Human TMPK Research Articles

Structural and functional analysis of human thymidylate kinase isoforms

Thymidylate kinase (TMPK) phosphorylates deoxythymidine monophosphate (dTMP) and plays an important role in genome stability. Deficiency in TMPK activity due to genetic alterations of DTYMK, i.e., the gene coding for TMPK, causes severe microcephaly in humans. However, no defects were observed in other tissues, suggesting the existence of a compensatory enzyme for dTTP synthesis. In search for this compensatory enzyme we analyzed 6 isoforms of TMPK mRNA deposited in the GenBank. Of these, only isoform 1 has been characterized and represents the known human TMPK. Our results reveal that isoform 2, 3, 4 and 5 lack essential structural elements for substrate binding and, thus, they are considered as nonfunctional isoforms. Isoform 6, however, has intact catalytic centers, i.e., dTMP-binding, DRX motif, ATP-binding p-loop and lid region, which are the key structural elements of an active TMPK, suggesting that isoform 6 may function as TMPK. When isoform 6 was expressed and purified, it showed only minimal activity (<0.1%) as compared with isoform 1. A putative isoform 6 was detected in a cancer cell line, in addition to the dominant isoform 1. However, because of its low activity, isoform 6 is unlikely be able to compensate for the loss of TMPK activity caused by deletions and/or point mutations of the DTYMK gene. Thereby, future studies to identify and characterize the compensatory TMPK enzyme found in patients with DTYMK mutations may contribute to the understanding of dTTP synthesis and of the pathophysiological role of DTYMK mutations in neurodegenerative disorders.

Biochemical Characterizations of Human TMPK Mutations Identified in Patients with Severe Microcephaly: Single Amino Acid Substitutions Impair Dimerization and Abolish Their Catalytic Activity.

Deoxythymidylate kinase (TMPK) is a key enzyme in the synthesis of deoxythymidine triphosphate (dTTP). Four TMPK variants (P81L, A99T, D128N, and a frameshift) have been identified in human patients who suffered from severe neurodegenerative diseases. However, the impact of these mutations on TMPK function has not been clarified. Here we show that in fibroblasts derived from a patient, the P81L and D128N mutations led to a complete loss of TMPK activity in mitochondria and extremely low and unstable TMPK activity in cytosol. Despite the lack of TMPK activity, the patient-derived fibroblasts apparently grew normal. To investigate the impact of the mutations on the enzyme function, the mutant TMPKs were expressed, purified, and characterized. The wild-type TMPK mainly exists as a dimer with high substrate binding affinity, that is, low KM value and high catalytic efficiency, that is, kcat/KM. In contrast, all mutants were present as monomers with dramatically reduced substrate binding affinity and catalytic efficiencies. Based on the human TMPK structure, none of the mutated amino acids interacted directly with the substrates. By structural analysis, we could explain why the respective amino acid substitutions could drastically alter the enzyme structure and catalytic function. In conclusion, TMPK mutations identified in patients represent loss of function mutations but surprisingly the proliferation rate of the patient-derived fibroblasts was normal, suggesting the existence of an alternative and hitherto unknown compensatory TMPK-like enzyme for dTTP synthesis. Further studies of the TMPK enzymes will help to elucidate the role of TMPK in neuropathology.

In Silico Studies of Potential Selective Inhibitors of Thymidylate Kinase from Variola virus.

Continuing the work developed by our research group, in the present manuscript, we performed a theoretical study of 10 new structures derived from the antivirals cidofovir and ribavirin, as inhibitor prototypes for the enzyme thymidylate kinase from Variola virus (VarTMPK). The proposed structures were subjected to docking calculations, molecular dynamics simulations, and free energy calculations, using the molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) method, inside the active sites of VarTMPK and human TMPK (HssTMPK). The docking and molecular dynamic studies pointed to structures 2, 3, 4, 6, and 9 as more selective towards VarTMPK. In addition, the free energy data calculated through the MM-PBSA method, corroborated these results. This suggests that these compounds are potential selective inhibitors of VarTMPK and, thus, can be considered as template molecules to be synthesized and experimentally evaluated against smallpox.

Chemical Inhibition of Human Thymidylate Kinase and Structural Insights into the Phosphate Binding Loop and Ligand-Induced Degradation.

Targeting thymidylate kinase (TMPK) that catalyzes the phosphotransfer reaction for formation of dTDP from dTMP is a new strategy for anticancer treatment. This study is to understand the inhibitory mechanism of a previously identified human TMPK (hTMPK) inhibitor YMU1 (1a) by molecular docking, isothermal titration calorimetry, and photoaffinity labeling. The molecular dynamics simulation suggests that 1a prefers binding at the catalytic site of hTMPK, whereas the hTMPK inhibitors that bear pyridino[d]isothiazolone or benzo[d]isothiazolone core structure in lieu of the dimethylpyridine-fused isothiazolone moiety in 1a can have access to both the ATP-binding and catalytic sites. The binding sites of hTMPK inhibitors were validated by photoaffinity labeling and mass spectrometric studies. Taking together, 1a and its analogues stabilize the conformation of ligand-induced degradation (LID) region of hTMPK and block the catalytic site or ATP-binding site, thus attenuating the ATP binding-induced closed conformation that is required for phosphorylation of dTMP.

Design of inhibitors of thymidylate kinase from Variola virus as new selective drugs against smallpox.

Recently we constructed a homology model of the enzyme thymidylate kinase from Variola virus (VarTMPK) and proposed it as a new target to the drug design against smallpox. In the present work, we used the antivirals cidofovir and acyclovir as reference compounds to choose eleven compounds as leads to the drug design of inhibitors for VarTMPK. Docking and molecular dynamics (MD) studies of the interactions of these compounds inside VarTMPK and human TMPK (HssTMPK) suggest that they compete for the binding region of the substrate and were used to propose the structures of ten new inhibitors for VarTMPK. Further docking and MD simulations of these compounds, inside VarTMPK and HssTMPK, suggest that nine among ten are potential selective inhibitors of VarTMPK.

Preventing the return of smallpox: molecular modeling studies on thymidylate kinase from Variola virus

Smallpox was one of the most devastating diseases in the human history and still represents a serious menace today due to its potential use by bioterrorists. Considering this threat and the non-existence of effective chemotherapy, we propose the enzyme thymidylate kinase from Variola virus (VarTMPK) as a potential target to the drug design against smallpox. We first built a homology model for VarTMPK and performed molecular docking studies on it in order to investigate the interactions with inhibitors of Vaccinia virus TMPK (VacTMPK). Subsequently, molecular dynamics (MD) simulations of these compounds inside VarTMPK and human TMPK (HssTMPK) were carried out in order to select the most promising and selective compounds as leads for the design of potential VarTMPK inhibitors. Results of the docking and MD simulations corroborated to each other, suggesting selectivity towards VarTMPK and, also, a good correlation with the experimental data.

Thymidylate Kinase: An Old Topic Brings New Perspectives

Thymidylate kinase (TMPK) is a key enzyme for pyrimidine synthesis that catalyzes the phosphorylation of thymidine 5'-monophosphate (dTMP) in the presence of ATP and Mg(2+) to form thymidine 5'-diphosphate (dTDP), which is then converted to thymidine 5'-triphosphate (dTTP) by nucleoside-diphosphate kinase (NDK). TMPK has an important function in cell proliferation and its enzyme kinetics and related structures have been determined in various organisms. TMPK is well recognized as a potential drug target, with the most notable function being in the activation of anti-HIV nucleoside prodrugs. Recent studies have shown that TMPK is a validated target for antibiotic development against gram-positive bacterium of Staphylococcus aureus. In addition, inhibition of human TMPK increases the potential of anticancer agent doxorubicin toward colon cancer cells regardless of p53 status. Following the rapid expanding knowledge on TMPKs and the rising interests in TMPKs as a drug target, in this review we try to describe current research on TMPKs in various organisms of eukaryotes, prokaryotes and viruses and to provide information for designing new potential inhibitors against TMPKs.

Engineered human Tmpk fused with truncated cell-surface markers: versatile cell-fate control safety cassettes

Cell-fate control gene therapy (CFCGT)-based strategies can augment existing gene therapy and cell transplantation approaches by providing a safety element in the event of deleterious outcomes. Previously, we described a novel enzyme/prodrug combination for CFCGT. Here, we present results employing novel lentiviral constructs harboring sequences for truncated surface molecules (CD19 or low-affinity nerve growth factor receptor) directly fused to that CFCGT cDNA (TmpkF105Y). This confers an enforced one-to-one correlation between cell marking and eradication functions. In-vitro analysis demonstrated the full functionality of the fusion product. Next, low-dose 3'-azido-3'-deoxythymidine (AZT) administration to non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice injected with transduced clonal K562 cells suppressed tumor growth; furthermore, one integrated vector on average was sufficient to mediate cytotoxicity. Further, in a murine xenogeneic leukemia-lymphoma model we also demonstrated in-vivo control over transduced Raji cells. Finally, in a proof-of-principle study to examine the utility of this cassette in combination with a therapeutic cDNA, we integrated this novel CFCGT fusion construct into a lentivector designed for treatment of Fabry disease. Transduction with this vector restored enzyme activity in Fabry cells and retained AZT sensitivity. In addition, human Fabry patient CD34(+) cells showed high transduction efficiencies and retained normal colony-generating capacity when compared with the non-transduced controls. These collective results demonstrated that this novel and broadly applicable fusion system may enhance general safety in gene- and cell-based therapies.

509. Truncated CD19 Fused with Mutant Human Tmpk: A Versatile Cell Fate Control Safety Cassette

509. Truncated CD19 Fused with Mutant Human Tmpk: A Versatile Cell Fate Control Safety Cassette

Transgenic cardiac-targeted overexpression of human thymidylate kinase

Thymidylate kinase (TMPK) is a nucleoside monophosphate kinase that catalyzes phosphorylation of thymidine monophosphate to thymidine diphosphate. TMPK also mediates phosphorylation of monophosphates of thymidine nucleoside analog (NA) prodrugs on the pathway to their active triphosphate antiviral or antitumor moieties. Novel transgenic mice (TG) expressing human (h) TMPK were genetically engineered using the α-myosin heavy chain promoter to drive its cardiac-targeted overexpression. In ‘2 by 2' protocols, TMPK TGs and wild-type (WT) littermates were treated with the NA zidovudine (a deoxythymidine analog, 3′-azido-3′deoxythymidine (AZT)) or vehicle for 35 days. Alternatively, TGs and WTs were treated with a deoxycytidine NA (racivir, RCV) or vehicle. Changes in mitochondrial DNA (mtDNA) abundance and mitochondrial ultrastructure were defined quantitatively by real-time PCR and transmission electron microscopy, respectively. Cardiac performance was determined echocardiographically. Results showed TMPK TGs treated with either AZT or RCV exhibited decreased cardiac mtDNA abundance. Cardiac ultrastructural changes were seen only with AZT. AZT-treated TGs exhibited increased left ventricle (LV) mass. In contrast, LV mass in RCV-treated TGs and WTs remained unchanged. In all cohorts, LV end-diastolic dimension remained unchanged. This novel cardiac-targeted overexpression of hTMPK helps define the role of TMPK in mitochondrial toxicity of antiretrovirals.

PD-L1 Immuno-Gene Therapy Under Cell Fate Control: Persistence of Cellular Delivery Vehicle and Transgene Expression.

Abstract 3580Poster Board III-517Immune cell expression of programmed death ligand-1 (PD-L1) represents a particularly important molecular mechanism responsible for control of auto- and allo-immunity mediated by effector memory T cells expressing PD1 receptor. As such, we have reasoned that an immuno-gene therapy approach that enables T cell expression of PD-L1 will represent a novel method of immune regulation. Advantageous features of this proposed therapy include a capacity to: (1) enforce long-term, stable expression of PD-L1; (2) build-in an independent surface marker to allow specific transduced cell enrichment; (3) utilize cellular delivery vehicles comprised of highly functional T cells that persist in vivo after adoptive transfer; and (4) incorporate an enhanced cell fate control or ‘suicide' gene to permit in vivo control of the immuno-gene therapy. Given these considerations, we developed a recombinant lentiviral vector (LV) incorporating an EF1-α promoter that first encodes the cDNA for a fusion protein consisting of human CD19 (truncated, non-signaling) combined with mutated human TMPK that efficiently activates AZT as a pro-drug (Sato et al; Mol Therapy, 2007); then, after an IRES element, the vector encodes full-length human PD-L1. LV was made after transfection of 293T cells and then concentrated and titered. Initial experiments used Jurkat cells to optimize virus infection and to confirm co-expression of CD19 and PD-L1 by flow cytometry. In previous work, we have demonstrated that ex vivo T cell expansion in rapamycin induces an anti-apoptotic phenotype that permits enhanced in vivo T cell persistence in murine models and human-into-mouse xenogeneic transplant models. As such, we established the goal of infecting primary human CD4+ T cells manufactured using ex vivo co-stimulation (anti-CD3, anti-CD28), Th1-type polarization (inclusion of IFN-α), and exposure to high-dose rapamycin (1 μM); using a 6-day culture system and subsequent anti-CD19 column purification, >90% of resultant transduced T cells expressed PD-L1. Next, we utilized a xenogeneic transplantation model (Rag2−/−γc−/− hosts) to assess in vivo persistence of the gene-modified T cells and transgene expression (10,000 T cells transferred i.v. into each host). In vivo experiment #1 demonstrated that recipients of gene-modified T cells had increased numbers of human T cells in the spleen that co-expressed CD19 and PD-L1 relative to recipients of non-transduced but identically expanded human T cells (harvested at day 5 after adoptive transfer; 38,000 cells/spleen vs. 1000 cells/spleen, p=0.02). Such in vivo harvested T cells were secondarily co-stimulated ex vivo and propagated for an additional 5 days: co-expression of CD19 and PD-L1 persisted in ∼ 50% of T cells harvested from the gene-modified T cell cohort, and T cell numbers were maintained ex vivo (yield of CD19+PD-L1+ cells, 28,600 vs. 1500; p=0.0001). In vivo experiment #2 confirmed and extended these results. At day 21 after adoptive transfer, recipients of gene-modified T cells had increased numbers of human T cells that co-expressed CD19 and PD-L1 relative to recipients of non-transduced but identically expanded human T cells in both the spleen (2800 cells/spleen vs. 390 cells/spleen, p=0.01; n=10 per cohort) and bone marrow (71,600 cells/marrow vs. 6500 cells/marrow, p=0.0001; n=10 per cohort). Such in vivo harvested T cells at day 21 after adoptive transfer were secondarily co-stimulated ex vivo and propagated for an additional 6 days: co-expression of CD19 and PD-L1 persisted in ∼ 50% of T cells harvested from the gene-modified T cell cohort, and T cell numbers were maintained ex vivo (yield of CD19+PD-L1+ cells harvested from spleen, 71,200 vs. 1800, p=0.0008; yield of CD19+PD-L1+ cells harvested from marrow, 226,000 vs. 1400, p=0.0001). Because the rapamycin-resistant T cell vehicle utilized in these experiments manifests an anti-apoptotic phenotype that confers long-term engraftment potential, it is likely that the demonstrated durability in transgene expression relates both to the efficiency of the LV method utilized and to a T cell pro-survival function. In conclusion, the LV-mediated transfer of this novel combination of CD19/TMPK fusion protein and PD-L1 results in stable transgene expression in primary human T cells in vitro and in vivo, thereby opening an avenue to assess PD-L1 mediated immuno-gene therapy under cell fate control. Disclosures:No relevant conflicts of interest to declare.

Behavior of Thymidylate Kinase toward Monophosphate Metabolites and Its Role in the Metabolism of 1-(2′-Deoxy-2′-Fluoro-β- l -Arabinofuranosyl)-5-Methyluracil (Clevudine) and 2′,3′-Didehydro-2′,3′-Dideoxythymidine in Cells

L-nucleoside analogs are a new class of antiviral and anticancer agents, several of which are currently used in the clinic. The phosphorylation of these agents to the triphosphate form is thought to be important for exertion of their pharmacological activities. 1-(2'-deoxy-2'-fluoro-beta-L-arabinofuranosyl)-5-methyluracil (L-FMAU; Clevudine) is a thymidine analog that is currently under phase III clinical trials as an anti-human hepatitis B virus agent. We examined the behavior of its monophosphate metabolite with human recombinant thymidylate kinase (TMPK) and showed that L-FMAU monophosphate (L-FMAUMP) is a poorer substrate than its D-configuration anomer (D-FMAUMP). The phosphorylation efficiency of l-FMAUMP is similar to that of the monophosphate of 2',3'-didehydro-2',3'-dideoxythymidine (d4T), an anti-human immunodeficiency virus analog, both of which are approximately 1% TMP. To clarify the role of human TMPK in the phosphorylation of L-FMAUMP to the diphosphate metabolite in cells, a Tet-On inducible human TMPK cell line system was established. In this system, the expression of TMPK is closely regulated in response to various concentrations of doxycycline. When the cells were treated with L-FMAU or d4T, the amounts of the diphosphate and triphosphate metabolites of these analogs were increased, in accordance with an increase in human TMPK activity in cells. In conclusion, this is the first demonstration of the behavior of TMPK toward L-FMAUMP. This study indicates that human TMPK can phosphorylate L-FMAUMP and play a critical role in L-FMAU metabolism in cells.

Structural requirements for efficient phosphorylation of nucleotide analogs by human thymidylate kinase.

Successive phosphorylation of nucleoside analog prodrugs to their triphosphate forms is required for the pharmacological activity of these compounds in the chemotherapeutic treatment of viral infections and cancer. Human thymidylate kinase (TMPK), apart from its essential physiological role in the biosynthesis of TTP, is also required for the activation of thymidine analogs, such as the clinically used anti-HIV prodrugs AZT and d4T. This enzyme is rate determining in the three-step cascade of AZT phosphorylation. Our structural work on human, yeast and E. coli TMPKs, in conjunction with sequence homology analyses and biochemical data, has demonstrated that three loops are crucial for the function of this enzyme: the first is the highly conserved P-loop motif, which binds and positions the phosphoryl groups of ATP, the second critical loop contains the DR(Y/H) motif that supplies a catalytic arginine and is also important for the binding and positioning of the magnesium ion complexed to ATP, and the third loop is the so-called Lid-region that is a flexible stretch which closes on ATP when it binds. Modifications of the sugar moieties of nucleoside monophosphates are shown to exert drastic effects on the enzyme's conformation and, thus, reduced activity. Our structural work on several TMPKs has formed the basis for generating mutants of human TMPK that are about 100 times more efficient in phosphorylating AZTMP. These enzyme variants could potentially be introduced into HIV-targeted cells in order to significantly improve AZT's antiviral activity.

Mycobacterium tuberculosis Thymidylate Kinase: Structural Studies of Intermediates along the Reaction Pathway

Mycobacterium tuberculosis TMP kinase (TMPK Mtub) represents a promising target for developing drugs against tuberculosis because the configuration of its active site is unique in the TMPK family. To help elucidate the phosphorylation mechanism employed by this enzyme, structural changes occurring upon binding of substrates and subsequent catalysis were investigated by protein crystallography. Six new structures of TMPK Mtub were solved at a resolution better than 2.3 Å, including the first structure of an apo-TMPK, obtained by triggering catalysis in a crystal of a TMPK Mtub–TMP complex, which resulted in the release of the TDP product. A series of snapshots along the reaction pathway is obtained, revealing the closure of the active site in going from an empty to a fully occupied state, suggestive of an induced-fit mechanism typical of NMPKs. However, in TMPK Mtub the LID closure couples to the binding with an unusual location for a magnesium ion coordinating TMP in the active site. Our data suggest strongly that this ion is required for catalysis, acting as a clamp, possibly in concert with Arg95, to neutralise electrostatic repulsion between the anionic substrates, optimise their proper alignment and activate them through direct and water-mediated interactions. The 3′-hydroxyl moiety of TMP, critical to metal stabilisation, appears to be a target of choice for the design of potent inhibitors. On the other hand, the usual NTP-bound magnesium is not seen in our structures and Arg14, a P-loop residue unique to TMPK Mtub, may take over its role. Therefore, TMPK Mtub seems to have swapped the use of a metal ion as compared with e.g. human TMPK. Finally, TTP was observed in crystals of TMPK Mtub, locked by Arg14, thus providing a structural explanation for the observed inhibitory effect of TTP putatively involved in a mechanism of feedback regulation of the enzymatic activity.

Insights into the phosphoryltransfer mechanism of human thymidylate kinase gained from crystal structures of enzyme complexes along the reaction coordinate

Background: Thymidylate kinase (TMPK) is a nucleoside monophosphate kinase that catalyzes the reversible phosphoryltransfer between ATP and TMP to yield ADP and TDP. In addition to its vital role in supplying precursors for DNA synthesis, human TMPK has an important medical role participating in the activation of a number of anti-HIV prodrugs. Results: Crystal structures of human TMPK in complex with TMP and ADP, TMP and the ATP analog AppNHp, TMP with ADP and the phosphoryl analog AlF 3, TDP and ADP, and the bisubstrate analog TP 5A were determined. The conformations of the P-loop, the LID region, and the adenine-binding loop vary according to the nature of the complex. Substitution of ADP by AppNHp results in partial closure of the P-loop and the rotation of the TMP phosphate group to a catalytically unfavorable position, which rotates back in the AlF 3 complex to a position suitable for in-line attack. In the fully closed state observed in the TP 5A and the TDP–ADP complexes, Asp15 interacts strongly with the 3′-hydroxyl group of TMP. Conclusions: The observed changes of nucleotide state and conformation and the corresponding protein structural changes are correlated with intermediates occurring along the reaction coordinate and show the sequence of events occurring during phosphate transfer. The low catalytic activity of human TMPK appears to be determined by structural changes required to achieve catalytic competence and it is suggested that a mechanism might exist to accelerate the activity.