N4-hydroxycytidine and inhibitors of dihydroorotate dehydrogenase synergistically suppress SARS-CoV-2 replication

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. SIGNIFICANCE The strong synergy displayed by DHODH inhibitors and the active compound of Molnupiravir might enable lower concentrations of each drug to antagonize virus replication, with less toxicity. Both Molnupiravir and DHODH inhibitors are currently being tested in advanced clinical trials or are FDA-approved for different purposes, raising the perspective of rapidly testing their combinatory efficacy in clinical studies. Molnupiravir is currently a promising candidate for treating early stages of COVID-19, under phase II/III clinical evaluation. However, like Remdesivir, it appears only moderately useful in treating severe COVID-19. Since the combination inhibits virus replication far more strongly, and since DHODH inhibitors may also suppress excessive immune responses, the combined clinical application bears the potential of alleviating the disease burden even at later stages.


SIGNIFICANCE 50
 The strong synergy displayed by DHODH inhibitors and the active compound 51 of Molnupiravir might enable lower concentrations of each drug to antagonize 52 virus replication, with less toxicity. 53  Both Molnupiravir and DHODH inhibitors are currently being tested in advanced 54 clinical trials or are FDA-approved for different purposes, raising the 55 perspective of rapidly testing their combinatory efficacy in clinical studies.

56
 Molnupiravir is currently a promising candidate for treating early stages of 57 COVID-19, under phase II/III clinical evaluation. However, like Remdesivir, it 58 appears only moderately useful in treating severe COVID-19. Since the 59 combination inhibits virus replication far more strongly, and since DHODH 60 inhibitors may also suppress excessive immune responses, the combined 61 clinical application bears the potential of alleviating the disease burden even at 62 later stages. 63

INTRODUCTION 64
In order to combat the COVID-19 pandemic, vaccines have successfully been 65 established, while efficient therapeutics are still under development (Doherty, 2021). 66 At present, the suppression of excessive inflammatory and immune responses by 67 steroids has been successfully applied in the clinics (Horby et al., 2021;Tomazini et 68 al., 2020). However, this strategy does not directly interfere with virus replication. So 69 far, the only FDA-approved antiviral drug for the treatment of COVID-19, the 70 adenosine analogue Remdesivir, has only been moderately successful in shortening 71 hospitalization by a few days, with variations reported between clinical studies (Beigel 72 et al., 2020;Goldman et al., 2020). Antiviral nucleoside analogues typically antagonize 73 the synthesis of viral genomes. Upon entry into the host cell, they are triphosphorylated 74 at their 5' positions. Subsequently, they interfere with the activity of the viral nucleic 75 acid polymerase or compromise the function of the newly synthesized viral genomes 76 (Pruijssers and Denison, 2019). 77 Next to Remdesivir, another nucleoside analogue showed promising performance 78 against SARS-CoV-2. Molnupiravir, also known as EIDD-2801 or MK-4482, is the 79 prodrug of N4-hydroxycytidine (NHC), or EIDD-1931(Cox et al., 2021Painter et al., 80 2021;Sheahan et al., 2020;Wahl et al., 2020;Wahl et al., 2021). NHC differs from 81 cytidine by a hydroxyl group at the pyrimidine base. According to current literature, this 82 does not impair the incorporation of triphosphorylated NHC into nascent RNA by the 83 viral RNA polymerase. However, due to a tautomeric interconversion within the NHC 84 base, RNA strands with incorporated NHC lead to erroneous RNA replication when 85 used as a template (Jena, 2020). NHC can basepair with guanosine, but also with 86 adenosine, thus leading to multiple errors in the subsequently synthesized viral RNA 87 genomes resulting in replication-deficient virus particles. Molnupiravir has been found 88 to be active against SARS-CoV-2 replication in in vitro and in vivo (Sheahan et al., 89 2020;Wahl et al., 2021). It has been shown to also prevent SARS-CoV-2 transmission 90 in vivo (Cox et al., 2021), and its clinical efficacy is currently being investigated in large-91 scale phase II clinical trials (Painter et al., 2021), NCT04575584, NCT04575597, 92

NCT04405739. 93
Besides immunosuppression and direct interference with virus replication, an 94 alternative approach of treatment against SARS-CoV-2 aims at reducing the cellular 95 synthesis of nucleotides, thereby indirectly impairing the synthesis of viral RNA. We 96 (Stegmann et al., 2021) and others (Caruso et al., 2021;Zhang et al., 2021) have 97 previously reported the high demand on cellular nucleotide biosynthesis during SARS-98 CoV-2 infection, resulting in an antiviral efficacy of folate antagonists that impair purine 99 synthesis. Moreover, in the context of nucleotide biosynthesis, the inhibition of 100 dihydroorotate dehydrogenase (DHODH) represents an attractive strategy to 101 (MOI 0.

Quantification and statistical analysis 217
Statistical testing was performed using Graph Pad Prism 6 (RRID:SCR_002798). A 218 two-sided unpaired Student's t-test was calculated, and significance was assumed 219 where p-values ≤ 0.05. Asterisks represent significance in the following way: ****, p ≤ 220 0.0001; ***, p ≤ 0.005; **, p ≤ 0.01; *, p ≤ 0.05. The biosynthesis of pyrimidines is crucial for RNA replication (Fig. 1A). The enzyme 227 dihydroorotate dehydrogenase (DHODH) catalyzes the oxidation of dihydroorotate to 228 orotate, which is a precursor of cytidine triphosphate (CTP). In the presence of N4-229 hydroxycytidine (NHC), its active metabolite NHCTP competes with CTP for 230 incorporation into nascent RNA. We hypothesized that the suppression of cellular CTP 231 synthesis by DHODH inhibitors favors the incorporation of NHCTP into newly 232 synthesized SARS-CoV-2 RNA, and thus potentiates the antiviral efficacy of NHC. To 233 test this, we combined both drugs for treatment of Vero E6 or Calu-3 cells, followed by 234 infection with SARS-CoV-2. We applied suboptimal concentrations of NHC and the 235 DHODH inhibitors BAY2402234, teriflunomide and IMU-838 to moderately diminish 236 virus replication. We treated cells with the drugs at these concentrations, alone or in 237 combination, 24 hours before and during infection with SARS-CoV-2. At these 238 concentrations, neither NHC nor DHODH inhibitors grossly affected the development 239 of a cytopathic effect (CPE) caused by SARS-CoV-2. Strikingly, however, the 240 combination of NHC and DHODH inhibitors was far more efficient in preventing the 241 CPE ( Fig. 1B) and reducing virus yield, as determined by the Median Tissue Culture 242 Infectious Dose (TCID50/mL) (Fig. 1C). Combining the drugs did not produce 243 morphologic signs of cytotoxicity in non-infected cells (Fig. 1B). To quantify a possible 244 increase in cytotoxicity, we measured the release of lactate dehydrogenase (LDH) into 245 the culture supernatant. Compared to the DMSO control, neither the single treatments 246 nor the combinations displayed major cytotoxicity (Fig. 1D). Hence, the drug 247 combination synergistically interferes with CPE and virus yield, without displaying 248 cytotoxicity on its own. 249 250

Combinations of NHC and DHODH inhibitors distinctly reduce viral RNA yield 251 upon infection with SARS-CoV-2. 252
We combined different concentrations of the DHODH inhibitor IMU-838 and NHC, and 253 determined their impact on the release of viral RNA, along with the combination index (Chou, 2010). When combined, the effective concentrations of IMU-838 and NHC 255 were between 5-10 µM and 100-300 nM, respectively ( Fig. 2A). Moreover, we 256 combined the DHODH inhibitors BAY2402234, teriflunomide, ASLAN003 and 257 brequinar with NHC and quantified the amount of viral RNA released into the cell 258 culture supernatant (Fig. 2B). Strikingly, the combination treatment diminished SARS-259 CoV-2 RNA progeny up to 400-fold as compared to single drug treatment, and up to 260 1000-fold as compared to untreated controls. This effect was not only seen in Vero E6 261 cells but also in Calu-3 cells (Fig. 2C), a human lung cancer cell line used to model 262 bronchial epithelia (Kreft et al., 2015).

DHODH inhibitors. 289
To elucidate the mechanism of interference with SARS-CoV-2 replication by drug 290 combinations, we performed rescue experiments by adding pyrimidine nucleosides. 291 First, we added uridine to Vero E6 cells along with the DHODH inhibitors IMU-838, 292 BAY2402234 or teriflunomide, combined with NHC (Fig. 5A). The addition of 2 µM 293 uridine did not prevent the inhibition of SARS-CoV-2 replication by the combination 294 treatment, but 10 µM uridine did. Similar results were obtained upon the addition of 295 cytidine (Fig. 5B). This strongly suggests that the synergism of NHC with DHODH 296 inhibitors can be explained by competition of NHC with endogenous pyrimidine 297 nucleosides for incorporation into nascent viral RNA, as we had hypothesized initially 298 ( Fig. 1A). 299

DISCUSSION 301
Our results demonstrate that the simultaneous application of NHC and DHODH 302 inhibitors suppresses the replication of SARS-CoV-2 far more profoundly than 303 treatment with single drugs. Since both classes of compounds are undergoing 304 advanced clinical evaluation for the treatment of COVID-19, our observations at least 305 raise the perspective of using both drugs as antiviral combination therapy. 306 On top of enhancing the incorporation of NHC into viral RNA, DHODH inhibitors also 307 suppress the immune response, e.g. by dampening the proliferation of B and T 308 lymphocytes. In fact, they are currently in clinical use to treat autoimmune diseases 309 Mechanistically, it is conceivable that reduced intracellular levels of cytidine 317 triphosphate reduce the competition for triphosphorylated NHC regarding their 318 incorporation into nascent virus RNA. This was further corroborated by the rescue of 319 virus replication by uridine and cytidine, each metabolic precursors of CTP. Notably, 320 the concentrations of uridine required for this rescue were in the range of 10 µM, which 321 is above physiological serum concentrations (Ashour et al., 2000;Deng et al., 2017;322 Karle et al., 1980;Traut, 1994), raising the hope that pyrimidines in body fluids would 323 not allow such rescue. NHC triphosphate is generated by the salvage pathway for 324 pyrimidines but not by de novo pyrimidine synthesis, suggesting that NHC 325 triphosphorylation is not impaired by DHODH inhibition. Thus, upon combined 326 treatment, virus RNA will contain a larger proportion of NHC vs cytidine. In subsequent 327 rounds of virus RNA replication, this will lead to misincorporations of adenine bases 328 instead of guanine (Janion, 1978;Janion and Glickman, 1980;Salganik et al., 1973)  suppressor of purine biosynthesis, as an antagonist to SARS-CoV-2 replication, and 333 this was confirmed and expanded recently (Caruso et al., 2021;Zhang et al., 2021). 334 The principle behind this approach is similar to that of DHODH inhibitors, which also 335 interfere with nucleotide biosynthesis. In our previous work, we also reported that 336 Methotrexate cooperates with the antiviral purine analogue Remdesivir (Stegmann et 337 al., 2021). However, this was not as pronounced as the synergy between NHC and 338 DHODH inhibitors reported here. We speculate that even a relatively subtle increase 339 in the ratio of NHC and cytidine in the template RNA strongly enhances the proportion 340 of defective virions (Graci andCameron, 2004, 2008), whereas the inhibitory effect of 341 Remdesivir is only mildly enhanced by a reduction in available ATP. We also consider 342 the possibility that the combined drugs might trigger signaling pathways that further 343 interfere with virus replication. For instance, DHODH inhibition was found to affect the NHC is a mutagen to bacteria (Janion, 1978;Janion and Glickman, 1980;Jena, 2020;372 Negishi et al., 1983;Popowska and Janion, 1974;Salganik et al., 1973). Presumably, 373 NHC is converted to its 2'-deoxy form and then incorporated into bacterial DNA, 374 Molnupiravir has not been reported to cause unacceptable levels of toxicities so far. 384 Perhaps even more remarkably, ribavirin was not found cancerogenic after being used 385 for decades in hepatitis C treatment, although its mechanism of action is also based 386 on mutagenesis of virus RNA (Crotty et al., 2000). Thus, there is reason for cautious 387 use of NHC-based drugs in the clinics, probably prohibiting their use during pregnancy; 388 but in the face of ongoing pandemics, we still propose that mutagenesis in bacteria 389 should not preclude the clinical evaluation of NHC and its prodrugs at least for the 390 treatment of severe cases of COVID-19. 391 Remarkably, NHC treatment of coronavirus-infected cells did not give rise to resistant 392 viruses, even after prolonged and repeated incubation (Agostini et al., 2019). Likewise, 393 DHODH-inhibitors have a cellular rather than a viral target, thus providing little if any 394 opportunity for resistant virus mutants to arise. Along with our finding that current virus 395 variants of concern (VOC) respond similarly to the original SARS-CoV-2 strain (Fig.  396  4), this raises the hope that the drug combination will be universally applicable to treat 397 most if not all SARS-CoV-2 variants, reducing disease burden and virus spread. Negishi, K., Harada, C., Ohara, Y., Oohara, K., Nitta, N., and Hayatsu, H. (1983). N4-516 aminocytidine, a nucleoside analog that has an exceptionally high mutagenic activity. 517 Nucleic acids research 11, 5223-5233. Tharnish, P.M., Ramesh, M., Chu, C.K., Jordan, R., Shi, J., et al. (2003). 548 Ribonucleoside analogue that blocks replication of bovine viral diarrhea and hepatitis 549 C viruses in culture. Antimicrobial agents and chemotherapy 47, 244-254. 550 Tegally, H., Wilkinson, E., Giovanetti, M., Iranzadeh, A., Fonseca, V., Giandhari, J., 551 Doolabh, D., Pillay, S., San, E.J., Msomi, N., et al. (2021). Detection of a SARS-CoV-552 2 variant of concern in South Africa. Nature 592, 438-443. RNA was isolated from the cell supernatant, followed by quantitative RT-PCR to detect 640 viral RNA and calculate the amount of SARS-CoV-2 RNA copies per mL (mean, n=3). 641 The combination index (CI) was calculated using the CompuSyn software to determine 642 the degree of drug synergism. 643

(B)
Reduced virus RNA progeny in the presence of NHC and DHODH inhibitors. 644 Vero E6 cells were treated with drugs and/or infected, followed by quantitative 645 detection of SARS-CoV-2 RNA. The amount of RNA found upon infection without drug 646 treatment was defined as 100%, and the other RNA quantities were normalized 647 accordingly. RNA was also isolated from the virus inoculum used to infect the cells. 648 The drug combination was found capable of reducing virus RNA yield by more than 649 100-fold as compared to single drug treatments (mean with SD, n=3). 650

(C)
In Calu-3 cells, the combination of NHC and the DHODH inhibitors IMU-838, 651 BAY2402234 or teriflunomide strongly reduced the amount of viral RNA released to 652 the supernatant. were stained with DAPI, and the SARS-CoV-2 Spike and Nucleoprotein were detected 660 by specific antibodies using immunofluorescence. 661

(D and E)
Reduced viral protein synthesis in the presence of NHC and DHODH 662 inhibitors. Upon drug treatment and/or infection of Vero E6 cells as in Fig. 1, the viral 663 Spike and Nucleoprotein as well as DHODH and HSC70 (loading control) were 664 detected by immunoblot analysis. The TCID50 of virus progeny was determined upon treatment with 669 NHC and DHODH inhibitors, upon infection with SARS-CoV-2 wildtype and two 670 variants of concern (VOC). Vero E6 cells were treated with drugs for 24 hrs before and 671 then throughout the infection. Cells were infected with SARS-CoV-2 (wildtype, (A)), 672 B.1.1.7 (Alpha, (B)) or B.1.351 (Beta, (C)) (MOI 0.1) and further incubated for 48 hrs. 673 The supernatant was titrated to determine the TCID50/mL. Note that both variants 674 responded similarly to the wildtype, indicating that the drug combination is effective 675 against newly emerged SARS-CoV-2 variants.