Identification of antiviral antihistamines for COVID-19 repurposing

Abstract

There is an urgent need to identify therapies that prevent SARS-CoV-2 infection and improve the outcome of COVID-19 patients. Although repurposed drugs with favorable safety profiles could have significant benefit, widely available prevention or treatment options for COVID-19 have yet to be identified. Efforts to identify approved drugs with in vitro activity against SARS-CoV-2 resulted in identification of antiviral sigma-1 receptor ligands, including antihistamines in the histamine-1 receptor binding class. We identified antihistamine candidates for repurposing by mining electronic health records of usage in population of more than 219,000 subjects tested for SARS-CoV-2. Usage of diphenhydramine, hydroxyzine and azelastine was associated with reduced incidence of SARS-CoV-2 positivity in subjects greater than age 61. We found diphenhydramine, hydroxyzine and azelastine to exhibit direct antiviral activity against SARS-CoV-2 in vitro. Although mechanisms by which specific antihistamines exert antiviral effects is not clear, hydroxyzine, and possibly azelastine, bind Angiotensin Converting Enzyme-2 (ACE2) and the sigma-1 receptor as off-targets. Clinical studies are needed to measure the effectiveness of diphenhydramine, hydroxyzine and azelastine for disease prevention, for early intervention, or as adjuvant therapy for severe COVID-19.

Keywords: Angiotensin converting Enzyme-2; Docking; Repurposing; SARS-CoV-2; Sigma-1 receptor.

Copyright © 2020 Elsevier Inc. All rights reserved.

Figures

Fig. 1

Antiviral activity of specific antihistamines against SARS-CoV-2 isolates in vitro. Panel A. An engineered GFP-expressing lentivirus pseudotyped with the SARS-CoV-2 surface glycoprotein was used to infect ACE2 expressing HEK293 cells in the presence and absence of histamine-1 receptor binding antihistamines. The number of GFP positive cells per image field 72 h after a single application of drugs in HEK293T cells overexpressing human ACE2 is shown. n = 6 independent experiments. ∗p = 0.0044 DMSO vs CET ∗; p = 0.0002 DMSO vs HYD; ∗p = 0.0006 DMSO vs DIPH; ∗p = 0.0052 DMSO vs AZ; p < 0.0001 DMSO vs SPK. Panel B. Number of SARS-CoV-2 isolate USA-UF-1/2020 viral plaques present in Vero E6 cells 3 dpi following exposure to antihistamines or DMSO vehicle control. n = 3 independent experiments. ∗p = 0.0003 DMSO vs HYD ∗; p = 0.0003 DMSO vs DIPH; ∗p < 0.0001 DMSO vs AZ. Dose response curves against SARS-CoV-2 isolate USA-UF-1/2020 in Vero E6 cells for hydroxyzine (C), diphenhydramine (D) and azelastine (E). n = 3 independent experiments. Dose response curve against SARS-CoV-2 isolate USA-WA1/2020 in human lung A549 cells for hydroxyzine (F). n = 2 independent experiments. Abbreviations: cetirizine (CET), hydroxyzine (HYD), diphenhydramine (DIPH), loratadine (LOR), azelastine (AZ), plaque forming units (PFU), spike protein (SPK). Drug concentrations in Panels A and B in μg/ml: hydroxyzine [10]; cetirizine [10]; loratadine [1.5]; diphenhydramine hydrochloride [25]; azelastine hydrochloride [7.0]. Spike protein used at 13.5 ng/ml. Data were analyzed by a one-way ANOVA followed by a Dunnett multiple comparison test. In panel A B, the median + interquartile range are shown.

Fig. 2

In silico modeling of the ACE2 catalytic inhibitor hydroxyzine suggests binding within the active site. A. Hydroxyzine was posed by molecular docking with AutoDock Vina. Hydroxyzine is shown as sticks, white for carbon, blue for nitrogen, red for oxygen, green for chlorine. Polar interactions (e.g., H bonds) shown as magenta dashes. B. The SARS-CoV-2 spike glycoprotein binds the open conformation of ACE2. The SARS-CoV-2 spike protein is shown in orange (from PDB 6M17). ACE2 is shown in grey in the open conformation (left, from PDB 6M17) and the closed conformation (right, PDB 1R4L). Inhibitor binding prevents intermolecular contacts between ACE2 and SARS-CoV-2 spike protein (shown as sticks in red). Hydroxyzine is shown as spheres in magenta, as posed by AutoDock Vina.

Fig. 3

Molecular docking predicts binding of diphenhydramine and azelastine to the ligand binding pocket of the Sigma-1 receptor. The crystal structure of the human sigma-1 receptor bound to a ligand was used as the basis for molecular docking with AutoDock Vina. The ligand PD144418 was extracted from the coordinates of PDB code 5HK1, and diphenhydramine was docked to this site (upper panel), estimated ΔG -8.2 kcal/mol. Azelastine was docked to the ligand binding site of the sigma 1 receptor (lower panel), estimated ΔG -11.3 kcal/mol.

Fig. 4

Model for antiviral mechanisms mediated by specific antihistamines. Hydroxyzine and related antihistamines have the potential to inhibit SARS-CoV-2 entry (by binding ACE2), and virus replication (by binding the sigma-1 receptor). Hydroxyzine (purple) exhibits off-target inhibitory ACE2 activity by forming intermolecular interactions with the active site, inducing a conformational change from the open conformation to the closed conformation. The conformational change shifts the position of ACE2 at positions that contact the SARS-CoV-2 spike glycoprotein receptor binding domain (RBD), shown in red, which results in loss of intermolecular interactions at the ACE2/RBD interface. The sigma-1 receptor is a membrane bound chaperone highjacked by SARS-CoV-2 to link the replicase/transcriptase complex to the endoplasmic reticulum by binding directly to nonstructural protein NSP6, which forms a complex with NSP3 and NSP4. Hydroxyzine binds the sigma-1 receptor with high affinity, potentially interfering with the virus life cycle by blocking protein-protein interactions with NSP6.