RESUMEN
Nicotinamide N-methyltransferase (NNMT) catalyzes the transfer of a methyl group from S-adenosylmethionine (SAM) to nicotinamide (NAM) and other pyridine-related compounds and is involved in various metabolic processes in the human body. In addition, abnormal expression of NNMT occurs under various pathological conditions such as cancer, diabetes, metabolic disorders, and neurodegenerative diseases, making it a promising drug target worthy of in-depth research. Small-molecule NNMT inhibitors with high potency and selectivity are necessary chemical tools to test biological hypotheses and potential therapies. In this study, we developed a series of highly active NNMT inhibitors by modifying N7 position of adenine. Among them, compound 3-12 (IC50 = 47.9 ± 0.6 nM) exhibited potent inhibitory activity and also had an excellent selectivity profile over a panel of human methyltransferases. We showed that the N7 position of adenine in the NNMT bisubstrate inhibitor was a modifiable site, thus offering insights into the development of NNMT inhibitors.
Asunto(s)
Nicotinamida N-Metiltransferasa , Tubercidina , Humanos , Nicotinamida N-Metiltransferasa/química , Nicotinamida N-Metiltransferasa/metabolismo , Tubercidina/metabolismo , Niacinamida/farmacología , Adenina , Metabolismo SecundarioRESUMEN
Protein kinase CK2a1 is a serine/threonine kinase that plays a crucial role in the growth, proliferation and survival of cells and is a well known target for tumour and glomerulonephritis therapies. Here, the crystal structure of the kinase domain of CK2a1 complexed with 5-iodotubercidin (5IOD), an ATP-mimetic inhibitor, was determined at 1.78â Å resolution. The structure shows distinct structural features and, in combination with a comparison of the crystal structures of five off-target kinases complexed with 5IOD, provides valuable information for the development of highly selective inhibitors.
Asunto(s)
Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/química , Secuencia de Aminoácidos , Quinasa de la Caseína II/antagonistas & inhibidores , Quinasa de la Caseína II/química , Quinasa de la Caseína II/metabolismo , Cristalografía por Rayos X , Humanos , Proteínas Serina-Treonina Quinasas/metabolismo , Estructura Secundaria de Proteína , Electricidad Estática , Tubercidina/análogos & derivados , Tubercidina/química , Tubercidina/metabolismoRESUMEN
BACKGROUND: Tubercidin (TBN), an adenosine analog with potent antimycobacteria and antitumor bioactivities, highlights an intriguing structure, in which a 7-deazapurine core is linked to the ribose moiety by an N-glycosidic bond. However, the molecular logic underlying the biosynthesis of this antibiotic has remained poorly understood. RESULTS: Here, we report the discovery and characterization of the TBN biosynthetic pathway from Streptomyces tubercidicus NBRC 13090 via reconstitution of its production in a heterologous host. We demonstrated that TubE specifically utilizes phosphoribosylpyrophosphate and 7-carboxy-7-deazaguanine for the precise construction of the deazapurine nucleoside scaffold. Moreover, we provided biochemical evidence that TubD functions as an NADPH-dependent reductase, catalyzing irreversible reductive deamination. Finally, we verified that TubG acts as a Nudix hydrolase, preferring Co2+ for the maintenance of maximal activity, and is responsible for the tailoring hydrolysis step leading to TBN. CONCLUSIONS: These findings lay a foundation for the rational generation of TBN analogs through synthetic biology strategy, and also open the way for the target-directed search of TBN-related antibiotics.
Asunto(s)
Streptomyces/metabolismo , Biología Sintética/métodos , Tubercidina/metabolismo , Tubercidina/biosíntesisRESUMEN
Efficient incorporation of modified nucleotides by DNA polymerases is essential for many cutting-edge biomolecular technologies. The present study compares the acceptance of either alkene- or alkyne-modified nucleotides by KlenTaq DNA polymerase and provides structural insights into how 7-deaza-adenosine and deoxyuridine with attached alkene-modifications are incorporated into the growing DNA strand. Thereby, we identified modified nucleotides that prove to be superior substrates for KlenTaq DNA polymerase compared with their natural analogues. The knowledge can be used to guide future design of functionalized nucleotide building blocks.
Asunto(s)
Alquenos/química , Alquinos/química , ADN Polimerasa Dirigida por ADN/metabolismo , Nucleótidos/metabolismo , Biocatálisis , Desoxiuridina/síntesis química , Desoxiuridina/química , Desoxiuridina/metabolismo , Electroforesis en Gel de Poliacrilamida , Técnicas de Amplificación de Ácido Nucleico , Nucleótidos/síntesis química , Nucleótidos/química , Tubercidina/síntesis química , Tubercidina/química , Tubercidina/metabolismoRESUMEN
7-(2-Thienyl)-7-deazaadenosine (AB61) showed nanomolar cytotoxic activities against various cancer cell lines but only mild (micromolar) activities against normal fibroblasts. The selectivity of AB61 was found to be due to inefficient phosphorylation of AB61 in normal fibroblasts. The phosphorylation of AB61 in the leukemic CCRF-CEM cell line proceeds well and it was shown that AB61 is incorporated into both DNA and RNA, preferentially as a ribonucleotide. It was further confirmed that a triphosphate of AB61 is a substrate for both RNA and DNA polymerases in enzymatic assays. Gene expression analysis suggests that AB61 affects DNA damage pathways and protein translation/folding machinery. Indeed, formation of large 53BP1 foci was observed in nuclei of AB61-treated U2OS-GFP-53BP1 cells indicating DNA damage. Random incorporation of AB61 into RNA blocked its translation in an in vitro assay and reduction of reporter protein expression was also observed in mice after 4-hour treatment with AB61. AB61 also significantly reduced tumor volume in mice bearing SK-OV-3, BT-549, and HT-29 xenografts. The results indicate that AB61 is a promising compound with unique mechanism of action and deserves further development as an anticancer agent. Mol Cancer Ther; 15(5); 922-37. ©2016 AACR.
Asunto(s)
Antineoplásicos/farmacología , Tubercidina/farmacología , Animales , Antineoplásicos/química , Antineoplásicos/metabolismo , Línea Celular Tumoral , Permeabilidad de la Membrana Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , ADN/genética , ADN/metabolismo , Daño del ADN/efectos de los fármacos , Modelos Animales de Enfermedad , Fibroblastos , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patología , Biosíntesis de Proteínas/efectos de los fármacos , Pliegue de Proteína/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Análisis de Supervivencia , Resultado del Tratamiento , Tubercidina/análogos & derivados , Tubercidina/química , Tubercidina/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Adenosine deaminases acting on RNA (ADAR1 and ADAR2) are human RNA-editing adenosine deaminases responsible for the conversion of adenosine to inosine at specific locations in cellular RNAs. Since inosine is recognized during translation as guanosine, this often results in the expression of protein sequences different from those encoded in the genome. While our knowledge of the ADAR2 structure and catalytic mechanism has grown over the years, our knowledge of ADAR1 has lagged. This is due, at least in part, to the lack of well defined, small RNA substrates useful for mechanistic studies of ADAR1. Here, we describe an ADAR1 substrate RNA that can be prepared by a combination of chemical synthesis and enzymatic ligation. Incorporation of adenosine analogs into this RNA and analysis of the rate of ADAR1 catalyzed deamination revealed similarities and differences in the way the ADARs recognize the edited nucleotide. Importantly, ADAR1 is more dependent than ADAR2 on the presence of N7 in the edited base. This difference between ADAR1 and ADAR2 appears to be dependent on the identity of a single amino acid residue near the active site. Thus, this work provides an important starting point in defining mechanistic differences between two functionally distinct human RNA editing ADARs.
Asunto(s)
Adenosina Desaminasa/metabolismo , Edición de ARN , Adenosina/análogos & derivados , Adenosina Desaminasa/genética , Secuencia de Aminoácidos , ADN Glicosilasas/genética , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Precursores del ARN/metabolismo , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Alineación de Secuencia , Transcripción Genética , Tubercidina/metabolismoRESUMEN
As part of an ongoing effort to explore the effect of major groove electrostatics on the thermodynamic stability and structure of DNA, a 7-deaza-2'-deoxyadenosine:dT (7-deaza-dA:dT) base pair in the Dickerson-Drew dodecamer (DDD) was studied. The removal of the electronegative N7 atom on dA and the replacement with an electropositive C-H in the major groove was expected to have a significant effect on major groove electrostatics. The structure of the 7-deaza-dA:dT base pair was determined at 1.1 Å resolution in the presence of Mg(2+). The 7-deaza-dA, which is isosteric for dA, had minimal effect on the base pairing geometry and the conformation of the DDD in the crystalline state. There was no major groove cation association with the 7-deaza-dA heterocycle. In solution, circular dichroism showed a positive Cotton effect centered at 280 nm and a negative Cotton effect centered at 250 nm that were characteristic of a right-handed helix in the B-conformation. However, temperature-dependent NMR studies showed increased exchange between the thymine N3 imino proton of the 7-deaza-dA:dT base pair and water, suggesting reduced stacking interactions and an increased rate of base pair opening. This correlated with the observed thermodynamic destabilization of the 7-deaza-dA modified duplex relative to the DDD. A combination of UV melting and differential scanning calorimetry experiments were conducted to evaluate the relative contributions of enthalpy and entropy in the thermodynamic destabilization of the DDD. The most significant contribution arose from an unfavorable enthalpy term, which probably results from less favorable stacking interactions in the modified duplex, which was accompanied by a significant reduction in the release of water and cations from the 7-deaza-dA modified DNA.
Asunto(s)
ADN/química , Timina/química , Tubercidina/análogos & derivados , Emparejamiento Base , Dicroismo Circular , ADN/metabolismo , Transición de Fase , Electricidad Estática , Temperatura , Termodinámica , Tubercidina/química , Tubercidina/metabolismo , Rayos UltravioletaRESUMEN
The protein kinase haspin/Gsg2 plays an important role in mitosis, where it specifically phosphorylates Thr-3 in histone H3 (H3T3). Its protein sequence is only weakly homologous to other protein kinases and lacks the highly conserved motifs normally required for kinase activity. Here we report structures of human haspin in complex with ATP and the inhibitor iodotubercidin. These structures reveal a constitutively active kinase conformation, stabilized by haspin-specific inserts. Haspin also has a highly atypical activation segment well adapted for specific recognition of the basic histone tail. Despite the lack of a DFG motif, ATP binding to haspin is similar to that in classical kinases; however, the ATP gamma-phosphate forms hydrogen bonds with the conserved catalytic loop residues Asp-649 and His-651, and a His651Ala haspin mutant is inactive, suggesting a direct role for the catalytic loop in ATP recognition. Enzyme kinetic data show that haspin phosphorylates substrate peptides through a rapid equilibrium random mechanism. A detailed analysis of histone modifications in the neighborhood of H3T3 reveals that increasing methylation at Lys-4 (H3K4) strongly decreases substrate recognition, suggesting a key role of H3K4 methylation in the regulation of haspin activity.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Modelos Moleculares , Conformación Proteica , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Adenosina Trifosfato/metabolismo , Histonas/metabolismo , Humanos , Fosforilación , Tubercidina/metabolismoRESUMEN
Replacement of a single dA nucleotide positioned at a programmed site in a DNA plasmid with its 7-deaza-analog is described together with its complete resistance to restriction enzymatic cleavage.
Asunto(s)
ADN/química , Plásmidos/química , Tubercidina/análogos & derivados , Roturas del ADN de Cadena Simple , ADN Ligasas/metabolismo , Enzimas de Restricción del ADN/metabolismo , Desoxiguanosina/análogos & derivados , Desoxiguanosina/química , Desoxiguanosina/metabolismo , Tubercidina/química , Tubercidina/metabolismoRESUMEN
TOR is an atypical multidrug resistance protein present in the human protozoan parasite, Leishmania. Resistance to the toxic adenosine analog tubercidin was brought about by redirecting the adenosine permease from the plasma membrane to the multivesicular tubule lysosome. The cells became resistant to tubercidin because they were unable to take up and accumulate this toxic purine. The domain, which was recognized by TOR in this internalization pathway, was identified by expressing portions of this transporter in Leishmania and assessing whether they were capable of hindering the multidrug resistance capability of TOR. This approach identified the adenosine permease region spanning Met289 to Trp305. This region was also the epitope recognized by the internalization mechanism. An internal deletion mutant lacking Met289-Trp305 was functionally active but could no longer be internalized in cells with high TOR levels. The internalization and altered trafficking of the adenosine permease by TOR was observed in yeast and human embryonic kidney cells co-expressing these two Leishmania proteins indicating that the internalization process was conserved in evolutionary diverse organisms. The inability of Saccharomyces with a temperature-sensitive ubiquitin ligase to internalize adenosine permease suggested that ubiquitination was involved in this altered trafficking.
Asunto(s)
Adenosina/metabolismo , Resistencia a Medicamentos/fisiología , Leishmania mexicana/efectos de los fármacos , Leishmania mexicana/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Proteínas Protozoarias/metabolismo , Factores de Transcripción/metabolismo , Adenosina/análogos & derivados , Animales , Línea Celular , Endocitosis/fisiología , Epítopos/fisiología , Humanos , Leishmania mexicana/genética , Lisosomas/metabolismo , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/genética , Mutación/fisiología , Estructura Terciaria de Proteína/fisiología , Transporte de Proteínas/efectos de los fármacos , Transporte de Proteínas/fisiología , Proteínas Protozoarias/genética , Saccharomyces cerevisiae , Tubercidina/metabolismo , Tubercidina/toxicidad , Ubiquitina/metabolismoRESUMEN
Chemotaxis of bacteria requires regulated methylation of chemoreceptors. However, despite considerable effort in the 1980s, transmethylation has never been established as a component of eukaryotic cell chemotaxis. S-adenosylhomocysteine (SAH), the product formed when the methyl group of the universal donor S-adenosylmethionine (SAM) is transferred to an acceptor molecule, is a potent inhibitor of all transmethylation reactions. In eukaryotic cells, this inhibition is relieved by hydrolysis of SAH to adenosine and homocysteine catalyzed by SAH hydrolase (SAHH). We now report that SAHH, which is diffuse in the cytoplasm of nonmotile Dictyostelium amoebae and human neutrophils, concentrates with F-actin in pseudopods at the front of motile, chemotaxing cells, but is not present in filopodia or at the very leading edge. Tubercidin, an inhibitor of SAHH, inhibits both chemotaxis and chemotaxis-dependent cell streaming of Dictyostelium, and chemotaxis of neutrophils at concentrations that have little effect on cell viability. Tubercidin does not inhibit starvation-induced expression of the cAMP receptor, cAR1, or G protein-mediated stimulation of adenylyl cyclase activity and actin polymerization in Dictyostelium. Tubercidin has no effect on either capping of Con A receptors or phagocytosis in Dictyostelium. These results add SAHH to the list of proteins that redistribute in response to chemotactic signals in Dictyostelium and neutrophils and strongly suggest a role for transmethylation in chemotaxis of eukaryotic cells.
Asunto(s)
Adenosilhomocisteinasa/metabolismo , Quimiotaxis , Actinas/metabolismo , Adenosilhomocisteinasa/genética , Animales , Dictyostelium/citología , Dictyostelium/enzimología , Dictyostelium/genética , Humanos , Metilación , Neutrófilos/citología , Neutrófilos/enzimología , Tubercidina/metabolismoRESUMEN
5'-Methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) is important in a number of cellular functions such as polyamine biosynthesis, methionine salvaging, biological methylation, and quorum sensing. The nucleosidase is found in many microbes but not in mammalian systems, thus making MTAN a broad-spectrum antimicrobial drug target. Substrate binding and catalytic residues were identified from the crystal structure of MTAN complexed with 5'-methylthiotubercidin [Lee, J. E., Cornell, K. A., Riscoe, M. K. and Howell, P. L. (2003) J. Biol. Chem. 278 (10) 8761-8770]. The roles of active site residues Met9, Glu12, Ile50, Ser76, Val102, Phe105, Tyr107, Phe151, Met173, Glu174, Arg193, Ser196, Asp197, and Phe207 have been investigated by site-directed mutagenesis and steady-state kinetics. Mutagenesis of residues Glu12, Glu174, and Asp197 completely abolished activity. The location of Asp197 and Glu12 in the active site is consistent with their having a direct role in enzyme catalysis. Glu174 is suggested to be involved in catalysis by stabilizing the transition state positive charge at the O3', C2', and C3' atoms and by polarizing the 3'-hydroxyl to aid in the flow of electrons to the electron withdrawing purine base. This represents the first indication of the importance of the 3'-hydroxyl in the stabilization of the transition state. Furthermore, mutation of Arg193 to alanine shows that the nucleophilic water is able to direct its attack without assistance from the enzyme. This mutagenesis study has allowed a reevaluation of the catalytic mechanism.
Asunto(s)
Sustitución de Aminoácidos/genética , Proteínas de Escherichia coli/química , Escherichia coli/enzimología , Homoserina/análogos & derivados , Mutagénesis Sitio-Dirigida , N-Glicosil Hidrolasas/química , Sitios de Unión/genética , Poliaminas Biogénicas/biosíntesis , Catálisis , Activación Enzimática/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Homoserina/biosíntesis , Cinética , Lactonas , Metionina/metabolismo , Metilación , N-Glicosil Hidrolasas/genética , N-Glicosil Hidrolasas/metabolismo , Unión Proteica/genética , Estructura Terciaria de Proteína/genética , Tionucleósidos/química , Tionucleósidos/metabolismo , Tubercidina/análogos & derivados , Tubercidina/química , Tubercidina/metabolismoRESUMEN
Sequence-specific 3-deazaadenosine (c(3)A)-substituted analogues of trimeric 2',5'-oligoadenylate, p5'A2'p5'A2'p5'A, were synthesized and evaluated for their ability to activate human RNase L (EC 3.1.2.6) aiming at the elucidation of the nitrogen-3 role in this biochemical process. Substitution of either 5'-terminal or 2'-terminal adenosine with c(3)A afforded the respective analogues p5'(c(3)A)2'p5'A2'p5'A and p5'A2'p5'A2'p5'(c(3)A) that were as effective as the natural tetramer itself as activators of RNase L (EC(50)=1nM). In contrast, p5'A2'p5'(c(3)A)2'p5'A showed diminished RNase L activation ability (EC(50)=10nM). The extensive conformational analysis of the c(3)A-substituted core trimers versus the parent natural core trimer by the (1)H and (13)C NMR, and CD spectroscopy displayed close stereochemical similarity between the natural core trimer and (c(3)A)2'p5'A2'p5'A and A2'p5'A2'p5'(c(3)A) analogues, thereby strong evidences for the syn base orientation about the glycosyl bond of the c(3)A residue of the latter were found. On the contrary, an analogue A2'p5'(c(3)A)2'p5'A displayed rather essential deviations from the spatial arrangement of the parent natural core trimer.
Asunto(s)
Adenina/química , Endorribonucleasas/química , Endorribonucleasas/metabolismo , Nitrógeno/química , Tubercidina/química , Tubercidina/farmacología , Animales , Cromatografía Líquida de Alta Presión , Dicroismo Circular , Activación Enzimática/efectos de los fármacos , Humanos , Espectroscopía de Resonancia Magnética , Ratones , Estructura Molecular , Oligonucleótidos/síntesis química , Oligonucleótidos/química , Fosfatos/química , Estereoisomerismo , Tubercidina/síntesis química , Tubercidina/metabolismoRESUMEN
Standard nucleobases all present electron density as an unshared pair of electrons to the minor groove of the double helix. Many heterocycles supporting artificial genetic systems lack this electron pair. To determine how different DNA polymerases use the pair as a substrate specificity determinant, three Family A polymerases, three Family B polymerases and three reverse transcriptases were examined for their ability to handle 3-deaza-2'-deoxyadenosine (c3dA), an analog of 2'-deoxyadenosine lacking the minor groove electron pair. Different polymerases differed widely in their interaction with c3dA. Most notably, Family A and Family B polymerases differed in their use of this interaction to exploit their exonuclease activities. Significant differences were also found within polymerase families. This plasticity in polymerase behavior is encouraging to those wishing to develop a synthetic biology based on artificial genetic systems. The differences also suggest either that Family A and Family B polymerases do not share a common ancestor, that minor groove contact was not used by that ancestor functionally or that this contact was not sufficiently critical to fitness to have been conserved as the polymerase families diverged. Each interpretation is significant for understanding the planetary biology of polymerases.
Asunto(s)
ADN Polimerasa Dirigida por ADN/metabolismo , ADN/genética , ADN/metabolismo , ADN Polimerasa Dirigida por ARN/metabolismo , Tubercidina/análogos & derivados , Tubercidina/metabolismo , Disparidad de Par Base , Secuencia de Bases , Sitios de Unión , ADN/química , ADN Polimerasa Dirigida por ADN/clasificación , Nucleótidos de Desoxiadenina/metabolismo , Exonucleasas/metabolismo , Especificidad por SustratoRESUMEN
S-adenosylhomocysteine hydrolase (SAHH) is a key regulator of S-adenosylmethionine-dependent methylation reactions and an interesting pharmacologic target. We cloned the SAHH gene from Plasmodium falciparum (PfSAHH), with an amino acid sequence agreeing with that of the PlasmoDB genomic database. Even though the expressed recombinant enzyme, PfSAHH, could use 3-deaza-adenosine (DZA) as an alternative substrate in contrast to the human SAHH, it has a unique inability to substitute 3-deaza-(+/-)aristeromycin (DZAri) for adenosine. Among the analogs of DZA, including neplanocin A, DZAri was the most potent inhibitor of the PfSAHH enzyme activity, with a K(i) of about 150 nM, whether Ado or DZA was used as a substrate. When the same DZA analogs were tested for their antimalarial activity, they also inhibited the in vitro growth of P. falciparum parasites potently. Homology-modeling analysis revealed that a single substitution (Thr60-Cys59) between the human and malarial PfSAHH, in an otherwise similar SAH-binding pocket, might account for the differential interactions with the nucleoside analogs. This subtle difference in the active site may be exploited in the development of novel drugs that selectively inhibit PfSAHH. We performed a comprehensive phylogenetic analysis of the SAHH superfamily and inferred that SAHH evolved in the common ancestor of Archaea and Eukaryota, and was subsequently horizontally transferred to Bacteria. Additionally, an analysis of the unusual and uncharacterized AHCYL1 family of the SAHH paralogs extant only in animals reveals striking divergence of its SAH-binding pocket and the loss of key conserved residues, thus suggesting an evolution of novel function(s).
Asunto(s)
Adenosina/análogos & derivados , Inhibidores Enzimáticos/metabolismo , Evolución Molecular , Hidrolasas/genética , Plasmodium falciparum/genética , Adenosina/metabolismo , Adenosilhomocisteinasa , Secuencia de Aminoácidos , Animales , Sitios de Unión , Clonación Molecular , ADN Protozoario/química , ADN Protozoario/genética , Electroforesis en Gel de Poliacrilamida , Hidrolasas/química , Hidrolasas/metabolismo , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Filogenia , Plasmodium falciparum/enzimología , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Tubercidina/metabolismoRESUMEN
A number of ligands for the adenosine binding sites has been obtained by using nucleoside convergent and divergent synthesis. Most of our nucleosides have been synthesized by coupling 2,6-dichloropurine (1), 2,6-dichloro-1-deazapurine (2), 2,6-dichloro-3-deazapurine (3) with ribose, 2- and 3-deoxyribose and 2,3-dideoxyribose derivatives. The use of these versatile synthons allowed the introduction of various substituents in 2- and/or 6-positions. The glycosylation site and the anomeric configuration of the obtained nucleosides were assigned on the basis of spectroscopic studies and confirmed by molecular models. A series of potent adenosine receptor ligands has been obtained by using divergent approaches, mostly starting from guanosine. Substitutions in 2, 6, 8, and 5' position of adenosine molecule led to ligands selective for the different adenosine receptor subtypes. Furthermore, we investigated the molecular bases of the different behavior of 2- and 8-alkynyl adenosines, by means of NMR experiments and molecular modeling studies. With docking experiments, we demonstrated that the two class of molecules should have different binding modes that explain their different degree of affinity and the shift of their activity from agonistic (2-substituted derivatives) to antagonistic (8-substituted derivatives).
Asunto(s)
Modelos Moleculares , Nucleósidos de Purina/síntesis química , Nucleósidos de Purina/metabolismo , Animales , Sitios de Unión/fisiología , Células CHO , Cricetinae , Humanos , Nucleósidos de Purina/química , Tubercidina/síntesis química , Tubercidina/química , Tubercidina/metabolismoRESUMEN
The A-minor motifs appear to be the most ubiquitous helix packing elements within RNA tertiary structures. These motifs have been identified throughout the ribosome and almost every other tertiary-folded RNA for which structural information is available. These motifs utilize the packing of the donor adenosine's N1, N3, and/or 2'-OH against the 2'-OHs and minor groove edge of the acceptor base pair. The ability to identify biochemically which adenosines form A-minor motifs and which base pairs they contact is an important experimental objective. Toward this goal, we report the synthesis and transcriptional incorporation of 5'-O-(1-thio)-3-deazaadenosine triphosphate and its use in Nucleotide Analogue Interference Mapping (NAIM) and Nucleotide Analogue Interference Suppression (NAIS). This analogue makes it possible for the first time to explore the functional importance of the N3 imino group of adenosine in RNA polymers. Interference analysis of the group I self-splicing introns from Tetrahymena and Azoarcus indicates that A-minor motifs are integral to the helix packing interactions that define the 5'-splice site of the intron. Specifically, Azoarcus A58 in the J4/5 region contacts the G.U wobble pair at the cleavage site in the P1 helix, and Azoarcus A167 in the J8/7 region contacts the C13-G37 base pair in the P2 helix. Both of these structural features are conserved between the eukaryotic and bacterial introns. These results suggest that nucleotide analogue interference patterns can identify and distinguish A-minor interactions in RNA tertiary structure, particularly the most prevalent type I and type II varieties. Furthermore, clustering of 3-deazaadenosine interferences is suggestive of A patches, in which a series of consecutive A-minor motifs mediate helix packing. Biochemical identification of these interactions may provide valuable constraints for RNA structure prediction.
Asunto(s)
Adenosina Trifosfato/análogos & derivados , Intrones , Conformación de Ácido Nucleico , Mapeo Nucleótido/métodos , ARN Bacteriano/química , ARN Catalítico/química , Tubercidina/química , Adenosina Trifosfato/síntesis química , Adenosina Trifosfato/metabolismo , Animales , Azoarcus/enzimología , Sitios de Unión/genética , Intrones/genética , Mutagénesis Sitio-Dirigida , Empalme del ARN , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , ARN Catalítico/genética , ARN Catalítico/metabolismo , Tetrahymena/enzimología , Tionucleótidos/síntesis química , Tionucleótidos/metabolismo , Transcripción Genética , Tubercidina/metabolismoRESUMEN
We previously reported that the rat organic cation transporter rOCT1 could transport the nucleoside analog deoxytubercidin (dTub) (Chen R, Nelson JA. Biochem Pharmacol 2000;60:215-9). The cationic form of dTub (dTub(+)) appeared to be the true substrate of rOCT1. We also reported that although rOCT2 is similar to rOCT1, it does not transport dTub at pH 7.4. In this study, we measured the K(m) and V(max) values of dTub(+) uptake at a reduced pH (pH 5.4) for both rOCT1 and rOCT2. The difference in substrate activity appears due, in large part, to a poor affinity of rOCT2 for dTub(+). The transport efficiency estimated by V(max)/K(m) values for rOCT2 was only 6% that of rOCT1. Chimeras constructed between rOCT1 and rOCT2 revealed that the difference in dTub binding lies within transmembrane domains 2-7. To evaluate the potential of OCT1 in the renal secretion of dTub, tissue distribution and urinary excretion of dTub in OCT1 knockout mice were measured. No significant difference was observed in renal elimination, plasma level, and tissue distribution of dTub between the knockout and the wild-type mice. Therefore, dTub is a good substrate for OCT1; however, OCT1 does not appear to be necessary for its renal secretion.
Asunto(s)
Proteínas Portadoras/metabolismo , Riñón/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Catión Orgánico/metabolismo , Transportador 1 de Catión Orgánico/metabolismo , Tubercidina/análogos & derivados , Tubercidina/metabolismo , Animales , Transporte Biológico , Transporte Iónico , Ratones , Ratones Noqueados , Proteínas de Transporte de Nucleósidos , Oocitos/metabolismo , Proteínas de Transporte de Catión Orgánico/deficiencia , Proteínas de Transporte de Catión Orgánico/genética , Transportador 1 de Catión Orgánico/deficiencia , Transportador 1 de Catión Orgánico/genética , Transportador 2 de Cátion Orgánico , Transfección , Xenopus laevisRESUMEN
Binding of the transition state analogue coformycin and the ground state analogue 1-deaazadenosine to bovine adenosine deaminase have been thermodynamically characterized. The heat capacity changes for coformycin and 1-deazaadenosine binding are -4.7 +/- 0.8 kJ/mole-K and -1.2 +/- 0.1 kJ/mole-K, respectively. Since the predominant source of heat capacity change in enzyme interactions are changes in the extent of exposure of nonpolar amino acid side chains to the aqueous environment and the hydrophobic effect is the predominant factor in native structure stabilization, we propose that the binding of either class of ligand is associated with a stabilizing enzyme conformational change with coformycin producing the far greater effect. Analysis of the T dependence of the second order rate constant for formation of the enzyme/coformycin complex further reveals that the conformational change is not rate limiting. We propose that the enzyme may facilitate catalysis via the formation of a stabilizing conformation at the reaction transition state.
Asunto(s)
Adenosina Desaminasa/metabolismo , Coformicina/metabolismo , Estructura Terciaria de Proteína , Tubercidina/metabolismo , Adenosina Desaminasa/química , Inhibidores de la Adenosina Desaminasa , Animales , Sitios de Unión , Bovinos , Coformicina/química , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Matemática , Estructura Molecular , Unión Proteica , Inhibidores de la Síntesis de la Proteína/química , Inhibidores de la Síntesis de la Proteína/metabolismo , Temperatura , Termodinámica , Tubercidina/químicaRESUMEN
The purine salvage pathway of parasitic protozoa is currently considered as a target for drug development because these organisms cannot synthesize purines de novo. Insight into the structure and mechanism of the involved enzymes can aid in the development of potent inhibitors, leading to new curative drugs. Nucleoside hydrolases are key enzymes in the purine salvage pathway of Trypanosomatidae, and they are especially attractive because they have no equivalent in mammalian cells. We cloned, expressed and purified a nucleoside hydrolase from Trypanosoma vivax. The substrate activity profile establishes the enzyme to be a member of the inosine-adenosine-guanosine-preferring nucleoside hydrolases (IAG-NH). We solved the crystal structure of the enzyme at 1.6 A resolution using MAD techniques. The complex of the enzyme with the substrate analogue 3-deaza-adenosine is presented. These are the first structures of an IAG-NH reported in the literature. The T. vivax IAG-NH is a homodimer, with each subunit consisting of ten beta-strands, 12 alpha-helices and three small 3(10)-helices. Six of the eight strands of the central beta-sheet form a motif resembling the Rossmann fold. Superposition of the active sites of this IAG-NH and the inosine-uridine-preferring nucleoside hydrolase (IU-NH) of Crithidia fasciculata shows the molecular basis of the different substrate specificity distinguishing these two classes of nucleoside hydrolases. An "aromatic stacking network" in the active site of the IAG-NH, absent from the IU-NH, imposes the purine specificity. Asp10 is the proposed general base in the reaction mechanism, abstracting a proton from a nucleophilic water molecule. Asp40 (replaced by Asn39 in the IU-NH) is positioned appropriately to act as a general acid and to protonate the purine leaving group. The second general acid, needed for full enzymatic activity, is probably part of a flexible loop located in the vicinity of the active site.