Drug repurposing of argatroban, glimepiride and ranolazine shows anti-SARS-CoV-2 activity via diverse mechanisms

Abstract

Despite the vast vaccination campaigns against SARS-CoV-2, vaccine-resistant variants have emerged, and COVID-19 is continuing to spread with the fear of emergence of new variants that are resistant to the currently available anti-viral drugs. Hence, there is an urgent need to discover potential host-directed - rather than virus-directed - therapies against COVID-19. SARS-CoV-2 enters host cells through binding of the viral spike (S)-protein to the host angiotensin-converting enzyme 2 (ACE2) receptor, rendering the viral port of entry an attractive therapeutic target. Accordingly, this study aimed to investigate FDA-approved drugs for their potential repurposing to inhibit the entry point of SARS-CoV-2. Accordingly, the FDA-approved drugs library was enrolled in docking simulations to identify drugs that bind to the Spike-ACE2 interface. The drugs list retrieved by the docking simulations was shortlisted to 19 drugs based on docking scores and safety profiles. These drugs were screened for their ability to prevent binding between ACE2 and S-protein using an ELISA-based Spike-ACE2 binding assay. Five drugs showed statistically significant inhibition of binding between ACE2 and S-protein, ranging from 4 % to 37 %. Of those five, argatroban, glimepiride and ranolazine showed potential antiviral activity at IC₅₀ concentrations well below their CC₅₀ assessed by the plaque assay. Their mode of antiviral action was then determined using the plaque assay with some modifications, which revealed that argatroban acted mainly through a direct virucidal mechanism, while glimepiride largely inhibited viral replication, and ranolazine exerted its antiviral impact primarily through inhibiting viral adsorption. In conclusion, this study has identified three FDA-approved drugs - argatroban, glimepiride and ranolazine - which could potentially be repurposed and used for the management of COVID-19.

Keywords: ACE2; Argatroban; COVID-19; Drug-repurposing; Glimepiride; Ranolazine.

Graphical abstract

Fig. 1

A Schematic illustration of the workflow of the study. (1) An initial phase of computational analysis to screen for the FDA-approved drugs targeting ACE2, followed by primary selection of drugs. (2) Then an experimental validation phase for the selected drugs achieved by (a) evaluating the ability of the selected drugs to interrupt the binding between the viral S-protein and the host ACE2 receptor, (b) assessing the antiviral activity and cytotoxicity of the promising candidates to determine the IC₅₀ and CC₅₀ values and selectivity indices of the drugs followed by (c) exploring the mechanism of action of the chosen drugs.

Fig. 2

Computational screening of drugs. Drugs that were computationally predicted to inhibit Spike-ACE2 interaction were first designated based on their docking scores. Out of 31 drugs showing docking scores < −10 kcal/mol, those with unfavorable safety profiles were excluded giving a final list of 19 drugs.

Fig. 3

Impact of drugs on binding of Spike protein to ACE2 receptor (A) Impact of drugs on binding of Spike protein to ACE2 receptor using the COVID-19 Spike-ACE2 binding assay kit with Spike-coated plates. (B) Impact of drugs on binding of Spike protein to ACE2 receptor using the COVID-19 Spike-ACE2 binding assay kit with ACE2-coated plates. The positive control represents 100 % binding between ACE2 receptor and Spike protein. Results are presented as mean ± SD (n = 4). Asterix (∗) indicate statistical significance, where ∗ = p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001.

Fig. 4

Cytotoxicity and SARS-CoV-2 antiviral activity of selected compounds. The percentage of cell viability (black line) and SARS-CoV-2 viral inhibition (red line) were assessed in Vero E6 cells to determine the cytotoxic concentration (CC₅₀) and the viral inhibitory concentration (IC₅₀) using serial dilutions of (A) argatroban, (B) ranolazine, and (C) glimepiride (n = 3). The chemical structure of each drug is shown above each graph.

Fig. 5

Mechanism of antiviral activity of argatroban, ranolazine, and glimepiride. The mechanism of anti-SARS-CoV-2 activity of argatroban, ranolazine, and glimepiride was assessed in terms of (A) virucidal effect, (B) inhibition of viral adsorption to Vero E6 cells, and (C) inhibition of intracellular viral replication. Results are expressed as mean ± SD (n = 3).

Fig. 6

Schematic representation of the mechanism of action of argatroban, ranolazine, and glimepiride. The figure shows the main site of action of each drug in the SARS-Cov-2 lifecycle, where argatroban acts directly on the viral particle inducing a virucidal effect. Ranolazine acts mainly by preventing the adsorption of the viral particles to the host cells, while glimepiride's main activity is via inhibiting viral replication.