key: cord-0890759-f9319ydm
authors: Stegmann, Kim M.; Dickmanns, Antje; Heinen, Natalie; Blaurock, Claudia; Karrasch, Tim; Breithaupt, Angele; Klopfleisch, Robert; Uhlig, Nadja; Eberlein, Valentina; Issmail, Leila; Herrmann, Simon T.; Schreieck, Amelie; Peelen, Evelyn; Kohlhof, Hella; Sadeghi, Balal; Riek, Alexander; Speakman, John R.; Groß, Uwe; Görlich, Dirk; Vitt, Daniel; Müller, Thorsten; Grunwald, Thomas; Pfaender, Stephanie; Balkema-Buschmann, Anne; Dobbelstein, Matthias
title: Inhibitors of dihydroorotate dehydrogenase cooperate with Molnupiravir and N4-hydroxycytidine to suppress SARS-CoV-2 replication
date: 2022-04-25
journal: iScience
DOI: 10.1016/j.isci.2022.104293
sha: 3662a9a686535aaac60029a7525cf1f7a6f14f66
doc_id: 890759
cord_uid: f9319ydm

The nucleoside analogue N4-hydroxycytidine (NHC) is the active metabolite of the prodrug Molnupiravir, which has been approved for the treatment of COVID-19. SARS-CoV-2 incorporates NHC into its RNA, resulting in defective virus genomes. Likewise, inhibitors of dihydroorotate dehydrogenase (DHODH) reduce virus yield upon infection, by suppressing the cellular synthesis of pyrimidines. Here we show that NHC and DHODH inhibitors strongly synergize in the inhibition of SARS-CoV-2 replication in vitro. We propose that the lack of available pyrimidine nucleotides upon DHODH inhibition increases the incorporation of NHC into nascent viral RNA. This concept is supported by the rescue of virus replication upon addition of pyrimidine nucleosides to the media. DHODH inhibitors increased the antiviral efficiency of Molnupiravir not only in organoids of human lung, but also in Syrian Gold hamsters and in K18-hACE2 mice. Combining Molnupiravir with DHODH inhibitors may thus improve available therapy options for COVID-19.

Besides immunosuppression and direct interference with virus replication, an 90 alternative approach of treatment against SARS-CoV-2 aims at reducing the cellular 91 synthesis of nucleotides, thereby indirectly impairing the synthesis of viral RNA. We 92 (Stegmann et al., 2021a) and others (Caruso et in vitro-evidence of drug synergism between NHC and DHODH inhibitors, supporting 119 our hypothesis and encouraging its further evaluation. We now found that the 120 combination of NHC and DHODH inhibitors resulted in profoundly synergistic 121 suppression of SARS-CoV-2 replication in vitro; DHODH inhibition also improved the 122 performance of Molnupiravir in two animal model systems, thus presenting a potential 123 treatment strategy. 124 J o u r n a l P r e -p r o o f primary sites for SARS-CoV-2 in humans. For a model closer to these primary infection 219 sites, we infected lung organoids derived from human induced pluripotent stem cells 220 (iPSCs) upon treatment with single and combined drugs. Here again, virus replication 221 was strongly reduced by the combination of both drugs 24, 48, and 72 h.p.i, (Fig. 6A) , 222 with tolerable cytotoxicity (Fig. 6B ). In parallel, the rate of cells that contained double-223 stranded RNA derived from the virus was drastically reduced by the drug combination 224 Our results demonstrate that the simultaneous application of NHC and DHODH 280 inhibitors suppresses the replication of SARS-CoV-2 in cultured cells far more 281 profoundly than treatment with single drugs. The combination is also effective in 282 animal models. Since both classes of compounds are singularly undergoing advanced 283 clinical evaluation for the treatment of COVID-19, our data raise the perspective of 284 using both drugs together as an antiviral combination therapy. 285

When using cell lines as an infection model, the two drugs displayed strong degrees 286 of statistically significant synergies (Fig. 2) . The results of in vivo models, while still 287 revealing the strongest effects when putting both drugs together, did not fulfill the strict 288 criteria of synergism. Perhaps, the uridine in the serum of the animals partially 289 compensates the effects of the DHODH inhibitors. Higher doses of DHODH inhibitors 290 might possibly overcome this in future studies. 291

Besides their cooperation, another advantage of both NHC and DHODH inhibitors is 292 their robustness towards virus variants, as exemplified (Fig. 4) . This was expected 293 since neither of the drugs works by inhibiting a viral enzyme. DHODH inhibitors target 294 a cellular, not a viral function, i.e. pyrimidine synthesis; NHC, in turn, works through 295 incorrect base pairing, not by inhibition of viral enzymes. Indeed, even prolonged NHC 296 treatment did not lead to resistance formation in other coronaviruses (Agostini et al., 297 2019) . This raises the expectation that even novel virus variants, e.g. Omicron, remain 298 susceptible to the drug combination. 299

On top of enhancing the incorporation of NHC into viral RNA, DHODH inhibitors also 300 induce metabolic stress signaling, leading to the induction of an innate immune 301 response independent of type I interferons. In addition, due to their selective inhibition 302 of hyperactive immune cells and excessive cytokine production, they may reduce the 303 hyperinflammation, termed "cytokine storm" by some authors (Fajgenbaum Along with our finding that current virus variants of concern (VOC) respond similarly 387 to the original SARS-CoV-2 strain (Fig. 4) , this raises the hope that the drug 388 combination will be universally applicable to treat most if not all SARS-CoV-2 variants. to detect viral RNA and determine the amount of SARS-CoV-2 RNA copies per mL 472 (mean, n=3). The synergy score was calculated using the Bliss independence model. 473

Data are presented as mean ± SEM. A Bliss score > 10 is generally considered to 474 reveal strong drug synergism. 475

Diminished virus RNA progeny by NHC and DHODH inhibitors even when 476 added 4 h after SARS-CoV-2 infection. Vero E6 cells were infected as described in 477 supernatant was titrated to determine the TCID50/mL (mean with SD, n=6). Statistical 543 evaluation was performed using the Mann-Whitney U test. 544

Cell viability of lung organoids was not detectably affected by NHC and/or the 545 DHODH inhibitor BAY2402234. The release of lactate dehydrogenase (LDH) to the 546 supernatant was quantified by bioluminescence as a read-out for cytotoxicity and cell 547 viability as in Fig. 1D . 548

Representative Gold hamsters (n=4) were treated with 250 mg/kg Molnupiravir alone, 10 mg/kg 559 Teriflunomide alone, or a combination of both, administered twice a day, starting 24 h 560 before inoculation until six days post inoculation with 1*10 4 TCID50 SARS-CoV-2. 561

Virus load within nasal washes, determined by TCID50. Nasal washes were 562 obtained at days 2 and 4 post infection (p.i.), and titrated to determine the content of 563 virus. On average, virus load was reduced by 1-2 orders of magnitude as compared 564

to non-treated animals, and the drug combination yielded the strongest reduction. 565

Statistical evaluation was performed using the Mann-Whitney U test. 

Further information and requests for resources and reagents should be directed to and 605 will be fulfilled by the lead contact, Matthias Dobbelstein (mdobbel@uni-606 goettingen.de). 607

This study did not generate new unique reagents. 609

• All data reported in this paper will be shared by the lead contact upon request. 611

• This paper does not report original code. 612

• Any additional information required to reanalyze the data reported in this paper 613 is available from the lead contact upon request. was defined as 100%, and the other RNA quantities were normalized accordingly. 674

Vero E6 cells were treated/infected as indicated and RNA within the cell culture 676 supernatant was isolated and quantified by qRT-PCR. The amount of SARS-CoV-2 677 RNA determined upon infection without any treatment was defined as 100% virus yield 678 (0% inhibition). The other samples were normalized accordingly and stated as 679 percentage of control. The Bliss independence model (Bliss, 1939) , calculated using 680 the synergyfinder (Ianevski et al., 2020) at https://synergyfinder.org/ was used to 681 quantify synergy between drug combinations. We did not assume mutual exclusivity 682 of the drug effects. 683

Vero E6 cells were seeded onto 8-well chamber slides (Nunc) and treated/infected as for 10 min, followed by luminometry using a Centro LB 960 luminometer (Berthold). 728

The extent of luminescence, reflecting relative ATP levels, was normalized to DMSO-729 treated cells. 730

Lung organoids were generated from CD34-positive human induced pluripotent stem 734

protocol. 120 days-old organoids were sliced using a Leica VT 100 S vibrating 736 microtome (Leica) and treated with β-D-N4-Hydroxycytidine and/or BAY2402234. 

The experiments were carried out according to the German Regulations for Animal 756

Welfare after obtaining the necessary approval from the ethics committee authorized 757 by Regierungspräsidium Tübingen. Experiments were performed at Synovo GmbH, 758

Tübingen, Germany. 759

Eight-week-old Syrian Gold hamsters (72 -96 g) were obtained from Janvier Labs 761 under BSL3 conditions. The treatment scheme was depicted using BioRender.com. 805

Extraction of total RNA from nose fluid samples from hamsters was done using the 807 "Viral RNA/DNA isolation" NucleoMag®VET kit (Machery & Nagel GmbH, Düren, 808

Germany) in a KingFisher Flex Purification System (ThermoFisher Scientific, USA). 809 SARS-CoV-2 RNA was detected using the AgPath-ID One-Step RT-PCR Kit (E-gene 810

Sarbeco 6-carboxyfluorescein quantitative RT-PCR) (Applied Biosystems) as 811 published previously (Corman et al., 2020) . 812

During the necropsy, the percentage of dark red discoloration per total lung tissue was 814 estimated. The left lung lobe was carefully removed, immersion-fixed in 10% neutral-815 buffered formalin, and paraffin-embedded. 2-3-μm-thick sections were stained with 816 hematoxylin and eosin (HE). Slides were scanned using a Hamamatsu S60 scanner, 817 and the evaluation was performed using the NDPview.2 plus software (Version 2.8.24, 818

Hamamatsu Photonics, K.K. Japan). For histopathology, the left lung lobe was 819 evaluated using a 500x500µm grid. The extent of pneumonia-associated consolidation 820 was recorded as percentage of affected lung fields. Further, the lung was examined 821

for the presence of SARS-CoV2-characteristic lesions described for hamsters, i.e. (LDH) to the supernatant was quantified by bioluminescence and displayed as percentage of total cellular LDH, as a read-out for cytotoxicity (mean with SD, n=3).

Lack of measurable cytotoxicity by IMU-838. The experiment was carried out as in A, using IMU-838 instead of NHC.

The combination of NHC and DHODH inhibitors does not affect cell viability.

Vero E6 cells were treated as in Fig. 1 and ATP levels were measured by the CellTiter-Glo® assay. The extent of luminescence, reflecting relative ATP levels, was normalized to untreated cells (mean with SD, n=3). Daily energy expenditure. Upon infection and treatment of hamsters as in Fig.   7A , the daily energy expenditure (DEE) was determined using the doubly labeled water (DLW) method. This method was only affordable to us to include animals that were either infected without treatment, or infected with combined treatment, to show that the drug combination improves the health of infected animals regardless of the contribution from each drug. 100 nM +BAY <0.0001 0.0084 n/a n/a <0.0001 n/a n/a n/a n/a n/a n/a n/a 100 nM +Terifl. <0.0001 n/a 0.0207 n/a <0.0001 n/a n/a n/a n/a n/a n/a n/a 100 nM +IMU <0.0001 n/a n/a 0.0260 <0.0001 n/a n/a n/a n/a n/a n/a n/a 300 nM +BAY <0.0001 0.0037 n/a n/a n/a n/a n/a n/a 0.0002 n/a n/a n/a 300 nM +Terifl. <0.0001 n/a 0.0081 n/a n/a n/a n/a n/a 0.0002 n/a n/a n/a 300 nM +IMU <0.0001 n/a n/a 0.0128 n/a n/a n/a n/a 0.0002 n/a n/a n/a 30 µM Terifl. 100 nM +BAY <0.0001 0.0066 n/a n/a 0.0049 n/a n/a n/a n/a n/a n/a n/a 100 nM +Terifl. <0.0001 n/a 0.0104 n/a 0.0045 n/a n/a n/a n/a n/a n/a n/a 100 nM +IMU <0.0001 n/a n/a 0.0260 0.0044 n/a n/a n/a n/a n/a n/a n/a 300 nM +BAY <0.0001 0.0050 n/a n/a n/a n/a n/a n/a 0.0019 n/a n/a n/a 300 nM +Terifl. <0.0001 n/a 0.0077 n/a n/a n/a n/a n/a 0.0018 n/a n/a n/a 300 nM +IMU <0.0001 n/a n/a 0.0124 n/a n/a n/a n/a 0.0018 n/a n/a n/a J o u r n a l P r e -p r o o f NHC + BAY <0.0001 0.0081 0.0157 n/a n/a n/a n/a n/a NHC + Terifl. <0.0001 0.0083 n/a 0.0008 n/a n/a n/a n/a NHC + IMU <0.0001 0.0080 n/a n/a 0.0248 n/a n/a n/a P values corresponding to Fig. S3A 

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