Synergistic and Antagonistic Drug Combinations against SARS-CoV-2

Abstract

Antiviral drug development for coronavirus disease 2019 (COVID-19) is occurring at an unprecedented pace, yet there are still limited therapeutic options for treating this disease. We hypothesized that combining drugs with independent mechanisms of action could result in synergy against SARS-CoV-2, thus generating better antiviral efficacy. Using in silico approaches, we prioritized 73 combinations of 32 drugs with potential activity against SARS-CoV-2 and then tested them in vitro. Sixteen synergistic and eight antagonistic combinations were identified; among 16 synergistic cases, combinations of the US Food and Drug Administration (FDA)-approved drug nitazoxanide with remdesivir, amodiaquine, or umifenovir were most notable, all exhibiting significant synergy against SARS-CoV-2 in a cell model. However, the combination of remdesivir and lysosomotropic drugs, such as hydroxychloroquine, demonstrated strong antagonism. Overall, these results highlight the utility of drug repurposing and preclinical testing of drug combinations for discovering potential therapies to treat COVID-19.

Keywords: COVID-19; CPE assay; SARS-CoV-2; combination therapy; drug combinations; drug repurposing; drug synergy; in silico design; knowledge mining; nitazoxanide remdesivir combo.

Graphical abstract

Figure 1

Performance of Matrix Screening (A) Z′ factor on different assays (CPE or Tox) and biological batches. (B) Reproducibility across all replicates (defined as a compound at certain concentration). Number of replicates (n) may vary, e.g., more single-agent replicates were performed due to matrix setting. (C–G) Dose response curves from an independent benchmark set performed at a different site. (H) Layout of a 6 × 6 dose matrix. Wells with (or without) bold border represent dose combination (or single agent alone).

Figure 2

Summary of Synergism or Antagonism across 73 Combinations Due to biphasic dose response, synergism was separated from antagonism. Synergism is calculated as the sum of HSA.neg values from non-toxic dose combinations (Tox > 50%) and vice versa. The size of circle reflected the confidence of the observed synergism/antagonism (bigger circle = less doses were excluded due to toxicity). The inconclusive blocks (n_non-toxic < 25 or rough activity landscape) were shown in transparent points and lines. Two dashed lines indicated the cutoff of HSA synergism (−100) or antagonism (100). Blue arrows highlighted the combinations between remdesivir and tertiary amine compounds from conclusive blocks.

Figure 3

Matrix Blocks from Remdesivir + Amine Drugs in CPE Assay The activity was normalized so that 100 corresponded to full cytopathic effect and 0 corresponded to no cytopathic effect. Red arrow, the concentrations that synergize with the partner compound. Blue arrow, the concentrations that antagonize against the partner compound. Chemical structures were shown on the right. (A) Hydroxychloroquine, (B) mefloquine, (C) amodiaquine, (D) arbidol. (E) CPE data from single-dose combination (5 μM GS-441524 or 20 μM remdesivir ± amine compounds; n_{single-agent RdRp inhibitor} = 12 and n_combination = 3). The lower and upper box hinges correspond to the first and third quartiles, and the whiskers extend from upper or lower hinges to 1.5 * IQR (inter-quartile range).

Figure 4

Heptagonal Polygonogram Depicting Some of the Binary Combinations Tested in the Study Degrees of synergism/antagonism were ascertained from Figure 3. The definitions were defined based on the degree of HSA synergism/antagonism determined in the CPE assay.

Figure 5

Summary of Synergism or Antagonism over Different Mechanism of Action (MoA) Combination Inconclusive blocks or singleton MoA were excluded. Two dashed lines indicated the cutoff of HSA synergism (−100) or antagonism (100). The lower and upper box hinges correspond to the first and third quartiles, and the whiskers extend from upper or lower hinges to 1.5 * IQR (inter-quartile range).

Figure 6

Matrix Blocks from Three Synergistic Combinations Involving Nitazoxanide (A) Nitazoxanide + remdesivir; (B) nitazoxanide + arbidol; (C) nitazoxanide + amodiaquine. Red arrow, the concentrations that synergize with the partner compound.

Figure 7

The Putative Mechanism of the Antagonism between Remdesivir and the Lysosomotropic Agent We hypothesize that lysosomotropic agents antagonize remdesivir by impairing its upstream activation (e.g., esterase-mediated hydrolysis) rather than the formation of triphosphate active agent.

Figure 8

Study Design for Selecting Possible Synergistic Drug Combinations In this study, we report only 73 binary combinations; 95 ternary combinations identified in a similar fashion will be reported in a future study.