Thymidine Kinase 2 Research Articles

Screening of FDA-approved Drugs against Protein Thymidine Kinase 2 Using Machine Learning Method Validated Applying Molecular Dynamics and Free Energy Landscape Calculation

Background: Thymidine kinase 2 (TK2) is a crucial enzyme in the mitochondrial pyrimidine salvage pathway, strongly associated with several mitochondrial diseases. Current treatments frequently damage mitochondria, leading to a decrease in cellular energy output. Objective: This study aimed to use computational approaches to identify inhibitors of TK2 that could prevent these harmful consequences. Method: The initial screening process entailed the application of machine learning algorithms, more particularly a Random Forest model, which was trained on 189 FDA-approved drugs and decoy datasets obtained from the DUD-E database. Its purpose was to identify potential inhibitors. The molecular docking technique was employed to evaluate the affinity of the chosen medicines towards TK2. Molecular dynamics (MD) simulations lasting 100 nanoseconds were employed to conduct additional validation by examining the dynamic interactions between the top-found compounds and TK2. Results: Three hit compounds (3168, 5209502, and 135402009) were identified through the screening process for their high affinity for TK2. Compound 5209502 had the most stable interaction with the lowest root-mean-square deviation (RMSD) in the molecular dynamics (MD) simulations and maintained 12 hydrogen bonds consistently. MM/GBSA computations verified that 5209502 exhibited the strongest binding affinity with a binding free energy of -62.14 kcal/mol, which was notably lower than that of the control ligand. Conclusion: Compound 5209502 is a promising candidate for additional experimental evaluation because of its notable stability and great affinity for TK2. This chemical may provide a focused and less harmful treatment for mitochondrial illnesses linked to TK2 malfunction.

Recent topics of spinocerebellar ataxia type 31

AbstractSpinocerebellar ataxia type 31 (SCA31) is an autosomal‐dominant neurodegenerative condition caused by a 2.5–3.8 kb‐long complex repeat containing (TGGAA/TTCCA)n in an intron shared by two genes called brain expressed, associated with Nedd4 (BEAN1) and thymidine kinase 2 (TK2) located in the human chromosome 16q22.1. Since BEAN1 and TK2 are transcribed in mutually opposite directions in human brains, two independently transcribed RNAs containing either (UGGAA)n or (UUCCA)n are likely to associate with the pathogenesis of SCA31. Recently, a minor TK2 mRNA isoform called TK2‐EXT was confirmed to be transcribed in human cerebellum, suggesting that (UUCCA)n is indeed expressed. The level of TK2 mRNA and TK2 protein expression levels was both preserved in SCA31 human cerebellum, suggesting that the expression of (UUCCA)n does not affect the expression of TK2, and hence, the function of TK2 seemed to be preserved. On the other hand, the other penta‐nucleotide RNA repeat (UGGAA)n, expressed through BEAN1 transcription, is likely to conform toxicity through forming abnormal RNA structures called RNA foci in the nucleus of expressing cells. In addition, three proteins TDP‐43, FUS, and hnRNPA2/B1 that commonly have a capacity to bind with (UGGAA)n reduced the number of RNA foci, and ameliorated the phenotype brought by (UGGAA)n in Drosophila. A small compound naphthyridine carbamate dimer that binds to (UGGAA)n dampened the (UGGAA)n toxicity in Drosophila, further supporting the idea that (UGGAA)n expressed by BEAN1 is pathogenic. Therefore, a plausible approach to treat SCA31 may be considered by administering agents with a capacity binding to (UGGAA)n.

Long term survival and abnormal liver fat accumulation in mice with specific thymidine kinase 2 deficiency in liver tissue.

Deficiency in thymidine kinase 2 (TK2) causes mitochondrial DNA depletion. Liver mitochondria are severely affected in Tk2 complete knockout models and have been suggested to play a role in the pathogenesis of the Tk2 knockout phenotype, characterized by loss of hypodermal fat tissue, growth retardation and reduced life span. Here we report a liver specific Tk2 knockout (KO) model to further study mechanisms contributing to the phenotypic changes associated with Tk2 deficiency. Interestingly, the liver specific Tk2 KO mice had a normal life span despite a much lower mtDNA level in liver tissue. Mitochondrial DNA encoded peptide COXI did not differ between the Tk2 KO and control mice. However, the relative liver weight was significantly increased in the male Tk2 KO mouse model. Histology analysis indicated an increased lipid accumulation. We conclude that other enzyme activities can partly compensate Tk2 deficiency to maintain mtDNA at a low but stable level throughout the life span of the liver specific Tk2 KO mice. The lower level of mtDNA was sufficient for survival but led to an abnormal lipid accumulation in liver tissue.

Mitochondrial dysfunction is associated with lipid metabolism disorder and upregulation of angiotensin-converting enzyme 2.

Thymidine kinase 2 (TK2) deficiency in humans leads to a myopathic form of mitochondrial DNA (mtDNA) deficiency. Here we present a skeletal and cardiac muscle specific TK2 knockout mouse (mTk2 KO). The mice showed dilated hearts and markedly reduced adipose tissue during week 12 to 16. A severe decrease of mtDNA was found only in skeletal muscle and heart tissue in mTk2 KO mice. Expression analysis of key metabolic genes of 16 weeks knockout mice showed significant changes of genes involved in lipid metabolism, with different patterns in heart and skeletal muscle. Our study further suggests that lipoprotein lipase (LPL) from liver supports the metabolism when heart and skeletal muscle were impaired due to mitochondrial dysfunction. The angiotensin-converting enzyme 2 (ACE2), which is involved in glucose homeostasis, was also affected by mtDNA deficiency in our study. Interestingly, both the gene and protein expression of ACE2 were increased in cardiac tissue of mTk2 KO mice. Since ACE2 is a receptor for the SARS-CoV-2 virus, its regulation in relation to mitochondrial function may have important clinical implications.

Research Progress on the Relationship Between Mitochondrial Deoxyguanosine Kinase and Apoptosis and Autophagy in Lung Adenocarcinoma Cells

<p>Lung cancer is the leading cause of cancer-related deaths. Non-small cell lung cancer (NSCLC) accounts for about 85% of all lung cancers, and lung adenocarcinoma is the most common NSCLC. Most patients with lung cancer eventually lead to local and metastatic recurrence, including many patients who have completely removed the primary tumor during surgery and have no noticeable metastasis. There are two different deoxynucleotide triphosphate (dNTP) libraries in eukaryotic cells. The de novo synthesis of dNTPs in the cytoplasm is coordinated with the cell cycle and reaches a peak in the S phase, thereby providing deoxynucleotides for the replication of genomic DNA. In contrast, the mitochondrial pool of dNTPs is maintained through the mitochondrial deoxynucleoside rescue pathway throughout the cell cycle and is essential for mtDNA replication. Mitochondria are vital cell powers in assimilation and catabolism. Oxidative phosphorylation (OXPHOS) of mitochondria is essential for the self-renewal of cancer stem-like cells in lung cancer, glioblastoma and leukemia. Thymidine kinase 2 (TK2) and deoxyguanosine kinase (DGUOK) are two mitochondrial deoxynucleoside kinases, which are responsible for the transport of pyrimidine and purine deoxynucleoside in mitochondria. Apoptosis and autophagy are important processes that regulate cell proliferation and death in normal cells and cancer cells. Inducing cancer cell apoptosis and autophagy is an effective means to treat malignant tumors. This review discusses the research progress of the relationship between mitochondrial deoxyguanosine kinase and lung adenocarcinoma cell apoptosis and autophagy.</p>

Advances in Thymidine Kinase 2 Deficiency: Clinical Aspects, Translational Progress, and Emerging Therapies.

Defects in the replication, maintenance, and repair of mitochondrial DNA (mtDNA) constitute a growing and genetically heterogeneous group of mitochondrial disorders. Multiple genes participate in these processes, including thymidine kinase 2 (TK2) encoding the mitochondrial matrix protein TK2, a critical component of the mitochondrial nucleotide salvage pathway. TK2 deficiency (TK2d) causes mtDNA depletion, multiple deletions, or both, which manifest predominantly as mitochondrial myopathy. A wide clinical spectrum phenotype includes a severe, rapidly progressive, early onset form (median survival: < 2 years); a less severe childhood-onset form; and a late-onset form with a variably slower rate of progression. Clinical presentation typically includes progressive weakness of limb, neck, facial, oropharyngeal, and respiratory muscle, whereas limb myopathy with ptosis, ophthalmoparesis, and respiratory involvement is more common in the late-onset form. Deoxynucleoside monophosphates and deoxynucleosides that can bypass the TK2 enzyme defect have been assessed in a mouse model, as well as under open-label compassionate use (expanded access) in TK2d patients, indicating clinical efficacy with a favorable side-effect profile. This treatment is currently undergoing testing in clinical trials intended to support approval in the US and European Union (EU). In the early expanded access program, growth differentiation factor 15 (GDF-15) appears to be a useful biomarker that correlates with therapeutic response. With the advent of a specific treatment and given the high morbidity and mortality associated with TK2d, clinicians need to know how to recognize and diagnose this disorder. Here, we summarize translational research about this rare condition emphasizing clinical aspects.

Thymidine Kinase 2 and Mitochondrial Protein COX I in the Cerebellum of Patients with Spinocerebellar Ataxia Type 31 Caused by Penta-nucleotide Repeats (TTCCA)n

Spinocerebellar ataxia type 31 (SCA31), an autosomal-dominant neurodegenerative disorder characterized by progressive cerebellar ataxia with Purkinje cell degeneration, is caused by a heterozygous 2.5–3.8 kilobase penta-nucleotide repeat of (TTCCA)n in intron 11 of the thymidine kinase 2 (TK2) gene. TK2 is an essential mitochondrial pyrimidine-deoxyribonucleoside kinase. Bi-allelic loss-of-function mutations of TK2 lead to mitochondrial DNA depletion syndrome (MDS) in humans through severe (~ 70%) reduction of mitochondrial electron-transport-chain activity, and tk2 knockout mice show Purkinje cell degeneration and ataxia through severe mitochondrial cytochrome-c oxidase subunit I (COX I) protein reduction. To clarify whether TK2 function is altered in SCA31, we investigated TK2 and COX I expression in human postmortem SCA31 cerebellum. We confirmed that canonical TK2 mRNA is transcribed from exons far upstream of the repeat site, and demonstrated that an extended version of TK2 mRNA (“TK2-EXT”), transcribed from exons spanning the repeat site, is expressed in human cerebellum. While canonical TK2 was conserved among vertebrates, TK2-EXT was specific to primates. Reverse transcription-PCR demonstrated that both TK2 mRNAs were preserved in SCA31 cerebella compared with control cerebella. The TK2 proteins, assessed with three different antibodies including our original polyclonal antibody against TK2-EXT, were detected as ~ 26 kilodalton proteins on western blot; their levels were similar in SCA31 and control cerebella. COX I protein level was preserved in SCA31 compared to nuclear DNA-encoded protein. We conclude that the expression and function of TK2 are preserved in SCA31, suggesting a mechanism distinct from that of MDS.

Generation of an induced pluripotent stem cell line from a compound heterozygous patient in TK2 gene

Autosomal recessive mutations in Thymidine kinase 2 (TK2) gene cause depletion and multiple deletions in mtDNA which normally lead to fatal and progressive neuromyopathy in infants and children. We have generated an induced pluripotent stem cell (iPSC) line by reprogramming fibroblasts derived from a patient carrying TK2 mutations. New iPSC line pluripotency was evaluated by verifying the expression of pluripotency-related genes and the in vitro differentiation into the three germ layers. This human-derived model will be useful for studying the pathogenic mechanisms triggered by these mutations and for testing therapies in cell types normally affected in patients.

Mutational analyses of human thymidine kinase 2 reveal key residues in ATP-Mg2+ binding and catalysis

Mitochondrial thymidine kinase 2 (TK2) is an essential enzyme for mitochondrial dNTP synthesis in many tissues. Deficiency in TK2 activity causes devastating mitochondrial diseases. Here we investigated several residues involved in substrate binding and catalysis. We showed that mutations of Gln-110 and Glu-133 affected Mg2+ and ATP binding, and thus are crucial for TK2 function. Furthermore, mutations of Gln-110 and Tyr-141 altered the kinetic behavior, suggesting their involvement in substrate binding through conformational changes. Since the 3 D structure of TK2 is still unknown, and thus, the identification of key amino acids for TK2 function may help to explain how TK2 mutations cause mitochondrial diseases.

Synergistic Deoxynucleoside and Gene Therapies for Thymidine Kinase 2 Deficiency.

Autosomal recessive human thymidine kinase 2 (TK2) mutations cause TK2 deficiency, which typically manifests as a progressive and fatal mitochondrial myopathy in infants and children. Treatment with pyrimidine deoxynucleosides deoxycytidine and thymidine ameliorates mitochondrial defects and extends the lifespan of Tk2 knock-in mouse (Tk2KI ) and compassionate use deoxynucleoside therapy in TK2 deficient patients have shown promising indications of efficacy. To augment therapy for Tk2 deficiency, we assessed gene therapy alone and in combination with deoxynucleoside therapy in Tk2KI mice. We generated pAAVsc CB6 PI vectors containing human TK2 cDNA (TK2). Adeno-associated virus (AAV)-TK2 was administered to Tk2KI , which were serially assessed for weight, motor functions, and survival as well as biochemical functions in tissues. AAV-TK2 treated mice were further treated with deoxynucleosides. AAV9 delivery of human TK2 cDNA to Tk2KI mice efficiently rescued Tk2 activity in all the tissues tested except the kidneys, delayed disease onset, and increased lifespan. Sequential treatment of Tk2KI mice with AAV9 first followed by AAV2 at different ages allowed us to reduce the viral dose while further prolonging the lifespan. Furthermore, addition of deoxycytidine and deoxythymidine supplementation to AAV9 + AAV2 treated Tk2KI mice dramatically improved mtDNA copy numbers in the liver and kidneys, animal growth, and lifespan. Our data indicate that AAV-TK2 gene therapy as well as combination deoxynucleoside and gene therapies is more effective in Tk2KI mice than pharmacological alone. Thus, combination of gene therapy with substrate enhancement is a promising therapeutic approach for TK2 deficiency and potentially other metabolic disorders. ANN NEUROL 2021;90:640-652.

Insight Into Spinocerebellar Ataxia Type 31 (SCA31) From Drosophila Model.

Spinocerebellar ataxia type 31 (SCA31) is a progressive neurodegenerative disease characterized by degeneration of Purkinje cells in the cerebellum. Its genetic cause is a 2.5- to 3.8-kb-long complex pentanucleotide repeat insertion containing (TGGAA)n, (TAGAA)n, (TAAAA)n, and (TAAAATAGAA)n located in an intron shared by two different genes: brain expressed associated with NEDD4-1 (BEAN1) and thymidine kinase 2 (TK2). Among these repeat sequences, (TGGAA)n repeat was the only sequence segregating with SCA31, which strongly suggests its pathogenicity. In SCA31 patient brains, the mutant BEAN1 transcript containing expanded UGGAA repeats (UGGAAexp) was found to form abnormal RNA structures called RNA foci in cerebellar Purkinje cell nuclei. In addition, the deposition of pentapeptide repeat (PPR) proteins, poly(Trp-Asn-Gly-Met-Glu), translated from UGGAAexp RNA, was detected in the cytoplasm of Purkinje cells. To uncover the pathogenesis of UGGAAexp in SCA31, we generated Drosophila models of SCA31 expressing UGGAAexp RNA. The toxicity of UGGAAexp depended on its length and expression level, which was accompanied by the accumulation of RNA foci and translation of repeat-associated PPR proteins in Drosophila, consistent with the observation in SCA31 patient brains. We also revealed that TDP-43, FUS, and hnRNPA2B1, motor neuron disease–linked RNA-binding proteins bound to UGGAAexp RNA, act as RNA chaperones to regulate the formation of RNA foci and repeat-associated translation. Further research on the role of RNA-binding proteins as RNA chaperones may also provide a novel therapeutic strategy for other microsatellite repeat expansion diseases besides SCA31.

Deoxynucleoside therapy for respiratory involvement in adult patients with thymidine kinase 2-deficient myopathy

BackgroundRecessive mutations in the thymidinekinase 2 (TK2) gene cause a rare mitochondrial myopathy, frequently with severe respiratory involvement. Deoxynucleoside therapy is currently under investigation.Research questionWhat is the impact of nucleosides...

MITOCHONDRIAL DISEASES & METABOLIC MYOPATHIES: P.320 Analysis of morbidity and mortality in untreated patients with thymidine kinase 2 deficiency

Thymidine kinase 2 (TK2) deficiency is an ultra-rare mitochondrial DNA replication disorder characterized by progressive muscle weakness that may lead to loss of ambulation, need for ventilatory support, and death. Severity varies by age of onset and there is no approved therapy. Understanding the course of untreated disease is important to evaluate new therapies under development. The objective of this analysis was to characterize the morbidity and mortality of patients with TK2 deficiency. A systematic literature review was conducted to identify unique patients with individual-level data and genetically confirmed TK2 deficiency to generate an untreated patient dataset (UPD).

Growth Differentiation Factor 15 is a potential biomarker of therapeutic response for TK2 deficient myopathy

GDF-15 is a biomarker for mitochondrial diseases. We investigated the application of GDF-15 as biomarker of disease severity and response to deoxynucleoside treatment in patients with thymidine kinase 2 (TK2) deficiency and compared it to FGF-21. GDF-15 and FGF-21 were measured in serum from 24 patients with TK2 deficiency treated 1–49 months with oral deoxynucleosides. Patients were grouped according to age at treatment and biomarkers were analyzed at baseline and various time points after treatment initiation. GDF-15 was elevated on average 30-fold in children and 6-fold in adults before the start of treatment. There was a significant correlation between basal GDF-15 and severity based on pretreatment distance walked (6MWT) and weight (BMI). During treatment, GDF-15 significantly declined, and the decrease was accompanied by relevant clinical improvements. The decline was greater in the paediatric group, which included the most severe patients and showed the greatest clinical benefit, than in the adult patients. The decline of FGF-21 was less prominent and consistent. GDF-15 is a potential biomarker of severity and of therapeutic response for patients with TK2 deficiency. In addition, we show evidence of clinical benefit of deoxynucleoside treatment, especially when treatment is initiated at an early age.

Basic biochemical characterization of cytosolic enzymes in thymidine nucleotide synthesis in adult rat tissues: implications for tissue specific mitochondrial DNA depletion and deoxynucleoside-based therapy for TK2-deficiency

BackgroundDeficiency in thymidine kinase 2 (TK2) or p53 inducible ribonucleotide reductase small subunit (p53R2) is associated with tissue specific mitochondrial DNA (mtDNA) depletion. To understand the mechanisms of the tissue specific mtDNA depletion we systematically studied key enzymes in dTMP synthesis in mitochondrial and cytosolic extracts prepared from adult rat tissues.ResultsIn addition to mitochondrial TK2 a cytosolic isoform of TK2 was characterized, which showed similar substrate specificity to the mitochondrial TK2. Total TK activity was highest in spleen and lowest in skeletal muscle. Thymidylate synthase (TS) was detected in cytosols and its activity was high in spleen but low in other tissues. TS protein levels were high in heart, brain and skeletal muscle, which deviated from TS activity levels. The p53R2 proteins were at similar levels in all tissues except liver where it was ~ 6-fold lower. Our results strongly indicate that mitochondria in most tissues are capable of producing enough dTTP for mtDNA replication via mitochondrial TK2, but skeletal muscle mitochondria do not and are most likely dependent on both the salvage and de novo synthesis pathways.ConclusionThese results provide important information concerning mechanisms for the tissue dependent variation of dTTP synthesis and explained why deficiency in TK2 or p53R2 leads to skeletal muscle dysfunctions. Furthermore, the presence of a putative cytosolic TK2-like enzyme may provide basic knowledge for the understanding of deoxynucleoside-based therapy for mitochondrial disorders.

P.60A retrospective study of the combination of pyrimidine nucleos(t)ides in patients with thymidine kinase 2 (TK2) deficiency

TK2 is a mitochondrial enzyme that phosphorylates thymidine (dT) and deoxycytidine (dC) to generate deoxycytidine and deoxythymidine monophosphates (dCMP/dTMP). TK2 deficiency, an ultra-rare autosomal recessive disorder, results in severe mitochondrial DNA depletion, deletions, or both due to unbalanced nucleotide pools. TK2 deficiency presents as a severe progressive proximal muscle weakness. Clinical presentations are heterogeneous with variable rates of progression, from rapidly progressive weakness in infants and young children leading to hypotonia and death from respiratory failure to a more slowly progressive adult-onset disease manifesting as ptosis, ophthalmoplegia, dysphagia, fatigue, progressive weakness, and respiratory insufficiency. Patients with TK2 deficiency were treated with dCMP/dTMP and dC/dT under compassionate use and investigator INDs. Use of dC/dT was supported by animal studies demonstrating that pyrimidine monophosphates are metabolized to pyrimidine nucleosides. Nucleoside substrate enhancement therapy is believed to act through residual TK2 activity as well as the cytosolic salvage pathways via dCK and TK1. This retrospective medical chart review systematically gathered data on the clinical course of 38 patients at 8 clinical sites in the US, Spain, Italy, and Israel from disease onset and after pyrimidine nucleos(t)ide therapy. Medical records from the time of onset of symptoms were collected to characterize the pretreatment disease course and allow comparison with post treatment disease course. Other analyses compared survival in treated patients with an untreated patient dataset gathered from all published reports of patients with TK2 deficiency. This study provides further knowledge about the safety and efficacy of treatment with pyrimidine nucelos(t)ides and the clinical manifestations and course of disease as well as demonstrates treatment has shifted the natural history of patients with TK2 deficiency.

Mechanisms of Chronic Fialuridine Hepatotoxicity as Revealed in Primary Human Hepatocyte Spheroids.

Drug hepatotoxicity is often delayed in onset. An exemplar case is the chronic nature of fialuridine hepatotoxicity, which resulted in the deaths of several patients in clinical trials as preclinical studies failed to identify this human-specific hepatotoxicity. Conventional preclinical in vitro models are mainly designed to evaluate the risk of acute drug toxicity. Here, we evaluated the utility of 3D spheroid cultures of primary human hepatocytes (PHHs) to assess chronic drug hepatotoxicity events using fialuridine as an example. Fialuridine toxicity was only detectable after 7 days of repeated exposure. Clinical manifestations, including reactive oxygen species formation, lipid accumulation, and induction of apoptosis, were readily identified. Silencing the expression or activity of the human equilibrative nucleoside transporter 1 (ENT1), implicated in the mitochondrial transport of fialuridine, modestly protected PHH spheroids from fialuridine toxicity. Interference with the phosphorylation of fialuridine into the active triphosphate metabolites by silencing of thymidine kinase 2 (TK2) provided substantial protection, whereas simultaneous silencing of ENT1 and TK2 provided near-complete protection. Fialuridine-induced mitochondrial dysfunction was suggested by a decrease in the expression of mtDNA-encoded genes, which correlated with the onset of toxicity and was prevented under the simultaneous silencing of ENT1 and TK2. Furthermore, interference with the expression or activity of ribonucleotide reductase (RNR), which is critical to deoxyribonucleoside triphosphate (dNTP) pool homeostasis, resulted in selective potentiation of fialuridine toxicity. Our findings demonstrate the translational applicability of the PHH 3D spheroid model for assessing drug hepatotoxicity events which manifest only under chronic exposure conditions.

Bioavailability and cytosolic kinases modulate response to deoxynucleoside therapy in TK2 deficiency

BackgroundTK2 is a nuclear gene encoding the mitochondrial matrix protein thymidine kinase 2 (TK2), a critical enzyme in the mitochondrial nucleotide salvage pathway. Deficiency of TK2 activity causes mitochondrial DNA (mtDNA) depletion, which in humans manifests predominantly as a mitochondrial myopathy with onset typically in infancy and childhood. We previously showed that oral treatment of the Tk2 H126N knock-in mouse model (Tk2−/−) with the TK2 substrates, deoxycytidine (dCtd) and thymidine (dThd), delayed disease onset and prolonged median survival by 3-fold. Nevertheless, dCtd + dThd treated Tk2−/− mice showed mtDNA depletion in brain as early as postnatal day 13 and in virtually all other tissues at age 29 days. MethodsTo enhance mechanistic understanding and efficacy of dCtd + dThd therapy, we studied the bioavailability of dCtd and dThd in various tissues as well as levels of the cytosolic enzymes, TK1 and dCK that convert the deoxynucleosides into dCMP and dTMP. FindingsParenteral treatment relative to oral treatment produced higher levels of dCtd and dThd and improved mtDNA levels in liver and heart, but did not ameliorate molecular defects in brain or prolong survival. Down-regulation of TK1 correlated with temporal- and tissue-specificity of response to dCtd + dThd. Finally, we observed in human infant and adult muscle expression of TK1 and dCK, which account for the long-term efficacy to dCtd + dThd therapy in TK2 deficient patients. InterpretationsThese data indicate that the cytosolic pyrimidine salvage pathway enzymes TK1 and dCK are critical for therapeutic efficacy of deoxynucleoside therapy for Tk2 deficiency. FundNational Institutes of Health P01HD32062.

Negative Cooperative Binding of Thymidine, Ordered Substrate Binding, and Product Release of Human Mitochondrial Thymidine Kinase 2 Explain Its Complex Kinetic Properties and Physiological Functions.

Mitochondrial thymidine kinase 2 (TK2) catalyzes the phosphorylation of thymidine (dT) and deoxycytidine (dC) and is essential for mitochondrial function in post-mitotic tissues. The phosphorylation of dT shows negative cooperativity, but the phosphorylation of dC follows classical Michaelis–Menten kinetics. The enzyme is feedback-inhibited by its end products deoxythymidine triphosphate (dTTP) and deoxycytidine triphosphate (dCTP). In order to better understand the reaction mechanism and the negative cooperative behavior, we conducted isothermal titration calorimetry (ITC) and intrinsic tryptophan fluorescence (ITF) quenching studies with purified recombinant human TK2. Cooperative binding was observed with dT but not dC by the ITC analysis in accordance with earlier enzyme kinetic studies. The phosphate donor adenosine triphosphate (ATP) did not bind to either dTTP-bound or dTTP-free enzymes but bound tightly to the dT– or dC–TK2 complexes with large differences in enthalpy and entropy changes, strongly suggesting an ordered binding of the substrates and different conformational states of the ATP and dT– and dC–TK2 ternary complexes. dTTP binding was endothermic; however, dCTP could not be shown to interact with the enzyme. ITF quenching studies also revealed tight binding of dT, dC, deoxythymidine monophosphate, deoxycytidine monophosphate, and dTTP but not adenosine 5′-diphosphate or ATP. These results strongly indicate an ordered sequential binding of the substrates and ordered release of the products as well as different conformational states of the active site of TK2. These results help to explain the different kinetics observed with dT and dC as substrates, which have important implications for TK2 regulation in vivo.

Clinical and molecular spectrum of thymidine kinase 2-related mtDNA maintenance defect

Mitochondrial DNA maintenance (mtDNA) defects have a wide range of causes, each with a set of phenotypes that overlap with many other neurological or muscular diseases. Clinicians face the challenge of narrowing down a long list of differential diagnosis when encountered with non-specific neuromuscular symptoms. Biallelic pathogenic variants in the Thymidine Kinase 2 (TK2) gene cause a myopathic form of mitochondrial DNA maintenance defect. Since the first description in 2001, there have been 71 patients reported with 42 unique pathogenic variants. Here we are reporting 11 new cases with 5 novel pathogenic variants. We describe and analyze a total of 82 cases with 47 unique TK2 pathogenic variants in effort to formulate a comprehensive molecular and clinical spectrum of TK2-related mtDNA maintenance disorders.