Nudix hydrolase 18 catalyzes the hydrolysis of active triphosphate metabolites of the antivirals remdesivir, ribavirin, and molnupiravir

Remdesivir and molnupiravir have gained considerable interest because of their demonstrated activity against SARS-CoV-2. These antivirals are converted intracellularly to their active triphosphate forms remdesivir-TP and molnupiravir-TP. Cellular hydrolysis of these active metabolites would consequently decrease the efficiency of these drugs; however, whether endogenous enzymes that can catalyze this hydrolysis exist is unknown. Here, we tested remdesivir-TP as a substrate against a panel of human hydrolases and found that only Nudix hydrolase (NUDT) 18 catalyzed the hydrolysis of remdesivir-TP with notable activity. The kcat/Km value of NUDT18 for remdesivir-TP was determined to be 17,700 s−1M−1, suggesting that NUDT18-catalyzed hydrolysis of remdesivir-TP may occur in cells. Moreover, we demonstrate that the triphosphates of the antivirals ribavirin and molnupiravir are also hydrolyzed by NUDT18, albeit with lower efficiency than Remdesivir-TP. Low activity was also observed with the triphosphate forms of sofosbuvir and aciclovir. This is the first report showing that NUDT18 hydrolyzes triphosphates of nucleoside analogs of exogenous origin, suggesting that NUDT18 can act as a cellular sanitizer of modified nucleotides and may influence the antiviral efficacy of remdesivir, molnupiravir, and ribavirin. As NUDT18 is expressed in respiratory epithelial cells, it may limit the antiviral efficacy of remdesivir and molnupiravir against SARS-CoV-2 replication by decreasing the intracellular concentration of their active metabolites at their intended site of action. Remdesivir and molnupiravir have gained considerable interest because of their demonstrated activity against SARS-CoV-2. These antivirals are converted intracellularly to their active triphosphate forms remdesivir-TP and molnupiravir-TP. Cellular hydrolysis of these active metabolites would consequently decrease the efficiency of these drugs; however, whether endogenous enzymes that can catalyze this hydrolysis exist is unknown. Here, we tested remdesivir-TP as a substrate against a panel of human hydrolases and found that only Nudix hydrolase (NUDT) 18 catalyzed the hydrolysis of remdesivir-TP with notable activity. The kcat/Km value of NUDT18 for remdesivir-TP was determined to be 17,700 s−1M−1, suggesting that NUDT18-catalyzed hydrolysis of remdesivir-TP may occur in cells. Moreover, we demonstrate that the triphosphates of the antivirals ribavirin and molnupiravir are also hydrolyzed by NUDT18, albeit with lower efficiency than Remdesivir-TP. Low activity was also observed with the triphosphate forms of sofosbuvir and aciclovir. This is the first report showing that NUDT18 hydrolyzes triphosphates of nucleoside analogs of exogenous origin, suggesting that NUDT18 can act as a cellular sanitizer of modified nucleotides and may influence the antiviral efficacy of remdesivir, molnupiravir, and ribavirin. As NUDT18 is expressed in respiratory epithelial cells, it may limit the antiviral efficacy of remdesivir and molnupiravir against SARS-CoV-2 replication by decreasing the intracellular concentration of their active metabolites at their intended site of action. The recent SARS-CoV-2 pandemic with its fast worldwide spread and high mortality rate led to put major efforts to repurpose already approved antiviral drugs in order to quickly control the outbreak and thereby save lives. Remdesivir (GS-5734) was originally developed for the treatment of Ebola but is a broad-acting antiviral which has been found to reduce the replication of coronaviruses both in cell culture and animal models (1de Wit E. Feldmann F. Cronin J. Jordan R. Okumura A. Thomas T. et al.Prophylactic and therapeutic remdesivir (GS-5734) treatment in the rhesus macaque model of MERS-CoV infection.Proc. Natl. Acad. Sci. U. S. A. 2020; 117: 6771-6776Crossref PubMed Scopus (554) Google Scholar). Remdesivir treatment was shown to decrease the severity of disease after MERS-CoV infection via decreasing virus replication and thereby reducing damage to the lungs when administered both before and after infection in a rhesus macaque model (1de Wit E. Feldmann F. Cronin J. Jordan R. Okumura A. Thomas T. et al.Prophylactic and therapeutic remdesivir (GS-5734) treatment in the rhesus macaque model of MERS-CoV infection.Proc. Natl. Acad. Sci. U. S. A. 2020; 117: 6771-6776Crossref PubMed Scopus (554) Google Scholar). Recently, remdesivir was tested in several clinical trials for treatment of COVID-19 caused by the coronavirus SARS-CoV-2. Remdesivir was shown to shorten the recovery time of hospitalized adults with COVID-19 as well as reducing the mortality rate (2Beigel J.H. Tomashek K.M. Dodd L.E. Mehta A.K. Zingman B.S. Kalil A.C. et al.Remdesivir for the treatment of Covid-19 - final report.N. Engl. J. Med. 2020; 383: 1813-1826Crossref PubMed Scopus (3427) Google Scholar) and suggested to be most effective if administered early after infection (3Ngo B.T. Marik P. Kory P. Shapiro L. Thomadsen R. Iglesias J. et al.The time to offer treatments for COVID-19.Expert Opin. Invest. Drugs. 2021; 30: 505-518Crossref PubMed Scopus (11) Google Scholar). Remdesivir was the first drug to be approved for the treatment of severe COVID-19 in July 2020 by the European Medicines Agency and later in the fall by the U.S Food and Drug Administration (2Beigel J.H. Tomashek K.M. Dodd L.E. Mehta A.K. Zingman B.S. Kalil A.C. et al.Remdesivir for the treatment of Covid-19 - final report.N. Engl. J. 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The monophosphate is subsequently phosphorylated by cellular kinases to its active triphosphate form (remdesivir-TP, aka GS-443902, Fig. 1, step 3). The antiviral effect of remdesivir-TP is mediated through its incorporation into the growing virus RNA chain by the viral RNA-dependent RNA polymerase (Fig. 1, step 4). Incorporation of the remdesivir metabolite causes the polymerase to stall and RNA replication to be terminated (Fig. 1, step 5), leading to a decrease in the production of viral RNA (7Agostini M.L. Andres E.L. Sims A.C. Graham R.L. Sheahan T.P. Lu X.T. et al.Coronavirus susceptibility to the antiviral remdesivir (GS-5734) is mediated by the viral polymerase and the Proofreading exoribonuclease.mBio. 2018; 9e00221-18Crossref PubMed Scopus (853) Google Scholar, 8Gordon C.J. Tchesnokov E.P. Feng J.Y. Porter D.P. Gotte M. The antiviral compound remdesivir potently inhibits RNA-dependent RNA polymerase from Middle East respiratory syndrome coronavirus.J. Biol. Chem. 2020; 295: 4773-4779Abstract Full Text Full Text PDF PubMed Scopus (490) Google Scholar). Molnupiravir (EIDD-2801) was, in November 2021, given a temporary authorization by the UK’s Medicines and Healthcare products Regulatory Agency for the treatment of mild to moderate COVID-19 in adults at risk for severe disease and one month later, an emergency use authorization was issued by the Food and Drug Administration agency. Administration of this orally active drug led to a clear reduction of SARS-CoV-2 replication as well as a quicker recovery time (9Fischer W. Eron J. Holman W. Cohen M.S. Fang L. Szewczyk L.J. et al.Molnupiravir, an oral antiviral treatment for COVID-19.medRxiv. 2021; https://doi.org/10.1101/2021.06.17.21258639Crossref Scopus (0) Google Scholar). In addition, early treatment with molnupiravir clearly reduced the risk of death or hospitalization in unvaccinated adults with COVID-19 at risk for severe disease (10Bernal A.J. Gomes da Silva M.M. Musungaie D.B. Kovalchuk E. Gonzalez A. Delos Reyes V. et al.Molnupiravir for oral treatment of Covid-19 in nonhospitalized patients.New Engl. J. Med. 2022; 386: 509-520Crossref PubMed Scopus (220) Google Scholar). Molnupiravir is a prodrug for β-d-N4-hydroxycytidine, which has previously been shown to be effective against various RNA viruses including influenza and other coronaviruses (11Toots M. Yoon J.J. Hart M. Natchus M.G. Painter G.R. Plemper R.K. Quantitative efficacy paradigms of the influenza clinical drug candidate EIDD-2801 in the ferret model.Transl. Res. 2020; 218: 16-28Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 12Sheahan T.P. Sims A.C. Zhou S. Graham R.L. Pruijssers A.J. Agostini M.L. et al.An orally bioavailable broad-spectrum antiviral inhibits SARS-CoV-2 in human airway epithelial cell cultures and multiple coronaviruses in mice.Sci. Med. 2020; PubMed Google Scholar) and is hydrolyzed to by in of its active metabolite G.R. G.R. J. V. et al.The and therapeutic activity of a active in a model of virus Res. PubMed Scopus Google Scholar). The incorporation of by RNA-dependent RNA polymerase into viral RNA causes a in the in a decrease in SARS-CoV-2 replication F. J. G. et of SARS-CoV-2 Biol. 2021; PubMed Scopus Google Scholar). is antiviral drug that was tested for activity SARS-CoV-2. the been to act as a broad-spectrum antiviral for both and RNA viruses J.H. R.K. antiviral activity of PubMed Google Scholar). has been in with to G. A. J.H. A. of with and ribavirin for The Full Text Full Text PDF PubMed Scopus Google Scholar) as well as other viral as D. J. for use of Opin. PubMed Scopus Google S. for patients with a and PubMed Scopus Google Scholar, A.K. N. for and the of ribavirin and PubMed Scopus Google Scholar) and virus N. J. S. M. S. J. et for virus infection in Engl. J. Med. PubMed Scopus Google Scholar). in with was shown to coronavirus replication in animal and human cell M. J. and coronavirus replication in animal and human cell Res. PubMed Scopus Google suggesting that ribavirin be a treatment for SARS-CoV-2. The of of ribavirin is and its metabolites have been suggested to several of which the antiviral may their intracellular J. L. S. J. et triphosphate ribavirin triphosphate and reduced activity in 2018; Scopus Google Scholar). it is that inhibits monophosphate leading to with cellular has been to viral RNA-dependent polymerase and may at also in the viral is hydrolyzed by the triphosphate and patients activity a reduced risk of suggesting that these patients have of the active ribavirin activity have been shown to against upon and ribavirin J. L. S. J. et triphosphate ribavirin triphosphate and reduced activity in 2018; Scopus Google Scholar, J. A.J. D. et against in patients for PubMed Scopus Google Scholar). The of ribavirin treatment the of cellular drug to several enzymes catalyze the hydrolysis of modified nucleoside triphosphates R. L. L. A. et the hydrolysis of Res. 2018; Google Scholar, L. A. U. T. et from the Biol. Chem. 2020; 295: Full Text Full Text PDF PubMed Scopus Google Scholar) and have been shown to the efficacy of various drugs including antivirals A. G. D. M. et hydrolyzes to the efficacy of Res. PubMed Scopus Google Scholar, D. A. N. et hydrolysis the efficacy of drug Chem. Biol. 2021; Full Text Full Text PDF PubMed Google Scholar). is Nudix hydrolase (NUDT) which we the hydrolysis of D. A. N. et hydrolysis the efficacy of drug Chem. Biol. 2021; Full Text Full Text PDF PubMed Google Scholar). In a hydrolysis of remdesivir-TP into catalyzed by a human hydrolase (Fig. 1, would reduce the cellular concentration of the active Moreover, of to the corresponding nucleoside through the of would its of the cell (Fig. 1, and decrease the antiviral of In order to if a hydrolase be found the of we tested remdesivir-TP as a substrate for a panel of human hydrolases and found that NUDT18 catalyzed the hydrolysis of by we tested NUDT18 for activity several antiviral triphosphates and found NUDT18 to also be an of and NUDT18 to only been to activity and has been suggested to the from their incorporation into D. R. M. hydrolyzes forms of and with and Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, M. M. The of human and in Res. 2018; PubMed Scopus Google Scholar). The of this that NUDT18 as a cellular sanitizer in a and to modified nucleotides of both endogenous and exogenous and the efficiency of the antiviral drugs remdesivir, molnupiravir, and ribavirin. In order to if human enzymes can the cellular concentration of the active metabolite of remdesivir and thereby reduce its efficacy as an antiviral we tested human and NUDT18 for hydrolysis activity with This of enzymes was showing that and NUDT18 have the to triphosphates or J. M. M. et and of the human hydrolase PubMed Scopus Google Scholar). and which are the most enzymes to the to which and NUDT18 catalyze the hydrolysis of nucleoside J. M. M. et and of the human hydrolase PubMed Scopus Google Scholar) and to the panel as to these only NUDT18 was found to catalyze the hydrolysis of remdesivir-TP (Fig. NUDT18 was shown to catalyze the hydrolysis of both the the and as well as the the and (Fig. and This was by an in the of in the of which hydrolyzes or activity is in the NUDT18 J. M. M. et and of the human hydrolase PubMed Scopus Google Scholar). the activity of with which one of the active metabolites of the antiviral we that may also other antiviral tested human as well as the human hydrolases and for activity with but activity with remdesivir-TP activity was with or (Fig. with NUDT18 activity an activity than remdesivir-TP at substrate concentration (Fig. The observed activity of NUDT18 with remdesivir-TP to other antiviral triphosphates as for found that is also hydrolyzed by NUDT18, albeit to the as remdesivir-TP (Fig. to NUDT18-catalyzed hydrolysis in the production of both ribavirin and as shown by an in the when was in the (Fig. NUDT18 was also shown to molnupiravir-TP. As by a production of in the of (Fig. hydrolysis of the the and to be In addition, previously shown to catalyze the hydrolysis of D. A. N. et hydrolysis the efficacy of drug Chem. Biol. 2021; Full Text Full Text PDF PubMed Google was found to catalyze the hydrolysis of albeit with lower efficiency than NUDT18, activity with this substrate (Fig. the activity of NUDT18 with these in and for with we of the NUDT18-catalyzed hydrolysis of and molnupiravir-TP. and these (Fig. and determined The value for NUDT18 was determined to be for which is in with a previously value of D. R. M. hydrolyzes forms of and with and Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The value for at was determined to be and the kcat/Km value was The value for remdesivir-TP was determined to be and the value was in a kcat/Km value of 17,700 The efficiency of NUDT18 for that would be the substrate remdesivir-TP at the the cellular remdesivir-TP concentration is in the order of than the concentration of an of the intracellular concentration of in T. et of the intracellular concentration of by 2020; Google and the that the cellular concentration of is in a or This that the activity of NUDT18 with remdesivir-TP may be of for the remdesivir-TP concentration in cells. The concentration of remdesivir in after treatment with a of was to be and the concentration of remdesivir-TP in was determined to be after of remdesivir M. P. V. S. L. et of remdesivir and in patients from 2020; PubMed Scopus Google Scholar, R. A. A. G. E. et and of remdesivir, an antiviral for treatment of in Sci. 2020; Google Scholar). The concentration of remdesivir-TP in to be than the remdesivir concentration in to the triphosphate in the cell cell the of that an in by the the efficiency of NUDT18 at is to be a concentration of remdesivir-TP in the cell of that one remdesivir triphosphate be NUDT18 that remdesivir-TP is hydrolyzed by NUDT18 in the of NUDT18 with triphosphates of antiviral of NUDT18 with and activity of NUDT18 with of triphosphates of the antivirals and the drug was tested by for at in and in the and of was the of a by of at of NUDT18 with and with and of determined from of phosphate at and in from Nudix of is and of determined from and with in in a is and of determined from and with in and as for NUDT18, remdesivir-TP to be the NUDT18 a value for hydrolysis that is and a value of in a efficiency value of s−1M−1, which is of the corresponding value for remdesivir-TP The efficiency of NUDT18 for was determined to s−1M−1, which is to the corresponding value for both the value and are lower for molnupiravir-TP. of the of remdesivir-TP and with of that the in the suggesting a of in the active site of In order to if NUDT18 can catalyze the hydrolysis of other antiviral we the substrate of NUDT18 and found that as well as are hydrolyzed by NUDT18 to to the NUDT18-catalyzed hydrolysis of remdesivir-TP and hydrolysis at both the and the phosphate in these with of the hydrolysis at of these (Fig. that the activity with the substrate in this is lower than the activity with with a kcat/Km value lower (Fig. and the kcat/Km value was than the corresponding kcat/Km value for The that a that the activity of NUDT18 with than in NUDT18, to the and the modified of in the active This in a of the phosphate for This is by a for than that of the are (Fig. whether the activity of NUDT18 with these antiviral triphosphates clinical for the of these antivirals a the of the (Fig. The that of the other tested activity with remdesivir-TP that only the active site of NUDT18 is and of this the of the for of human NUDT18 have been and both of to of these that the to the the is and in to other M. E. et of the hydrolase as a for Chem. 2020; PubMed Scopus Google Scholar). of remdesivir-TP to this site with of the of In the and druggable which and T. S. et by of the PubMed Scopus Google Scholar, M. A. D. M. et of a against Chem. Biol. 2020; PubMed Scopus Google Scholar). This that considerable are for NUDT18 to the of remdesivir-TP and other to a an as by et of the with of Biol. PubMed Scopus Google Scholar) also to a for remdesivir-TP with In order to to the of NUDT18 we trials of NUDT18 or with or This and from to and and we to in of the As only of the are for NUDT18, it is that the and the for a the L. P. in Res. PubMed Scopus Google which that is most to human and other from and of As remdesivir-TP is an of we in the with of to L. A. U. T. et from the Biol. Chem. 2020; 295: Full Text Full Text PDF PubMed Scopus Google Scholar) and S. T. M. T. et and of the human Nudix hydrolase the for its substrate Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of these that from the that the for substrate hydrolysis the (Fig. This of as to where the of remdesivir-TP would be in and the of the the of the a (Fig. and are clearly to the of and in that the forms an with the In NUDT18, the corresponding is an however, a in which would be of an is in (Fig. In the NUDT18 with the when to the of the in the occur in order to for the of this would the in a that is of and remdesivir-TP with its (Fig. The lower value of for NUDT18 it the active site of the than In the of a NUDT18 a of the NUDT18 to the substrate or to remdesivir-TP would be for the that occur the site upon the of these In an to which of the that influence their as NUDT18 we their of efficiency and of NUDT18-catalyzed hydrolysis for the tested to the corresponding for and the of the tested (Fig. NUDT18 has previously been to catalyze the hydrolysis of and with the efficiency D. R. M. hydrolyzes forms of and with and Biol. Chem. Full Text Full Text PDF PubMed Scopus Google that the can be in the active site of This a this of the nucleoside the tested the is for suggesting that NUDT18-catalyzed hydrolysis or is for remdesivir-TP than substrate the value is lower for the and active site of The lower which into efficiency for may in be to nucleoside triphosphates with lower to the active site to nucleoside The in efficiency remdesivir-TP and can be to in for hydrolysis their are The lower value for than remdesivir-TP a of for The NUDT18-catalyzed hydrolysis of to at the phosphate in the of hydrolysis at both the and the phosphate to the (Fig. and suggesting that of the of in the active site is in to of the of the the of remdesivir-TP and NUDT18 may to a in the of it is to the the that the active site of NUDT18 can with and that hydrolysis at with with the of a for these remdesivir-TP is a substrate for NUDT18, we that remdesivir and its metabolites may NUDT18 and tested and for by remdesivir, the remdesivir metabolite from cellular hydrolysis of and tested NUDT18 for by remdesivir and was found to be by remdesivir as well as by (Fig. was by of NUDT18 by of the tested remdesivir was The effect of remdesivir and its metabolites that remdesivir treatment is to have a effect the cellular activity of these The that NUDT18 can remdesivir-TP to and in the and are for be phosphorylated and a cellular the production of remdesivir-TP by kinases and hydrolysis catalyzed by NUDT18 is the of intracellular the concentration of In a the cellular of the metabolites of molnupiravir and ribavirin be by the of and NUDT18 and in the of and also NUDT18 to a in remdesivir it has to be in the that are by the NUDT18 is and is expressed in the lungs and in respiratory epithelial This that NUDT18 would be to the effective concentration of remdesivir-TP and at their intended site of action. of NUDT18 would be a to the efficacy of these NUDT18 are to the of The of intracellular for the efficiency of antivirals is by the activity of human with J. L. S. J. et triphosphate ribavirin triphosphate and reduced activity in 2018; Scopus Google Scholar). a of the human activity J. L. S. J. et triphosphate ribavirin triphosphate and reduced activity in 2018; Scopus Google Scholar) and it is clear that cellular activity the antiviral efficacy of ribavirin as well as its of NUDT18 in respiratory epithelial in a COVID-19 in considerable remdesivir-TP and may in lower efficiency of the corresponding antivirals than in a with lower NUDT18 This may in to remdesivir treatment Here, we that human NUDT18 can catalyze the hydrolysis of the active metabolites of the antivirals remdesivir, and These NUDT18 may be and the for of the cellular NUDT18 and in the efficacy of these antiviral In we a cellular of NUDT18 as a sanitizer of modified including antiviral NUDT18, and as and expressed and by the by or as previously for and its use in PubMed Scopus Google Scholar). and as at in and and as and expressed and as T. S. et by of the PubMed Scopus Google Scholar). activity of NUDT18, and with remdesivir-TP was in by in of clear at for was by of W. T. R. for Med. Chem. PubMed Scopus Google Scholar) and of at of NUDT18, and with remdesivir-TP also tested in the of E. which to of the human and with remdesivir-TP in the of was and to activity of NUDT18 with with and and the was from the In order to if NUDT18 can also and to its activity remdesivir-TP and or was with NUDT18 in and with for at after which was by the of and of at in a In order to the activity of NUDT18 with the active metabolite of the orally active antiviral molnupiravir, and to if the enzymes and can this substrate hydrolysis activity of NUDT18, and was tested with in the and of E. at for and the was by of with the of at of NUDT18 determined in at from to for remdesivir-TP and from to for and molnupiravir-TP. for and was by and of at was the and to to also with which has been to be the endogenous substrate of NUDT18, the concentration and and and against substrate The was to the and of NUDT18 determined from in at substrate and The that NUDT18 activity with the antiviral triphosphates and to also the activity of NUDT18 with an panel of antiviral triphosphates that are to be the active antiviral The panel the triphosphates of the sofosbuvir for the treatment of to infection treatment and for virus infection also the active metabolite of the drug NUDT18 was with of and for at in and in the and of was by of by of at of NUDT18 determined in and at from to for and by the for and was by and of at The concentration of was a the also for Healthcare a concentration and against substrate concentration and the was to the of NUDT18 to be in and The of human NUDT18 was the the was by to and with in the with the by of and was the an of was for the was to the NUDT18 for The site of with a at or from the site The of remdesivir-TP was as from and as a into was to the of remdesivir-TP for The was for at and and at most one was was to NUDT18 the The site of site by to the The of the was to was for the at remdesivir-TP as a substrate of NUDT18, we to if remdesivir or its metabolites act as of NUDT18 or its and of and was tested in and with with in the of or with of remdesivir-TP remdesivir or remdesivir metabolite NUDT18 activity was NUDT18 and in The was for at after which was by of by at in a are from the upon This The that have of interest with the of this The the at for for and and for for E. R. E. and M. M. E. and M. M. E. R. and E. E. R. and E. E. R. and E. P. T. and M. M. E. R. E. M. P. T. and M. M. and and M. M. P. T. and M. M. A. S. J. This was by the European European to T. the to T. and to P. the and T. the and to T. the to T. and to P. to A. S. the P. the for to M. This has from the Medicines The from the European 2020 and and and for for the of This the of the and the is for use that may be of the for The

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