RESUMO
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.
Assuntos
Desoxicitidina Quinase/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Tiazóis/farmacologia , Células A549 , Antineoplásicos/química , Antineoplásicos/farmacologia , Benzamidas , Linhagem Celular Tumoral , Cristalografia por Raios X , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Desoxicitidina Quinase/química , Desenho de Fármacos , Resistencia a Medicamentos Antineoplásicos , Ativação Enzimática/efeitos dos fármacos , Humanos , Mesilato de Imatinib/química , Mesilato de Imatinib/farmacologia , Modelos Biológicos , Modelos Moleculares , Fosforilação , Piperidinas , Polifarmacologia , Inibidores de Proteínas Quinases/química , Proteômica , Piridinas , Tiazóis/química , GencitabinaRESUMO
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.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/fisiologia , Reparo do DNA/efeitos da radiação , Desoxicitidina Quinase/metabolismo , Animais , Linfócitos B/citologia , Linfócitos B/fisiologia , Linhagem Celular Tumoral , Dano ao DNA , Reparo do DNA/efeitos dos fármacos , Desoxicitidina/metabolismo , Desoxicitidina Quinase/química , Desoxicitidina Quinase/genética , Desoxirribonucleosídeos/metabolismo , Instabilidade Genômica , Hematopoese/genética , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Mutagênese Sítio-Dirigida , Fosforilação , Processamento de Proteína Pós-Traducional , Especificidade por Substrato , Linfócitos T/citologia , Linfócitos T/fisiologiaRESUMO
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 1.5â 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.
Assuntos
Desoxicitidina Quinase/antagonistas & inibidores , Desoxicitidina Quinase/química , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Cristalografia por Raios X , Humanos , Modelos Moleculares , Ligação Proteica , Conformação Proteica/efeitos dos fármacos , Difosfato de Uridina/metabolismoRESUMO
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.
Assuntos
Proteína Quinase CDC2/metabolismo , Dano ao DNA , Desoxicitidina Quinase/metabolismo , Pontos de Checagem da Fase G2 do Ciclo Celular , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Desoxicitidina Quinase/química , Desoxicitidina Quinase/fisiologia , Células HeLa , Humanos , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Radiação Ionizante , Serina/metabolismo , Proteínas Supressoras de Tumor/metabolismoRESUMO
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.
Assuntos
Antimetabólitos Antineoplásicos/uso terapêutico , Desoxicitidina/análogos & derivados , Neoplasias/tratamento farmacológico , Domínio Catalítico , Linhagem Celular Tumoral , Desoxicitidina/uso terapêutico , Desoxicitidina Quinase/antagonistas & inibidores , Desoxicitidina Quinase/química , Humanos , Modelos Moleculares , Nucleosídeos/química , Nucleosídeos/uso terapêutico , Fosforilação , GencitabinaRESUMO
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.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Desoxicitidina Quinase/metabolismo , Medicamentos de Ervas Chinesas/farmacologia , Sequência de Aminoácidos , Antineoplásicos/metabolismo , Domínio Catalítico , Desoxicitidina Quinase/química , Desoxicitidina Quinase/genética , Medicamentos de Ervas Chinesas/metabolismo , Ativação Enzimática/efeitos dos fármacos , Células HL-60 , Humanos , Simulação de Acoplamento Molecular , Dados de Sequência Molecular , Mutagênese , MutaçãoRESUMO
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.
Assuntos
Desoxicitidina Quinase/química , Desoxicitidina/análogos & derivados , Desoxicitidina/metabolismo , Timidina/metabolismo , Substituição de Aminoácidos , Cristalografia por Raios X , Desoxicitidina Quinase/genética , Desoxicitidina Quinase/metabolismo , Humanos , Cinética , Fosforilação , Especificidade por Substrato , Timidina/análogos & derivadosRESUMO
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.
Assuntos
Caseína Quinase Idelta/metabolismo , Desoxicitidina Quinase/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Caseína Quinase Idelta/antagonistas & inibidores , Caseína Quinase Idelta/genética , Linhagem Celular , Desoxicitidina Quinase/química , Desoxicitidina Quinase/genética , Ativação Enzimática , Humanos , Técnicas In Vitro , Cinética , Mutagênese Sítio-Dirigida , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Processamento de Proteína Pós-Traducional , Interferência de RNA , RNA Interferente Pequeno/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Serina/químicaRESUMO
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.
Assuntos
Desoxicitidina Quinase/metabolismo , Serina/metabolismo , Treonina/metabolismo , Linhagem Celular , Desoxicitidina Quinase/química , Desoxicitidina Quinase/genética , Estabilidade Enzimática , Humanos , Mutagênese Sítio-Dirigida , Fosforilação , Serina/química , Relação Estrutura-Atividade , Treonina/químicaRESUMO
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.
Assuntos
Antineoplásicos/farmacologia , Desoxicitidina Quinase/genética , Desoxicitidina/análogos & derivados , Núcleosídeo-Fosfato Quinase/genética , Farmacogenética , Desoxicitidina/farmacologia , Desoxicitidina Quinase/química , Haplótipos , Humanos , Cinética , Desequilíbrio de Ligação , Modelos Moleculares , Núcleosídeo-Fosfato Quinase/química , GencitabinaRESUMO
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.
Assuntos
Fosfoglicerato Quinase/química , Pró-Fármacos/química , Difosfato de Adenosina/química , Antivirais/química , Antivirais/metabolismo , Cistina Difosfato/química , Desoxicitidina Quinase/química , Humanos , Ligantes , Modelos Moleculares , Núcleosídeo-Fosfato Quinase/química , Fosfoglicerato Quinase/metabolismo , Pró-Fármacos/metabolismo , Estrutura Terciária de Proteína , EstereoisomerismoRESUMO
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.
Assuntos
Desoxicitidina Quinase/metabolismo , Desoxicitidina/análogos & derivados , Desoxicitidina/metabolismo , Mimetismo Molecular , Fosfosserina/metabolismo , Transporte Ativo do Núcleo Celular , Catálise , Linhagem Celular , Núcleo Celular/enzimologia , Desoxicitidina Quinase/química , Humanos , Espaço Intracelular/enzimologia , Cinética , Proteínas Mutantes/metabolismo , Fosforilação , Estrutura Secundária de Proteína , Transporte Proteico , Especificidade por SubstratoRESUMO
Human deoxycytidine kinase (dCK) is involved in the nucleotide-biosynthesis salvage pathway and has also been shown to phosphorylate several antitumor and antiviral prodrugs. The structures of dCK alone and the dead-end complex of dCK with substrate nucleoside and product ADP or UDP have previously been reported; however, there is currently no structure available for a substrate or product complex. Here, the structures of dCK complexes with the products dCMP, UDP and Mg2+ ion, and with dAMP, UDP and Mg2+ ion are reported. Structural comparisons show that the product complexes with UDP and a dead-end complex with substrate and UDP have similar active-site conformations.
Assuntos
Desoxicitidina Quinase/química , Desoxicitidina Monofosfato/química , Complexos Multiproteicos/química , Proteínas Recombinantes/química , Difosfato de Uridina/química , Cristalografia por Raios X , Desoxicitidina Quinase/genética , Desoxicitidina Quinase/metabolismo , Desoxicitidina Monofosfato/genética , Desoxicitidina Monofosfato/metabolismo , Humanos , Modelos Moleculares , Conformação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Relação Estrutura-Atividade , Especificidade por Substrato , Difosfato de Uridina/genética , Difosfato de Uridina/metabolismoRESUMO
L-nucleoside analogs represent an important class of small molecules for treating both viral infections and cancers. These pro-drugs achieve pharmacological activity only after enzyme-catalyzed conversion to their tri-phosphorylated forms. Herein, we report the crystal structures of human deoxycytidine kinase (dCK) in complex with the L-nucleosides (-)-beta-2',3'-dideoxy-3'-thiacytidine (3TC)--an approved anti-human immunodeficiency virus (HIV) agent--and troxacitabine (TRO)--an experimental anti-neoplastic agent. The first step in activating these agents is catalyzed by dCK. Our studies reveal how dCK, which normally catalyzes phosphorylation of the natural D-nucleosides, can efficiently phosphorylate substrates with non-physiologic chirality. The capability of dCK to phosphorylate both D- and L-nucleosides and nucleoside analogs derives from structural properties of both the enzyme and the substrates themselves. First, the nucleoside-binding site tolerates substrates with different chiral configurations by maintaining virtually all of the protein-ligand interactions responsible for productive substrate positioning. Second, the pseudo-symmetry of nucleosides and nucleoside analogs in combination with their conformational flexibility allows the L- and D-enantiomeric forms to adopt similar shapes when bound to the enzyme. This is the first analysis of the structural basis for activation of L-nucleoside analogs, providing further impetus for discovery and clinical development of new agents in this molecular class.
Assuntos
Fármacos Anti-HIV/química , Antineoplásicos/química , Citosina/análogos & derivados , Desoxicitidina Quinase/química , Dioxolanos/química , Lamivudina/química , Modelos Moleculares , Cristalografia por Raios X , Citosina/química , Humanos , Pró-Fármacos/química , Pró-Fármacos/metabolismo , Estereoisomerismo , Especificidade por SubstratoRESUMO
Methotrexate (MTX) is the anticancer and antirheumatoid drug that is believed to block nucleotide synthesis and cell cycle by inhibiting dihydrofolate reductase activity. We have developed novel affinity matrices, termed SG beads, that are easy to manipulate and are compatible with surface functionalization. Using the matrices, here we present evidence that deoxycytidine kinase (dCK), an enzyme that acts in the salvage pathway of nucleotide biosynthesis, is another target of MTX. MTX modulates dCK activity differentially depending on substrate concentrations. 1-beta-D-Arabinofuranosylcytosine (ara-C), a chemotherapy agent often used in combination with MTX, is a nucleoside analog whose incorporation into chromosome requires prior phosphorylation by dCK. We show that, remarkably, MTX enhances incorporation and cytotoxicity of ara-C through regulation of dCK activity in Burkitt's lymphoma cells. Thus, this study provides new insight into the mechanisms underlying MTX actions and demonstrates the usefulness of the SG beads.
Assuntos
Cromatografia de Afinidade/métodos , Ensaios de Seleção de Medicamentos Antitumorais/instrumentação , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Metotrexato/farmacologia , Microesferas , Sequência de Aminoácidos , Arabinofuranosilcitosina Trifosfato/metabolismo , Morte Celular/efeitos dos fármacos , Cromossomos Humanos/genética , Desoxicitidina Quinase/química , Desoxicitidina Quinase/isolamento & purificação , Humanos , Células Jurkat , Dados de Sequência Molecular , Peso Molecular , Tetra-Hidrofolato Desidrogenase/metabolismoRESUMO
Various D- and L-thietanose nucleosides were synthesized from D- and L-xylose. The four-membered thietane ring was efficiently synthesized by the cyclization of 1-thioacetyl-3-mesylate (4/38) under basic conditions. Condensation with various heterocyclic bases was conducted via Pummerer-type rearrangement to afford various nucleoside derivatives. Among the synthesized nucleosides, D-uridine (23), D-cytidine (24), D-5-fluorocytidine (25), and L-cytidine (52) analogues showed moderate anti-HIV activity, with EC50 = 6.9, 1.3, 5.8, and 14.1 microM, respectively. However, these four nucleoside analogues are cytotoxic in peripheral blood mononuclear and CEM cells. The other nucleosides are neither active nor cytotoxic. Interestingly, the oxetanocin A analogue 33 was not active. Comparison of the minimized reverse transcriptases (RTs) complexed with the corresponding triphosphates of the cytidine analogue 24 and the adenosine analogue 33 by molecular modeling studies showed that there is no difference in the binding mode of the triphosphate of the cytidine analogue 24 to the active site of HIV-1 RT from that of the triphosphate of the adenosine analogue 33. Modeling studies on the initial monophosphorylation step by deoxycytidine kinase showed that the catalytic efficiency of phosphorylation through a nucleophilic attack of the 4'-hydroxyl group of thietanose on the gamma-phosphate of ATP is diminished in the case of L-cytidine analogue (52) due to the increased distance between the 4'-hydroxyl group and the gamma-phosphate.
Assuntos
Fármacos Anti-HIV/síntese química , HIV-1/efeitos dos fármacos , Nucleosídeos de Pirimidina/síntese química , Sulfetos/síntese química , Adenina/análogos & derivados , Adenina/química , Fármacos Anti-HIV/química , Fármacos Anti-HIV/farmacologia , Sítios de Ligação , Catálise , Linhagem Celular , Desoxicitidina Quinase/química , Transcriptase Reversa do HIV/química , Humanos , Modelos Moleculares , Conformação Molecular , Fosforilação , Nucleosídeos de Pirimidina/química , Nucleosídeos de Pirimidina/farmacologia , Estereoisomerismo , Relação Estrutura-Atividade , Sulfetos/química , Sulfetos/farmacologia , Xilose/químicaRESUMO
Human deoxycytidine kinase (dCK) phosphorylates both pyrimidine and purine deoxynucleosides, including numerous nucleoside analogue prodrugs. Energy transfer studies of transfer between Trp residues of dCK and the fluorescent probe N-(1-pyrene)maleimide (PM), which specifically labels Cys residues in proteins, were performed. Two of the six Cys residues in dCK were labeled, yielding a protein that was functionally active. We determined the average distances between PM-labeled Cys residues and Trp residues in dCK in the absence and presence of various pyrimidine and purine nucleoside analogues with the Trp residues as energy donors and PM-labeled Cys residues as acceptors. The transfer efficiency was determined from donor intensity quenching and the Förster distance R(0) at which the efficiency of energy transfer is 50%, which was 19.90 A for dCK-PM. The average distance R between the Trp residues and the labeled Cys residues in dCK-PM was 18.50 A, and once substrates bound, this distance was reduced, demonstrating conformational changes. Several of the Cys residues of dCK were mutated to Ala, and the properties of the purified mutant proteins were studied. PM labeled a single Cys residue in Cys-185-Ala dCK, suggesting that one of the two Cys residues labeled in wild-type dCK was Cys 185. The distance between the single PM-labeled Cys residue and the Trp residues in Cys-185-Ala dCK was 20.75 A. Binding of nucleosides had no effect on the pyrene fluorescence of Cys-185-Ala dCK, indicating that the conformational changes observed upon substrate binding to wild-type dCK-PM involved the "lid region" of which Cys 185 is a part. The substrate specificity of Cys-185-Ala dCK was altered in that dAdo and UTP were better substrates for the mutant than for the wild-type enzyme.
Assuntos
Cisteína/metabolismo , Desoxicitidina Quinase/química , Transferência Ressonante de Energia de Fluorescência/métodos , Conformação Proteica , Alanina/química , Alanina/genética , Alanina/metabolismo , Sítios de Ligação , Cisteína/química , Cisteína/genética , Desoxicitidina Quinase/genética , Desoxicitidina Quinase/metabolismo , Transferência de Energia , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/genética , Corantes Fluorescentes/metabolismo , Corantes Fluorescentes/farmacologia , Humanos , Cinética , Maleimidas/metabolismo , Maleimidas/farmacologia , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação , Nucleosídeos/metabolismo , Nucleosídeos/farmacologia , Fosfatos/metabolismo , Ligação Proteica , Especificidade por Substrato/efeitos dos fármacos , Especificidade por Substrato/genética , Triptofano/químicaRESUMO
Human deoxycytidine kinase (dCK) uses nucleoside triphosphates to phosphorylate several clinically important prodrugs in addition to its natural substrates. Although UTP is the preferred phosphoryl donor for this reaction, our previous studies reported dCK structures solely containing ADP in the phosphoryl donor binding site. To determine the molecular basis of the kinetically observed phosphoryl donor preference, we solved crystal structures of a dCK variant lacking a flexible insert (residues 65-79) but having similar catalytic properties as wild type, in complex with deoxycytidine (dC) and UDP, and in the presence of dC but the absence of UDP or ADP. These structures reveal major changes in the donor base binding loop (residues 240-247) between the UDP-bound and ADP-bound forms, involving significant main-chain rearrangement. This loop is disordered in the dCK-dC structure, which lacks a ligand at the phosphoryl donor site. In comparison with the ADP-bound form, in the presence of UDP this loop is shifted inward to make closer contact to the smaller uracil base. These structures illuminate the phosphoryl donor binding and preference mechanisms of dCK.
Assuntos
Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Desoxicitidina Quinase/química , Desoxicitidina Quinase/metabolismo , Uridina Trifosfato/química , Uridina Trifosfato/metabolismo , Sequência de Aminoácidos , Animais , Desoxicitidina Quinase/genética , Drosophila melanogaster/enzimologia , Drosophila melanogaster/genética , Humanos , Cinética , Dados de Sequência Molecular , Estrutura Terciária de Proteína/genética , Alinhamento de Sequência , Deleção de Sequência , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
Clofarabine [2-chloro-9-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-9H-purin-6-amine] is a hybrid of the widely used anticancer drugs cladribine and fludarabine. It is the precursor of an effective chemotherapeutic agent for leukemias and other hematological malignancies and received accelerated approval by the FDA for the treatment of pediatric patients with relapsed or refractory acute lymphoblastic leukemia. Clofarabine is phosphorylated intracellularly by human deoxycytidine kinase (dCK) to the 5'-monophosphate, which is the rate-limiting step in activation of the prodrug. dCK has a broad substrate specificity, with a much higher activity to deoxycytidine than to deoxyadenosine and deoxyguanosine. As a purine-nucleoside analog, clofarabine is a better substrate of dCK than deoxycytidine. The crystal structure of dCK has been solved previously in complex with pyrimidine nucleosides and ADP [Sabini et al. (2003), Nature Struct. Biol. 10, 513-519]. In the current study, the crystal structure of clofarabine- and ADP-bound dCK was solved to 2.55 angstroms by molecular replacement. It appears that the enzyme takes the same conformation as in the structures of the pyrimidine nucleoside-bound complexes. The interactions between 2-Cl and its surrounding hydrophobic residues contribute to the high catalytic efficiency of dCK for clofarabine.
Assuntos
Arabinonucleosídeos/metabolismo , Desoxicitidina Quinase/química , Desoxicitidina Quinase/metabolismo , Nucleotídeos de Adenina , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Sítios de Ligação , Catálise , Clofarabina , Desoxicitidina Quinase/isolamento & purificação , Humanos , Modelos Moleculares , Conformação Proteica , Relação Estrutura-AtividadeRESUMO
Deoxycytidine kinase (dCK) catalyzes the rate-limiting step of the deoxyribonucleoside salvage pathway in mammalian cells and plays a key role in the activation of numerous nucleoside analogues used in anti-cancer and antiviral chemotherapy. Although compelling evidence indicated that dCK activity might be regulated by phosphorylation/dephosphorylation, direct demonstration was lacking. Here we showed that dCK overexpressed in HEK 293T cells was labeled after incubating the cells with [32P]orthophosphate. Sorbitol, which was reported to decrease dCK activity, also decreased the labeling of dCK. These results indicated that dCK may exist as a phosphoprotein in vivo and that its activity can be correlated with its phosphorylation level. After purification of 32P-labeled dCK, digestion by trypsin, and analysis of the radioactive peptides by tandem mass spectrometry, the following four in vivo phosphorylation sites were identified: Thr-3, Ser-11, Ser-15, and Ser-74, the latter being the major phosphorylation site. Site-directed mutagenesis and use of an anti-phospho-Ser-74 antibody demonstrated that Ser-74 phosphorylation was crucial for dCK activity in HEK 293T cells, whereas phosphorylation of other identified sites did not seem essential. Phosphorylation of Ser-74 was also detected on endogenous dCK in leukemic cells, in which the Ser-74 phosphorylation state was increased by agents that enhanced dCK activity. Our study provided direct evidence that dCK activity can be controlled by phosphorylation in intact cells and highlights the importance of Ser-74 for dCK activity.