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Abstract

Effective therapeutics to inhibit the replication of SARS-CoV-2 in infected individuals are still under development. The nucleoside analogue N4-hydroxycytidine (NHC), also known as EIDD-1931, interferes with SARS-CoV-2 replication in cell culture. It is the active metabolite of the prodrug Molnupiravir (MK-4482), which is currently being evaluated for the treatment of COVID-19 in advanced clinical studies. Meanwhile, inhibitors of dihydroorotate dehydrogenase (DHODH), by reducing the cellular synthesis of pyrimidines, counteract virus replication and are also being clinically evaluated for COVID-19 therapy. Here we show that the combination of NHC and DHODH inhibitors such as teriflunomide, IMU-838/vidofludimus, and BAY2402234, strongly synergizes to inhibit SARS-CoV-2 replication. While single drug treatment only mildly impaired virus replication, combination treatments reduced virus yields by at least two orders of magnitude. We determined this by RT-PCR, TCID50, immunoblot and immunofluorescence assays in Vero E6 and Calu-3 cells infected with wildtype and the Alpha and Beta variants of SARS-CoV-2. We propose that the lack of available pyrimidine nucleotides upon DHODH inhibition increases the incorporation of NHC in nascent viral RNA, thus precluding the correct synthesis of the viral genome in subsequent rounds of replication, thereby inhibiting the production of replication competent virus particles. This concept was further supported by the rescue of replicating virus after addition of pyrimidine nucleosides to the media. Based on our results, we suggest combining these drug candidates, which are currently both tested in clinical studies, to counteract the replication of SARS-CoV-2, the progression of COVID-19, and the transmission of the disease within the population.