Eidd 1931

All compounds except for ciclesonide and EIDD-1931 were purchased from MedChemExpress (Monmouth Junction, NJ, USA). ciclesonide and EIDD-1931 were purchased from Cayman Chemical (Ann Arbor, MI, USA). The following are the lot numbers and purities of each compound: aprotinin (lot, 62009; purity of ≥98.0%), bromhexine hydrochloride (lot, 15159; purity of 99.39%), niclosamide (lot, 15718; purity of 98.68%), ciclesonide (lot, 0472344-2; purity of ≥98.0%), remdesivir (lot, 46182; purity of 99.78%), EIDD-2801 (lot, 67548; purity of 99.94%), and EIDD-1931 (lot, 0590872-1; purity of ≥95.0%). Stock solution was dissolved in dimethyl sulfoxide (DMSO) at a 10 mM concentration. Anti‐SARS‐CoV‐2 N protein antibody was purchased from Sino Biological Inc. (Beijing, China). Alexa Fluor 488 goat anti‐rabbit IgG (H + L) secondary antibody and Hoechst 33342 were purchased from Molecular Probes. Paraformaldehyde (PFA) (32% aqueous solution) and normal goat serum were purchased from Electron Microscopy Sciences (Hatfield, PA, USA) and Vector Laboratories, Inc. (Burlingame, CA, USA), respectively.

EIDD-1931, EIDD2801, and Nirmatrelvir (PF-073213332) were purchased from MedChemExpress (Shanghai, China). Stock solutions of the compounds were freshly prepared in dimethyl sulfoxide (DMSO). Non-radiolabeled chemicals were obtained from Merck/Sigma-Aldrich (St. Louis, MO, USA). All chemicals were of analytical grade. ³H-Uridine ([5-³H], 20.9 Ci/mmol), and ³H-Adenosine ([2,8-³H], 29.3 Ci/mmol) were purchased from Moravek Biochemicals Inc. (Brea, CA, USA). Ultima Gold XR scintillation fluid was purchased from PerkinElmer (Waltham, MA, USA).

The P-glycoprotein inhibitor CP-100356 (cat. HY-108347) and the DAA, including RDV (cat. HY-104077), NRM (cat. HY-138687) and EIDD-1931 (the active metabolite of MNP; cat. HY-125033), were supplied by MedChemExpress (Monmouth Junction, NJ, USA) as powder and dissolved in 100% dimethyl sulfoxide (DMSO). Biosimilar mAb, including bebtelovimab (BEB; ref. PXTA1750-100), sotrovimab (SOT; ref. PXTA1637-100), tixagevimab (TIX; ref. PXTA1032-100) and cilgavimab (CIL; ref. PXTA1033-100) were supplied by ProteoGenix SAS (Schiltigheim, France) as powder and dissolved in phosphate-buffered saline (PBS).
The cytotoxicity of all investigated compounds was assayed in VERO E6 cells as previously described [22 (link)]. Briefly, cell viability was calculated using the CellTiter Glo 2.0 Luminescent Cell Viability Assay (Promega, Madison, WI, USA), and luminescence values were measured using the GloMax^® Discover Multimode Microplate Reader (Promega, Madison, WI, USA) and elaborated with the GraphPad PRISM software version 9.0 (La Jolla, CA, USA). The half-maximal cytotoxic concentration (CC₅₀) and the compound concentration allowing 90% cell viability (CC₁₀) were calculated using a non-linear regression analysis of the dose–response curves and the ECanything GraphPad function. The CC₁₀ was used for each compound as the starting concentration in the antiviral activity assay.

Active components of remdesivir and molnupiravir (i.e., GS-441524 and EIDD-1931), and nirmatrelvir (PF-07321332) were purchased from MedChemExpress. S-217622 (ensitrelvir) was kindly provided by Shionogi Co., Ltd. Compounds were dissolved in dimethyl sulfoxide (in vitro) or 0.5% methylcellulose (in vivo) prior to use.