Molnupiravir combined with different repurposed drugs further inhibits SARS-CoV-2 infection in human nasal epithelium in vitro

Abstract

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a worldwide pandemic with unprecedented economic and societal impact. Currently, several vaccines are available and multitudes of antiviral treatments have been proposed and tested. Although many of the vaccines show clinical efficacy, they are not equally accessible worldwide. Additionally, due to the continuous emergence of new variants and generally short duration of immunity, the development of effective antiviral treatments remains of the utmost importance. Since the emergence of SARS-CoV-2, substantial efforts have been undertaken to repurpose existing drugs for accelerated clinical testing and emergency use authorizations. However, drug-repurposing studies using cellular assays often identify hits that later prove ineffective clinically, highlighting the need for more complex screening models. To this end, we evaluated the activity of single compounds that have either been tested clinically or already undergone extensive preclinical profiling, using a standardized in vitro model of human nasal epithelium. Furthermore, we also evaluated drug combinations based on a sub-maximal concentration of molnupiravir. We report the antiviral activity of 95 single compounds and 30 combinations. We show that only a few single agents are highly effective in inhibiting SARS-CoV-2 replication while selected drug combinations containing 10 µM molnupiravir boosted antiviral activity compared to single compound treatment. These data indicate that molnupiravir-based combinations are worthy of further consideration as potential treatment strategies against coronavirus disease 2019 (COVID-19).

Keywords: Air-liquid interface; Antivirals; COVID-19; Drug repurposing; SARS-CoV-2.

Graphical abstract

Fig. 1

Overview over experimental design and layout. Toxicity of 104 single compounds was determined in reconstituted nasal epithelium in vitro to determine the maximum tolerated concentration (≤30 µM). Compounds were applied basally, mimicking systemic administration. Three endpoints were assessed, trans-epithelial electrical resistance (TEER), cytotoxicity, and the ciliary beating frequency of ciliated cells. Subsequently, 99 single compounds were screened for antiviral activity against SARS-CoV-2. Additionally, 30 combinations based on the orally bioactive RdRp inhibitor molnupiravir were tested for antiviral activity with additional parameters (infectious virus and cytokine secretion) determined for top candidates.

Fig. 2

Antiviral activity of single compounds against SARS-CoV-2. Antiviral activity of single compounds against SARS-CoV-2 as detected by qPCR in a) apical wash and b) intracellular lysate at 72 hpi. Top 16 compounds based on the viral content of apical wash at 72 hpi (n = 2–3, except for conditions tested at both investigatory sites: n = 5 for 10 µM nelfinavir, camostat; n = 6 for 30 µM molnupiravir; and n = 12 for 10 µM remdesivir and molnupiravir. HHT: homoharringtonine. Data are represented as mean ± standard deviation (SD). Dashed line: limit of what is considered significant antiviral activity in the current study (>1 log).

Fig. 3

Dose-response of molnupiravir treatment against SARS-CoV-2 (a) Antiviral activity of molnupiravir at 48 and 72 hpi as detected by qPCR in apical wash. Data are represented as mean ± SEM, n = 6, 6, 2, 6, 2, 12, 4 and 6 for 0.36, 1.15, 2.5, 3.3, 5, 10, 20 and 30 µM, respectively. Dashed line: limit of what is considered significant antiviral activity in the current study (>1 log). (b) Remaining infectivity in apical wash after treatment with molnupiravir (TCID₅₀/ml) (n = 2–4). Data are represented as mean ± standard deviation (SD). Dashed line: limit of detection (LOD).

Fig. 4

Antiviral activity of compound combinations against SARS-CoV-2. Antiviral activity of molnupiravir-based combinations against SARS-CoV-2 as determined by (a) qPCR in apical wash or intracellularly at 72 hpi (n = 2–4, and n = 12 for molnupiravir) and (b) TCID₅₀/ml in apical wash at 72 hpi (n = 2–4). Dashed lines represent the limit of detection (LOD) and vehicle control (VC). Black column: 10 µM molnupiravir alone, gray columns: single compound treatments, striped columns: molnupiravir-based combinations. *p < 0.05, **p < 0.01, ***p < 0.001, **** p < 0.0001, representing significance over molnupiravir alone. Statistical significance between single agents and combinations are shown numerically. Data are represented as mean ± SD.