A viral RNA-dependent RNA polymerase inhibitor VV116 broadly inhibits human coronaviruses and has synergistic potency with 3CLpro inhibitor nirmatrelvir

Fig. 1. VV116 and its parent nucleoside X1 broadly inhibited human coronavirus. a The chemical structures of VV116 and X1. VV116 is efficiently metabolized to X1 after oral uptake, and then X1 is metabolized to active triphosphate X1-NTP in the cells. b–k The activity of VV116 in inhibiting SARS-CoV-2 variants (Delta, Omicron BA.1, and Omicron BA.5), HCoV-OC43, and HCoV-229E. The curves were fitted with a nonlinear regression model. VV116, X1, GS-441524, remdesivir (RDV), and NHC were compared head-to-head in Vero E6, RD, and Huh-7 cells (b, d, f, h, j), and then the antiviral activity of VV116, X1, and GS-441524 against the SARS-CoV-2 variants (Delta, Omicron BA.1, and Omicron BA.5), HCoV-OC43, and HcoV-229E were validated and compared in HEK293T-hACE2-TMPRSS2, Huh-7, and MRC-5 cells, respectively. Error bars denote mean ± sd of 3–6 independent replicates (c, e, g, I, and k). l The half-maximum effective concentration (EC50) values of compounds against HCoVs. The EC50 values were calculated using nonlinear regression in Prism version 7.00 (GraphPad software). The bar indicates the standard deviation (SD) from 3–6 independent experiments

Fig. 2. In vitro quantification of the antiviral activity of combinations of two drugs to screen high potency drug combinations against HCoV-OC43. a–d The instantaneous inhibitory potential (IIP) of combinations of nucleoside analogs and proteinase inhibitors. The concentrations of the drug combinations were normalized by IC50 values. D drug concentration. e–h Experimental IIP values (IIPcom) and corresponding theoretical IIP values predicted by Bliss independence (IIPBcom) of double drug combinations at 4 × IC50. i–l IIPcom and IIPBcom values of double drug combinations at 2 × IC50. The columns represent one of three independent experiments. m and n The landscapes for the interaction of VV116 and nirmatrelvir against HCoV-OC43 (m) and the SARS-CoV-2 Delta variant (n). The synergy δ-score was calculated using SynergyFinder with the zero-interaction potency (ZIP) model. Each point and column bar represent one of three independent experiments

Fig. 3. In vivo efficacy of VV116, nirmatrelvir, and the VV116 plus nirmatrelvir combination in 5-day-old suckling mice infected with HCoV-OC43. a Schematic of the experimental design for therapeutic treatment in suckling mice. Mice were intranasally challenged with 104 TCID50 of HCoV-OC43. Mice were divided into 9 groups (n = 5 for each group): the vehicle group, the group receiving VV116 10 mpk, the group receiving VV116 25 mpk, the group receiving VV116 50 mpk, the group receiving nirmatrelvir 10 mpk with ritonavir 50 mpk, the group receiving nirmatrelvir 25 mpk with ritonavir 50 mpk, the group receiving drug combination of VV116 10 mpk and nirmatrelvir 10 mpk with ritonavir 50 mpk (Combo 1), the group receiving drug combination of VV116 25 mpk and nirmatrelvir 25 mpk with ritonavir 50 mpk (Combo 2), or the group receiving EIDD-2801 200 mpk. Vehicle or drug was administered at 2 h post-infection, and then quaque die (q.d.) from day 1 to day 4. Lung tissues were collected at 5 days post-infection (n = 5). b Determination of viral RNA copies targeting nucleoprotein genes in the brains, spinal cords, lungs, and kidneys collected on day 5 by real-time fluorescence quantitative PCR. c…

Fig. 4. In vivo efficacy of VV116, nirmatrelvir, and the VV116 plus nirmatrelvir combination in K18-hACE2 mice infected with the SARS-CoV-2 Delta variant. a Schematic of the experimental design for therapeutic treatment in K18-hACE2 mice. The mice were intranasally challenged with 1000 pfu of the SARS-CoV-2 Delta variant. The mice were divided into five groups (n = 9 for each group): the vehicle group, the group receiving VV116 100 mpk, the group receiving VV116 50 mpk, the group receiving nirmatrelvir 100 mpk with ritonavir 50 mpk, and the group receiving the drug combination (Combo) of VV116 50 mpk and nirmatrelvir 100 mpk with ritonavir 50 mpk. The vehicle or drugs were orally administered at 2 h post-infection and were then administered bis in die (b.i.d.) at 8-h intervals from day 1 to day 4. Lung tissues were collected at 2 days post-infection (n = 5) and 4 days post-infection (n = 4). b and c Determination of viral RNA copies targeting the gene of receptor binding domain in the lungs collected at day 2 and day 4 by real-time fluorescence quantitative PCR. d and e Determination of viral titers in lungs collected at day 2 and day 4 by plaque assay. f and g Cytokine gene expression…