R E V I E W S
`
`Advances in the development of
`nucleoside and nucleotide analogues
`for cancer and viral diseases
`
`Lars Petter Jordheim1,2, David Durantel3, Fabien Zoulim2,3 and Charles Dumontet1,2
`
`(cid:35)(cid:68)(cid:85)(cid:86)(cid:84)(cid:67)(cid:69)(cid:86)(cid:2)(cid:94)(cid:2)(cid:48)(cid:87)(cid:69)(cid:78)(cid:71)(cid:81)(cid:85)(cid:75)(cid:70)(cid:71)(cid:2)(cid:67)(cid:80)(cid:67)(cid:78)(cid:81)(cid:73)(cid:87)(cid:71)(cid:85)(cid:2)(cid:74)(cid:67)(cid:88)(cid:71)(cid:2)(cid:68)(cid:71)(cid:71)(cid:80)(cid:2)(cid:75)(cid:80)(cid:2)(cid:69)(cid:78)(cid:75)(cid:80)(cid:75)(cid:69)(cid:67)(cid:78)(cid:2)(cid:87)(cid:85)(cid:71)(cid:2)(cid:72)(cid:81)(cid:84)(cid:2)(cid:67)(cid:78)(cid:79)(cid:81)(cid:85)(cid:86)(cid:2)(cid:23)(cid:18)(cid:124)(cid:91)(cid:71)(cid:67)(cid:84)(cid:85)(cid:2)(cid:67)(cid:80)(cid:70)(cid:2)(cid:74)(cid:67)(cid:88)(cid:71)(cid:2)
`become cornerstones of treatment for patients with cancer or viral infections. The approval
`(cid:81)(cid:72)(cid:2)(cid:85)(cid:71)(cid:88)(cid:71)(cid:84)(cid:67)(cid:78)(cid:2)(cid:67)(cid:70)(cid:70)(cid:75)(cid:86)(cid:75)(cid:81)(cid:80)(cid:67)(cid:78)(cid:2)(cid:70)(cid:84)(cid:87)(cid:73)(cid:85)(cid:2)(cid:81)(cid:88)(cid:71)(cid:84)(cid:2)(cid:86)(cid:74)(cid:71)(cid:2)(cid:82)(cid:67)(cid:85)(cid:86)(cid:2)(cid:70)(cid:71)(cid:69)(cid:67)(cid:70)(cid:71)(cid:2)(cid:70)(cid:71)(cid:79)(cid:81)(cid:80)(cid:85)(cid:86)(cid:84)(cid:67)(cid:86)(cid:71)(cid:85)(cid:2)(cid:86)(cid:74)(cid:67)(cid:86)(cid:2)(cid:86)(cid:74)(cid:75)(cid:85)(cid:2)(cid:72)(cid:67)(cid:79)(cid:75)(cid:78)(cid:91)(cid:2)(cid:85)(cid:86)(cid:75)(cid:78)(cid:78)(cid:2)(cid:82)(cid:81)(cid:85)(cid:85)(cid:71)(cid:85)(cid:85)(cid:71)(cid:85)(cid:2)
`strong potential. Here, we review new nucleoside analogues and associated compounds
`(cid:86)(cid:74)(cid:67)(cid:86)(cid:2)(cid:67)(cid:84)(cid:71)(cid:2)(cid:69)(cid:87)(cid:84)(cid:84)(cid:71)(cid:80)(cid:86)(cid:78)(cid:91)(cid:2)(cid:75)(cid:80)(cid:2)(cid:82)(cid:84)(cid:71)(cid:69)(cid:78)(cid:75)(cid:80)(cid:75)(cid:69)(cid:67)(cid:78)(cid:2)(cid:81)(cid:84)(cid:2)(cid:69)(cid:78)(cid:75)(cid:80)(cid:75)(cid:69)(cid:67)(cid:78)(cid:2)(cid:70)(cid:71)(cid:88)(cid:71)(cid:78)(cid:81)(cid:82)(cid:79)(cid:71)(cid:80)(cid:86)(cid:2)(cid:72)(cid:81)(cid:84)(cid:2)(cid:86)(cid:74)(cid:71)(cid:2)(cid:86)(cid:84)(cid:71)(cid:67)(cid:86)(cid:79)(cid:71)(cid:80)(cid:86)(cid:2)(cid:81)(cid:72)(cid:2)(cid:69)(cid:67)(cid:80)(cid:69)(cid:71)(cid:84)(cid:2)(cid:67)(cid:80)(cid:70)(cid:2)(cid:88)(cid:75)(cid:84)(cid:67)(cid:78)(cid:2)
`infections, and that aim to provide increased response rates and reduced side effects.
`We also highlight the different approaches used in the development of these drugs and the
`(cid:82)(cid:81)(cid:86)(cid:71)(cid:80)(cid:86)(cid:75)(cid:67)(cid:78)(cid:2)(cid:81)(cid:72)(cid:2)(cid:82)(cid:71)(cid:84)(cid:85)(cid:81)(cid:80)(cid:67)(cid:78)(cid:75)(cid:92)(cid:71)(cid:70)(cid:2)(cid:86)(cid:74)(cid:71)(cid:84)(cid:67)(cid:82)(cid:91)(cid:16)
`
`issues of resistance, poor oral bioavailability, long-term
`toxicity and inter-individual variability requiring dose
`adaptation.
`In this Review, we first highlight approved agents,
`their mechanisms of action and mechanisms of resist-
`ance, and then focus on recent progress in the develop-
`ment of new nucleoside and nucleotide analogues for the
`treatment of cancer and viral disease, which are two of
`the main indications for this drug class.
`
`© 2013 Macmillan Publishers Limited. All rights reserved
`
` VOLUME 12 | JUNE 2013 | 447
`
`Nucleosides and nucleotides are endogenous compounds
`that are involved in several cellular processes such as
`DNA and RNA synthesis, cell signalling, enzyme regula-
`tion and metabolism. Nucleoside and nucleotide ana-
`logues are synthetic, chemically modified compounds
`that have been developed to mimic their physio logical
`counterparts (FIG. 1) in order to exploit cellular metabo-
`lism and subsequently be incorporated into DNA and
`RNA to inhibit cellular division and viral replication.
`This action has potential therapeutic benefits — for
`example, in the inhibition of cancer cell growth, the
`Approved agents
`Since the initial approval of cytarabine in 1969 by the
`inhibition of viral replication as well as other indica-
`tions (BOX 1). In addition to their incorporation into
`US Food and Drug Administration (FDA) for the treat-
`nucleic acids, nucleoside and nucleotide analogues
`ment of acute myeloid leukaemia, numerous nucleo-
`can interact with and inhibit essential enzymes such as
`side analogues have been synthesized and evaluated in
`human and viral polymerases (that is, DNA-dependent
`patients for the treatment of cancers. There are currently
`DNA polymerases, RNA-dependent DNA polymerases
`six FDA- and European Medicines Agency (EMA)-
`or RNA-dependent RNA polymerases), kinases, ribo-
`approved cytotoxic nucleoside analogues, all of which
`nucleotide reductase, DNA methyltransferases, purine
`are nucleosides, that are derivatives of deoxycytidine,
`and pyrimidine nucleoside phosphorylase and thymi-
`deoxyadenosine or deoxyguanosine (Supplementary
`dylate synthase.
`information S1 (table)). Two compounds, azacitidine
`The seminal work of Gertrude B. Elion and George
`(approved in 2004) and decitabine (approved in 2006),
`are used as demethylating agents but have also shown anti-
`H. Hitchings led to the development of agents such
`as the nucleobase 6-mercaptopurine and the antiviral
`proliferative activity against cancer cells.
`nucleoside analogue acyclovir 1,2. Further pioneering
`The first antiviral nucleoside analogue, edoxudine,
`work by Erik De Clercq and Antonín Holý allowed the
`which is not used in the clinic anymore, was also approved
`development of several of the nucleoside and nucleotide
`by the FDA in 1969; there are currently over 25 approved
`analogues that are currently in clinical use3,4. However,
`nucleoside and nucleotide analogues that are used as
`despite the availability of several nucleoside and nucleo-
`antiviral agents for several indications such as hepatitis,
`tide analogues in the clinic, the development of newer
`HIV and herpesvirus infections (Supplementary infor-
`agents with improved properties is needed to overcome
`mation S1 (table)). In addition, antiviral nucleoside
`Gilead 2011 - I-MAK v. Gilead - IPR2018-00125
`NATURE REVIEWS | DRUG DISCOVERY
`
`1Anticancer Antibody Team,
`Institut National de la Santé
`et de la Recherche Médicale
`(INSERM) U1052, Centre
`national de la recherche
`scientifique UMR 5286,
`Cancer Research Center of
`Lyon, Faculté Rockefeller,
`8 Ave Rockefeller, 69008
`Lyon, France.
`2Hematology Laboratory,
`Hospices Civils de Lyon,
`F-69000 Lyon, France.
`3Pathogenesis of hepatitis B
`and C infection team, Institut
`National de la Santé et de la
`Recherche Médicale
`(INSERM) U1052, Centre
`national de la recherche
`scientifique UMR 5286,
`Cancer Research Center of
`Lyon, 151 cours Albert
`Thomas, 69424 Lyon
`Cedex 03, France.
`e-mails: lars-petter.
`jordheim@univ-lyon1.fr;
`david.durantel@inserm.fr;
`fabien.zoulim@inserm.fr;
`charles.dumontet@chu-lyon.fr
`doi:10.1038/nrd4010
`
`

`

`R E V I E W S
`
`Nucleotide
`Nucleoside
`Nucleobase
`
`Azotation
`
`Halogenation
`
`Protection by
`polar groups
`
`Base
`
`N-conjugation
`
`P
`
`Sugar
`
`Halogenation
`
`Replacement of P–O
`bond by P–N bond
`
`Methylation
`
`Saturation
`
`Hydroxylation or
`dehydroxylation
`
`Ring opening
`
`Figure 1 | General structural and chemical modifications of nucleoside and
`nucleotide analogues. Nucleoside and nucleotide analogues consist of a nucleobase
`(cid:10)(cid:67)(cid:2)(cid:82)(cid:87)(cid:84)(cid:75)(cid:80)(cid:71)(cid:2)(cid:81)(cid:84)(cid:2)(cid:82)(cid:91)(cid:84)(cid:75)(cid:79)(cid:75)(cid:70)(cid:75)(cid:80)(cid:71)(cid:2)(cid:70)(cid:71)(cid:84)(cid:75)(cid:88)(cid:67)(cid:86)(cid:75)(cid:88)(cid:71)(cid:11)(cid:2)(cid:78)(cid:75)(cid:80)(cid:77)(cid:71)(cid:70)(cid:2)(cid:86)(cid:81)(cid:2)(cid:67)(cid:2)(cid:85)(cid:87)(cid:73)(cid:67)(cid:84)(cid:2)(cid:79)(cid:81)(cid:75)(cid:71)(cid:86)(cid:91)(cid:14)(cid:2)(cid:67)(cid:80)(cid:70)(cid:2)(cid:80)(cid:87)(cid:69)(cid:78)(cid:71)(cid:81)(cid:86)(cid:75)(cid:70)(cid:71)(cid:2)(cid:67)(cid:80)(cid:67)(cid:78)(cid:81)(cid:73)(cid:87)(cid:71)(cid:85)(cid:2)
`(cid:74)(cid:67)(cid:88)(cid:71)(cid:2)(cid:67)(cid:2)(cid:82)(cid:74)(cid:81)(cid:85)(cid:82)(cid:74)(cid:67)(cid:86)(cid:71)(cid:2)(cid:73)(cid:84)(cid:81)(cid:87)(cid:82)(cid:2)(cid:10)(cid:50)(cid:11)(cid:2)(cid:78)(cid:75)(cid:80)(cid:77)(cid:71)(cid:70)(cid:2)(cid:86)(cid:81)(cid:2)(cid:86)(cid:74)(cid:71)(cid:2)(cid:85)(cid:87)(cid:73)(cid:67)(cid:84)(cid:2)(cid:79)(cid:81)(cid:75)(cid:71)(cid:86)(cid:91)(cid:16)(cid:2)(cid:54)(cid:74)(cid:71)(cid:2)(cid:69)(cid:74)(cid:71)(cid:79)(cid:75)(cid:69)(cid:67)(cid:78)(cid:2)(cid:70)(cid:75)(cid:88)(cid:71)(cid:84)(cid:85)(cid:75)(cid:86)(cid:91)(cid:2)(cid:81)(cid:72)(cid:2)(cid:86)(cid:74)(cid:71)(cid:85)(cid:71)(cid:2)
`(cid:69)(cid:81)(cid:79)(cid:82)(cid:81)(cid:87)(cid:80)(cid:70)(cid:85)(cid:2)(cid:75)(cid:85)(cid:2)(cid:68)(cid:67)(cid:85)(cid:71)(cid:70)(cid:2)(cid:81)(cid:80)(cid:2)(cid:79)(cid:81)(cid:70)(cid:75)(cid:72)(cid:75)(cid:69)(cid:67)(cid:86)(cid:75)(cid:81)(cid:80)(cid:85)(cid:2)(cid:85)(cid:87)(cid:69)(cid:74)(cid:2)(cid:67)(cid:85)(cid:2)(cid:74)(cid:67)(cid:78)(cid:81)(cid:73)(cid:71)(cid:80)(cid:67)(cid:86)(cid:75)(cid:81)(cid:80)(cid:14)(cid:2)(cid:67)(cid:92)(cid:81)(cid:86)(cid:67)(cid:86)(cid:75)(cid:81)(cid:80)(cid:14)(cid:2)(cid:82)(cid:84)(cid:81)(cid:86)(cid:71)(cid:69)(cid:86)(cid:75)(cid:81)(cid:80)(cid:2)(cid:68)(cid:91)(cid:2)
`(cid:82)(cid:81)(cid:78)(cid:67)(cid:84)(cid:2)(cid:73)(cid:84)(cid:81)(cid:87)(cid:82)(cid:85)(cid:2)(cid:67)(cid:80)(cid:70)(cid:2)(cid:81)(cid:86)(cid:74)(cid:71)(cid:84)(cid:2)(cid:79)(cid:81)(cid:70)(cid:75)(cid:72)(cid:75)(cid:69)(cid:67)(cid:86)(cid:75)(cid:81)(cid:80)(cid:85)(cid:2)(cid:85)(cid:74)(cid:81)(cid:89)(cid:80)(cid:2)(cid:75)(cid:80)(cid:2)(cid:86)(cid:74)(cid:75)(cid:85)(cid:2)(cid:72)(cid:75)(cid:73)(cid:87)(cid:84)(cid:71)(cid:16)(cid:2)(cid:48)(cid:14)(cid:2)(cid:80)(cid:75)(cid:86)(cid:84)(cid:81)(cid:73)(cid:71)(cid:80)(cid:29)(cid:2)(cid:49)(cid:14)(cid:2)(cid:81)(cid:90)(cid:91)(cid:73)(cid:71)(cid:80)(cid:16)
`
`and nucleotide analogues are structurally more diverse
`than anticancer nucleoside analogues, as they consist of
`nucleosides, nucleotides and acyclic nucleosides5. A major
`difference between anticancer nucleoside analogues
`and antiviral nucleoside and/or nucleotide analogues is
`that antiviral nucleoside and/or nucleotide analogues
`have low activity on mammalian enzymes, which results
`in a better tolerance profile than anticancer nucleoside
`analogues.
`
`Mechanisms of action of nucleoside analogues
`Currently used therapeutic nucleoside and nucleo-
`tide analogues exploit the same metabolic pathways as
`endogenous nucleosides or nucleotides, and they also
`act as antimetabolites (FIG. 2). Nucleoside and nucleotide
`analogues enter cells through specific nucleoside trans-
`porters6,7 and there is growing evidence that organic
`anion or cation transporters as well as peptide transport-
`ers are involved in the cellular uptake of certain antiviral
`analogues. Inside the cells, the drugs are subsequently
`phosphorylated by a nucleoside kinase and a nucleoside
`monophosphate kinase, and then a nucleoside diphos-
`phate kinase, creatine kinase or 3-phosphoglycerate
`kinase catalyses the last phosphorylation step. This
`leads to the accumulation of di- and triphosphorylated
`nucleoside analogues in cancer or virus-infected cells.
`In cells infected by some DNA viruses (such as
`herpesvirus-infected cells) the first and second phos-
`phorylation steps of thymidine are also performed
`by a virus-encoded thymidine kinase8. Herpesvirus
`thymidine kinases have broader substrate specificity
`than mammalian counterparts; this difference in
`
`Ribonucleotide reductase
`A complex intracellular enzyme
`that converts ribonucleoside
`diphosphates into deoxyribo-
`nucleoside diphosphates, and
`is targeted by anticancer
`agents such as gemcitabine.
`
`Nucleobase
`A nitrogen-containing
`heterocyclic compound that
`can be grouped into purines
`(adenine and guanine) and
`pyrimidines (cytosine, thymine
`and uracil).
`
`Dose adaptation
`Determination of the dose that
`should be administered to a
`patient based on predicted or
`observed toxicity.
`
`Demethylating agents
`Compounds that modify the
`methylation status of
`regulatory sequences in DNA,
`thereby modifying the levels
`of expression of the
`corresponding gene.
`
`Nucleoside transporters
`Membrane pumps that allow
`the uptake and/or the efflux
`of nucleosides by cells.
`
`substrate specificity forms the basis for the selectivity
`of nucleoside and nucleotide analogues as anti-herpes
`molecules9,10. Mono-, di- and triphosphorylated nucleo-
`sides are the active forms of these drugs and they act by
`inhibiting intracellular enzymes, such as viral or human
`polymerases or ribonucleotide reductase, as well as by
`being incorporated into newly synthesized DNA and
`RNA. The incorporation of nucleoside or nucleotide
`analogues into DNA may induce either the termina-
`tion of chain elongation, the accumulation of mutations
`in viral progeny or the induction of apoptosis (BOX 2).
`Supplementary information S2 (table) details the conse-
`quences of inhibiting viral enzymes using nucleoside or
`nucleotide analogues of four different viruses that affect
`human health.
`An in-depth knowledge of the mechanism of action
`of currently used compounds is of great value for the
`development of new compounds. These data have led to
`the development of compounds that act independently of
`membrane transporters or activating kinases and are less
`susceptible to degradation. A better understanding of the
`mechanism of action of these compounds will also con-
`tribute to the rational development of synergistic combi-
`nations of nucleoside or nucleotide analogues with drugs
`that have different and/or complementary mechanisms
`of action.
`
`Mechanisms of resistance
`Understanding the mechanisms that cause resistance to
`currently used nucleoside and nucleotide analogues is
`a prerequisite for the development of novel agents that
`could circumvent these mechanisms and therefore be
`prescribed to patients with relapsing or refractory dis-
`ease. Resistance of cancer cells to the effects of nucleo-
`side analogues is thought to be largely due to somatic
`changes in the tumour cells. Resistance to the inhibitory
`effects of nucleoside and nucleotide analogues on viral
`replication seems to be due to specific mutations in the
`viral genomes but may also be partly due to mutations
`and/or single nucleotide polymorphisms in the host
`genome; however, this needs to be further investigated.
`For example, the interferon λ3 (IFNL3; also known as
`IL28B) polymorphism strongly predicted the response
`to interferon and ribavirin in patients with hepatitis C
`virus (HCV) genotype 1 infection, via a mechanism that
`may involve the responsiveness of the infected host to
`interferon11.
`
`Metabolic resistance profile. Studying the pathways
`involved in the transport, activation or inactivation of
`nucleoside analogues has allowed the identification
`of mechanisms of resistance to these drugs, many of
`which have subsequently been clinically validated12. In
`cancer cells, a deficiency in nucleoside transporters such
`as equilibrative nucleoside transporter 1 (ENT1; also
`known as SLC29A1) or intracellular nucleoside kinases
`such as deoxycytidine kinase (DCK), as well as increased
`activity of ribonucleotide reductase and expression of
`5ʹ-nucleotidases, are all correlated with a reduced cyto-
`toxicity of nucleoside analogues in cell models and in
`clinical samples12–15.
`
`448 | JUNE 2013 | VOLUME 12
`
` www.nature.com/reviews/drugdisc
`
`© 2013 Macmillan Publishers Limited. All rights reserved
`
`

`

`R E V I E W S
`
`Box 1 | Other indications and actions of nucleoside and nucleotide analogues
`
`Besides their classical use in cancer and virology, some nucleoside and nucleotide analogues, and related compounds
`such as xanthine derivatives, have been used in various other indications.
`Hyperuricaemia
`Allopurinol, a structural isomer of hypoxanthine, is an inhibitor of xanthine oxidase and has been used for the treatment
`of chronic hyperuricaemia since 1966.
`Immunosuppression
`Azathioprine, a purine analogue that is a derivative of mercaptopurine, is used as an immunosuppressive drug in organ
`transplantation and autoimmune disease. Bone marrow suppression can be life-threatening in patients with low levels
`of thiopurine S-methyltransferase and so screening of this enzyme is recommended before azathioprine is prescribed.
`Cladribine also possesses specific activity on lymphocytes and has therefore been evaluated in patients with autoimmune
`diseases such as rheumatoid arthritis and multiple sclerosis.
`Phosphodiesterase inhibitors
`Theophylline, a methylxanthine analogue, acts as a nonselective inhibitor of phosphodiesterases, leads to an increase in
`intracellular cyclic AMP and is indicated in several situations including the treatment of chronic obstructive pulmonary
`disease and asthma.
`Epigenetic modulators
`Decitabine and azacitidine are DNA methyltransferase inhibitors and act as demethylating agents. Decitabine and
`azacitidine are currently approved for the treatment of myelodysplastic syndromes.
`Neuroprotection and cardioprotection
`The cellular uptake of adenosine as well as the activity of the intracellular enzyme adenosine kinase have been
`considered as potential targets for the protection of neurons and cardiac cells159,160.
`
`An increase in the activity of cytidine deaminase
`has also been correlated with decreased activity of cyti-
`dine derivatives (cytarabine and gemcitabine) in vitro16.
`Conversely, decreased cytidine deaminase activity in
`clinical blood samples was associated with increased
`exposure to the parental compound (gemcitabine),
`which subsequently resulted in the induction of toxic-
`ity17. Accordingly, an assessment of cytidine deaminase
`activity in the serum can be routinely performed before
`gemcitabine is administered to patients with cancer, and
`this could help clinicians to use a dose of gemcitabine that
`reduces the risk of severe toxicity18.
`
`Genomic resistance profile. The rate of spontaneous
`mutations within viral genomes is far higher than in
`mammalian genomes owing to absent or limited proof-
`reading capabilities of viral polymerases. RNA viruses
`exist as complex quasi-species (that is, a wide population
`of related genomes that differ by less than 5%), which
`evolve over time depending on the selective pressure of
`the environment or exposure to therapy. This property
`is, at least in part, responsible for the high adaptability
`of viruses and represents a major hurdle to overcoming
`resistance because mutants that confer resistance to a
`given drug may appear or pre-exist in the population of
`genomes. Generally, resistance to a given nucleoside or
`nucleotide analogue is caused by a limited number of
`mutations (usually fewer than five) in a viral genome,
`which mainly affect the catalytic site of the polymerase
`to which the normal nucleotides or nucleotide ana-
`logues bind. Supplementary information S3 (table) lists
`the main mutations — within the genes encoding HCV,
`hepatitis B virus (HBV) and HIV polymerases — that
`confer resistance to the nucleoside or nucleotide ana-
`logues that are approved or in late-stage development.
`
`We believe that the determination of resistance and
`toxicity factors before initiating treatment will become
`a major parameter that will, in the near future, enable
`the selection of the most appropriate nucleoside or
`nucleotide analogues for therapy. As selected mutations
`in viral genomes and clinically relevant polymorphisms
`in genes encoding proteins that are involved in nucleo-
`tide metabolism are increasingly being validated, these
`analyses — performed before initiating treatment — will
`contribute to better efficacy and tolerance.
`
`Predicting response
`The ability to choose a treatment that has the highest
`probability of invoking a positive response would be
`beneficial to patients, their environment (in the case of
`contagious diseases) and the health-care system overall
`as it would reduce costs. As illustrated below, our current
`ability to predict patient response is essentially based on
`our knowledge of drug metabolism and targeting as well
`as mechanisms of resistance.
`
`Genetic polymorphisms and somatic phenotypes. The
`response to currently used regimens based on nucleoside
`or nucleotide analogues can be partially predicted by the
`genetic make-up of patients. Many studies that aimed
`to identify biological markers of treatment outcome
`have collectively highlighted the link between genetic
`polymorphisms and patient response to a nucleoside or
`nucleotide analogue; the main results of these studies
`are presented in Supplementary information S4 (table).
`
`Target cell phenotype. Studies performed directly on
`tumour cells to identify markers that predict the activity
`of nucleoside and nucleotide analogue-based treatments
`have shown that clinically relevant markers include
`
`Chain elongation
`The increase in length of DNA
`or RNA strands during
`replication or transcription.
`
`NATURE REVIEWS | DRUG DISCOVERY
`
` VOLUME 12 | JUNE 2013 | 449
`
`© 2013 Macmillan Publishers Limited. All rights reserved
`
`

`

`R E V I E W S
`
`Nucleoside analogue
`
`Nucleoside transporter
`
`Nucleoside analogue
`
`5′-nucleotidase
`
`Nucleoside
`kinase
`
`Deaminase
`
`Nucleoside analogue
`
`Deamination
`
`P
`
`Nucleoside
`monophosphate
`kinase
`
`Nucleoside analogue
`
`P P
`
`RRM1
`
`Nucleoside
`diphosphate
`kinase
`
`DNA incorporation
`
`RNA incorporation
`
`Nucleoside analogue
`
`P P P
`
`DNA synthesis
`
`Figure 2 | Mechanism of action of nucleoside analogues. (cid:37)(cid:71)(cid:78)(cid:78)(cid:87)(cid:78)(cid:67)(cid:84)(cid:2)(cid:87)(cid:82)(cid:86)(cid:67)(cid:77)(cid:71)(cid:2)(cid:81)(cid:72)(cid:2)
`nucleoside analogues is an active process involving concentrative nucleoside
`(cid:86)(cid:84)(cid:67)(cid:80)(cid:85)(cid:82)(cid:81)(cid:84)(cid:86)(cid:71)(cid:84)(cid:85)(cid:2)(cid:10)(cid:37)(cid:48)(cid:54)(cid:85)(cid:11)(cid:2)(cid:67)(cid:80)(cid:70)(cid:2)(cid:71)(cid:83)(cid:87)(cid:75)(cid:78)(cid:75)(cid:68)(cid:84)(cid:67)(cid:86)(cid:75)(cid:88)(cid:71)(cid:2)(cid:80)(cid:87)(cid:69)(cid:78)(cid:71)(cid:81)(cid:85)(cid:75)(cid:70)(cid:71)(cid:2)(cid:86)(cid:84)(cid:67)(cid:80)(cid:85)(cid:82)(cid:81)(cid:84)(cid:86)(cid:71)(cid:84)(cid:85)(cid:2)(cid:10)(cid:39)(cid:48)(cid:54)(cid:85)(cid:11)(cid:16)(cid:2)(cid:54)(cid:74)(cid:71)(cid:84)(cid:71)(cid:2)(cid:67)(cid:84)(cid:71)(cid:2)
`(cid:86)(cid:74)(cid:84)(cid:71)(cid:71)(cid:2)(cid:37)(cid:48)(cid:54)(cid:85)(cid:2)(cid:67)(cid:80)(cid:70)(cid:2)(cid:72)(cid:81)(cid:87)(cid:84)(cid:2)(cid:39)(cid:48)(cid:54)(cid:85)(cid:2)(cid:70)(cid:71)(cid:85)(cid:69)(cid:84)(cid:75)(cid:68)(cid:71)(cid:70)(cid:2)(cid:75)(cid:80)(cid:2)(cid:74)(cid:87)(cid:79)(cid:67)(cid:80)(cid:85)(cid:16)(cid:2)(cid:49)(cid:80)(cid:69)(cid:71)(cid:2)(cid:75)(cid:80)(cid:85)(cid:75)(cid:70)(cid:71)(cid:2)(cid:86)(cid:74)(cid:71)(cid:2)(cid:69)(cid:71)(cid:78)(cid:78)(cid:14)(cid:2)(cid:86)(cid:74)(cid:71)(cid:2)(cid:80)(cid:87)(cid:69)(cid:78)(cid:71)(cid:81)(cid:85)(cid:75)(cid:70)(cid:71)(cid:2)
`(cid:67)(cid:80)(cid:67)(cid:78)(cid:81)(cid:73)(cid:87)(cid:71)(cid:2)(cid:87)(cid:80)(cid:70)(cid:71)(cid:84)(cid:73)(cid:81)(cid:71)(cid:85)(cid:2)(cid:67)(cid:80)(cid:2)(cid:75)(cid:80)(cid:75)(cid:86)(cid:75)(cid:67)(cid:78)(cid:2)(cid:84)(cid:67)(cid:86)(cid:71)(cid:15)(cid:78)(cid:75)(cid:79)(cid:75)(cid:86)(cid:75)(cid:80)(cid:73)(cid:2)(cid:82)(cid:74)(cid:81)(cid:85)(cid:82)(cid:74)(cid:81)(cid:84)(cid:91)(cid:78)(cid:67)(cid:86)(cid:75)(cid:81)(cid:80)(cid:2)(cid:85)(cid:86)(cid:71)(cid:82)(cid:2)(cid:68)(cid:91)(cid:2)(cid:67)(cid:2)(cid:80)(cid:87)(cid:69)(cid:78)(cid:71)(cid:81)(cid:85)(cid:75)(cid:70)(cid:71)(cid:2)
`(cid:77)(cid:75)(cid:80)(cid:67)(cid:85)(cid:71)(cid:14)(cid:2)(cid:89)(cid:74)(cid:75)(cid:69)(cid:74)(cid:2)(cid:78)(cid:71)(cid:67)(cid:70)(cid:85)(cid:2)(cid:86)(cid:81)(cid:2)(cid:86)(cid:74)(cid:71)(cid:2)(cid:82)(cid:84)(cid:81)(cid:70)(cid:87)(cid:69)(cid:86)(cid:75)(cid:81)(cid:80)(cid:2)(cid:81)(cid:72)(cid:2)(cid:67)(cid:2)(cid:79)(cid:81)(cid:80)(cid:81)(cid:82)(cid:74)(cid:81)(cid:85)(cid:82)(cid:74)(cid:67)(cid:86)(cid:71)(cid:2)(cid:79)(cid:71)(cid:86)(cid:67)(cid:68)(cid:81)(cid:78)(cid:75)(cid:86)(cid:71)(cid:16)(cid:2)(cid:35)(cid:2)(cid:85)(cid:71)(cid:69)(cid:81)(cid:80)(cid:70)(cid:2)
`(cid:82)(cid:74)(cid:81)(cid:85)(cid:82)(cid:74)(cid:81)(cid:84)(cid:91)(cid:78)(cid:67)(cid:86)(cid:75)(cid:81)(cid:80)(cid:2)(cid:85)(cid:86)(cid:71)(cid:82)(cid:2)(cid:75)(cid:85)(cid:2)(cid:86)(cid:74)(cid:71)(cid:80)(cid:2)(cid:82)(cid:71)(cid:84)(cid:72)(cid:81)(cid:84)(cid:79)(cid:71)(cid:70)(cid:2)(cid:68)(cid:91)(cid:2)(cid:80)(cid:87)(cid:69)(cid:78)(cid:71)(cid:81)(cid:85)(cid:75)(cid:70)(cid:71)(cid:2)(cid:79)(cid:81)(cid:80)(cid:81)(cid:82)(cid:74)(cid:81)(cid:85)(cid:82)(cid:74)(cid:67)(cid:86)(cid:71)(cid:2)(cid:77)(cid:75)(cid:80)(cid:67)(cid:85)(cid:71)(cid:14)(cid:2)
`(cid:67)(cid:80)(cid:70)(cid:2)(cid:86)(cid:74)(cid:71)(cid:2)(cid:86)(cid:74)(cid:75)(cid:84)(cid:70)(cid:2)(cid:82)(cid:74)(cid:81)(cid:85)(cid:82)(cid:74)(cid:81)(cid:84)(cid:91)(cid:78)(cid:67)(cid:86)(cid:75)(cid:81)(cid:80)(cid:2)(cid:85)(cid:86)(cid:71)(cid:82)(cid:2)(cid:75)(cid:85)(cid:2)(cid:82)(cid:71)(cid:84)(cid:72)(cid:81)(cid:84)(cid:79)(cid:71)(cid:70)(cid:2)(cid:68)(cid:91)(cid:2)(cid:80)(cid:87)(cid:69)(cid:78)(cid:71)(cid:81)(cid:85)(cid:75)(cid:70)(cid:71)(cid:2)(cid:70)(cid:75)(cid:82)(cid:74)(cid:81)(cid:85)(cid:82)(cid:74)(cid:67)(cid:86)(cid:71)(cid:2)(cid:77)(cid:75)(cid:80)(cid:67)(cid:85)(cid:71)(cid:16)(cid:2)
`Triphosphates can be incorporated in nucleic acids, in competition with their normal
`(cid:69)(cid:81)(cid:87)(cid:80)(cid:86)(cid:71)(cid:84)(cid:82)(cid:67)(cid:84)(cid:86)(cid:85)(cid:14)(cid:2)(cid:81)(cid:84)(cid:2)(cid:86)(cid:74)(cid:71)(cid:91)(cid:2)(cid:69)(cid:67)(cid:80)(cid:2)(cid:75)(cid:80)(cid:74)(cid:75)(cid:68)(cid:75)(cid:86)(cid:2)(cid:80)(cid:87)(cid:69)(cid:78)(cid:71)(cid:75)(cid:69)(cid:2)(cid:67)(cid:69)(cid:75)(cid:70)(cid:2)(cid:85)(cid:91)(cid:80)(cid:86)(cid:74)(cid:71)(cid:85)(cid:75)(cid:85)(cid:2)(cid:68)(cid:91)(cid:2)(cid:75)(cid:80)(cid:74)(cid:75)(cid:68)(cid:75)(cid:86)(cid:75)(cid:80)(cid:73)(cid:2)(cid:71)(cid:85)(cid:85)(cid:71)(cid:80)(cid:86)(cid:75)(cid:67)(cid:78)(cid:2)(cid:71)(cid:80)(cid:92)(cid:91)(cid:79)(cid:71)(cid:85)(cid:2)
`(cid:85)(cid:87)(cid:69)(cid:74)(cid:2)(cid:67)(cid:85)(cid:2)(cid:82)(cid:81)(cid:78)(cid:91)(cid:79)(cid:71)(cid:84)(cid:67)(cid:85)(cid:71)(cid:85)(cid:16)(cid:2)(cid:52)(cid:75)(cid:68)(cid:81)(cid:80)(cid:87)(cid:69)(cid:78)(cid:71)(cid:81)(cid:86)(cid:75)(cid:70)(cid:71)(cid:2)(cid:84)(cid:71)(cid:70)(cid:87)(cid:69)(cid:86)(cid:67)(cid:85)(cid:71)(cid:2)(cid:47)(cid:19)(cid:2)(cid:10)(cid:52)(cid:52)(cid:47)(cid:19)(cid:11)(cid:14)(cid:2)(cid:67)(cid:2)(cid:77)(cid:71)(cid:91)(cid:2)(cid:71)(cid:80)(cid:92)(cid:91)(cid:79)(cid:71)(cid:2)(cid:75)(cid:80)(cid:88)(cid:81)(cid:78)(cid:88)(cid:71)(cid:70)(cid:2)
`(cid:75)(cid:80)(cid:2)(cid:80)(cid:87)(cid:69)(cid:78)(cid:71)(cid:81)(cid:86)(cid:75)(cid:70)(cid:71)(cid:2)(cid:79)(cid:71)(cid:86)(cid:67)(cid:68)(cid:81)(cid:78)(cid:75)(cid:85)(cid:79)(cid:14)(cid:2)(cid:69)(cid:67)(cid:80)(cid:2)(cid:68)(cid:71)(cid:2)(cid:75)(cid:80)(cid:74)(cid:75)(cid:68)(cid:75)(cid:86)(cid:71)(cid:70)(cid:2)(cid:68)(cid:81)(cid:86)(cid:74)(cid:2)(cid:68)(cid:91)(cid:2)(cid:70)(cid:75)(cid:82)(cid:74)(cid:81)(cid:85)(cid:82)(cid:74)(cid:81)(cid:84)(cid:91)(cid:78)(cid:67)(cid:86)(cid:71)(cid:70)(cid:2)(cid:67)(cid:80)(cid:70)(cid:2)
`(cid:86)(cid:84)(cid:75)(cid:82)(cid:74)(cid:81)(cid:85)(cid:82)(cid:74)(cid:81)(cid:84)(cid:91)(cid:78)(cid:67)(cid:86)(cid:71)(cid:70)(cid:2)(cid:67)(cid:80)(cid:67)(cid:78)(cid:81)(cid:73)(cid:87)(cid:71)(cid:85)(cid:16)(cid:2)(cid:37)(cid:67)(cid:86)(cid:67)(cid:68)(cid:81)(cid:78)(cid:75)(cid:69)(cid:2)(cid:71)(cid:80)(cid:92)(cid:91)(cid:79)(cid:71)(cid:85)(cid:2)(cid:79)(cid:67)(cid:91)(cid:2)(cid:84)(cid:71)(cid:70)(cid:87)(cid:69)(cid:71)(cid:2)(cid:86)(cid:74)(cid:71)(cid:2)(cid:67)(cid:79)(cid:81)(cid:87)(cid:80)(cid:86)(cid:2)(cid:81)(cid:72)(cid:2)(cid:67)(cid:69)(cid:86)(cid:75)(cid:88)(cid:71)(cid:2)
`metabolites, including deaminases and 5ʹ-nucleotidases. The cellular effects induced
`(cid:68)(cid:91)(cid:2)(cid:80)(cid:87)(cid:69)(cid:78)(cid:71)(cid:81)(cid:85)(cid:75)(cid:70)(cid:71)(cid:2)(cid:67)(cid:80)(cid:70)(cid:2)(cid:80)(cid:87)(cid:69)(cid:78)(cid:71)(cid:81)(cid:86)(cid:75)(cid:70)(cid:71)(cid:2)(cid:67)(cid:80)(cid:67)(cid:78)(cid:81)(cid:73)(cid:87)(cid:71)(cid:85)(cid:2)(cid:67)(cid:84)(cid:71)(cid:2)(cid:70)(cid:71)(cid:85)(cid:69)(cid:84)(cid:75)(cid:68)(cid:71)(cid:70)(cid:2)(cid:75)(cid:80)(cid:2)BOX 2.
`
`ribonucleotide reductase subunit M1 (RRM1)13,19, the
`membrane transporter ENT115,20, the activating kinase
`DCK21,22, the cytosolic 5ʹ nucleotidase II23,24 as well as
`uridine phosphorylase and dihydropyrimidine dehydro-
`genase. A routinely applicable functional assay of mem-
`brane transport or enzymatic activity would therefore
`certainly be of great interest for predicting the activity of
`these drugs. A clinical trial evaluating ENT1 transport
`activity with a fluorescent nucleoside probe is currently
`being carried out in patients receiving gemcitabine
`for pancreatic cancer (ClinicalTrials.gov identifier:
`NCT00414570)25.
`
`Search for known mutations or resistant profile in
`circulating or intracellular viral strains. In viral diseases,
`mutations that are associated with resistance to nucleo-
`side analogues are systematically identified during the
`preclinical development of a drug using sequencing-
`based technologies or in vitro phenotypic assays26–28.
`
`Phenotypic assays
`Assays in which biological
`function or cell response is
`measured as an index of
`drug action.
`
`Pronucleotides
`Phosphorylated nucleosides
`in which the phosphate is
`linked to a protective group
`to increase diffusion of the
`nucleoside across the cell
`membrane.
`
`Indeed, the identification of drug-resistant viral strains
`is a prerequisite for obtaining investigational new drug
`(IND) status from the FDA or the EMA29–31. In addition,
`genotypic and phenotypic assays, such as sequencing-
`based or hybridization-based technologies, or assays
`based on the replication of isolated circulating viruses
`or molecular clones, are important for the management
`of patient treatment, particularly for patients with HIV.
`Moreover, as a result of the establishment of comprehen-
`sive databases on drug resistance, such as the Stanford
`University HIV Drug Resistance Database, virtual
`pheno typing can also be used by clinicians to make deci-
`sions on the treatment to be prescribed26.
`In current medical practice, physicians routinely
`take into account specific mutations associated with
`resistance to certain antiviral nucleoside and nucleo-
`tide analogues. However, the patient’s genotype seldom
`influences the choice of anticancer nucleoside analogues
`prescribed in the clinic. A sobering example is that of
`thio purine methyltransferase genotyping in patients
`receiving the nucleobase drug 6-mercaptopurine32.
`Although specific genotypes are associated with severe
`drug toxicity, genotyping (which has been routinely avail-
`able for over a decade) has yet to gain widespread accept-
`ance33. Several factors, such as the absence of a clear-cut
`impact on response rates or the toxicity observed in most
`cases, may explain why there is a relative lack of interest
`in genotyping patients with cancer34.
`
`Novel agents
`The development of new nucleoside and nucleotide
`analogues is based on the need to identify new agents
`that have different mechanisms of action compared to
`existing agents, the need to provide drugs with improved
`bioavailability and solubility as well as the need to over-
`come resistance mechanisms and to improve the balance
`between efficacy and long-term toxicity for drugs that are
`administered over a long period of time (for example,
`patients with HIV or HBV require lifelong treatment).
`In this article, we have chosen to divide these novel
`drug candidates into the following categories: new
`nucleosides that have important modifications in their
`base and sugar moieties; pronucleotides; conjugates com-
`posed of nucleoside or nucleotide analogues and other
`chemical entities; liposomal formulations; and orally
`administered formulations of approved drugs. As there
`is a vast amount of inform