Skip to main content
NCBI home page
Wiley - PMC COVID-19 Collection logoLink to Wiley - PMC COVID-19 Collection
. 2008 May 5;28(5):797–820. doi: 10.1002/med.20124

Structure, physiological role, and specific inhibitors of human thymidine kinase 2 (TK2): Present and future

María‐Jesús Pérez‐Pérez 1,✉, Eva‐María Priego 1, Ana‐Isabel Hernández 1, Olga Familiar 1, María‐José Camarasa 1, Ana Negri 2, Federico Gago 2, Jan Balzarini 3
PMCID: PMC7168489  PMID: 18459168

Abstract

Human mitochondrial thymidine kinase (TK2) is a pyrimidine deoxynucleoside kinase (dNK) that catalyzes the phosphorylation of pyrimidine deoxynucleosides to their corresponding deoxynucleoside 5′‐monophosphates by γ‐phosphoryl transfer from ATP. In resting cells, TK2 is suggested to play a key role in the mitochondrial salvage pathway to provide pyrimidine nucleotides for mitochondrial DNA (mtDNA) synthesis and maintenance. However, recently the physiological role of TK2turned out to have direct clinical relevance as well. Point mutations in the gene encoding TK2 have been correlated to mtDNA disorders in a heterogeneous group of patients suffering from the so‐called mtDNA depletion syndrome (MDS). TK2 activity could also be involved in mitochondrial toxicity associated to prolonged treatment with antiviral nucleoside analogues like AZT and FIAU. Therefore, TK2 inhibitors can be considered as valuable tools to unravel the role of TK2 in the maintenance and homeostasis of mitochondrial nucleotide pools and mtDNA, and to clarify the contribution of TK2 activity to mitochondrial toxicity of certain antivirals. Highly selective TK‐2 inhibitors having an acyclic nucleoside structure and efficiently discriminating between TK‐2 and the closely related TK‐1 have already been reported. It is actually unclear whether these agents efficiently reach the inner mitochondrial compartment. In the present review article,structural features of TK2, MDS‐related mutations observed in TK2 and their role in MDS will be discussed. Also, an update on novel and selective TK2 inhibitors will be provided. © 2008 Wiley Periodicals, Inc. Med Res Rev, 28, No. 5, 797–820, 2008

Keywords: thymidine kinase 2 (TK2), deoxynucleoside kinases, inhibitors, mitochondrial depletion syndrome (MDS), acyclic nucleosides

REFERENCES

  • 1. Arner ESJ, Eriksson S. Mammalian deoxyribonucleoside kinases. Pharmacol Ther 1995; 67: 155–186. [DOI] [PubMed] [Google Scholar]
  • 2. Eriksson S, Munch‐Petersen B, Johansson K, Eklund H. Structure and function of cellular deoxyribonucleoside kinases. Cell Mol Life Sci 2002; 59: 1327–1346. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3. Van Rompay AR, Johansson M, Karlsson A. Substrate specificity and phosphorylation of antiviral and anticancer nucleoside analogues by human deoxyribonucleoside kinases and ribonucleoside kinases. Pharmacol Ther 2003; 100: 119–139. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4. Pérez‐Pérez MJ, Hernández AI, Priego EM, Rodríguez‐Barrios F, Gago F, Camarasa MJ, Balzarini J. Mitochondrial thymidine kinase inhibitors. Curr Top Med Chem 2005; 5: 1205–1219. [DOI] [PubMed] [Google Scholar]
  • 5. Al‐Madhoun AS, Tjarks W, Eriksson S. The role of thymidine kinases in the activation of pyrimidine nucleoside analogues. Mini Rev Med Chem 2004; 4: 341–350. [DOI] [PubMed] [Google Scholar]
  • 6. Johansson M, Karlsson A. Cloning of the cDNA and chromosome localization of the gene for human thymidine kinase 2. J Biol Chem 1997; 272: 8454–8458. [DOI] [PubMed] [Google Scholar]
  • 7. Wang L, Munch‐Petersen B, Sjoberg AH, Hellman U, Bergman T, Jornvall H, Eriksson S. Human thymidine kinase 2: Molecular cloning and characterisation of the enzyme activity with antiviral and cytostatic nucleoside substrates. FEBS Lett 1999; 443: 170–174. [DOI] [PubMed] [Google Scholar]
  • 8. Eriksson S, Kierdaszuk B, Munch‐Petersen B, Oberg B, Johansson NG. Comparison of the substrate specificities of human thymidine kinase‐1 and kinase‐2 and deoxycytidine kinase toward antiviral and cytostatic nucleoside analogs. Biochem Biophys Res Commun 1991; 176: 586–592. [DOI] [PubMed] [Google Scholar]
  • 9. Munch‐Petersen B, Cloos L, Tyrsted G, Eriksson S. Diverging substrate‐specificity of pure human thymidine kinase‐1 and kinase‐2 against antiviral dideoxynucleosides. J Biol Chem 1991; 266: 9032–9038. [PubMed] [Google Scholar]
  • 10. Arner ESJ, Valentin A, Eriksson S. Thymidine and 3′‐azido‐3′‐deoxythymidine metabolism in human peripheral‐blood lymphocytes and monocyte‐derived macrophages—A study of both anabolic and catabolic pathways. J Biol Chem 1992; 267: 10968–10975. [PubMed] [Google Scholar]
  • 11. Verri A, Priori G, Spadari S, Tondelli L, Focher F. Relaxed enantioselectivity of human mitochondrial thymidine kinase and chemotherapeutic uses of L‐nucleoside analogues. Biochem J 1997; 328: 317–320. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12. Sabini E, Hazra S, Konrad M, Lavie A. Nonenantioselectivity property of human deoxycytidine kinase explained by structures of the enzyme in complex with L‐ and D‐nucleosides. J Med Chem 2007; 50: 3004–3014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13. Saada A. Deoxyribonucleotides and disorders of mitochondrial DNA integrity. DNA Cell Biol 2004; 23: 797–806. [DOI] [PubMed] [Google Scholar]
  • 14. Gallinaro L, Crovatto K, Rampazzo C, Pontarin G, Ferraro P, Milanesi E, Reichard P, Bianchi V. Human mitochondrial 5′‐deoxyribonucleotidase—Overproduction in cultured cells and functional aspects. J Biol Chem 2002; 277: 35080–35087. [DOI] [PubMed] [Google Scholar]
  • 15. Elpeleg O, Mandel H, Saada A. Depletion of the other genome‐mitochondrial DNA depletion syndromes in humans. J Mol Med 2002; 80: 389–396. [DOI] [PubMed] [Google Scholar]
  • 16. Bourdon A, Minai L, Serre V, Jais JP, Sarzi E, Aubert S, Chrétien D, de Lonlay P, Paquis‐Flucklinger V, Arakawa H, Nakamura Y, Munnich A, Rötig A. Mutation of RRM2B, encoding p53‐controlled ribonucleotide reductase (p53R2), causes severe mitochondrial DNA depletion. Nat Genet 2007; 39: 776–780. [DOI] [PubMed] [Google Scholar]
  • 17. Saada‐Reisch A. Deoxyribonucleoside kinases in mitochondrial DNA depletion. Nucleos Nucleot Nucl 2004; 23: 1205–1215. [DOI] [PubMed] [Google Scholar]
  • 18. Mathews CK, Song SW. Maintaining precursor pools for mitochondrial DNA replication. FASEB J 2007; 21: 2294–2303. [DOI] [PubMed] [Google Scholar]
  • 19. Saada A, Shaag A, Mandel H, Nevo Y, Eriksson S, Elpeleg O. Mutant mitochondrial thymidine kinase in mitochondrial DNA depletion myopathy. Nat Genet 2001; 29: 342–344. [DOI] [PubMed] [Google Scholar]
  • 20. Saada A, Ben‐Shalom E, Zyslin R, Miller C, Mandel H, Elpeleg O. Mitochondrial deoxyribonucleoside triphosphate pools in thymidine kinase 2 deficiency. Biochem Biophys Res Commun 2003; 310: 963–966. [DOI] [PubMed] [Google Scholar]
  • 21. Tulinius M, Moslemi AR, Darin N, Holme E, Oldfors A. Novel mutations in the thymidine kinase 2 gene (TK2) associated with fatal mitochondrial myopathy and mitochondrial DNA depletion. Neuromuscular Disord 2005; 15: 412–415. [DOI] [PubMed] [Google Scholar]
  • 22. Galbiati S, Bordoni A, Papadimitriou D, Toscano A, Rodolico C, Katsarou E, Sciacco M, Garufi A, Prelle A, Aguennouz Mh. New mutations in TK2 gene associated with mitochondrial DNA depletion. Pedriatr Neurol 2006; 34: 177–185. [DOI] [PubMed] [Google Scholar]
  • 23. Oskoui M, Davidzon G, Pascual J, Erazo R, Gurgel‐Giannetti J, Krishna S, Bonilla E, De Vivo DC, Shanske S, DiMauro S. Clinical spectrum of mitochondrial DNA depletion due to mutations in the thymidine kinase 2 gene. Arch Neurol 2006; 63: 1122–1126. [DOI] [PubMed] [Google Scholar]
  • 24. Carrozzo R, Bornstein B, Lucioli S, Campos Y, de la Pena P, Petit N, Dionisi‐Vici C, Vilarinho L, Rizza T, Bertini E, Garesse R, Santorelli FM, Arenas J. Mutation analysis in 16 patients with mtDNA depletion. Hum Mutat 2003; 21: 453–454. [DOI] [PubMed] [Google Scholar]
  • 25. Mancuso M, Salviati L, Sacconi S, Otaegui D, Camano P, Marina A, Bacman S, Moraes CT, Carlo JR, Garcia M, Garcia‐Alvarez M, Monzon L, Naini AB, Hirano M, Bonilla E, Taratuto AL, DiMauro S, Vu TH. Mitochondrial DNA depletion—Mutations in thymidine kinase gene with myopathy and SMA. Neurology 2002; 59: 1197–1202. [DOI] [PubMed] [Google Scholar]
  • 26. Vilà MR, Segovia‐Silvestre T, Gamez J, Marina A, Naini AB, Meseguer A, Lombes A, Bonilla E, DiMauro S, Hirano M, Andreu AL. Reversion of mtDNA depletion in a patient with TK2 deficiency. Neurology 2003; 60: 1203–1205. [DOI] [PubMed] [Google Scholar]
  • 27. Leshinsky‐Silver E, Michelson M, Cohen S, Ginsberg M, Sadeh M, Barash V, Lerman‐Sagie T, Lev D. A defect in the thymidine kinase 2 gene causing isolated mitochondrial myopathy without mtDNA depletion. Eur J Paediatr Neuro (in press); 10.1016/j.ejpn.2007.09.005. [DOI] [PubMed] [Google Scholar]
  • 28. Saada A, Shaag A, Elpeleg O. mtDNA depletion myopathy: Elucidation of the tissue specificity in the mitochondrial thymidine kinase (TK2) deficiency. Mol Genet Metab 2003; 79: 1–5. [DOI] [PubMed] [Google Scholar]
  • 29. Lewis W, Day BJ, Copeland WC. Mitochondrial toxicity of NRTI antiviral drugs: An integrated cellular perspective. Nat Rev Drug Discov 2003; 2: 812–822. [DOI] [PubMed] [Google Scholar]
  • 30. Kohler JJ, Lewis W. A brief overview of mechanisms of mitochondrial toxicity from NRTIs. Environ Mol Mutagen 2007; 48: 166–172. [DOI] [PubMed] [Google Scholar]
  • 31. Lewis W, Dalakas MC. Mitochondrial toxicity of antiviral drugs. Nat Med 1995; 1: 417–422. [DOI] [PubMed] [Google Scholar]
  • 32. Susan‐Resiga D, Bentley AT, Lynx MD, LaClair DD, McKee EE. Zidovudine inhibits thymidine phosphorylation in the isolated perfused rat heart. Antimicrob Agents Chemother 2007; 51: 1142–1149. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33. Lynx MD, McKee EE. 3′‐Azido‐3′‐deoxythymidine (AZT) is a competitive inhibitor of thymidine phosphorylation in isolated rat heart and liver mitochondria. Biochem Pharmacol 2006; 72: 239–243. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34. Hosseini SH, Kohler JJ, Haase CP, Tioleco N, Stuart T, Keebaugh E, Ludaway T, Russ R, Green E, Long R, Wang LY, Eriksson S, Lewis W. Targeted transgenic overexpression of mitochondrial thymidine kinase (TK2) alters mitochondrial DNA (mtDNA) and mitochondrial polypeptide abundance—Transgenic TK2, mtDNA, and antiretrovirals. Am J Pathol 2007; 170: 865–874. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35. Bradshaw PC, Li JX, Samuels DC. A computational model of mitochondrial AZT metabolism. Biochem J 2005; 392: 363–373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36. Lee EW, Lai YR, Zhang HX, Unadkat JD. Identification of the mitochondrial targeting signal of the human equilibrative nucleoside transporter 1 (hENT1)—Implications for interspecies differences in mitochondrial toxicity of fialuridine. J Biol Chem 2006; 281: 16700–16706. [DOI] [PubMed] [Google Scholar]
  • 37. Johansson K, Ramaswamy S, Ljungcrantz C, Knecht W, Piskur J, Munch‐Petersen B, Eriksson S, Eklund H. Structural basis for substrate specificities of cellular deoxyribonucleoside kinases. Nat Struct Biol 2001; 8: 616–620. [DOI] [PubMed] [Google Scholar]
  • 38. Barroso JF, Elholm M, Flatmark T. Tight binding of deoxyribonucleotide triphosphates to human thymidine kinase 2 expressed in Escherichia coli. Purification and partial characterization of its dimeric and tetrameric forms. Biochemistry 2003; 42: 15158–15169. [DOI] [PubMed] [Google Scholar]
  • 39. Hernández AI, Familiar O, Negri A, Rodríguez‐Barrios F, Gago F, Karlsson A, Camarasa MJ, Balzarini J, Pérez‐Pérez MJ. N‐1‐substituted thymine derivatives as mitochondrial thymidine kinase (TK‐2) inhibitors. J Med Chem 2006; 49: 7766–7773. [DOI] [PubMed] [Google Scholar]
  • 40. Johansson M, Van Rompay AR, Degreve B, Balzarini J, Karlsson A. Cloning and characterization of the multisubstrate deoxyribonucleoside kinase of Drosophila melanogaster . J Biol Chem 1999; 274: 23814–23819. [DOI] [PubMed] [Google Scholar]
  • 41. Munch‐Petersen B, Knecht W, Lenz C, Sondergaard L, Piskur J. Functional expression of a multisubstrate deoxyribonucleoside kinase from Drosophila melanogaster and its C‐terminal deletion mutants. J Biol Chem 2000; 275: 6673–6679. [DOI] [PubMed] [Google Scholar]
  • 42. Mikkelsen NE, Johansson K, Karlsson A, Knecht W, Andersen G, Piskur J, Munch‐Petersen B, Eklund H. Structural basis for feedback inhibition of the deoxyribonucleoside salvage pathway: Studies of the Drosophila deoxyribonucleoside kinase. Biochemistry 2003; 42: 5706–5712. [DOI] [PubMed] [Google Scholar]
  • 43. Gerth ML, Lutz S. Mutagenesis of non‐conserved active site residues improves the activity and narrows the specificity of human thymidine kinase 2. Biochem Biophys Res Commun 2007; 354: 802–807. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44. Wild K, Bohner T, Folkers G, Schulz GE. The structures of thymidine kinase from Herpes simplex virus type 1 in complex with substrates and a substrate analogue. Protein Sci 1997; 6: 2097–2106. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45. Egeblad‐Welin L, Sonntag Y, Eklund H, Munch‐Petersen B. Functional studies of active‐site mutants from Drosophila melanogaster deoxyribonucleoside kinase—Investigations of the putative catalytic glutamate‐arginine pair and of residues responsible for substrate specificity. FEBS J 2007; 274: 1542–1551. [DOI] [PubMed] [Google Scholar]
  • 46. Kierdaszuk B, Krawiec K, Kazimierczuk Z, Jacobsson U, Johansson NG, Munch‐Petersen B, Eriksson S, Shugar D. Substrate/inhibitor properties of human deoxycytidine kinase (dCK) and thymidine kinases (TK1 and TK2) towards the sugar moiety of nucleosides, including O′‐alkyl analogues. Nucleos Nucleot 1999; 18: 1883–1903. [DOI] [PubMed] [Google Scholar]
  • 47. Beck RA, Munch‐Petersen B, Dölker M, Cloos L, Tyrsted G, Eger K. Ligands for the affinity chromatography of mammalian thymidine kinase 1: Strategy, synthesis and evaluation. Pharm Acta Helv 1996; 71: 279–291. [DOI] [PubMed] [Google Scholar]
  • 48. Balzarini J, Zhu CY, De Clercq E, Pérez‐Pérez MJ, Chamorro C, Camarasa MJ, Karlsson A. Novel ribofuranosylnucleoside lead compounds for potent and selective inhibitors of mitochondrial thymidine kinase‐2. Biochem J 2000; 351: 167–171. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49. Manfredini S, Baraldi PG, Durini E, Porcu L, Angusti A, Vertuani S, Solaroli N, De Clercq E, Karlsson A, Balzarini J. Design, synthesis and enzymatic activity of highly selective human mitochondrial thymidine kinase inhibitors. Bioorg Med Chem Lett 2001; 11: 1329–1332. [DOI] [PubMed] [Google Scholar]
  • 50. Ciliberti N, Manfredini S, Angusti A, Durini E, Solaroli N, Vertuani S, Buzzoni L, Bonache MC, Ben‐Shalom E, Karlsson A, Saada A, Balzarini J. Novel selective human mitochondrial kinase inhibitors: Design, synthesis and enzymatic activity. Bioorg Med Chem 2007; 15: 3065–3081. [DOI] [PubMed] [Google Scholar]
  • 51. Balzarini J, Degrève B, Zhu CY, Durini E, Porcu L, De Clercq E, Karlsson A, Manfredini S. 2′‐O‐acyl/alkyl‐substituted arabinosyl nucleosides as inhibitors of human mitochondrial thymidine kinase. Biochem Pharmacol 2001; 61: 727–732. [DOI] [PubMed] [Google Scholar]
  • 52. Hernández AI, Balzarini J, Karlsson A, Camarasa MJ, Pérez‐Pérez MJ. Acyclic nucleoside analogues as novel inhibitors of human mitochondrial thymidine kinase. J Med Chem 2002; 45: 4254–4263. [DOI] [PubMed] [Google Scholar]
  • 53. Balzarini J, Hernández AI, Roche P, Esnouf R, Karlsson A, Camarasa MJ, Pérez‐Pérez MJ. Non‐nucleoside inhibitors of mitochondrial thymidine kinase (TK‐2) differentially inhibit the closely related herpes simplex virus type 1 TK and Drosophila melanogaster multifunctional deoxynucleoside kinase. Mol Pharmacol 2003; 63: 263–270. [DOI] [PubMed] [Google Scholar]
  • 54. Hernández AI, Balzarini J, Rodríguez‐Barrios F, San‐Félix A, Karlsson A, Gago F, Camarasa MJ, Pérez‐Pérez MJ. Improving the selectivity of acyclic nucleoside analogues as inhibitors of human mitochondrial thymidine kinase: Replacement of a triphenylmethoxy moiety with substituted amines and carboxamides. Bioorg Med Chem Lett 2003; 13: 3027–3030. [DOI] [PubMed] [Google Scholar]
  • 55. Priego EM, Balzarini J, Karlsson A, Camarasa MJ, Pérez‐Pérez MJ. Synthesis and evaluation of thymine‐derived carboxamides against mitochondrial thymidine kinase (TK‐2) and related enzymes. Bioorg Med Chem 2004; 12: 5079–5090. [DOI] [PubMed] [Google Scholar]
  • 56. Knecht W, Petersen GE, Munch‐Petersen B, Piskur J. Deoxyribonucleoside kinases belonging to the thymidine kinase 2 (TK2)‐like group vary significantly in substrate specificity, kinetics and feed‐back regulation. J Mol Biol 2002; 315: 529–540. [DOI] [PubMed] [Google Scholar]
  • 57. Marco E, Gago F. Overcoming the inadequacies or limitations of experimental structures as drug targets by using computational modeling tools and molecular dynamics simulations. ChemMedChem 2007; 2: 1388–1401. [DOI] [PubMed] [Google Scholar]
  • 58. Balzarini J, McGuigan C. Chemotherapy of varicella‐zoster virus by a novel class of highly specific anti‐VZV bicyclic pyrimidine nucleosides. Biochim Biophys Acta Mol Basis Dis 2002; 1587: 287–295. [DOI] [PubMed] [Google Scholar]
  • 59. Ponomarev V, Doubrovin M, Shavrin A, Serganova I, Beresten T, Ageyeva L, Cai CD, Balatoni J, Alauddin M, Gelovani J. A human‐derived reporter gene for noninvasive imaging in humans: Mitochondrial thymidine kinase type 2. J Nucl Med 2007; 48: 819–826. [DOI] [PubMed] [Google Scholar]
  • 60. Degrève B, Johansson M, De Clercq E, Karlsson A, Balzarini J. Differential intracellular compartmentalization of herpetic thymidine kinases (TKs) in TK gene‐transfected tumor cells. Molecular characterization of the nuclear localization signal of herpes simplex virus type 1 TK. J Virol 1998; 72: 9535–9543. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 61. Franzolin E, Rampazzo C, Pérez‐Pérez MJ, Hernández AI, Balzarini J, Bianchi V. Bromovinyl‐deoxyuridine: A selective substrate for mitochondrial thymidine kinase in cell extracts. Biochem Biophys Res Commun 2006; 344: 30–36. [DOI] [PubMed] [Google Scholar]
  • 62. Gerth ML, Lutz S. Non‐homologous recombination of deoxyribonucleoside kinases from human and Drosophila melanogaster yields human‐like enzymes with novel activities. J Mol Biol 2007; 370: 742–751. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 63. O'Sullivan O, Suhre K, Abergel C, Higgins DG, Notredame C. 3DCoffee: Combining protein sequences and structures within multiple sequence alignments. J Mol Biol 2004; 340: 385–395. [DOI] [PubMed] [Google Scholar]

Articles from Medicinal Research Reviews are provided here courtesy of Wiley

RESOURCES