key: cord-0899572-u6aukcos
authors: Ogando, Natacha S.; Zevenhoven-Dobbe, Jessika C.; Jarhad, Dnyandev B.; Tripathi, Sushil Kumar; Lee, Hyuk Woo; Jeong, Lak Shin; Snijder, Eric J.; Posthuma, Clara C.
title: 6′,6′-Difluoro-aristeromycin is a potent inhibitor of MERS-coronavirus replication
date: 2021-09-04
journal: bioRxiv
DOI: 10.1101/2021.05.20.445077
sha: feb35a276fa39b24cfd22eab79baf114f396083e
doc_id: 899572
cord_uid: u6aukcos

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has highlighted the lack of treatments to combat infections with human or (potentially) zoonotic CoVs. Thus, it is critical to develop and evaluate antiviral compounds that either directly target CoV functions or modulate host functions involved in viral replication. Here, we demonstrate that low-micromolar concentrations of 6′,6′-difluoro-aristeromycin (DFA), an adenosine nucleoside analogue, strongly inhibit the replication of Middle East respiratory syndrome coronavirus (MERS-CoV) in a cell-based infection assay. DFA was designed to target S-adenosylhomocysteine (SAH) hydrolase and, consequently, may affect intracellular levels of the methyl donor S-adenosylmethionine, which is used by two CoV methyltransferases involved in the capping of the 5’ end of the viral mRNAs. Passaging of wild-type MERS-CoV in the presence of DFA selected a virus population with a ∼100-fold decreased DFA sensitivity, which carried various amino acid substitutions in viral nonstructural proteins (nsps). Specifically, mutations were present in the RNA polymerase subunit (nsp12) and in nsp13, the helicase subunit containing a nucleoside triphosphate hydrolase activity that has been implicated in CoV capping. We hypothesize that DFA directly or indirectly affects viral cap methylation, either by inhibiting the viral enzymes involved or by binding to SAH hydrolase. We also evaluated the antiviral activity of DFA against other betacoronaviruses, but found it to have limited impact on their replication, while being quite cytotoxic to the Calu-3 cells used for this comparison. Nevertheless, our results justify the further characterization of DFA derivatives as an inhibitor of MERS-CoV replication. Importance Currently, there is a lack of antiviral drugs with proven efficacy against human CoV infections including the MERS-CoV that is endemic in the Middle East, the pandemic SARS-CoV-2 and potential future zoonotic CoV. This highlights the importance to investigate new drug targets and identify compounds that can be used to inhibit CoV replication. In this study, we characterize the inhibitory effect of DFA on MERS-CoV replication by phenotypic studies, time-of-addition studies, and the generation and genotyping of a DFA-resistant virus population. Our results revealed that DFA needs further improvement to reduce its cytotoxic side-effects and potentially enhance its broad-spectrum activity. Despite this observation, we think that DFA can be used to understand the function and metabolic interactions of the CoV RNA-synthesizing machinery, or as a starting point for the design of new compounds of the same class.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has highlighted the 22 lack of treatments to combat infections with human or (potentially) zoonotic CoVs. Thus, it is 23 critical to develop and evaluate antiviral compounds that either directly target CoV functions or 24 modulate host functions involved in viral replication. Here, we demonstrate that low-micromolar 25 concentrations of 6′,6′-difluoro-aristeromycin (DFA), an adenosine nucleoside analogue, strongly 26 inhibit the replication of Middle East respiratory syndrome coronavirus (MERS-CoV) in a cell-27 based infection assay. DFA was designed to target S-adenosylhomocysteine (SAH) hydrolase and, 28 consequently, may affect intracellular levels of the methyl donor S-adenosylmethionine, which is 29 used by two CoV methyltransferases involved in the capping of the 5' end of the viral mRNAs. 30

Passaging of wild-type MERS-CoV in the presence of DFA selected a virus population with a ~100-31 fold decreased DFA sensitivity, which carried various amino acid substitutions in viral 32 nonstructural proteins (nsps). Specifically, mutations were present in the RNA polymerase 33 subunit (nsp12) and in nsp13, the helicase subunit containing a nucleoside triphosphate 34 hydrolase activity that has been implicated in CoV capping. We hypothesize that DFA directly or 35

indirectly affects viral cap methylation, either by inhibiting the viral enzymes involved or by 36 binding to SAH hydrolase. We also evaluated the antiviral activity of DFA against other 37 betacoronaviruses, but found it to have limited impact on their replication, while being quite 38 cytotoxic to the Calu-3 cells used for this comparison. Nevertheless, our results justify the further 39 characterization of DFA derivatives as an inhibitor of MERS-CoV replication. 40

Previously, the emergence of severe acute respiratory syndrome coronavirus (SARS-CoV; in 2003 54 in China) and Middle East respiratory syndrome coronavirus (MERS-CoV; in including compounds directly targeting viral functions, like viral proteases and the RNA 64 polymerase, and host factor-targeting inhibitors (reviewed in (9-12)). 65

Coronaviruses are positive-stranded RNA (+RNA) viruses with a single genomic RNA of 66 approximately 30 kb that is replicated in the cytoplasm of infected cells. Following entry, the 5'-67 capped viral genome is recognized and translated by host ribosomes to yield the replicase 68 polyproteins pp1a and pp1ab (13). Subsequently, these large precursors are processed into 16 69 individual nonstructural proteins (nsp 1 to 16), which are released following polyprotein cleavage 70 by two or three internal proteases. Together, the nsps form a multi-enzyme complex that ensures 71 the replication of the viral genome and the transcription of a set of subgenomic mRNAs (reviewed 72 in (14, 15) ). The enzymatic core of this complex is formed by the nsp12 RNA-dependent RNA 73 polymerase (RdRp) that synthesizes RNA with the help of the auxiliary factors nsp7 and nsp8 (16, 74 17), the nsp13 helicase that unwinds RNA duplexes (18) (19) (20) , and several other RNA-processing 75 enzymes residing in nsp12-nsp16 (reviewed in (15, 21, 22) ). These also include a 3'-to-5' 76 exoribonuclease (nsp14-ExoN) that is thought to increase replication fidelity by correcting 77 mismatches sustained during RNA synthesis (reviewed in (23-26) ). The viral structural and 78 accessory proteins, encoded by smaller open reading frames located in the 3'-proximal part of 79 the genome, are expressed from a set of 5'-capped and 3'-polyadenylated subgenomic mRNAs 80 (reviewed in (15, 22) ). Apart from ensuring mRNA recognition during formation of the ribosomal 81 preinitiation complex, the 5'-terminal cap structure protects the viral mRNAs from degradation 82 by cellular ribonucleases and prevents detection by the host's intracellular pathogen recognition 83 receptors, which would trigger innate immune responses (reviewed in (27)). 84

The CoV capping mechanism is thought to consist of four sequential reactions: (i) an RNA 85 triphosphatase activity residing in nsp13 removes the γ-phosphate group from the 5′-86 triphosphorylated RNA (28, 29); (ii) a guanosine monophosphate (GMP) is transferred to the 5'-87 diphosphate terminus by a yet to be confirmed guanylyltransferase (GTase)(30), which was 88 recently proposed to reside in the N-terminal nucleotidyl transferase (NiRAN) domain of nsp12 89 (31); (iii) the nsp14 methyltransferase (MTase) methylates the cap's 5'-terminal guanine at the 90 N7-position, producing the so-called cap-0 structure, 7me GpppN (32); (iv) finally, a cap-1 structure 91 is formed when nsp16, in complex with its nsp10 co-factor, methylates the ribose 2'-O-position 92 of the first transcribed nucleotide of each viral RNA, converting 7me GpppN into 7me GpppN2'me (33) . Recently, using cell-based assays for MERS-CoV, SARS-CoV, chikungunya and Zika virus 109 replication, we described the inhibitory potential of a set of adenosine and selenoadenosine 110 analogues (38). These compounds were derived from aristeromycin, a well-known carbocyclic 111 nucleoside compound that inhibits SAH hydrolase and exhibits anti-viral, anti-cancer and anti-112 toxoplasma activities (reviewed in (42)). These aristeromycin derivatives are nucleoside 113 analogues designed to directly target viral RdRp activity and/or indirectly target the methylation 114 of viral RNA by inhibiting the host SAH hydrolase (38) . From this library, we identified 6′,6′- Having established the strong inhibition of MERS-CoV replication by DFA, we also tested its 155 monophosphoramidate pro-drug (pDFA; Fig. 2A ) in a CPE-reduction assay. This compound was 156 synthesized in order to circumvent the rate-limiting first phosphorylation step that presumably 157 restricts the efficient metabolization of nucleoside analogues like DFA following their uptake by 158 the cell (reviewed in (47)). Unfortunately, in this case the pro-drug was less active than DFA itself, 159 independent of the cell line used (Fig. 2B) . Although the chemical and structural modifications of 160 the prodrug decreased its cytotoxicity, the calculated EC50 values, 9 µM in Vero cells and 36 µM 161 in MRC-5 cells, were more than 10 times higher than the ones measured for DFA (Fig. 1) . 162

Therefore, pDFA was not included in subsequent experiments. 163

To characterize the mechanism of action of DFA in more detail, a time-of-addition assay was 166 performed to determine which stage of the viral replication cycle was inhibited by the compound. 167 In order to identify mutations that contribute to DFA resistance, we sequenced the wtP10 and 208 L3P10 virus populations by Illumina next-generation sequencing. Subsequently, sequencing reads 209 were mapped to the reference sequence of MERS-CoV strain EMC/2012 (NC_019843.3; (3)). 210

Sequence variants constituting less than 10% of the total population of viral reads were excluded 211 from further analysis. Compared to the original viral sequence, a total of 14 changes were 212 identified: five synonymous and nine non-synonymous mutations distributed across genes 213 encoding nine different viral proteins. Two of the identified non-synonymous mutations were 214 present both in wtP10 and L3P10, suggesting they were cell culture adaptations acquired during 215 repeated passaging. These mutations, G12033-to-A and C21068-to-U, resulted in D73N and L56F 216 substitutions in nsp7 and nsp16, respectively. Likewise, the five translationally silent mutations 217 in the L3P10 population were considered unlikely to be relevant for its phenotypic profile. Of the 218 remaining seven (Table 1) non-synonymous mutations, one mapped to the accessory protein 219 encoded by ORF5, which is not essential for viral replication in cell culture (51)-(52), and one to 220 the Spike protein (53), which also is an unlikely target for inhibition by nucleoside analogues. This 221 left five L3P10-specific mutations leading to amino acid substitutions in the viral replicase 222 subunits nsp1, 3, 12, and 13 that may be associated with DFA resistance. As shown in Table 1 , all 223 of these were present in only part of the viral population (in 37% to 55% of the total reads), 224 suggesting a complex pattern of virus evolution with DFA resistance possibly relying on (different) 225 combinations of mutations (see Discussion). The short NGS read-length (150 nucleotides) did not 226 allow us to determine which mutations were combined in the same genome. Since further 227 optimization of this compound class is needed to improve its selectivity index (Table 2) , we did 228 not perform follow-up experiments to elucidate its mode of action at this stage. 229 230

To explore the potential of DFA as a broad-spectrum antiviral, Calu-3 cells, human lung cells that 232 supports MERS-CoV, SARS-CoV and SARS-CoV-2 replication (54, 55), were treated with increasing 233 concentrations of DFA and infected with each of these viruses in a dose response assay. By using 234 the same cell line for all three CoVs, differences in DFA up-take or metabolic conversion to its 235 triphosphate form were eliminated. The results showed a dose-dependent decrease in the 236 production of viral progeny for MERS-CoV (Fig. 5A ) and SARS-CoV-2 (Fig. 5C ) that followed the 237 cytotoxicity of the compound. At a DFA concentration of 3.2 µM, only a small reduction of MERS-238

CoV and SARS-CoV-2 progeny was observed, 0.5 to 1log10. Surprisingly, the antiviral activity of 239 DFA against MERS-CoV in Calu-3 cells was severely reduced when compared to results obtained 240 in other cell lines, including another human lung cell line MRC-5 (Fig. 1D-F) . In the case of SARS-241

CoV infection, a minor inhibitory effect was observed at concentrations that appeared to be 242 somewhat cytotoxic (Fig. 5B and 5D) , contrary to what was demonstrated in Vero E6 cells ((38) 243

and Table 2 ). Unfortunately, in Calu-3 cells cytotoxicity was detected at low compound 244 concentrations (>6.2 µM) and the inhibitory effects observed could thus be associated with an 245 overall decrease in relative cell viability. This indicates that the design of improved DFA 246 derivatives is needed to decrease cytotoxicity and improve inhibitory potency. 247 248

This study describes that treatment with low-micromolar DFA concentrations exhibits a strong 250 antiviral effect on MERS-CoV replication in cell culture-based infection models (Fig. 1) . Time-of-251 addition assays indicated that DFA reduced MERS-CoV progeny production when cells were 252 treated prior to, at the time of, or within 4 h after infection (Fig. 3) , suggesting that DFA interferes 253 with the early stage of replication. Propagation of MERS-CoV in the presence of DFA led to the 254 selection of a virus population with strongly enhanced resistance to this compound (Fig. 4) . 255

Subsequent sequence analysis revealed a potentially complex pattern of resistance evolution, 256 exhibiting multiple mutations that are present in only part of the virus population, including 257 several that map to enzymes involved in viral RNA synthesis and mRNA capping (Table 1) . 258 DFA was originally designed to target the host SAH hydrolase directly and was demonstrated to 259 inhibit this enzyme in vitro with an IC50 (50% inhibitory concentration) of 1.06 µM (38). The 260

compound is a carbocyclic adenosine analogue based on the parental inhibitor aristeromycin (56, 261 57), which was further modified by incorporation of a difluorine group at the 6' (top) position of 262 its sugar ring ((38) and Fig. 2A ). This modification improved the binding affinity of the compound 263 for human SAH hydrolase and, consequently, the inhibition of its enzymatic activity. Previous 264 studies demonstrated that treatment of cells with high-affinity SAH hydrolase inhibitors, such as 265 neplanocin A and aristeromycin, increases the intracellular SAH concentration, preventing the 266 metabolic conversion of SAH to adenosine and L-homocysteine (reviewed in (58)). Therefore, SAH 267 hydrolase inhibitors reduce or deplete the intracellular pools of homocysteine and adenosine, 268 the latter being produced exclusively by SAH hydrolysis. As the SAM methyl donor is formed via 269 homocysteine trans-sulfuration or the adenosine kinase pathway, SAH hydrolase regulates the 270 intracellular SAM levels and consequently the cell's SAM-dependent methylation reactions. 271

Moreover, SAH accumulation can also reduce the activity of SAM-dependent methyltransferases 272 by feed-back inhibition, as SAH can bind to their active site with higher affinity than SAM itself 273 domain was proposed to function as the capping GTase (31), while also nsp13 has been 294 implicated in the CoV capping pathway (see Introduction; reviewed in (30)). As the identified 295 mutations have not been characterized in structural or biochemical studies, one can only 296 speculate about their potential role in viral replication and DFA resistance 297

Further phenotypic and mechanistic studies will be needed to better understand the mode of 298 action of DFA. Additionally, cloning of L3P10 viruses by plaque picking could help to define the 299 combination(s) of mutations that are the basis for DFA resistance, by evaluating their frequency 300 of occurrence and associated replication and plaque phenotype. 301

As a nucleoside analogue, DFA was also considered to be a potential RdRp inhibitor. This would 302 require uptake by the cell's nucleoside transporters, and subsequent phosphorylation into a 303 triphosphorylated product that could be incorporated into the RNA chain during viral RNA 304 synthesis (reviewed on (47)). In order to improve absorption of the compound by the cells and 305 metabolism into its active form, a prodrug of DFA was synthesized and its antiviral activity was 306 evaluated. In theory, the monophosphoramidate mask would promote the second 307 phosphorylation to occur once the compound enters the cytoplasm by circumventing the rate-308 limiting step of the first phosphorylation. However, when compared to DFA, the EC50 of the 309 prodrug was more than 10 times higher (Fig. 2) , in contrast to results obtained with prodrugs of 310 other nucleoside analogues (46, 68). In previous work, structure-activity studies and tests of 311 several purine and pyrimidine analogues of DFA suggested that DFA is most likely not targeting 312 the RdRp (38, 61, 69 ). This notion is also supported by the fact that the genotypic profile obtained 313 for L3P10 did not reveal mutations in the RdRp domain of nsp12. 314

In this study, we demonstrate that DFA can inhibit the replication of MERS-CoV, but that the 315 design and development of DFA-based derivatives will be required to reduce cytotoxic side 316 effects. Combining our results in this study with our previous report (38), showing that DFA can 317 inhibit chikungunya and Zika virus, DFA appears to be an interesting compound for further 318 development as a broad-spectrum antiviral agent. 319 320

Cell culture and viruses 322

Vero cells were a kind gift from the Department of Viroscience, Erasmus Medical Center, 323

Rotterdam, the Netherlands, and Huh7 cells were provided by Dr. Ralf Bartenschlager, Heidelberg 324

University, Germany. Vero, Vero E6, Huh7, MRC-5 and Calu-3 were cultured as described before 325 (48, 49, (70) (71) (72) . All cells were incubated at 37°C with 5% CO2. Infections were carried out in Eagle's "mock"-infected with medium to monitor the (potential) cytotoxicity of the compound. Plates 350 were incubated for three days (or as mentioned) at 37°C, after which cell viability was measured 351 using the colorimetric CellTiter 96® Aqueous Non-Radioactive Cell Proliferation kit (Promega). 352

The absorption at 495 nm was measured using a monochromatic filter in a multimode plate 353 reader (Envision; Perkin Elmer). Data were normalized to the "mock"-infected control, after 354 which EC50 and CC50 values were calculated using non-linear regression with Graph-Pad Prism 355 V8.0. Each experiment was performed at least in quadruplicate and repeated at least twice. Confluent monolayers of MRC-5 or Vero cells were seeded in 12-well plates in 1 ml/well of the 375 appropriate medium (see above), and were grown overnight at 37⁰C. Treatment of cells (before, 376 during or after infection) was performed using 0.6 µM (for Vero) and 12.5 µM (for MRC-5) of 377 compound solution freshly prepared in EMEM-2%FCS medium. Cells were infected with MERS-378

CoV inoculum (MOI of 5) for 1h and washed three times with PBS. Subsequently, EMEM-2%FCS 379 medium was added to the cells and supplemented with compound solution in 2-h intervals to a 380 final concentration as mentioned above. Supernatants were collected 16 h p.i. and viral titers 381 were determined by plaque assay. with cell culture adaptation. Additionally, a "mock"-infected well treated with the same 389 concentration of compound in each passage was evaluated for cytotoxicity by light microcopy. 390

Supernatants were harvested when 80% to full CPE was observed (usually at 3 d p.i.). Three 391 lineages were generated by serial passaging, but only lineage 3 was used for next-generation 392 sequencing. To this end, RNA was isolated from 200 µl of virus-containing cell culture 393 supernatants using TriPure isolation reagent (Roche Applied Science) and purified according to 394 manufacturer's instructions. The RNA concentration was measured using a Qubit fluorometer 395 and RNA High Sensitivity kit (Thermo Fisher Scientific). NGS sample preparation and analysis were 396 performed as described previously (75). After filtration and trimming of data, the remaining reads 397 were mapped to the MERS-CoV GenBank reference sequence (NC_019843; (3, 4) Calu-3 ~2 >25 >12

Calu-3 ~5 >25 >5

Calu-3 ~2 >25 >12

EC50s values were calculated based on results obtained in dose response assay, while CC50s values were determined in a cell viability assay as described in materials and methods. SI, selectivity index was calculated by comparing CC50 with EC50 values.

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