key: cord-0265871-3kpsmdo4 authors: Stegmann, Kim M.; Dickmanns, Antje; Gerber, Sabrina; Nikolova, Vella; Klemke, Luisa; Manzini, Valentina; Schlösser, Denise; Bierwirth, Cathrin; Freund, Julia; Sitte, Maren; Lugert, Raimond; Salinas, Gabriela; Görlich, Dirk; Wollnik, Bernd; Groß, Uwe; Dobbelstein, Matthias title: The folate antagonist methotrexate diminishes replication of the coronavirus SARS-CoV-2 and enhances the antiviral efficacy of remdesivir in cell culture models date: 2020-07-20 journal: bioRxiv DOI: 10.1101/2020.07.18.210013 sha: 23a31dad8e28ffe8df92d7ee2aaa0daa22463c08 doc_id: 265871 cord_uid: 3kpsmdo4 The search for successful therapies of infections with the coronavirus SARS-CoV-2 is ongoing. We tested inhibition of host cell nucleotide synthesis as a promising strategy to decrease the replication of SARS-CoV-2-RNA, thus diminishing the formation of virus progeny. Methotrexate (MTX) is an established drug for cancer therapy and to induce immunosuppression. The drug inhibits dihydrofolate reductase and other enzymes required for the synthesis of nucleotides. Strikingly, the replication of SARS-CoV-2 was inhibited by MTX in therapeutic concentrations around 1 μM, leading to more than 1000-fold reductions in virus progeny in Vero C1008 (Vero E6) as well as Calu-3 cells. Virus replication was more sensitive to equivalent concentrations of MTX than of the established antiviral agent remdesivir. MTX strongly diminished the synthesis of viral structural proteins and the amount of released virus RNA. Virus replication and protein synthesis were rescued by folinic acid (leucovorin) and also by inosine, indicating that purine depletion is the principal mechanism that allows MTX to reduce virus RNA synthesis. The combination of MTX with remdesivir led to synergistic impairment of virus replication, even at 300 nM MTX. The use of MTX in treating SARS-CoV-2 infections still awaits further evaluation regarding toxicity and efficacy in infected organisms, rather than cultured cells. Within the frame of these caveats, however, our results raise the perspective of a two-fold benefit from repurposing MTX for treating COVID-19. Firstly, its previously known ability to reduce aberrant inflammatory responses might dampen respiratory distress. In addition, its direct antiviral activity described here would limit the dissemination of the virus. SIGNIFICANCE MTX is one of the earliest cancer drugs to be developed, giving rise to seven decades of clinical experience. It is on the World Health Organization’s List of Essential Medicines, can be administered orally or parenterally, and its costs are at single digit € or $ amounts/day for standard treatment. In case of its successful further preclinical evaluation for treating SARS-CoV-2 infections, its repurposing to treat COVID-19 would thus be feasible, especially under low-resource conditions. Additional drugs exist to interfere with the synthesis of nucleotides, e.g. additional folate antagonists, inhibitors of GMP synthetase, or inhibitors of dihydroorotate dehydrogenase (DHODH). Such inhibitors have been approved as drugs for different purposes and might represent further therapeutic options against infections with SARS-CoV-2 Remdesivir is currently the most established drug for treating COVID-19. Our results argue that MTX and remdesivir, even at moderate concentrations, can act in a synergistic fashion to repress virus replication to a considerably greater extent than either drug alone. COVID-19, in its severe forms, is characterized by pneumonia and acute respiratory distress syndrome, and additional organ involvements. These manifestations are not necessarily a direct consequence of virus replication and cytopathic effects, but rather a result of an uncontrolled inflammatory and immune response. Anti-inflammatory drugs such as glucocorticoids are thus being evaluated for treating COVID-19. However, this bears the risk of re-activating virus spread by suppressing a sufficient and specific immune response. In this situation, it is tempting to speculate that MTX might suppress both excessive inflammation as well as virus replication at the same time, thus limiting both the pathogenesis of pneumonia and also the spread of virus within a patient. The SARS-CoV-2 pandemic has so far infected > 12 million people, with > 500,000 85 deaths ascribed to COVID-19 so far (July 2020). The fast spread of the virus, currently 86 most notable in the Americas and south Asia, raises the need for readily available 87 therapies, preferably through drug repurposing. In addition to viral proteins, cellular 88 pathways represent attractive targets, with fewer opportunities for viruses to develop 89 drug resistance. of action is to prevent the synthesis of folic acid in bacteria (Estrada et al., 2016) . 107 Methotrexate (MTX), along with the closely related aminopterin, represents the most 108 established antifolate to act on eukaryotic cells. They belong to the earliest cancer 109 drugs whatsoever, since they were first characterized by Sidney Farber and 110 colleagues in the forties of the last century (Farber et al., 1948) , and have been in 111 clinical use ever since. They were first applied at high doses to treat leukemia as well 112 as solid malignancies. Later, however, they were found to suppress the inflammatory 113 and immune response even at low doses (Cronstein and Aune, 2020) . Until today, 114 MTX is broadly used to treat rheumatoid arthritis and other autoimmune diseases, and 115 it is also part of current standard anti-cancer regimens. It is bioavailable orally and 116 parenterally, inexpensive, and, with seven decades of clinical use, one of the best-117 characterized drugs in the clinics. Its disadvantages are often a result of long-term 118 treatment, which would not be necessary in the case of treating an acute virus 119 infection. Of note, however, pregnancy is a contraindication for MTX, due to its 120 For RNA isolation, the SARS-CoV-2 containing cell culture supernatant was mixed 164 Vero E6 cells were seeded into 24-well-plates and treated/infected as indicated. After 214 fixation of the cells and Hoechst staining to visualize nuclear DNA, the numbers of 215 nuclei were counted using the Celígo ® S Imaging Cytometer (Nexcelom Bioscience). SAM files were processed using SAMTOOLS/1.9 (Li, 2011) . In short, SAM files were 244 converted into BAM files using samtools view, followed by merging of technical 245 replicates using samtools merge. The resulting BAM files were sorted according to 246 read names using samtools sort with the -n option and mate-scores were added to 247 each read pair using samtools fixmate. The resulting BAM files were subsequently re-248 sorted according to genomic coordinates using samtools sort, PCR duplicates were 249 removed using samtools markdup with default settings, and the corresponding index 250 files were generated using samtools index. Sequencing depth was determined using We isolated SARS-CoV-2 from a patient sample taken in March 2020 at Göttingen, 272 Germany. Throat swab material was used to inoculate Vero C1008 cells (i.e. Vero E6 273 cells; shortly termed Vero cells from here on). The supernatant was passaged twice 274 more to expand the virus, and cell supernatant was subjected to deep sequencing 275 analysis. More than 80*10 6 reads, 2*150bp each, were taken and aligned with the 276 Next, we detected and quantified the viral RNA released from infected cells. With 287 decreasing virus inoculate, the yield of virus progeny after two days first remained at 288 a maximum and then steeply decreased (Fig. 1B) . The decrease in virus yield was 289 more than linear compared to the decrease in inoculum. This could not solely be 290 explained by the presence of defective virus particles. Rather, we propose that more 291 than one virus particle might be required for the productive infection of a cell, leading 292 to a more than proportional decrease in infectivity with lower virus concentrations. 293 Quantitative RT-PCR with comparison to a standard (kind gift by the M. L. Schmidt, 294 Robert Koch Institute, Berlin, Germany) revealed that up to 4*10 6 copies of viral 295 genomic RNA were released from each cell in the infected culture. Given the length of 296 the viral genome of roughly 3*10 4 nucleotides, this suggests that one infected cell 297 needs to produce at least 3*4*10 10 = 1,2*10 11 nucleotides for virus RNA synthesis -298 and this does not include subgenomic virus RNA to encode virus proteins, nor negative 299 strand RNA intermediates. Hence, virus production requires about 20 times more 300 nucleotides than the replication of the cellular genome (6*10 9 nucleotides in a diploid 301 human cell). By this calculation, the demand of virus replication on nucleotide 302 biosynthesis corresponds to more than three times of what is needed for the re-303 synthesis of all ribosomal RNAs, assuming 5*10 6 ribosomes per cell (Lewin, 1994; 304 Milo et al., 2010) and 7*10 3 ribonucleotides per ribosome. Considering these numbers, 305 we reasoned that nucleotide biosynthesis might represent a limiting factor for virus 306 replication, and hence a suitable drug target. 307 308 As a strategy to interfere with nucleotide biosynthesis, and hence with virus replication, 310 we treated Vero cells with MTX before and during infection with SARS-CoV-2. 311 Strikingly, the treatment with MTX strongly antagonized the infection. MTX largely 312 prevented the cytopathic effect (CPE) observed in infected cells, whereas up to 10 µM 313 MTX did not produce obvious morphologic signs of cytotoxicity ( Fig. 2A) , in agreement 314 with a previous preliminary report (Xing et al., 2020) . The amount of virus RNA 315 released into the media was reduced up to 1000-fold by MTX in Vero cells (Fig. 2B) , 316 and the effective concentration of MTX was around 1 µM, which was lower than the 317 effective concentration of the established antiviral drug remdesivir, i.e. 3 µM (Fig. 2B) . 318 MTX diminished SARS-CoV-2 progeny even at 100 nM concentration when using 319 Calu-3 cells (Fig. 2C) , a cell line that can be used to model bronchial epithelia (Foster 320 et al., 2000) . Taken together, these results indicate that MTX is a potent inhibitor of dsRNA, were severely reduced by MTX (Fig. 3A, B ). This argues that not just the 330 release of virus particles is diminished by MTX, but also the earlier steps of the 331 infectious cycle, i.e. the synthesis of RNA and virus proteins. 332 CoV-2-infected cells, but it strongly decreases the amount of cellular ATP. 335 can affect the levels of DHFR, we performed immunoblot analyses of Vero cells that 337 were infected with SARS-CoV-2 and/or treated with MTX. We found infected cells to 338 express the virus N and S proteins, whereas the amounts of these virus proteins were 339 strongly diminished by MTX (Fig. 4A) monophosphate (Chu et al., 1990) . This then reduces the availability of 378 deoxythymidine triphosphate, which is required for the synthesis of DNA but not RNA. 379 Finally, MTX also acts as an inhibitor of methylene tetrahydrofolate-reductase 380 (MTHFR) (Chabner et al., 1985) . In addition to the shortage of the MTHFR substrate 381 5,10 methylene THF, this direct inhibition reduces the regeneration of methionine from 382 homocysteine. Methionine is required to synthesize S-adenosylmethionine (SAM), a 383 necessary substrate of methylations of proteins (e.g. histones) as well as the RNA 5'-384 cap. Given these diverse mechanisms of action by MTX, we performed rescue 385 experiments to elucidate the mechanism(s) required to interfere with virus replication. 386 First, we added folinic acid (leucovorin) to the cells along with the MTX treatment. 387 Remarkably, this fully rescued virus replication. Folinic acid is readily converted to 5,10 388 methylene THF, thus re-supplying the source of methylation required by all MTX-389 inhibited reactions (Chan and Cronstein, 2010) . The rescue strongly suggests that the 390 impact of MTX on virus replication is indeed due to its competition with endogenous 391 folic acid and its metabolism (Fig. 5B) . 392 Moreover, the addition of inosine restored virus replication in the presence of MTX 393 (Fig. 5C ). Both leucovorin and inosine were also capable of re-establishing the 394 synthesis of virus N and S proteins (Fig. 5D) . Inosine is rapidly converted to inosine (reflecting viral progeny) by roughly 50%, the combination of both drugs led to a 412 reduction by more than 90% (Fig. 6A) . Similarly, virus proteins were still detectable 413 when the cells had been treated with single drugs at these concentrations, but the drug 414 combination virtually extinguished the corresponding immunofluorescence signals 415 (Fig. 6B) . We conclude that MTX enhances the efficacy of remdesivir, leading to 416 synergistic therapeutic efficacy. the observed reduction in spermidine, at least suggested that SAM levels were 451 reduced in infected cells (Gassen et al., 2020) . However, based on the fact that the 452 purine nucleoside inosine was capable of rescuing virus replication (Fig. 5C) , we 453 propose that the lack of purine synthesis represents the major mechanism of how MTX 454 limits SARS-CoV-2 replication. 455 Using MTX against other viruses has been considered previously. As early as in 1957, 456 it was reported that a folate antagonist was capable of protecting mice from infections 457 by single intramuscular application of 50 mg MTX, which is a common procedure to 486 induce abortions at early stages of pregnancy (Creinin and Vittinghoff, 1994 So far, the use of MTX in patients with rheumatoid arthritis was at least reported to be 517 of no detrimental effect when these patients had COVID-19 (Sanchez-Piedra et al., 518 2020). However, none of these studies was aiming at a direct antiviral effect of the 519 drugs. According to the results presented here, MTX would have the additional 520 advantage of interfering with virus replication itself. 521 The clinical efficacy of MTX against SARS-CoV-2 replication might be further 522 enhanced through the combination with antivirals such as remdesivir. We propose that 523 this combination might work in a more than additive fashion, but by a mechanistic 524 synergism. Specifically, the reduction in available purine nucleotides might enhance 525 the likelihood of incorporating remdesivir or similar antiviral drugs and their active 526 metabolites into the nascent viral RNA. In any case, treatment with remdesivir andMTX 527 might not only be tolerable but serve to potentiate antiviral efficacy. 528 In summary, albeit solely based on cell culture experiments so far, our study raises 529 the possibility of using MTX, alone or in combination with remdesivir, to limit the Polyglutamation of methotrexate. Is methotrexate 603 a prodrug? Methotrexate--how does it really work? Nature 605 reviews Mechanism of 607 thymidylate synthase inhibition by methotrexate in human neoplastic cell lines 608 and normal human myeloid progenitor cells. The Journal of biological 609 chemistry 611 High dose methotrexate in adult patients with osteosarcoma: clinical and 612 pharmacokinetic results Detection of 2019 novel coronavirus (2019-nCoV) by real-time 618 RT-PCR. 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