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

Abstract

During the ongoing pandemic, providing treatment consisting of effective, low-cost oral antiviral drugs at an early stage of SARS-CoV-2 infection has been a priority for controlling COVID-19. Although Paxlovid and molnupiravir have received emergency approval from the FDA, some side effect concerns have emerged, and the possible oral agents are still limited, resulting in optimized drug development becoming an urgent requirement. An oral remdesivir derivative, VV116, has been reported to have promising antiviral effects against SARS-CoV-2 and positive therapeutic outcomes in clinical trials. However, whether VV116 has broad-spectrum anti-coronavirus activity and potential synergy with other drugs is not clear. Here, we uncovered the broad-spectrum antiviral potency of VV116 against SARS-CoV-2 variants of concern (VOCs), HCoV-OC43, and HCoV-229E in various cell lines. In vitro drug combination screening targeted RdRp and proteinase, highlighting the synergistic effect of VV116 and nirmatrelvir on HCoV-OC43 and SARS-CoV-2. When co-administrated with ritonavir, the combination of VV116 and nirmatrelvir showed significantly enhanced antiviral potency with noninteracting pharmacokinetic properties in mice. Our findings will facilitate clinical treatment with VV116 or VV116+nirmatrelvir combination to fight coronavirus infection.

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 (IIP^com) and corresponding theoretical IIP values predicted by Bliss independence (IIP^Bcom) of double drug combinations at 4 × IC₅₀. i–l IIP^com and IIP^Bcom 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 Determination of viral titers in the brains, spinal cords, lungs, and kidneys collected on day 5 by immunoplaque assay. d Cytokine gene expression was measured in the brain, spinal cord, lungs, and kidneys at day 5. The relative gene expression of IL-1β, IL-6, IFNAR, TNF-α, CCL2, CXCL10, and ISG15 was compared to that of unchallenged mice. e–v Immunofluorescence staining of brains and spinal cords to detect the SARS-CoV-2 antigen. e, n Vehicle, f, o EIDD2801-200 mpk, g, p VV116-50 mpk, h, q VV116-10 mpk, i, r nirmatrelvir-10 mpk + rito-50 mpk, j, s Combo 1, k, t VV116-25 mpk, l, u nirmatrelvir-25 mpk with ritonavir-50 mpk, and m, v Combo 2. The scale bars on the pictures of tissue slides indicate 1000 µm. The data on viral copies and viral titers were statistically analyzed with Student’s t-test. *P < 0.05; **P < 0.01; ***P < 0.001; and ns, not significant

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 was measured in the lungs at days 2 and 4. The relative gene expression of IL-1β, IL-6, IFNAR, TNF-α, CCL2, CXCL10, and ISG15 was compared to that of unchallenged mice. h–l Histopathological analysis (hematoxylin-eosin) and immunofluorescence staining to detect the SARS-CoV-2 antigen in lung tissues of the vehicle (h), VV116 100 mpk (i), VV116 50 mpk (j), nirmatrelvir 100 mpk with ritonavir 50 mpk (k), and drug combination (Combo) VV116 50 mpk and nirmatrelvir 100 mpk with ritonavir 50 mpk (l) groups. Scale bars indicate 500 µm. m and n Plasma pharmacokinetic analysis of C57BL/6 J mice that received oral doses of VV116 (single administration), nirmatrelvir + ritonavir (single administration), and VV116 + nirmatrelvir + ritonavir (co-administration) at 50, 100 + 50, and 50 + 100 + 50 mg/kg, respectively. Data on the viral copies and viral titers were statistically analyzed with Student’s t-test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; and ns, not significant