RESUMO
A homo-dimeric enzyme, thymidylate synthase (TS), has been a long-standing molecular target in chemotherapy. To further elucidate properties and interactions with ligands of wild-type mouse thymidylate synthase (mTS) and its two single mutants, H190A and W103G, spectroscopic and theoretical investigations have been employed. In these mutants, histidine at position 190 and tryptophan at position 103 are substituted with alanine and glycine, respectively. Several emission-based spectroscopy methods used in the paper demonstrate an especially important role for Trp 103 in TS ligands binding. In addition, the Advanced Poisson-Boltzmann Solver (APBS) results show considerable differences in the distribution of electrostatic potential around Trp 103, as compared to distributions observed for all remaining Trp residues in the mTS family of structures. Together, spectroscopic and APBS results reveal a possible interplay between Trp 103 and His190, which contributes to a reduction in enzymatic activity in the case of H190A mutation. Comparison of electrostatic potential for mTS complexes, and their mutants, with the substrate, dUMP, and inhibitors, FdUMP and N4-OH-dCMP, suggests its weaker influence on the enzyme-ligand interactions in N4OH-dCMP-mTS compared to dUMP-mTS and FdUMP-mTS complexes. This difference may be crucial for the explanation of the "abortive reaction" inhibitory mechanism of N4OH-dCMP towards TS. In addition, based on structural analyses and the H190A mutant capacity to form a denaturation-resistant complex with N4-OH-dCMP in the mTHF-dependent reaction, His190 is apparently responsible for a strong preference of the enzyme active center for the anti rotamer of the imino inhibitor form.
Assuntos
Nucleotídeos de Desoxiuracil/metabolismo , Modelos Teóricos , Espectrometria de Fluorescência/métodos , Eletricidade Estática , Timidilato Sintase/metabolismo , Substituição de Aminoácidos , Animais , Desoxicitidina Monofosfato/análogos & derivados , Desoxicitidina Monofosfato/metabolismo , Nucleotídeos de Desoxiuracil/química , Fluordesoxiuridilato/metabolismo , Camundongos , Modelos Moleculares , Análise Multivariada , Conformação Proteica , Timidilato Sintase/químicaRESUMO
BACKGROUND: FOLFOX is a combinational regimen of folinic acid (FnA, FOL), fluorouracil (5-Fu, F) and oxaliplatin (OxP, OX), and has been long considered as the standard treatment of colorectal cancer (CRC) and hepatocellular carcinoma (HCC). Recent developments of nano delivery systems have provided profound promise for improving anticancer efficacy and alleviating side effects of FOLFOX. Previously, a nanoformulation (termed Nano-Folox) containing OxP derivative and FnA was developed in our laboratory using nanoprecipitation technique. Nano-Folox induced OxP-mediated immunogenic cell death (ICD)-associated antitumor immunity, which significantly suppressed tumor growth in the orthotopic CRC mouse model when administrated in combination with free 5-Fu. METHODS: A nanoformulation (termed Nano-FdUMP) containing FdUMP (5-Fu active metabolite) was newly developed using nanoprecipitation technique and used in combination with Nano-Folox for CRC and HCC therapies. RESULTS: Synergistic efficacy was achieved in orthotopic CRC and HCC mouse models. It resulted mainly from the fact that Nano-FdUMP mediated the formation of reactive oxygen species (ROS), which promoted the efficacy of ICD elicited by Nano-Folox. In addition, combination of Nano-Folox/Nano-FdUMP and anti-PD-L1 antibody significantly inhibited CRC liver metastasis, leading to long-term survival in mice. CONCLUSION: This study provides proof of concept that combination of two nano delivery systems can result in successful FOLFOX-associated CRC and HCC therapies. Further optimization in terms of dosing and timing will enhance clinical potential of this combination strategy for patients.
Assuntos
Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/imunologia , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/imunologia , Morte Celular Imunogênica , Neoplasias Hepáticas/tratamento farmacológico , Nanopartículas/química , Espécies Reativas de Oxigênio/metabolismo , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Antígeno B7-H1/antagonistas & inibidores , Antígeno B7-H1/metabolismo , Linhagem Celular Tumoral , Modelos Animais de Doenças , Composição de Medicamentos , Sinergismo Farmacológico , Feminino , Fluordesoxiuridilato/metabolismo , Fluoruracila/farmacologia , Fluoruracila/uso terapêutico , Morte Celular Imunogênica/efeitos dos fármacos , Imunoterapia , Leucovorina/farmacologia , Leucovorina/uso terapêutico , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/patologia , Camundongos Endogâmicos BALB C , Camundongos Nus , Nanopartículas/ultraestrutura , Metástase Neoplásica , Compostos Organoplatínicos/farmacologia , Compostos Organoplatínicos/uso terapêutico , Distribuição TecidualRESUMO
In view of previous crystallographic studies, N4-hydroxy-dCMP, a slow-binding thymidylate synthase inhibitor apparently caused "uncoupling" of the two thymidylate synthase-catalyzed reactions, including the N5,10-methylenetetrahydrofolate one-carbon group transfer and reduction, suggesting the enzyme's capacity to use tetrahydrofolate as a cofactor reducing the pyrimidine ring C(5) in the absence of the 5-methylene group. Testing the latter interpretation, a possibility was examined of a TS-catalyzed covalent self-modification/self-inactivation with certain pyrimidine deoxynucleotides, including 5-fluoro-dUMP and N4-hydroxy-dCMP, that would be promoted by tetrahydrofolate and accompanied with its parallel oxidation to dihydrofolate. Electrophoretic analysis showed mouse recombinant TS protein to form, in the presence of tetrahydrofolate, a covalently bound, electrophoretically separable 5-fluoro-dUMP-thymidylate synthase complex, similar to that produced in the presence of N5,10-methylenetetrahydrofolate. Further studies of the mouse enzyme binding with 5-fluoro-dUMP/N4-hydroxy-dCMP by TCA precipitation of the complex on filter paper showed it to be tetrahydrofolate-promoted, as well as to depend on both time in the range of minutes and the enzyme molecular activity, indicating thymidylate synthase-catalyzed reaction to be responsible for it. Furthermore, the tetrahydrofolate- and time-dependent, covalent binding by thymidylate synthase of each 5-fluoro-dUMP and N4-hydroxy-dCMP was shown to be accompanied by the enzyme inactivation, as well as spectrophotometrically confirmed dihydrofolate production, the latter demonstrated to depend on the reaction time, thymidylate synthase activity and temperature of the incubation mixture, further documenting its catalytic character.
Assuntos
Fluordesoxiuridilato/metabolismo , Tetra-Hidrofolatos/metabolismo , Timidilato Sintase/metabolismo , Animais , Desoxicitidina Monofosfato/análogos & derivados , Desoxicitidina Monofosfato/metabolismo , Inibidores Enzimáticos/metabolismo , Ácido Fólico/análogos & derivados , Ácido Fólico/metabolismo , Camundongos , Ligação Proteica , Espectrofotometria UltravioletaRESUMO
Thymidylate synthase (TS) is an enzyme of paramount importance as it provides the only de novo source of deoxy-thymidine monophosphate (dTMP). dTMP, essential for DNA synthesis, is produced by the TS-catalyzed reductive methylation of 2'-deoxyuridine-5'-monophosphate (dUMP) using N5,N10-methylenetetrahydrofolate (mTHF) as a cofactor. TS is ubiquitous and a validated drug target. TS enzymes from different organisms differ in sequence and structure, but are all obligate homodimers. The structural and mechanistic differences between the human and bacterial enzymes are exploitable to obtain selective inhibitors of bacterial TSs that can enrich the currently available therapeutic tools against bacterial infections. Enterococcus faecalis is a pathogen fully dependent on TS for dTMP synthesis. In this study, we present four new crystal structures of Enterococcus faecalis and human TSs in complex with either the substrate dUMP or the inhibitor FdUMP. The results provide new clues about the half-site reactivity of Enterococcus faecalis TS and the mechanisms underlying the conformational changes occurring in the two enzymes. We also identify relevant differences in cofactor and inhibitor binding between Enterococcus faecalis and human TS that can guide the design of selective inhibitors against bacterial TSs.
Assuntos
Enterococcus faecalis/enzimologia , Fluordesoxiuridilato/química , Conformação Proteica , Timidina Monofosfato/química , Timidilato Sintase/química , Sítios de Ligação , Domínio Catalítico , Fluordesoxiuridilato/metabolismo , Humanos , Modelos Moleculares , Ligação Proteica , Multimerização Proteica , Relação Estrutura-Atividade , Especificidade por Substrato , Timidina Monofosfato/metabolismo , Timidilato Sintase/metabolismoRESUMO
Multidrug resistance protein 8 (ABCC11) is an efflux transporter for anionic lipophilic compounds, conferring resistance to antiviral and anticancer agents like 5-fluorouracil (5-FU). ABCC11 missense variants may contribute to variability in drug response but functional consequences, except for the 'earwax variant' c.538G>A, are unknown. Using the 'Screen and Insert' technology, we generated human embryonic kidney 293 cells stably expressing ABCC11 missense variants frequently occurring in different ethnic populations: c.57G>A, c.538G>A, c.950C>A, c.1637C>T, c.1942G>A, c.4032A>G. A series of in silico prediction analyses and in vitro plasma membrane vesicle uptake, immunoblotting and immunolocalization experiments were undertaken to investigate functional consequences. We identified c.1637C>T (T546M), previously associated with 5-FU-related toxicity, as a novel functionally damaging ABCC11 variant exhibiting markedly reduced transport function of 5-FdUMP, the active cytotoxic metabolite of 5-FU. Detailed analysis of 14 subpopulations revealed highest allele frequencies of c.1637C>T in Europeans and Americans (up to 11%) compared with Africans and Asians (up to 3%).
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Transportadores de Cassetes de Ligação de ATP/metabolismo , Adenosina Trifosfatases/metabolismo , Antineoplásicos/metabolismo , Povo Asiático , Transporte Biológico , População Negra , Linhagem Celular , Simulação por Computador , Sulfato de Desidroepiandrosterona/metabolismo , Estrona/análogos & derivados , Estrona/metabolismo , Fluordesoxiuridilato/metabolismo , Frequência do Gene , Humanos , Desequilíbrio de Ligação , Mutação de Sentido Incorreto , População BrancaRESUMO
5-Fluorouracil (5-FU) and its oral prodrug capecitabine are among the most widely used chemotherapeutics. For cytotoxic activity, 5-FU requires cellular uptake and intracellular metabolic activation. Three intracellular formed metabolites are responsible for the antineoplastic effect of 5-FU: 5-fluorouridine 5'-triphosphate (FUTP), 5-fluoro-2'-deoxyuridine 5'-triphosphate (FdUTP) and 5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP). In this paper, we describe the development of an LC-MS/MS assay for quantification of these active 5-FU nucleotides in peripheral blood mononuclear cells (PBMCs). Because the intracellular 5-FU nucleotide concentrations were very low, maximization of the release from the cell matrix and minimization of interference were critical factors. Therefore, a series of experiments was performed to select the best method for cell lysis and nucleotide extraction. Chromatography was optimized to obtain separation from endogenous nucleotides, and the effect of different cell numbers was examined. The assay was validated for the following concentration ranges in PBMC lysate: 0.488-19.9 nM for FUTP, 1.66-67.7 nM for FdUTP and 0.748-30.7 nM for FdUMP. Accuracies were between -2.2 and 7.0% deviation for all analytes, and the coefficient of variation values were ≤ 4.9%. The assay was successfully applied to quantify 5-FU nucleotides in PBMC samples from patients treated with capecitabine and patients receiving 5-FU intravenously. FUTP amounts up to 3054 fmol/10(6) PBMCs and FdUMP levels up to 169 fmol/10(6) PBMCs were measured. The FdUTP concentrations were below the lower limit of quantification. To our knowledge, this is the first time that 5-FU nucleotides were quantified in cells from patients treated with 5-FU or capecitabine without using a radiolabel.
Assuntos
Antineoplásicos/metabolismo , Cromatografia Líquida , Monitoramento de Medicamentos/métodos , Fluoruracila/metabolismo , Leucócitos Mononucleares/metabolismo , Espectrometria de Massas em Tandem , Antineoplásicos/farmacocinética , Transporte Biológico , Biotransformação , Calibragem , Cromatografia Líquida/normas , Nucleotídeos de Desoxiuracil/metabolismo , Monitoramento de Medicamentos/normas , Floxuridina/análogos & derivados , Floxuridina/metabolismo , Fluordesoxiuridilato/metabolismo , Fluoruracila/farmacocinética , Humanos , Modelos Lineares , Padrões de Referência , Reprodutibilidade dos Testes , Espectrometria de Massas em Tandem/normas , Uridina Trifosfato/análogos & derivados , Uridina Trifosfato/metabolismoRESUMO
BACKGROUND: The Multidrug Resistance Protein ABCC11/MRP8 is expressed in physiological barriers and tumor breast tissues in which it secretes various substrates including cGMP (cyclic guanosine monophosphate) and 5FdUMP (5-fluoro-2'-deoxyuridine-5'-monophosphate), the active metabolite of the anticancer drug 5-FluoroUracil (frequently included to anticancer therapy).Previously, we described that ABCC11 high levels are associated to the estrogen receptor (ER) expression level in breast tumors and in cell lines resistant to tamoxifen. Consequently, by lowering the intracellular concentration of anticancer drugs, ABCC11 likely promotes a multidrug resistance (MDR) phenotype and decreases efficiency of anticancer therapy of 5FdUMP. Since no experimental data about binding sites of ABCC11 substrate are available, we decided to in silico localize putative substrate interaction sites of the nucleotide derivatives. Taking advantage of molecular dynamics simulation, we also analysed their evolution under computational physiological conditions and during the time. RESULTS: Since ABCC11 crystal structure is not resolved yet, we used the X-ray structures of the mouse mdr3 (homologous to human ABCB1) and of the bacterial homolog Sav1866 to generate two independent ABCC11 homology models in inward- and outward-facing conformations. Based on docking analyses, two putative binding pockets, for cGMP and 5FdUMP, were localized in both inward- and outward-facing conformations. Furthermore, based on our 3D models, and available biochemical data from homologous transporters, we identified several residues, potentially critical in ABCC11 transport function. Additionally, molecular dynamics simulation on our inward-facing model revealed for the first time conformation changes assumed to occur during transport process. CONCLUSIONS: ABCC11 would present two binding sites for cGMP and for 5FdUMP. Substrates likely first bind at the intracellular side of the transmembrane segment while ABCC11 is open forward the cytoplasm (inward-facing conformation). Then, along with conformational changes, it would pass through ABCC11 and fix the second site (close to the extracellular side), until the protein open itself to the extracellular space and allow substrate release.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Antineoplásicos/metabolismo , GMP Cíclico/metabolismo , Fluordesoxiuridilato/metabolismo , Simulação de Dinâmica Molecular , Subfamília B de Transportador de Cassetes de Ligação de ATP/química , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/genética , Sequência de Aminoácidos , Animais , Antineoplásicos/química , Sítios de Ligação , GMP Cíclico/química , Bases de Dados de Proteínas , Fluordesoxiuridilato/química , Humanos , Camundongos , Dados de Sequência Molecular , Polimorfismo de Nucleotídeo Único , Estrutura Terciária de Proteína , Alinhamento de Sequência , Eletricidade EstáticaRESUMO
Thymidylate synthase (TS) plays a major role in the response to 5-fluorouracil (5-FU) by binding directly to the 5-FU metabolite, 5-fluoro-dUMP (FdUMP). The change in the TS expression levels after 5-FU administration was examined in parallel to 5-FU responsiveness in six human gastric adenocarcinoma cell lines to elucidate the source of variability of 5-FU sensitivity. MKN-1, SH-10-TC and MKN-74 cells were more resistant to 5-FU than MKN-28, KATO III and MKN-45 cells. Western blotting analysis revealed that the 5-FU sensitivity of these cells did not correlate with the basal TS expression levels but did correlate with rapid detection of the TS-FdUMP complex after exposure to 5-FU. In 5-FU-resistant cells, very low levels of the TS-FdUMP complex early after 5-FU exposure were elevated by pretreatment with calpain inhibitors such as benzyloxycarbonyl-leucyl-leucinal (ZLLH), benzyloxycarbonyl-leucyl-leucyl-leucinal (ZLLLH) and ALLN, but not by other protease inhibitors. In contrast, ONO-3403, which causes calpain activation, stimulated downregulation of the TS-FdUMP complex in 5-FU-sensitive cells. The expression levels of calpastatin, an endogenous calpain inhibitor, were higher in 5-FU-sensitive cells than in 5-FU-resistant cells. ZLLH increased the 5-FU sensitivity of 5-FU-resistant cells, whereas ONO-3403 decreased the sensitivity of 5-FU-sensitive cells. In addition, knockdown of m-calpain by siRNA increased the 5-FU sensitivity in 5-FU-resistant cells, while knockdown of calpastatin reduced the sensitivity in 5-FU-sensitive cells. These results suggest that calpain might reduce the chemosensitivity of human gastric cancer cells to 5-FU possibly by rapid degradation of the TS-FdUMP complex, a finding that is considered to have novel therapeutic implications.
Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Calpaína/fisiologia , Fluordesoxiuridilato/metabolismo , Fluoruracila/farmacologia , Neoplasias Gástricas/tratamento farmacológico , Timidilato Sintase/metabolismo , Animais , Calpaína/análise , Linhagem Celular Tumoral , Humanos , Camundongos , Células NIH 3T3 , Inibidores de Proteases/farmacologia , Neoplasias Gástricas/metabolismoRESUMO
Thymidylate synthase (TS) is a well validated target in cancer chemotherapy. Here, a new crystal form of the R163K variant of human TS (hTS) with five subunits per asymmetric part of the unit cell, all with loop 181-197 in the active conformation, is reported. This form allows binding studies by soaking crystals in artificial mother liquors containing ligands that bind in the active site. Using this approach, crystal structures of hTS complexes with FdUMP and dUMP were obtained, indicating that this form should facilitate high-throughput analysis of hTS complexes with drug candidates. Crystal soaking experiments using oxidized glutathione revealed that hTS binds this ligand. Interestingly, the two types of binding observed are both asymmetric. In one subunit of the physiological dimer covalent modification of the catalytic nucleophile Cys195 takes place, while in another dimer a noncovalent adduct with reduced glutathione is formed in one of the active sites.
Assuntos
Nucleotídeos de Desoxiuracil/química , Fluordesoxiuridilato/química , Glutationa/química , Mutação , Timidilato Sintase/química , Cristalografia por Raios X , Nucleotídeos de Desoxiuracil/metabolismo , Fluordesoxiuridilato/metabolismo , Glutationa/metabolismo , Humanos , Ligantes , Modelos Moleculares , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Especificidade por Substrato , Timidilato Sintase/genética , Timidilato Sintase/metabolismoRESUMO
Prodrugs can have the advantage over parent drugs in increased activation and cellular uptake. The multidrug ETC-L-FdUrd and the duplex drug ETC-FdUrd are composed of two different monophosphate-nucleosides, 5-fluoro-2'deoxyuridine (FdUrd) and ethynylcytidine (ETC), coupled via a glycerolipid or phosphodiester, respectively. The aim of the study was to determine cytotoxicity levels and mode of drug cleavage. Moreover, we determined whether a liposomal formulation of ETC-L-FdUrd would improve cytotoxic activity and/or cleavage. Drug effects/cleavage were studied with standard radioactivity assays, HPLC and LC-MS/MS in FM3A/0 mammary cancer cells and their FdUrd resistant variants FM3A/TK(-). ETC-FdUrd was active (IC(50) of 2.2 and 79 nM) in FM3A/0 and TK(-) cells, respectively. ETC-L-FdUrd was less active (IC(50): 7 nM in FM3A/0 vs 4500 nM in FM3A/TK(-)). Although the liposomal formulation was less active than ETC-L-FdUrd in FM3A/0 cells (IC(50):19.3 nM), resistance due to thymidine kinase (TK) deficiency was greatly reduced. The prodrugs inhibited thymidylate synthase (TS) in FM3A/0 cells (80-90%), but to a lower extent in FM3A/TK(-) (10-50%). FdUMP was hardly detected in FM3A/TK(-) cells. Inhibition of the transporters and nucleotidases/phosphatases resulted in a reduction of cytotoxicity of ETC-FdUrd, indicating that this drug was cleaved outside the cells to the monophosphates, which was verified by the presence of FdUrd and ETC in the medium. ETC-L-FdUrd and the liposomal formulation were neither affected by transporter nor nucleotidase/phosphatase inhibition, indicating circumvention of active transporters. In vivo, ETC-FdUrd and ETC-L-FdURd were orally active. ETC nucleotides accumulated in both tumor and liver tissues. These formulations seem to be effective when a lipophilic linker is used combined with a liposomal formulation.
Assuntos
Citidina/análogos & derivados , Floxuridina/farmacologia , Animais , Linhagem Celular Tumoral , Citidina/administração & dosagem , Citidina/química , Citidina/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Espaço Extracelular/efeitos dos fármacos , Espaço Extracelular/metabolismo , Floxuridina/administração & dosagem , Floxuridina/química , Fluordesoxiuridilato/metabolismo , Humanos , Concentração Inibidora 50 , Lipossomos/metabolismo , Camundongos , Proteínas de Transporte de Nucleosídeos/metabolismo , Nucleosídeos/metabolismo , Timidilato Sintase/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
To specifically quantify several metabolites of 5-fluorouracil (5-FU) and two endogenous monophosphate nucleotides, we developed an original method based on a liquid chromatography-tandem mass spectrometry (LC-MS/MS). This assay allowed the determination of: (i) the intracellular production of 5-fluoro-2'-deoxyuridine-5'-monophosphate (5-FdUMP) from 5-FU or 5-fluoro-2'-deoxyuridine (5-FdUrd), (ii) the impact of 5-FdUMP concentration on the intracellular 2'-deoxyuridine-5'-monophosphate (dUMP)/thymidine-5'-monophosphate (TMP) ratio, and (iii) the secretion extent of 5-FdUMP and 5-FU from human cultured cells by ABC transporters. Under our experimental conditions, cells were incubated with 5-FU or 5-FUrd. Then, cellular proteins were precipitated by methanol. This procedure provided high extraction recovery. In addition, to measure 5-FU and 5-FdUMP secretion from cells, we carried out quantification of these molecules in culture medium. Media were either directly injected (5-FU) or underwent a solid phase extraction using Oasis Wax extraction cartridge (5-FdUMP). Separation of analytes was performed on a dC18 Atlantis 3.5microm, (100mmx2.1mm i.d) column with isocratic mode using ammonium formate buffer/methanol/water (5/5/90, v/v) as mobile phase. The run time did not exceed 6.2min. The analytes were ionized in an electrospray interface under negative ion mode. We validated the method over a range of 2.5-150ngmL(-1) according to the compounds. Intra- and inter-assay variability was lower than 10% over seven days. All compounds were stable in cells or in culture medium when samples were stored at -20 degrees C for at least two weeks, and after three freeze-thaw cycles. No matrix effect was observed in both media.
Assuntos
Cromatografia Líquida/métodos , Floxuridina/análogos & derivados , Floxuridina/análise , Fluordesoxiuridilato/análise , Fluoruracila/análise , Espectrometria de Massas em Tandem/métodos , Linhagem Celular , Linhagem Celular Tumoral , Nucleotídeos de Desoxiuracil/análise , Nucleotídeos de Desoxiuracil/metabolismo , Estabilidade de Medicamentos , Floxuridina/metabolismo , Fluordesoxiuridilato/metabolismo , Fluoruracila/metabolismo , Humanos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Extração em Fase Sólida , Timidina Monofosfato/análise , Timidina Monofosfato/metabolismoRESUMO
The interaction of nucleotide excision repair factors--xeroderma pigmentosum complementation group C protein in complex with human homolog of yeast Rad23 protein (XPC-HR23B), replication protein A (RPA), and xeroderma pigmentosum complementation group A protein (XPA)--with 48-mer DNA duplexes imitating damaged DNA structures was investigated. All studied proteins demonstrated low specificity in binding to damaged DNA compared with undamaged DNA duplexes. RPA stimulates formation of XPC-HR23B complex with DNA, and when XPA and XPC-HR23B are simultaneously present in the reaction mixture a synergistic effect in binding of these proteins to DNA is observed. RPA crosslinks to DNA bearing photoreactive 5I-dUMP residue on one strand and fluorescein-substituted dUMP analog as a lesion in the opposite strand of DNA duplex and also stimulates cross-linking with XPC-HR23B. Therefore, RPA might be one of the main regulation factors at various stages of nucleotide excision repair. The data are in agreement with the cooperative binding model of nucleotide excision repair factors participating in pre-incision complex formation with DNA duplexes bearing damages.
Assuntos
Dano ao DNA , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Proteína de Replicação A/metabolismo , Proteína de Xeroderma Pigmentoso Grupo A/metabolismo , Sequência de Bases , DNA/genética , DNA/efeitos da radiação , Reparo do DNA , Proteínas de Ligação a DNA/genética , Nucleotídeos de Desoxiuracil/química , Nucleotídeos de Desoxiuracil/metabolismo , Fluordesoxiuridilato/química , Fluordesoxiuridilato/metabolismo , Humanos , Luz , Substâncias Macromoleculares/metabolismo , Substâncias Macromoleculares/efeitos da radiação , Dados de Sequência Molecular , Estrutura Molecular , Proteína de Replicação A/genética , Alinhamento de Sequência , Proteína de Xeroderma Pigmentoso Grupo A/genéticaRESUMO
Molecular dynamics simulations and free energy calculations are presented, exploring previously described experimentally studied interactions of a series of 2'-fluoro-substituted dUMP/FdUMP analogues with thymidylate synthase (TS). The results show the inhibitory behaviors of 2'-F-ara-UMP, 2',2''-diF-dUMP and 2',5-diF-ara-UMP to be dependent upon the binding positions and orientations adopted by the molecules of these compounds in the active site of TS. The binding mode of 2',5-diF-ara-UMP suggests a novel role of the active site residue Trp 80, stabilizing through hydrophobic stacking the binding position of the pyrimidine ring in 2',5-diF-ara-UMP.
Assuntos
Fluordesoxiuridilato/análogos & derivados , Fluordesoxiuridilato/metabolismo , Timidilato Sintase/química , Timidilato Sintase/metabolismo , Fluordesoxiuridilato/síntese química , Fluordesoxiuridilato/química , Modelos Moleculares , Estrutura Molecular , Especificidade por SubstratoRESUMO
Plasmodium falciparum thymidylate synthase-dihydrofolate reductase (TS-DHFR) is an essential enzyme in nucleotide biosynthesis and a validated molecular drug target in malaria. Because P. falciparum TS and DHFR are highly homologous to their human counterparts, existing active-site antifolate drugs can have dose-limiting toxicities. In humans, TS and DHFR are two separate proteins. In P. falciparum, however, TS-DHFR is bifunctional, with both TS and DHFR active sites on a single polypeptide chain of the enzyme. Consequently, P. falciparum TS-DHFR contains unique distant or nonactive regions that might modulate catalysis: (1) an N-terminal tail and (2) a linker region tethering DHFR to TS, and encoding a crossover helix that forms critical electrostatic interactions with the DHFR active site. The role of these nonactive sites in the bifunctional P. falciparum TS-DHFR is unknown. We report the first in-depth, pre-steady-state kinetic characterization of the full-length, wild-type (WT) P. falciparum TS-DHFR enzyme and probe the role of distant, nonactive regions through mutational analysis. We show that the overall rate-limiting step in the WT P. falciparum TS-DHFR enzyme is TS catalysis. We further show that if TS is in an activated (liganded) conformation, the DHFR rate is 2-fold activated, from 60 s-1 to 130 s-1 in the WT enzyme. The TS rate is also reciprocally activated by approximately 1.5-fold if DHFR is in an activated, ligand-bound conformation. Mutations to the linker region affect neither catalytic rate nor domain-domain communication. Deletion of the N-terminal tail, although in a location remote from the active site, decreases the DHFR single rate and the bifunctional TS-DHFR rate by a factor of 2. The 2-fold activation of the DHFR rate by TS ligands remains intact, although even the activated N-terminal mutant has just half the DHFR activity of the WT enzyme. However, the reciprocal communication between TS active site and DHFR ligands is impaired in N-terminal mutants. Surprisingly, deletion of the analogous N-terminal tail in Leishmania major TS-DHFR causes a 3-fold enhancement of the DHFR rate from approximately 14 s-1 to approximately 40 s-1. In summary, our results demonstrate a complex interplay of domain-domain communication and nonactive-site modulation of catalysis in P. falciparum TS-DHFR. Furthermore, each parasitic TS-DHFR is activated by unique mechanisms, modulated by their nonactive site regions. Finally, our studies suggest the N-terminal tail of P. falciparum TS-DHFR is a highly selective, novel target for potential antifolate development in malaria.
Assuntos
Complexos Multienzimáticos/metabolismo , Plasmodium falciparum/enzimologia , Tetra-Hidrofolato Desidrogenase/metabolismo , Timidilato Sintase/metabolismo , Animais , Catálise , Ativação Enzimática , Fluordesoxiuridilato/metabolismo , Cinética , Leishmania major/enzimologia , Modelos Moleculares , Complexos Multienzimáticos/química , Complexos Multienzimáticos/genética , Tetra-Hidrofolato Desidrogenase/química , Tetra-Hidrofolato Desidrogenase/genética , Tetra-Hidrofolatos/metabolismo , Timidilato Sintase/química , Timidilato Sintase/genéticaRESUMO
Saccharomyces cerevisiae cells (strain W303-1A) treated with 5-fluorouracil and grown in 2% (fermentative conditions) or in 0.1% glucose (oxidative conditions) accumulated two types of 5-fluoro-UDP-sugars (FUDP-sugars): FUDP-N-acetylglucosamine and FUDP-glucose. No difference was observed in both conditions of culture. The viability of yeast cells on treatment with 5-fluorouracil was also followed. Both FUDP-sugars were partially purified by column chromatography (on Hypersil ODS and Mono Q columns) and characterized by: (i) treatment with alkaline phosphatase (EC 3.1.3.1), snake venom phosphodiesterase (EC 3.1.4.1) and UDP-glucose dehydrogenase (EC 1.1.1.22); (ii) UV spectra; and (iii) matrix-assisted laser desorption/ionization-time of flight mass analysis and 1H-nuclear magnetic resonance spectrometry. The syntheses of both FUDP-sugars were inversely related to the concentration of uracil and directly related to the concentration of 5-fluorouracil in the culture medium. The strain W303-1A, requiring uracil for growth, was useful as a tool to analyze the effect of 5-fluorouracil on nucleotide metabolism.
Assuntos
Antimetabólitos/farmacologia , Fluordesoxiuridilato/análogos & derivados , Fluoruracila/farmacologia , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Uridina Difosfato Glucose/análogos & derivados , Uridina Difosfato N-Acetilglicosamina/análogos & derivados , Uridina Difosfato N-Acetilglicosamina/metabolismo , Aerobiose , Fosfatase Alcalina/metabolismo , Cromatografia Líquida , Meios de Cultura/química , Fermentação , Fluordesoxiuridilato/química , Fluordesoxiuridilato/isolamento & purificação , Fluordesoxiuridilato/metabolismo , Espectroscopia de Ressonância Magnética , Viabilidade Microbiana , Fosfodiesterase I/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Análise Espectral , Uracila/análise , Uridina Difosfato Glucose/química , Uridina Difosfato Glucose/isolamento & purificação , Uridina Difosfato Glucose/metabolismo , Uridina Difosfato Glucose Desidrogenase/metabolismo , Uridina Difosfato N-Acetilglicosamina/química , Uridina Difosfato N-Acetilglicosamina/isolamento & purificaçãoRESUMO
Multidrug-resistance-associated protein, MRP8/ABCC11 (ABCC11), is an efflux pump for nucleotide analogues and 5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP). To test whether ABCC11 directly confers 5-fluorouracil (5-FU) resistance, we used the 5-FU-resistant subline PC-6/FU23-26 selected from PC-6 human small-cell lung cancer cells by 5-FU and found that it increases the resistance by approximately 25-fold. The intracellular FdUMP accumulation was reduced in PC-6/FU23-26 cells concomitant with the overexpression of the ABCC11 gene. These findings suggest that ABCC11 confers 5-FU resistance in the sublines by enhancing the efflux for the active metabolite FdUMP. Previously, methotrexate also increased the efflux by ABCC11, and we found cross-resistance to methotrexate in PC-6/FU23-26 cells. To confirm our hypothesis, we examined whether decreasing the expression of ABCC11 in PC-6/FU23-26 cells by small interfering RNA altered the cytotoxicity to 5-FU and methotrexate and found that this enhanced 5-FU and methotrexate cytotoxicity in PC-6/FU23-26 cells. These data indicate that expression of the ABCC11 gene is induced by 5-FU, and that ABCC11 is directly involved in 5-FU resistance by the efflux transport of the active metabolite FdUMP.
Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Calgranulina A/metabolismo , Resistencia a Medicamentos Antineoplásicos , Fluoruracila/farmacologia , Calgranulina A/genética , Morte Celular/efeitos dos fármacos , Fluordesoxiuridilato/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Neoplasias Pulmonares/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismoRESUMO
Thymidylate synthase (TS) of Trichinella spiralis, a parasitic nematode causing trichinellosis, was found to bind its own mRNA and repress translation of the latter, similar to its human counter-part [Chu, Koeller, Casey, Drake, Chabner, Elwood, Zinn and Allegra (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 8977-8981]. However, in striking contrast with human TS, the parasite enzyme's interaction with mRNA was not affected by any of the substrate (deoxyuridylate or N(5,10)-methylenetetrahydrofolate) nor by the inhibitor (fluorodeoxyuridylate; used alone or in the presence of N(5,10)-methylenetetrahydrofolate) similar to that shown for the bifunctional enzyme from Plasmodium falciparum [Zhang and Rathod (2002) Science 296, 545-547]. Moreover, repression of the translation of the parasite enzyme was enhanced by the same ligands that were shown by others (Chu et al., 1991) to prevent human TS from impairing its translation. On comparing the capacity of TS to bind to its cognate mRNA, relative to its ability to inhibit its translation, the same enzyme preparation was active as translational repressor at a considerably lower protein/mRNA ratio, suggesting the two phenomena to be disconnected. Of interest is the fact that the presence of the enzyme protein N-terminal methionine proved to be critical for binding, but not for repression of its translation, indicating that mRNA binding requires a methionine or an adduct (i.e. methionine-histidine) at the N-terminus of TS, but that the translational repression effect does not. Notably, chicken liver dihydrofolate reductase, which is incapable of binding to T. spiralis TS mRNA, repressed the translation of TS.
Assuntos
Biossíntese de Proteínas/genética , RNA Mensageiro/metabolismo , Timidilato Sintase/genética , Timidilato Sintase/metabolismo , Trichinella spiralis/enzimologia , Trichinella spiralis/genética , Animais , Galinhas , Nucleotídeos de Desoxiuracil/genética , Nucleotídeos de Desoxiuracil/metabolismo , Fluordesoxiuridilato/metabolismo , Regulação Enzimológica da Expressão Gênica , Humanos , Mercaptoetanol , Ligação Proteica , RNA Mensageiro/genética , Especificidade da Espécie , Tetra-Hidrofolatos/metabolismo , Timidilato Sintase/biossínteseRESUMO
Phosphoramidate pronucleotides have proven to be an effective strategy for the intracellular delivery of nucleoside 5'-monophosphates. This review will summarize our efforts to understand the in vitro and in vivo behavior of phosphoramidate monoesters of 3'-azido-3'-deoxythymidine (AZT), 3'-fluoro-3'-deoxythymidine (FLT) and 5-fluoro-2'-deoxyuridine (FUdR). Insights drawn from these studies have proved valuable for the future design of phosphoramidate-based pronucleotides.
Assuntos
Antineoplásicos/química , Antivirais/química , Nucleotídeos/química , Pró-Fármacos/química , Pirimidinas/química , Amidas/química , Aminoácidos/química , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Antivirais/metabolismo , Antivirais/farmacologia , Didesoxinucleosídeos/química , Didesoxinucleosídeos/metabolismo , Didesoxinucleosídeos/farmacologia , Desenho de Fármacos , Fluordesoxiuridilato/química , Fluordesoxiuridilato/metabolismo , Fluordesoxiuridilato/farmacologia , Humanos , Estrutura Molecular , Nucleotídeos/metabolismo , Nucleotídeos/farmacologia , Ácidos Fosfóricos/química , Pró-Fármacos/metabolismo , Pró-Fármacos/farmacologia , Pirimidinas/metabolismo , Pirimidinas/farmacologia , Fatores de Tempo , Zidovudina/química , Zidovudina/metabolismo , Zidovudina/farmacologiaRESUMO
Following an i.p. dose of 150 mg x kg(-1) 5-fluorouracil (5-FU), drug uptake and metabolism over a 2-h period were studied by in vivo (19)F magnetic resonance spectroscopy (MRS) for the murine colon carcinoma lines C26-B (5-FU-insensitive; n=11) and C26-10 (5-FU-sensitive; n=15) implanted s.c. in Balb/C mice. Time courses for tumour growth, intracellular levels of FdUMP, thymidylate synthase (TS) activity, and 5-FU in RNA were also determined, and the effects of a 9.5-min period of carbogen breathing, starting 1 min before drug administration, on MRS-detected 5-FU metabolism and tumour growth curves were examined. Both tumour variants generated MRS-detectable 5-FU nucleotides and showed similar initial growth inhibition after treatment. However, the growth rate of C26-B tumours returned to normal, while the sensitive C26-10 tumours, which produced larger fluoronucleotide pools, still showed moderate growth inhibition. Carbogen breathing did not significantly influence 5-FU uptake or fluoronucleotide production but did significantly enhance growth inhibition in C26-10 tumours. While both tumour variants exhibited incorporation of 5-FU into RNA and inhibition of TS via FdUMP, clearance of 5-FU from RNA and recovery of TS activity were greater for the insensitive C26-B line, indicating that these processes, in addition to 5-FU uptake and metabolism, may be important determinants of drug sensitivity and treatment response.
Assuntos
Antimetabólitos Antineoplásicos/farmacocinética , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/metabolismo , Fluoruracila/farmacocinética , Administração por Inalação , Animais , Dióxido de Carbono/administração & dosagem , Feminino , Fluordesoxiuridilato/metabolismo , Espectroscopia de Ressonância Magnética/métodos , Camundongos , Camundongos Endogâmicos BALB C , Transplante de Neoplasias , Oxigênio/administração & dosagem , RNA Neoplásico/metabolismo , Taxa de Sobrevida , Timidilato Sintase/metabolismoRESUMO
In this study we used site-directed mutagenesis to test the hypothesis that substrate channeling in the bifunctional thymidylate synthase-dihydrofolate reductase enzyme from Leishmania major occurs via electrostatic interactions between the negatively charged dihydrofolate produced at thymidylate synthase and a series of lysine and arginine residues on the surface of the protein. Accordingly, 12 charge reversal or charge neutralization mutants were made, with up to 6 putative channel residues changed at once. The mutants were assessed for impaired channeling using two criteria: a lag in product formation at dihydrofolate reductase and an increase in dihydrofolate accumulation. Surprisingly, none of the mutations produced changes consistent with impaired channeling, so our findings do not support the electrostatic channeling hypothesis. Burst experiments confirmed that the mutants also did not interfere with intermediate formation at thymidylate synthase. One mutant, K282E/R283E, was found to be thymidylate synthase-dead because of an impaired ability to form the covalent enzyme-methylene tetrahydrofolate-deoxyuridate complex prerequisite for chemical catalysis.