Exploring the Mutated Kinases for Chemoenzymatic Synthesis of N4-Modified Cytidine Monophosphates.

Nucleosides, nucleotides, and their analogues are an important class of molecules that are used as substrates in research of enzymes and nucleic acid, or as antiviral and antineoplastic agents. Nucleoside phosphorylation is usually achieved with chemical methods; however, enzymatic phosphorylation is a viable alternative. Here, we present a chemoenzymatic synthesis of modified cytidine monophosphates, where a chemical synthesis of novel N4-modified cytidines is followed by an enzymatic phosphorylation of the nucleosides by nucleoside kinases. To enlarge the substrate scope, multiple mutant variants of Drosophila melanogaster deoxynucleoside kinase (DmdNK) (EC:2.7.1.145) and Bacillus subtilis deoxycytidine kinase (BsdCK) (EC:2.7.1.74) have been created and tested. It has been determined that certain point mutations in the active sites of the kinases alter their substrate specificities noticeably and allow phosphorylation of compounds that had been otherwise not phosphorylated by the wild-type DmdNK or BsdCK.

TMSOTf assisted synthesis of 2'-deoxy-2'-[18F]fluoro-ß-D-arabinofuranosylcytosine ([18F]FAC).

[18F]FAC (2'-deoxy-2'-[18F]fluoro-ß-D-arabinofuranosylcytosine, 1) is a versatile probe for imaging deoxycytidine kinase (dCK) expression levels in vivo. dCK is responsible for phosphorylation of deoxycytidine (dC, 2) and other nucleoside analogs, plays a key role in immune activation and has demonstrated to be one of the key enzymes in activating nucleoside based drugs including gemcitabine. Reported synthesis of [18F]FAC is high yielding but is quite challenging requiring bromination using HBr and careful drying of excess HBr which is critical for successful synthesis. Here in we report a simplified trimethylsilyl trifluoromethanesulfonate (TMSOTf) assisted synthesis of [18F]FAC eliminating the need of bromination and drying. [18F]FAC (ß-anomer) was synthesized with average isolated decay corrected yield of 10.59 + 4.2% (n = 6) with radiochemical purity of >98% and total synthesis time of 158 + 19 min.

Dual protein kinase and nucleoside kinase modulators for rationally designed polypharmacology.

Masitinib, a highly selective protein kinase inhibitor, can sensitise gemcitabine-refractory cancer cell lines when used in combination with gemcitabine. Here we report a reverse proteomic approach that identifies the target responsible for this sensitisation: the deoxycytidine kinase (dCK). Masitinib, as well as other protein kinase inhibitors, such as imatinib, interact with dCK and provoke an unforeseen conformational-dependent activation of this nucleoside kinase, modulating phosphorylation of nucleoside analogue drugs. This phenomenon leads to an increase of prodrug phosphorylation of most of the chemotherapeutic drugs activated by this nucleoside kinase. The unforeseen dual activity of protein kinase inhibition/nucleoside kinase activation could be of great therapeutic benefit, through either reducing toxicity of therapeutic agents by maintaining effectiveness at lower doses or by counteracting drug resistance initiated via down modulation of dCK target.

Redesigning human 2'-deoxycytidine kinase enantioselectivity for L-nucleoside analogues as reporters in positron emission tomography.

Recent advances in nuclear medicine have allowed for positron emission tomography (PET) to track transgenes in cell-based therapies using PET reporter gene/probe pairs. A promising example for such reporter gene/probe pairs are engineered nucleoside kinases that effectively phosphorylate isotopically labeled nucleoside analogues. Upon expression in target cells, the kinase facilitates the intracellular accumulation of radionuclide monophosphate, which can be detected by PET imaging. We have employed computational design for the semi-rational engineering of human 2'-deoxycytidine kinase to create a reporter gene with selectivity for L-nucleosides including L-thymidine and 1-(2'-fluoro-5-methyl-ß-L-arabinofuranosyl) uracil. Our design strategy relied on a combination of preexisting data from kinetic and structural studies of native kinases, as well as two small, focused libraries of kinase variants to generate an in silico model for assessing the effects of single amino acid changes on favorable activation of L-nucleosides over their corresponding D-enantiomers. The approach identified multiple amino acid positions distal to the active site that conferred desired L-enantioselectivity. Recombination of individual amino acid substitutions yielded orthogonal kinase variants with significantly improved catalytic performance for unnatural L-nucleosides but reduced activity for natural D-nucleosides.

Deoxycytidine kinase augments ATM-Mediated DNA repair and contributes to radiation resistance.

Efficient and adequate generation of deoxyribonucleotides is critical to successful DNA repair. We show that ataxia telangiectasia mutated (ATM) integrates the DNA damage response with DNA metabolism by regulating the salvage of deoxyribonucleosides. Specifically, ATM phosphorylates and activates deoxycytidine kinase (dCK) at serine 74 in response to ionizing radiation (IR). Activation of dCK shifts its substrate specificity toward deoxycytidine, increases intracellular dCTP pools post IR, and enhances the rate of DNA repair. Mutation of a single serine 74 residue has profound effects on murine T and B lymphocyte development, suggesting that post-translational regulation of dCK may be important in maintaining genomic stability during hematopoiesis. Using [(18)F]-FAC, a dCK-specific positron emission tomography (PET) probe, we visualized and quantified dCK activation in tumor xenografts after IR, indicating that dCK activation could serve as a biomarker for ATM function and DNA damage response in vivo. In addition, dCK-deficient leukemia cell lines and murine embryonic fibroblasts exhibited increased sensitivity to IR, indicating that pharmacologic inhibition of dCK may be an effective radiosensitization strategy.

Structural characterization of new deoxycytidine kinase inhibitors rationalizes the affinity-determining moieties of the molecules.

Deoxycytidine kinase (dCK) is a key enzyme in the nucleoside salvage pathway that is also required for the activation of several anticancer and antiviral nucleoside analog prodrugs. Additionally, dCK has been implicated in immune disorders and has been found to be overexpressed in several cancers. To allow the probing and modulation of dCK activity, a new class of small-molecule inhibitors of the enzyme were developed. Here, the structural characterization of four of these inhibitors in complex with human dCK is presented. The structures reveal that the compounds occupy the nucleoside-binding site and bind to the open form of dCK. Surprisingly, a slight variation in the nature of the substituent at the 5-position of the thiazole ring governs whether the active site of the enzyme is occupied by one or two inhibitor molecules. Moreover, this substituent plays a critical role in determining the affinity, improving it from >700 to 1.5 nM in the best binder. These structures lay the groundwork for future modifications that would result in even tighter binding and the correct placement of moieties that confer favorable pharmacodynamics and pharmacokinetic properties.

Deoxycytidine kinase regulates the G2/M checkpoint through interaction with cyclin-dependent kinase 1 in response to DNA damage.

Deoxycytidine kinase (dCK) is a rate limiting enzyme critical for phosphorylation of endogenous deoxynucleosides for DNA synthesis and exogenous nucleoside analogues for anticancer and antiviral drug actions. dCK is activated in response to DNA damage; however, how it functions in the DNA damage response is largely unknown. Here, we report that dCK is required for the G2/M checkpoint in response to DNA damage induced by ionizing radiation (IR). We demonstrate that the ataxia-telangiectasia-mutated (ATM) kinase phosphorylates dCK on Serine 74 to activate it in response to DNA damage. We further demonstrate that Serine 74 phosphorylation is required for initiation of the G2/M checkpoint. Using mass spectrometry, we identified a protein complex associated with dCK in response to DNA damage. We demonstrate that dCK interacts with cyclin-dependent kinase 1 (Cdk1) after IR and that the interaction inhibits Cdk1 activity both in vitro and in vivo. Together, our results highlight the novel function of dCK and provide molecular insights into the G2/M checkpoint regulation in response to DNA damage.

Gemcitabine: a critical nucleoside for cancer therapy.

Gemcitabine (dFdC, 2',2'-difluorodeoxycytidine) is a deoxycytidine nucleoside analogue of deoxycytidine in which two fluorine atoms have been inserted into the deoxyribose ring. Like other nucleoside analogues, gemcitabine is a prodrug. It is inactive in its original form, and depends on the intracellular machinery to gain pharmacological activity. What makes gemcitabine different from other nucleoside analogues is that it is actively transported across the cell membrane, it is phosphorylated more efficiently and it is eliminated at a slower rate. These differences, together with self-potentiation mechanisms, masked DNA chain termination and extensive inhibitory efficiency against several enzymes, are the source of gemcitabine's cytotoxic activity against a wide variety of tumors. This unique combination of metabolic properties and mechanistic characteristics is only found in very few other anticancer drugs, and both the FDA and the EMEA have already approved its use for clinical purposes, for the treatment of several types of tumors. In spite of the promising results associated with gemcitabine, the knowledge of its mode of action and of the enzymes it interacts with is still not fully documented. In this article we propose to review all these aspects and summarize the path of gemcitabine inside the cell.

The apoptotic effects of toosendanin are partially mediated by activation of deoxycytidine kinase in HL-60 cells.

Triterpenoid toosendanin (TSN) exhibits potent cytotoxic activity through inducing apoptosis in a variety of cancer cell lines. However, the target and mechanism of the apoptotic effects by TSN remain unknown. In this study, we captured a specific binding protein of TSN in HL-60 cells by serial affinity chromatography and further identified it as deoxycytidine kinase (dCK). Combination of direct activation of dCK and inhibition of TSN-induced apoptosis by a dCK inhibitor confirmed that dCK is a target for TSN partially responsible for the apoptosis in HL-60 cells. Moreover, the activation of dCK by TSN was a result of conformational change, rather than auto-phosphorylation. Our results further imply that, in addition to the dATP increase by dCK activation in tumor cells, dCK may also involve in the apoptotic regulation.

Post-translational phosphorylation of serine 74 of human deoxycytidine kinase favors the enzyme adopting the open conformation making it competent for nucleoside binding and release.

Deoxycytidine kinase (dCK) uses either ATP or UTP as a phosphoryl donor to catalyze the phosphorylation of nucleoside acceptors. The kinetic properties of human dCK are modulated in vivo by phosphorylation of serine 74. This residue is a part of the insert region and is distant from the active site. Replacing the serine with a glutamic acid (S74E variant) can mimic phosphorylation of Ser74. To understand how phosphorylation affects the catalytic properties of dCK, we examined the S74E variant of dCK both structurally and kinetically. We observe that the presence of a glutamic acid at position 74 favors the adoption by the enzyme of the open conformation. Glu74 stabilizes the open conformation by directly interacting with the indole side chain of Trp58, a residue that is in the proximity of the base of the nucleoside substrate. The open dCK conformation is competent for the binding of nucleoside but not for phosphoryl transfer. In contrast, the closed conformation is competent for phosphoryl transfer but not for product release. Thus, dCK must make the transition between the open and closed states during the catalytic cycle. We propose a reaction scheme for dCK that incorporates the transition between the open and closed states, and this serves to rationalize the observed kinetic differences between wild-type dCK and the S74E variant.

The sugar ring of the nucleoside is required for productive substrate positioning in the active site of human deoxycytidine kinase (dCK): implications for the development of dCK-activated acyclic guanine analogues.

The low toxicity of acyclovir (ACV) is mainly due to the fact that human nucleoside kinases have undetectable phosphorylation rates with this acyclic guanine analogue. In contrast, herpes virus thymidine kinase (HSV1-TK) readily activates ACV. We wanted to understand why human deoxycytidine kinase (dCK), which is related to HSV1-TK and phosphorylates deoxyguanosine, does not accept acyclic guanine analogues as substrates. Therefore, we crystallized dCK in complex with ACV at the nucleoside phosphoryl acceptor site and UDP at the phosphoryl donor site. The structure reveals that while ACV does bind at the dCK active site, it does so adopting a nonproductive conformation. Despite binding ACV, the enzyme remains in the open, inactive state. In comparison to ACV binding to HSV1-TK, in dCK, the nucleoside base adopts a different orientation related by about a 60 degrees rotation. Our analysis suggests that dCK would phosphorylate acyclic guanine analogues if they can induce a similar rotation.

Structural and kinetic characterization of human deoxycytidine kinase variants able to phosphorylate 5-substituted deoxycytidine and thymidine analogues .

The physiological role of human deoxycytidine kinase (dCK) is to phosphorylate deoxynucleosides required for DNA synthesis, with the exception of thymidine. Previous structural analysis of dCK implicated steric factors, specifically the thymine methyl group at the 5-position, that prevent thymidine phosphorylation by dCK. This hypothesis is supported by the observation that mutations that enlarge the active site cavity in proximity to the nucleoside 5-position endow dCK with the ability to phosphorylate thymidine. However, in conflict with this hypothesis was our discovery that the cytidine analogue 5-methyldeoxycytidine (5-Me-dC), an isostere of thymidine, can indeed be phosphorylated by wild-type (WT) dCK. To reconcile this seemingly contradicting observation, and to better understand the determinants preventing thymidine phosphorylation by WT dCK, we solved the crystal structure of dCK in complex with 5-Me-dC. The structure reveals the active site adjustments required to accommodate the methyl group at the 5-position. Combination of kinetic, mutagenesis, and structural data suggested that it is in fact residue Asp133 of dCK that is most responsible for discriminating against the thymine base. dCK variants in which Asp133 is replaced by an alanine and Arg104 by select hydrophobic residues attain significantly improved activity with 5-substituted deoxycytidine and thymidine analogues. Importantly, the ability of the designer enzymes to activate 5-substitued pyrimidines makes it possible to utilize such nucleoside analogues in suicide gene therapy or protein therapy applications that target cancer cells.

Casein kinase 1delta activates human recombinant deoxycytidine kinase by Ser-74 phosphorylation, but is not involved in the in vivo regulation of its activity.

Deoxycytidine kinase (dCK) is a key enzyme in the salvage of deoxynucleosides and in the activation of several anticancer and antiviral nucleoside analogues. We recently showed that dCK was activated in vivo by phosphorylation of Ser-74. However, the protein kinase responsible was not identified. Ser-74 is located downstream a Glu-rich region, presenting similarity with the consensus phosphorylation motif of casein kinase 1 (CKI), and particularly of CKI delta. We showed that recombinant CKI delta phosphorylated several residues of bacterially overexpressed dCK: Ser-74, but also Ser-11, Ser-15, and Thr-72. Phosphorylation of dCK by CKI delta correlated with increased activity reaching at least 4-fold. Site-directed mutagenesis demonstrated that only Ser-74 phosphorylation was involved in dCK activation by CKI delta, strengthening the key role of this residue in the control of dCK activity. However, neither CKI delta inhibitors nor CKI delta siRNA-mediated knock-down modified Ser-74 phosphorylation or dCK activity in cultured cells. Moreover, these approaches did not prevent dCK activation induced by treatments enhancing Ser-74 phosphorylation. Taken together, the data preclude a role of CKI delta in the regulation of dCK activity in vivo. Nevertheless, phosphorylation of dCK by CKI delta could be a useful tool for elucidating the influence of Ser-74 phosphorylation on the structure-activity relationships in the enzyme.

Influence of phosphorylation of THR-3, SER-11, and SER-15 on deoxycytidine kinase activity and stability.

Deoxycytidine kinase (dCK) is a key enzyme in the salvage of deoxyribonucleosides and in the activation of several anticancer and antiviral nucleoside analogues. We have recently shown that dCK is a phosphoprotein. Four in vivo phosphorylation sites were identified: Thr-3, Ser-11, Ser-15, and Ser-74. Site-directed mutagenesis demonstrated that phosphorylation of Ser-74, the major phosphorylated residue, strongly influences dCK activity in eucaryotic cells. Here, we show that phosphorylation of the three other sites, located in the N-terminal extremity of the protein, does not significantly modify dCK activity, but phosphorylation of Thr-3 could promote dCK stability.

Elucidation of different binding modes of purine nucleosides to human deoxycytidine kinase.

Purine nucleoside analogues of medicinal importance, such as cladribine, require phosphorylation by deoxycytidine kinase (dCK) for pharmacological activity. Structural studies of ternary complexes of human dCK show that the enzyme conformation adjusts to the different hydrogen-bonding properties between dA and dG and to the presence of substituent at the 2-position present in dG and cladribine. Specifically, the carbonyl group in dG elicits a previously unseen conformational adjustment of the active site residues Arg104 and Asp133. In addition, dG and cladribine adopt the anti conformation, in contrast to the syn conformation observed with dA. Kinetic analysis reveals that cladribine is phosphorylated at the highest efficiency with UTP as donor. We attribute this to the ability of cladribine to combine advantageous properties from dA (favorable hydrogen-bonding pattern) and dG (propensity to bind to the enzyme in its anti conformation), suggesting that dA analogues with a substituent at the 2-position are likely to be better activated by human dCK.

Gemcitabine pharmacogenomics: deoxycytidine kinase and cytidylate kinase gene resequencing and functional genomics.

Gemcitabine and other cytidine antimetabolites require metabolic activation by phosphorylation. Deoxycytidine kinase (DCK) and cytidine monophosphate kinase (CMPK) catalyze these reactions. We have applied a genotype-to-phenotype strategy to study DCK and CMPK pharmacogenomics. Specifically, we resequenced DCK and CMPK using 240 DNA samples, 60 each from African-American, Caucasian-American, Han Chinese-American, and Mexican-American subjects. We observed 28 DCK polymorphisms and 28 polymorphisms in CMPK, 33 of which were novel. Expression in COS-1 cells showed that variant allozyme enzyme activities ranged from 32 to 105% of the wild type (WT) for DCK and from 78 to 112% of WT for CMPK--with no significant differences in apparent K(m) values for either enzyme except for a DCK Val24/Ser122 double variant allozyme. Relative levels of DCK and CMPK immunoreactive protein in the COS-1 cells paralleled relative levels of enzyme activity and were significantly correlated for DCK (R(p) = 0.89, P = 0.0004) but not for CMPK (R(p) = 0.82, P = 0.095). The results of an analysis of DCK and CMPK structural models were compatible with the observed functional consequences of sequence alterations in variant allozymes. We also confirmed that the CMPK protein expressed in COS-1 cells and in a rabbit reticulocyte lysate was 196 rather than 228 amino acids in length. In summary, we determined common sequence variations in DCK and CMPK and systematically evaluated their functional implications. These gene sequence differences may contribute to variations in the metabolic activation of gemcitabine and other cytidine antimetabolites.

Molecular basis for the lack of enantioselectivity of human 3-phosphoglycerate kinase.

Non-natural L-nucleoside analogues are increasingly used as therapeutic agents to treat cancer and viral infections. To be active, L-nucleosides need to be phosphorylated to their respective triphosphate metabolites. This stepwise phosphorylation relies on human enzymes capable of processing L-nucleoside enantiomers. We used crystallographic analysis to reveal the molecular basis for the low enantioselectivity and the broad specificity of human 3-phosphoglycerate kinase (hPGK), an enzyme responsible for the last step of phosphorylation of many nucleotide derivatives. Based on structures of hPGK in the absence of nucleotides, and bound to L and d forms of MgADP and MgCDP, we show that a non-specific hydrophobic clamp to the nucleotide base, as well as a water-filled cavity behind it, allows high flexibility in the interaction between PGK and the bases. This, combined with the dispensability of hydrogen bonds to the sugar moiety, and ionic interactions with the phosphate groups, results in the positioning of different nucleotides so to expose their diphosphate group in a position competent for catalysis. Since the third phosphorylation step is often rate limiting, our results are expected to alleviate in silico tailoring of L-type prodrugs to assure their efficient metabolic processing.

Structural basis for substrate promiscuity of dCK.

Deoxycytidine kinase (dCK) is an essential nucleoside kinase critical for the production of nucleotide precursors for DNA synthesis. This enzyme catalyzes the initial conversion of the nucleosides deoxyadenosine (dA), deoxyguanosine (dG), and deoxycytidine (dC) into their monophosphate forms, with subsequent phosphorylation to the triphosphate forms performed by additional enzymes. Several nucleoside analog prodrugs are dependent on dCK for their pharmacological activation, and even nucleosides of the non-physiological L-chirality are phosphorylated by dCK. In addition to accepting dC and purine nucleosides (and their analogs) as phosphoryl acceptors, dCK can utilize either ATP or UTP as phosphoryl donors. To unravel the structural basis for substrate promiscuity of dCK at both the nucleoside acceptor and nucleotide donor sites, we solved the crystal structures of the enzyme as ternary complexes with the two enantiomeric forms of dA (D-dA, or L-dA), with either UDP or ADP bound to the donor site. The complexes with UDP revealed an open state of dCK in which the nucleoside, either D-dA or L-dA, is surprisingly bound in a manner not consistent with catalysis. In contrast, the complexes with ADP, with either D-dA or L-dA, adopted a closed and catalytically competent conformation. The differential states adopted by dCK in response to the nature of the nucleotide were also detected by tryptophan fluorescence experiments. Thus, we are in the unique position to observe differential effects at the acceptor site due to the nature of the nucleotide at the donor site, allowing us to rationalize the different kinetic properties observed with UTP to those with ATP.

Systematic exploration of active site mutations on human deoxycytidine kinase substrate specificity.

Human deoxycytidine kinase (dCK) is responsible for the phosphorylation of a number of clinically important nucleoside analogue prodrugs in addition to its natural substrates, 2'-deoxycytidine, 2'-deoxyguanosine, and 2'-deoxyadenosine. To improve the low catalytic activity and tailor the substrate specificity of dCK, we have constructed libraries of mutant enzymes and tested them for thymidine kinase (tk) activity. Random mutagenesis was employed to probe for amino acid positions with an impact on substrate specificity throughout the entire enzyme structure, identifying positions Arg104 and Asp133 in the active site as key residues for substrate specificity. Kinetic analysis indicates that Arg104Gln/Asp133Gly creates a "generalist" kinase with broader specificity and elevated turnover for natural and prodrug substrates. In contrast, the substitutions of Arg104Met/Asp133Thr, obtained via site-saturation mutagenesis, yielded a mutant with reversed substrate specificity, elevating the specific constant for thymidine phosphorylation by over 1000-fold while eliminating activity for dC, dA, and dG under physiological conditions. The results illuminate the key contributions of these two amino acid positions to enzyme function by demonstrating their ability to moderate substrate specificity.

Mimicking phosphorylation of Ser-74 on human deoxycytidine kinase selectively increases catalytic activity for dC and dC analogues.

Intracellular phosphorylation of dCK on Ser-74 results in increased nucleoside kinase activity. We mimicked this phosphorylation by a Ser-74-Glu mutation in bacterially produced dCK and investigated kinetic parameters using various nucleoside substrates. The S74E mutation increases the k(cat) values 11-fold for dC, and 3-fold for the anti-cancer analogues dFdC and AraC. In contrast, the rate is decreased for the purine substrates. In HEK293 cells, we found that by comparing transiently transfected dCK(S74E)-GFP and wild-type dCK-GFP, mimicking the phosphorylation of Ser-74 has no effect on cellular localisation. We note that phosphorylation may represent a mechanism to enhance the catalytic activity of the relatively slow dCK enzyme.