Fig. 6: Molecular basis of cordycepin and ADEP-42 activity.

A Pretreatment assay comparing cordycepin to remdesivir with assay timeline given in schematic. Compounds were added 24 h before infection and removed at time of infection. Media were harvested 24 hpi and extracellular RNA release was then analyzed. Mean ± SD are shown from n = 3 independent experiments, each performed in quadruplicates, ****p < 0.0001. B Dose-response of cordycepin and cordycepin-monophosphate (cordycepin-MP) in 229E infected Huh-7 cells (MOI 1) after 24 hpi. Extracellular viral RNA released was measured by RT-qPCR and normalized to DMSO control. Mean ± SEM shown are generated from n = 3 independent experiments, each performed in triplicates. C Western blot analysis of AMPK, mTOR, and Akt in cordycepin treated SARS-CoV-2 infected samples compared to DMSO control. Huh-7 cells were infected with SARS-CoV-2 at MOI 2, treated with DMSO or 30 µM cordycepin after 1 hour, and samples were harvested 48 hpi. Mock-infected samples were treated with DMSO. Lysates were stained with AMPKα, phospho-AMPKα (Thr172), mTOR, phospho-mTOR (Ser2448), Akt, phospho-Akt (Ser473), and GAPDH antibodies. MW markers are given on the right in kDa. D RT-qPCR quantification of extracellular 229E N RNA levels after infection (MOI 1) and treatment with various concentrations of metformin for 24 hours in Huh-7 cells. Mean ± SD shown from 4 replicates. E Representative western blot of wild type (WT) and CLPP knockout (CLPP^KO) cells generated by CRISPR/Cas9. Lysates were stained with ClpP and GAPDH antibodies. MW markers are given on the right in kDa. WT and CLPP^KO Huh-7 cells were infected at MOI 2 with F 229E, G OC43 or H SARS-CoV-2. After 1 hour adsorption, cells were treated with DMSO (blue), or 10 µM ADEP-42 (red). Media samples were harvested at 24, 72 or 48 hpi for (F), (G) and (H), respectively, and analyzed by RT-qPCR.