key: cord-0957146-qpdiiwh9
authors: Demarest, James F.; Kienle, Maryline; Boytz, RuthMabel; Ayres, Mary; Kim, Eun Jung; Chung, Donghoon; Gandhi, Varsha; Davey, Robert; Sykes, David B.; Shohdy, Nadim; Pottage, John C.; Kumar, Vikram S.
title: Brequinar and Dipyridamole in Combination Exhibits Synergistic Antiviral Activity Against SARS-CoV-2 in vitro: Rationale for a host-acting antiviral treatment strategy for COVID-19
date: 2022-04-04
journal: bioRxiv
DOI: 10.1101/2022.03.30.486499
sha: db65f663fd19e193dc2d6801b7472e44cd560702
doc_id: 957146
cord_uid: qpdiiwh9

The continued evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has compromised the efficacy of currently available vaccines and monoclonal antibody (mAb)-based treatment options for COVID-19. The limited number of authorized small-molecule direct-acting antivirals present challenges with pill burden, the necessity for intravenous administration or potential drug interactions. There remains an unmet medical need for effective and convenient treatment options for SARS-CoV-2 infection. SARS-CoV-2 is an RNA virus that depends on host intracellular ribonucleotide pools for its replication. Dihydroorotate dehydrogenase (DHODH) is a ubiquitous host enzyme that is required for de novo pyrimidine synthesis. The inhibition of DHODH leads to a depletion of intracellular pyrimidines, thereby impacting viral replication in vitro. Brequinar (BRQ) is an orally available, selective, and potent low nanomolar inhibitor of human DHODH that has been shown to exhibit broad spectrum inhibition of RNA virus replication. However, host cell nucleotide salvage pathways can maintain intracellular pyrimidine levels and compensate for BRQ-mediated DHODH inhibition. In this report, we show that the combination of BRQ and the salvage pathway inhibitor dipyridamole (DPY) exhibits strong synergistic antiviral activity in vitro against SARS-CoV-2 by enhanced depletion of the cellular pyrimidine nucleotide pool. The combination of BRQ and DPY showed antiviral activity against the prototype SARS-CoV-2 as well as the Beta (B.1.351) and Delta (B.1.617.2) variants. These data support the continued evaluation of the combination of BRQ and DPY as a broad-spectrum, host-acting antiviral strategy to treat SARS-CoV-2 and potentially other RNA virus infections.

(nirmatrelvir also requires the pharmacologic booster ritonavir in order to achieve sufficient 50 plasma drug levels). There remains therefore a high unmet medical need for safe, efficacious, 51 and patient-friendly treatments for SARS-CoV-2 infection. 52 While the therapeutic small molecules described above are direct-acting antivirals (DAAs) that 53 target virus-specific proteins, an alternative treatment strategy is to develop host-acting antivirals 54 (HAAs) that target host pathways essential for the viral lifecycle. A host-based mechanism of 55 action may provide unique advantages over DAAs including a greater likelihood of broad-56 spectrum antiviral activity against several families of RNA viruses. In addition, HAAs may 57 possess an inherently higher barrier to the development of resistance when compared to DAAs as 58 host-targets typically remain unchanged in contrast to the rapid emergence of viral variants 59 containing mutations that decrease the efficacy of DAAs. Dihydroorotate dehydrogenase (DHODH) is a host enzyme that is essential for de novo 61 pyrimidine synthesis and has emerged as a candidate target of HAAs. Brequinar (BRQ) is an 62 orally available, selective, and potent low nanomolar human DHODH inhibitor (DHODHI) 63 shown to deplete intracellular uridine, cytidine, and thymidine levels in vitro and in vivo [2, 3, 4] . 64 As an antiviral approach, DHODHIs such as BRQ block the host production of cellular 65 pyrimidine nucleotide triphosphates (NTPs) required by viruses for replication [5, 6, 7, 8] was <50% in the MTT assay (Supplemental Figure S1 ). 174 To determine whether BRQ+DPY had additive, synergistic, or antagonistic interactions, the 175 antiviral data were analyzed using a combination of Loewe and Bliss models [16] . The 2-drug 176 combination of BRQ+DPY exhibited a strong synergistic interaction ( Figure 4B and Table 1) . 177 The average synergy score from three replicates was 22.6, with the most synergistic area A major area of concern is the development of viral resistance, as seen with selective pressures 266 by DAAs, or viral escape from immune pressures that lead to the emergence of novel SARS-267

CoV-2 VOCs, as has been observed after vaccination and the development of natural immunity. 268 As BRQ and DPY target host rather than viral proteins, the antiviral activity of BRQ+DPY 

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ACKNOWLEDGEMENTS 355 We thank Cindy Motaka for assistance in editing, Dr Olga Kharchenko for assistance with