HCQ were compared head-to-head at the same concentration [15 µM ]. For the 0-minute time point, test or control article were added immediately following virus pre-adsorption. Plates were incubated at 37°C in 5% Yeo et al, [18] . A pH of 6 was selected, since slightly acidic pH is anticipated for COVID-19 patients.
The input assumptions for various parameters are described in Table 2 The urgency of identifying therapeutic options for COVID-19 patients, and the absence of widely available animal models, has meant that repositioning programs have embraced candidate prioritization through comparison of SARS-CoV-2 in vitro susceptibility in VERO E6 cells to anticipated plasma or unbound lung concentrations of proposed or approved clinical dosing regimens [3, 18, 23] . It is apparent that the lack/modest level of activity of HCQ in both animal models and clinical studies [5, 14] was not predicted from VERO cells and evidence of extensive lung penetration of HCQ. There is much speculation about the mechanistic basis and generalizability of this incongruence, and a growing consensus on the need to move towards including additional in vitro cell systems evaluations when evaluating COVID-19 candidates, such as lung epithelial cells and organoids that may more closely represent the in vivo situation. However, in the absence of international guidance and harmonization on appropriate in vitro cell systems to evaluate antiviral activity of SARS-CoV-2, VERO cells will continue to be the standard.
We have demonstrated antiviral activity of TQ against SARS-CoV-2 in in vitro VERO E6 cell system at concentrations deemed to be pharmacologically relevant and achievable in lung tissue via PBPK modelling at doses of TQ which have been approved. Furthermore, we have noted indirect evidence that is suggestive of a differentiated mechanism of action of this 8AQ against SARS-CoV-2. Based on these data, we believe that there is pharmacological plausibility and proof of hope that TQ may have potential to be effective in the treatment pathway for COVID-19, and would encourage further evaluation. 