RESUMO
We report the successful one-pot synthesis of adenosine mono-, di-, and triphosphate in the confined space of a mordenite zeolite. This is also the first report of ATP synthesized onto a porous mineral surface. The results revealed a plausible prebiotic route to ribonucleotides and highlighted the contribution of microporous minerals in the origins of life.
Assuntos
Difosfato de Adenosina/síntese química , Monofosfato de Adenosina/síntese química , Trifosfato de Adenosina/síntese química , Silicatos de Alumínio/química , Nanopartículas/química , Técnicas de Síntese em Fase Sólida/métodos , Zeolitas/química , Adenina/química , Organofosfatos/química , Origem da Vida , Porosidade , Ribose/química , Propriedades de Superfície , Termodinâmica , Fatores de TempoRESUMO
GPR17 is believed to be a novel target for the development of new therapeutic approaches to human stroke and multiple sclerosis. Hence, the selection of GPR17 ligands may be a potent way to reduce the progression of ischemic damage. New potential ligands for GPR17, mono-, di-, and triphosphate adenosine nucleotides substituted at N6-position with a methyl and a cyclopentyl group were synthesized. The ability of new ligands to bind GPR17 was evaluated using frontal affinity chromatography-mass spectrometry (FAC-MS) method. Cangrelor, MRS2179, and uridine diphosphate were selected as the reference compounds. The new triphosphate derivatives 9 and 10 were considered as the new GPR17 ligands. The compound 10 was eluted with breakthrough time (bt) between cangrelor and MRS 2179 (compound 10, bt=12.25; cangrelor, bt=24.55, and MRS 2179, bt=7.10), while the breakthrough volume of compound 9 was similar to that of MRS 2179 (compound 9, bt=7.53 and MRS 2179, bt=7.10). N6-cyclopentyATP 10 is medium-high affinity ligand of GPR17, while the corresponding N6-methyl derivative 9 is a medium affinity ligand similar to MRS 2179. Hence, the new N6-cyclopentylATP 10 might be a good candidate for the pharmacological characterization of GPR17.
Assuntos
Monofosfato de Adenosina/análogos & derivados , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptores Acoplados a Proteínas G/metabolismo , Difosfato de Adenosina/análogos & derivados , Difosfato de Adenosina/síntese química , Difosfato de Adenosina/química , Difosfato de Adenosina/farmacologia , Monofosfato de Adenosina/química , Monofosfato de Adenosina/farmacologia , Sítios de Ligação , Cromatografia de Afinidade , Humanos , Proteínas Imobilizadas/química , Ligantes , Modelos Moleculares , Ligação Proteica , Receptores Acoplados a Proteínas G/biossínteseRESUMO
BACKGROUND AND PURPOSE: Ectonucleotidases control extracellular nucleotide levels and consequently, their (patho)physiological responses. Among these enzymes, nucleoside triphosphate diphosphohydrolase-1 (NTPDase1), -2, -3 and -8 are the major ectonucleotidases responsible for nucleotide hydrolysis at the cell surface under physiological conditions, and NTPDase1 is predominantly located at the surface of vascular endothelial cells and leukocytes. Efficacious inhibitors of NTPDase1 are required to modulate responses induced by nucleotides in a number of pathological situations such as thrombosis, inflammation and cancer. EXPERIMENTAL APPROACH: Here, we present the synthesis and enzymatic characterization of five 8-BuS-adenine nucleotide derivatives as potent and selective inhibitors of NTPDase1. KEY RESULTS: The compounds 8-BuS-AMP, 8-BuS-ADP and 8-BuS-ATP inhibit recombinant human and mouse NTPDase1 by mixed type inhibition, predominantly competitive with Ki values <1 µM. In contrast to 8-BuS-ATP which could be hydrolyzed by other NTPDases, the other BuS derivatives were resistant to hydrolysis by either NTPDase1, -2, -3 or -8. 8-BuS-AMP and 8-BuS-ADP were the most potent and selective inhibitors of NTPDase1 expressed in human umbilical vein endothelial cells as well as in situ in human and mouse tissues. As expected, as a result of their inhibition of recombinant human NTPDase1, 8-BuS-AMP and 8-BuS-ADP impaired the ability of this enzyme to block platelet aggregation. Importantly, neither of these two inhibitors triggered platelet aggregation nor prevented ADP-induced platelet aggregation, in support of their inactivity towards P2Y1 and P2Y12 receptors. CONCLUSIONS AND IMPLICATIONS: The 8-BuS-AMP and 8-BuS-ADP have therefore potential to serve as drugs for the treatment of pathologies regulated by NTPDase1.
Assuntos
Difosfato de Adenosina/farmacologia , Monofosfato de Adenosina/farmacologia , Trifosfato de Adenosina/farmacologia , Apirase/antagonistas & inibidores , Difosfato de Adenosina/análogos & derivados , Difosfato de Adenosina/síntese química , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/síntese química , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/síntese química , Animais , Antígenos CD , Células COS , Chlorocebus aethiops , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Agregação Plaquetária/efeitos dos fármacos , Ratos , Especificidade da EspécieRESUMO
Extracellular nucleotides can modify the production or drainage of the aqueous humor via activation of P2 receptors and therefore affect the intraocular pressure (IOP). We have synthesized slowly hydrolyzable nucleoside di- and triphosphate analogues, 1, and 8-14. Analogues 8-14 were completely resistant to hydrolysis by alkaline phosphatase over 30 min at 37 degrees C. In human blood serum, analogues 8-14 exhibited high stability, e.g., analogues 9 and 10-14 were only 15% and 0% degraded after 24 h, respectively. Moreover, analogues 8-14 were highly stable at pH 1.4 (t(1/2) 1 h-30 days). Analogues 8-14 were agonists of the P2Y(1) receptor (EC(50) 0.57-9.54 muM). Ocular administration of most analogues into rabbits reduced IOP, e.g., analogue 9 reduced IOP by 32% (EC(50) 95.5 nM). Analogue 9 was more effective at reducing IOP than several common glaucoma drugs and represents a promising alternative to timolol maleate, which cannot be used for the treatment of patients suffering from asthma or cardiac problems.
Assuntos
Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/síntese química , Pressão Intraocular/efeitos dos fármacos , Difosfato de Adenosina/análogos & derivados , Difosfato de Adenosina/síntese química , Difosfato de Adenosina/farmacologia , Trifosfato de Adenosina/farmacologia , Animais , Linhagem Celular Tumoral , Estabilidade de Medicamentos , Humanos , Hidrólise , Masculino , Agonistas do Receptor Purinérgico P2 , Coelhos , Receptores Purinérgicos P2Y1 , Relação Estrutura-Atividade , TurquiaRESUMO
Thiamine and its three phosphorylated derivatives (mono-, di- and triphosphate) occur naturally in most cells. Recently, we reported the presence of a fourth thiamine derivative, adenosine thiamine triphosphate, produced in Escherichia coli in response to carbon starvation. Here, we show that the chemical synthesis of adenosine thiamine triphosphate leads to another new compound, adenosine thiamine diphosphate, as a side product. The structure of both compounds was confirmed by MS analysis and 1H-, 13C- and 31P-NMR, and some of their chemical properties were determined. Our results show an upfield shifting of the C-2 proton of the thiazolium ring in adenosine thiamine derivatives compared with conventional thiamine phosphate derivatives. This modification of the electronic environment of the C-2 proton might be explained by a through-space interaction with the adenosine moiety, suggesting U-shaped folding of adenosine thiamine derivatives. Such a structure in which the C-2 proton is embedded in a closed conformation can be located using molecular modeling as an energy minimum. In E. coli, adenosine thiamine triphosphate may account for 15% of the total thiamine under energy stress. It is less abundant in eukaryotic organisms, but is consistently found in mammalian tissues and some cell lines. Using HPLC, we show for the first time that adenosine thiamine diphosphate may also occur in small amounts in E. coli and in vertebrate liver. The discovery of two natural thiamine adenine compounds further highlights the complexity and diversity of thiamine biochemistry, which is not restricted to the cofactor role of thiamine diphosphate.
Assuntos
Adenina/análogos & derivados , Difosfato de Adenosina/química , Trifosfato de Adenosina/química , Tiamina Pirofosfato/química , Tiamina Trifosfato/química , Células 3T3 , Adenina/análise , Adenina/síntese química , Adenina/química , Difosfato de Adenosina/análise , Difosfato de Adenosina/síntese química , Trifosfato de Adenosina/análise , Trifosfato de Adenosina/síntese química , Animais , Química Encefálica , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , Escherichia coli/química , Fibroblastos/química , Humanos , Rim/química , Fígado/química , Espectroscopia de Ressonância Magnética , Camundongos , Modelos Moleculares , Estrutura Molecular , Músculo Esquelético/química , Miocárdio/química , Codorniz , Espectrometria de Fluorescência , Espectrometria de Massas por Ionização por Electrospray , Tiamina Pirofosfato/análise , Tiamina Pirofosfato/síntese química , Tiamina Trifosfato/análise , Tiamina Trifosfato/síntese químicaRESUMO
Ribose-based nucleoside 5'-diphosphates and triphosphates and related nucleotides were compared in their potency at the P2Y receptors with the corresponding nucleoside 5'-phosphonate derivatives. Phosphonate derivatives of UTP and ATP activated the P2Y(2) receptor but were inactive or weakly active at P2Y(4) receptor. Uridine 5'-(diphospho)phosphonate was approximately as potent at the P2Y(2) receptor as at the UDP-activated P2Y(6) receptor. These results suggest that removal of the 5'-oxygen atom from nucleotide agonist derivatives reduces but does not prevent interaction with the P2Y(2) receptor. Uridine 5'-(phospho)phosphonate as well as the 5'-methylenephosphonate equivalent of UMP were inactive at the P2Y(4) receptor and exhibited maximal effects at the P2Y(2) receptor that were 50% of that of UTP suggesting novel action of these analogues.
Assuntos
Nucleotídeos/síntese química , Agonistas do Receptor Purinérgico P2 , Difosfato de Adenosina/análogos & derivados , Difosfato de Adenosina/síntese química , Difosfato de Adenosina/química , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/síntese química , Trifosfato de Adenosina/química , Linhagem Celular Tumoral , Humanos , Nucleotídeos/química , Receptores Purinérgicos P2/metabolismo , Proteínas Recombinantes/agonistas , Proteínas Recombinantes/metabolismo , Difosfato de Uridina/análogos & derivados , Difosfato de Uridina/síntese química , Difosfato de Uridina/química , Uridina Trifosfato/análogos & derivados , Uridina Trifosfato/síntese química , Uridina Trifosfato/químicaRESUMO
We have prepared a caged form (MRS2703) of a potent dual agonist of the P2Y(1) and P2Y(12) nucleotide receptors, 2-MeSADP, by blocking the beta-phosphate group with a 1-(3,4-dimethyloxyphenyl)eth-1-yl phosphoester. Although MRS2703 is itself inactive at human P2Y(1) and P2Y(12) receptors expressed heterologously in 1321N1 astrocytoma cells or in washed human platelets, this derivative readily regenerates the parent agonist upon mild irradiation with long-wave UV light (360 nm). The functional effect of the regenerated agonist was demonstrated by a rise in intracellular calcium mediated by either P2Y(1) or P2Y(12) receptors in transfected cells. Washed human platelets exposed to a solution of MRS2703 were induced to aggregate upon UV irradiation. At 1.0 microM MRS2703, full aggregation was achieved within 1 min of irradiation. Thus, this caged nucleotide promises to be a useful probe for potent P2Y receptor activation with light-directed spatial and temporal control.
Assuntos
Difosfato de Adenosina/análogos & derivados , Agregação Plaquetária/efeitos dos fármacos , Agonistas do Receptor Purinérgico P2 , Tionucleotídeos/farmacologia , Difosfato de Adenosina/síntese química , Difosfato de Adenosina/farmacologia , Difosfato de Adenosina/efeitos da radiação , Cálcio/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Humanos , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2Y1 , Receptores Purinérgicos P2Y12 , Tionucleotídeos/efeitos da radiação , Transfecção , Raios UltravioletaRESUMO
A fundamental question in protein chemistry is how the native energy landscape of enzymes enables efficient catalysis of chemical reactions. Adenylate kinase is a small monomeric enzyme that catalyzes the reversible conversion of AMP and ATP into two ADP molecules. Previous structural studies have revealed that substrate binding is accompanied by large rate-limiting spatial displacements of both the ATP and AMP binding motifs. In this report a solution-state NMR approach was used to probe the native energy landscape of adenylate kinase in its free form, in complex with its natural substrates, and in the presence of a tight binding inhibitor. Binding of ATP induces a dynamic equilibrium in which the ATP binding motif populates both the open and the closed conformations with almost equal populations. A similar scenario is observed for AMP binding, which induces an equilibrium between open and closed conformations of the AMP binding motif. These ATP- and AMP-bound structural ensembles represent complexes that exist transiently during catalysis. Simultaneous binding of AMP and ATP is required to force both substrate binding motifs to close cooperatively. In addition, a previously unknown unidirectional energetic coupling between the ATP and AMP binding sites was discovered. On the basis of these and previous results, we propose that adenylate kinase belongs to a group of enzymes whose substrates act to shift pre-existing equilibria toward catalytically active states.
Assuntos
Difosfato de Adenosina/síntese química , Monofosfato de Adenosina/química , Trifosfato de Adenosina/química , Adenilato Quinase/química , Espectroscopia de Ressonância Magnética/métodos , Difosfato de Adenosina/química , Catálise , Espectroscopia de Ressonância Magnética/normas , Mutagênese Sítio-Dirigida , Conformação Proteica , Estrutura Terciária de Proteína , Padrões de ReferênciaRESUMO
We report that N(6)-(1-naphthyl)-ADP inhibits the Escherichia coli RecA protein in vitro. A novel rapid screen identified it as a potent inhibitor of RecA nucleoprotein filament formation, and further characterization established it as an ATP-competitive inhibitor of RecA-catalyzed ATP hydrolysis. This and other inhibitors of RecA activities represent a new approach for understanding the molecular targets and pathways involved in the evolution of antibiotic resistance in bacteria.
Assuntos
1-Naftilamina/análogos & derivados , Difosfato de Adenosina/análogos & derivados , Farmacorresistência Bacteriana , Proteínas de Escherichia coli/química , Recombinases Rec A/antagonistas & inibidores , Recombinases Rec A/química , 1-Naftilamina/síntese química , 1-Naftilamina/química , Difosfato de Adenosina/síntese química , Difosfato de Adenosina/química , Trifosfato de Adenosina/química , DNA de Cadeia Simples/química , Hidrólise , Cinética , Modelos Moleculares , Ligação Proteica , Relação Estrutura-AtividadeRESUMO
We have synthesized a set of ATP and ADP analogues that have a fluorophore linked to the nucleotide via the 3'-position of the ribose moiety. Combinations of three different coumarins are each attached via different length linkers. A linker based on propylenediamine increases the separation between the nucleotide and fluorophore relative to that of the previously reported ethylenediamine-linked coumarin nucleotides [Webb, M. R., and Corrie, J. E. T. (2001) Biophys. J. 81, 1562-1569]. A synthesis of 3'-amino-3'-deoxyATP is described using a combination of chemical and enzymatic procedures, mostly from published methods for synthesis of this compound but with some modifications that improved the convenience of the experimental procedures. This compound is used as a basis of a series of analogues with effectively a zero-length linker. Fluorescence properties of all these analogues are described, together with the kinetics of their interaction with rabbit skeletal myosin subfragment 1 in the presence and absence of actin. One particular analogue, deac-aminoATP [3'-(7-diethylaminocoumarin-3-carbonylamino)-3'-deoxyadenosine 5'-triphosphate], shows a 17-fold enhancement of fluorescence upon binding to this (skeletal) myosin II. As the diphosphate, it exhibits a large signal change upon dissociation from the actomyosin, with kinetics similar to those of natural ADP. The ability of this set of analogues to produce large signals indicated potential uses when scarce proteins are studied in small amounts.
Assuntos
Actomiosina/química , Difosfato de Adenosina/análogos & derivados , Difosfato de Adenosina/metabolismo , Adenosina Trifosfatases/química , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/metabolismo , Corantes Fluorescentes/metabolismo , Actomiosina/metabolismo , Difosfato de Adenosina/síntese química , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/síntese química , Animais , Azidas/síntese química , Azidas/metabolismo , Cumarínicos/síntese química , Cumarínicos/metabolismo , Nucleotídeos de Desoxiadenina/síntese química , Nucleotídeos de Desoxiadenina/metabolismo , Desoxiadenosinas/síntese química , Desoxiadenosinas/metabolismo , Didesoxinucleotídeos , Corantes Fluorescentes/síntese química , Fluorometria , Subfragmentos de Miosina/química , Subfragmentos de Miosina/metabolismo , Coelhos , Espectrometria de Fluorescência/métodos , EspectrofotometriaRESUMO
A previous study around adenine nucleotides afforded the reference N(6)-methyl-2'-deoxyadenosine-3',5'-bisphosphate (1a, MRS 2179) as a selective human P2Y(1) receptor antagonist (pA(2)=6.55+/-0.05) with antithrombotic properties. In the present paper, we have synthesized and tested in vitro various 2-substituted derivatives with the goal of exploring the 2-position binding region and developing more potent P2Y(1) receptor antagonists. Thus, we have adopted a novel and versatile chemical pathway using a palladium-catalyzed cross-coupling reaction with the 2-iodinated derivative 7 as a common intermediate for a very efficient synthesis of the 2-alkyl-N(6)-methyl-2'-deoxyadenosine-3',5'-bisphosphate nucleotides 1e-i. The biological activity was evaluated through the ability of compounds to inhibit ADP-induced platelet aggregation, intracellular calcium rise and to displace the specific binding of [(33)P]2-MeSADP. 2-Ethyl and 2-propyl groups appeared to be tolerated, whereas a bulky group or a C(3) linear substituent dramatically decreased potency of antagonists. The 2-ethynyl derivative 1h (pA(2)=7.54+/-0.10) was significantly more potent (10-fold) as an antagonist when compared to the reference 1a, revealing a potential electronic interaction highly favorable between triple bond orbitals and the P2Y(1) receptor at this position.
Assuntos
Difosfato de Adenosina/análogos & derivados , Nucleotídeos de Desoxiadenina/síntese química , Nucleotídeos de Desoxiadenina/farmacologia , Antagonistas do Receptor Purinérgico P2 , Difosfato de Adenosina/antagonistas & inibidores , Difosfato de Adenosina/síntese química , Difosfato de Adenosina/química , Difosfato de Adenosina/farmacologia , Inibidores de Adenilil Ciclases , Adenilil Ciclases/metabolismo , Ligação Competitiva/efeitos dos fármacos , Plaquetas/metabolismo , Cálcio/metabolismo , Nucleotídeos de Desoxiadenina/química , Humanos , Proteínas de Membrana/antagonistas & inibidores , Estrutura Molecular , Paládio/química , Agregação Plaquetária/efeitos dos fármacos , Receptores Purinérgicos P2Y1 , Receptores Purinérgicos P2Y12 , Relação Estrutura-AtividadeRESUMO
Preference for the northern (N) ring conformation of the ribose moiety of adenine nucleotide 3',5'-bisphosphate antagonists of P2Y(1) receptors was established by using a ring-constrained methanocarba (a bicyclo[3.1.0]hexane) ring as a ribose substitute (Nandanan et al. J. Med. Chem. 2000, 43, 829-842). We have now combined the ring-constrained (N)-methanocarba modification with other functionalities at the 2-position of the adenine moiety. A new synthetic route to this series of bisphosphate derivatives was introduced, consisting of phosphorylation of the pseudoribose moiety prior to coupling with the adenine base. The activity of the newly synthesized analogues was determined by measuring antagonism of 2-methylthio-ADP-stimulated phospholipase C (PLC) activity in 1321N1 human astrocytoma cells expressing the recombinant human P2Y(1) receptor and by using the radiolabeled antagonist [(3)H]2-chloro-N(6)-methyl-(N)-methanocarba-2'-deoxyadenosine 3',5'-bisphosphate 5 in a newly developed binding assay in Sf9 cell membranes. Within the series of 2-halo analogues, the most potent molecule at the hP2Y(1) receptor was an (N)-methanocarba N(6)-methyl-2-iodo analogue 12, which displayed a K(i) value in competition for binding of [(3)H]5 of 0.79 nM and a K(B) value of 1.74 nM for inhibition of PLC. Thus, 12 is the most potent antagonist selective for the P2Y(1) receptor yet reported. The 2-iodo group was substituted with trimethyltin, thus providing a parallel synthetic route for the introduction of an iodo group in this high-affinity antagonist. The (N)-methanocarba-2-methylthio, 2-methylseleno, 2-hexyl, 2-(1-hexenyl), and 2-(1-hexynyl) analogues bound less well, exhibiting micromolar affinity at P2Y(1) receptors. An enzymatic method of synthesis of the 3',5'-bisphosphate from the corresponding 3'-monophosphate, suitable for the preparation of a radiophosphorylated analogue, was explored.
Assuntos
Nucleotídeos de Adenina/síntese química , Difosfato de Adenosina/síntese química , Antagonistas do Receptor Purinérgico P2 , Nucleotídeos de Adenina/química , Nucleotídeos de Adenina/farmacologia , Difosfato de Adenosina/análogos & derivados , Difosfato de Adenosina/química , Difosfato de Adenosina/farmacologia , Ligação Competitiva , Linhagem Celular , Linhagem Celular Tumoral , Humanos , Conformação Molecular , Ensaio Radioligante , Receptores Purinérgicos P2/metabolismo , Receptores Purinérgicos P2Y1 , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/metabolismo , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
Polyadenosine diphosphoribose glycohydrolase (PARG) catalyzes the intracellular hydrolysis of adenosine diphosphoribose polymers. Because structure-activity data are lacking for PARG, the specific inhibitor adenosine diphosphate (hydroxymethyl)pyrrolidinediol (ADP-HPD) was utilized to determine the effects of structure on inhibitor potency using PARG isolated from bovine thymus (bPARG) and recombinant bovine PARG catalytic fragment (rPARG-CF). Both enzymes were strongly inhibited by submicromolar levels of ADP-HPD, but ADP and the phosphorylated pyrrolidine displayed no activity. Utilizing ADP-HPD analogues containing 2-, N(6), or 8-adenosyl substituents or guanine instead of adenine, the importance of adenine ring recognition as well as a correlation between loss of PARG inhibition and the length and bulkiness of 8-adenosyl substituents was shown. Utilization of ADP-HPD analogues lacking one or both pyrrolidine cis-hydroxyls demonstrated their importance for inhibitor binding. Last, the similarity between naturally occurring bPARG and heterologously expressed rPARG-CF was demonstrated. Therefore, readily available rPARG-CF is suitable for use in future studies to determine the structural aspects of PARG.
Assuntos
Difosfato de Adenosina/análogos & derivados , Difosfato de Adenosina/química , Difosfato de Adenosina/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Glicosídeo Hidrolases/antagonistas & inibidores , Pirrolidinas/química , Pirrolidinas/farmacologia , Difosfato de Adenosina/síntese química , Animais , Sítios de Ligação , Bovinos , Inibidores Enzimáticos/síntese química , Glicosídeo Hidrolases/genética , Concentração Inibidora 50 , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/genética , Poli Adenosina Difosfato Ribose/metabolismo , Pirrolidinas/síntese química , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/genética , Relação Estrutura-AtividadeRESUMO
Sulfotransferases catalyze the transfer of a sulfuryl group from the eukaryotic sulfate donor 3'-phosphoadenosine 5'-phosphosulfate to an acceptor biomolecule. Sulfotransferases have been linked with several disease states, prompting our investigation of specific sulfotransferase inhibitors. Presented herein is the synthesis and evaluation of a bisubstrate analogue designed to inhibit estrogen sulfotransferase. The synthesis utilizes a novel, orthogonally protected 3'-phosphoadenosine 5'-phosphate (PAP) derivative allowing the selective functionalization of the 5'-phosphate with a sulfate acceptor mimic. Kinetic studies revealed significant inhibitory activity and provide guidance for improved inhibitor design.
Assuntos
Difosfato de Adenosina/síntese química , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Sulfotransferases/antagonistas & inibidores , Sulfotransferases/química , Difosfato de Adenosina/farmacologia , Catálise , Cinética , Modelos Moleculares , Mimetismo Molecular , Estrutura Molecular , Especificidade por SubstratoRESUMO
Activation by ADP of both P2Y(1) and P2Y(12) receptors in platelets contributes to platelet aggregation, and antagonists at these receptor subtypes have antithrombotic properties. In an earlier publication, we have characterized the SAR as P2Y(1) receptor antagonists of acyclic analogues of adenine nucleotides, containing two phosphate groups on a symmetrically branched aliphatic chain, attached at the 9-position of adenine. In this study, we have focused on antiaggregatory effects of P2Y antagonists related to a 2-chloro-N(6)-methyladenine-9-(2-methylpropyl) scaffold, containing uncharged substitutions of the phosphate groups. For the known nucleotide (cyclic and acyclic) bisphosphate antagonists of P2Y(1) receptors, there was a significant correlation between inhibition of aggregation induced by 3.3 microM ADP in rat platelets and inhibition of P2Y(1) receptor-induced phospholipase C (PLC) activity previously determined in turkey erythrocytes. Substitution of the phosphate groups with nonhydrolyzable phosphonate groups preserved platelet antiaggregatory activity. Substitution of one of the phosphate groups with O-acyl greatly reduced the inhibitory potency, which tended to increase upon replacement of both phosphate moieties of the acyclic derivatives with uncharged (e.g., ester) groups. In the series of nonsymmetrically substituted monophosphates, the optimal antagonist potency occurred with the phenylcarbamate group. Among symmetrical diester derivatives, the optimal antagonist potency occurred with the di(phenylacetyl) group. A dipivaloyl derivative, a representative uncharged diester, inhibited ADP-induced aggregation in both rat (K(I) 3.6 microM) and human platelets. It antagonized the ADP-induced inhibition of the cyclic AMP pathway in rat platelets (IC(50) 7 microM) but did not affect hP2Y(1) receptor-induced PLC activity measured in transfected astrocytoma cells. We propose that the uncharged derivatives are acting as antagonists of a parallel pro-aggregatory receptor present on platelets, that is, the P2Y(12) receptor. Thus, different substitution of the same nucleoside scaffold can target either of two P2Y receptors in platelets.
Assuntos
Difosfato de Adenosina/análogos & derivados , Difosfato de Adenosina/síntese química , Inibidores da Agregação Plaquetária/síntese química , Antagonistas do Receptor Purinérgico P2 , Difosfato de Adenosina/química , Difosfato de Adenosina/farmacologia , Animais , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Linhagem Celular , AMP Cíclico/metabolismo , Humanos , Técnicas In Vitro , Fosfatos de Inositol/metabolismo , Nucleosídeos/síntese química , Nucleosídeos/química , Nucleosídeos/farmacologia , Inibidores da Agregação Plaquetária/química , Inibidores da Agregação Plaquetária/farmacologia , Ratos , Ratos Sprague-Dawley , Receptores Purinérgicos P2Y1 , Relação Estrutura-Atividade , Fosfolipases Tipo C/metabolismoRESUMO
2-Chloro-N(6)-methyl-(N )-methanocarba-2'-deoxyadenosine-3',5'- bisphosphate (MRS2279) was developed previously as a selective high-affinity, non-nucleotide P2Y(1) receptor (P2Y1-R) antagonist (J Med Chem 43:829-842, 2002; Br J Pharmacol 135:2004-2010, 2002). We have taken advantage of the N(6)-methyl substitution in the adenine base to incorporate [(3)H]methylamine into the synthesis of [(3)H]MRS2279 to high (89 Ci/mmol) specific radioactivity and have used this molecule as a radioligand for the P2Y1-R. [(3)H]MRS2279 bound to membranes from Sf9 insect cells expressing recombinant human P2Y1-R but not to membranes from wild-type Sf9 cells or Sf9 cells expressing high levels of recombinant P2Y(2) or P2Y(12) receptors. Equilibrium binding of [(3)H]MRS2279 to P2Y1-R expressed in Sf9 membranes was with a high affinity (K(d) = 8 nM) essentially identical to the apparent affinity of MRS2279 determined previously in studies of P2Y1-R-promoted inositol phosphate accumulation or platelet aggregation. A kinetically derived K(d) calculated from independent determinations of the rate constants of association (7.15 x 10(7) M(-1) min(-1)) and dissociation (0.72 min(-1)) of [(3)H]MRS2279 also was in good agreement with the K(d) derived from equilibrium binding studies. Competition binding assays with [(3)H]MRS2279 and P2Y1-R expressing Sf9 cell membranes revealed K(i) values for the P2Y1-R antagonists MRS2279 (K(i) = 13 nM), N(6)-methyl-2'-deoxyadenosine-3',5'-bisphosphate (MRS2179; K(i) = 84 nM), adenosine-3', 5'-bisphosphate (K(i)=900 nM), and pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (K(i) = 6 microM) that were in good agreement with antagonist activities of these molecules previously determined at the P2Y1-R in intact tissues. Moreover, [(3)H]MRS2279 also bound with high affinity (K(d) = 4-8 nM) to Chinese hamster ovary (CHO) or 1321N1 human astrocytoma cells stably expressing the human P2Y1-R, but specific binding was not observed in wild-type CHO or 1321N1 cells. [(3)H]MRS2279 bound with high affinity (K(d) = 16 nM) to a binding site on out-dated human platelets (5-35 receptors/platelet) and rat brain membranes (210 fmol/mg protein) that fit the expected drug selectivity of a P2Y1-R. Taken together, these results indicate that [(3)H]MRS2279 is the first broadly applicable antagonist radioligand for a P2Y receptor.
Assuntos
Difosfato de Adenosina/análogos & derivados , Difosfato de Adenosina/farmacologia , Compostos Radiofarmacêuticos/farmacologia , Receptores Purinérgicos P2/análise , Difosfato de Adenosina/síntese química , Difosfato de Adenosina/química , Animais , Células Cultivadas , Feminino , Humanos , Insetos/citologia , Antagonistas do Receptor Purinérgico P2 , Compostos Radiofarmacêuticos/síntese química , Compostos Radiofarmacêuticos/química , Ratos , Ratos Sprague-Dawley , Receptores Purinérgicos P2Y1 , TrítioRESUMO
The human P2Y(1) receptor is widely distributed in many tissues and has a classical structure of a G protein-coupled receptor. Activated by adenosine-5'-diphosphate (ADP), this receptor is essential for platelet aggregation. In the present paper, we describe the synthesis of novel P2Y(1) antagonists that could be of interest at least as tools to define the physiological roles of the P2Y(1) receptor, at best as new antithrombotic agents. Thus, we prepared the 2,N(6)-dimethyl-2'-deoxyadenosine-3',5'-bisphosphate derivative, 1e. The biological activity was demonstrated by the ability of compound 1e to inhibit ADP-induced platelet aggregation, shape change, and intracellular calcium rise. This compound was a full antagonist at the P2Y(1) receptor with a pA(2) value of 7.11 +/- 0.11 and was found to be 4-fold more potent than the reference N(6)-methyl-2'-deoxyadenosine-3',5'-bisphosphate (1a, pA(2) = 6.55 +/- 0.05), revealing the potency-enhancing effects of the 2-methyl group. The better activity of 1e as compared to 1a was analyzed using both potentiometric and nuclear magnetic resonance titration techniques, which highlighted specific conformational features of this compound. These results clearly indicate the preference for both compounds for an anti conformation at the N-glycosyl linkage. Furthermore, the percentage of S conformer of 1e is close to that of 1a, which is nearly 70% at pH = 2.8 and increases dramatically when pH increases. From the macroprotonation constants, it can be noted that compound 1e is significantly more basic than 1a. This is indeed expected for the N1 adenine nitrogen due to the electron-donating character of the methyl moiety. By considering the microconstants of the phosphate groups, the higher basicity of P3 and P5 for 1e may be due to the decrease in the local dielectric constant induced by the substitution of the hydrogen atom by a more lipophilic methyl group. Thus, it may be suggested that the gain in activity of 1e when compared to the reference compound 1a would result from its gain in basicity rather than steric and conformational modifications. The synthesis of the first selective radioligand acting at the P2Y(1) receptor ([(33)P]-N(6)-methyl-2'-deoxyadenosine-3',5'-bisphosphate, 17) is also reported and will be used in the future for efficient screening needed for drug optimization.
Assuntos
Difosfato de Adenosina/síntese química , Fibrinolíticos/síntese química , Antagonistas do Receptor Purinérgico P2 , Difosfato de Adenosina/análogos & derivados , Difosfato de Adenosina/química , Difosfato de Adenosina/farmacologia , Inibidores de Adenilil Ciclases , Plaquetas/metabolismo , Cálcio/metabolismo , Fibrinolíticos/química , Fibrinolíticos/farmacologia , Humanos , Técnicas In Vitro , Espectroscopia de Ressonância Magnética , Conformação Molecular , Radioisótopos de Fósforo , Agregação Plaquetária/efeitos dos fármacos , Potenciometria , Receptores Purinérgicos P2Y1 , Relação Estrutura-AtividadeRESUMO
The activation of P2Y1 receptors in platelets contributes to platelet aggregation, and selective antagonists are sought as potential antithrombotic agents. We reported (Kim et al. J. Med. Chem. 2000, 43, 746-755) that acyclic analogues of adenine nucleotides, containing two phosphate groups on a symmetrically branched aliphatic chain, attached at the 9-position of adenine, are moderately potent P2Y1 receptor antagonists. In this study we have varied the chain structure, to include asymmetric substitution, olefinic, and cyclopropyl groups. These antagonists inhibited the stimulation of phospholipase C in turkey erythrocyte membranes induced by 30 nM 2-MeS-ADP in the micromolar range. In the series of symmetrically branched aliphatic groups substituted with two phosphate groups, the optimal antagonist potency occurred with the 2-methylpropyl group. A 2-chloro-N(6)-methyladenine derivative, 2-[2-(2-chloro-6-methylaminopurin-9-yl)methyl]propane-1,3-bisoxy(diammoniumphosphate) (7), was a full antagonist at the P2Y1 receptor with an IC(50) value of 0.48 microM. Esterification of one of the phosphate groups or substitution with O-acetyl greatly reduced the antagonist potency at the P2Y1 receptor. Removal of a methylene group of 7 or inclusion of an olefinic or cyclopropyl group also reduced potency. A pair of enantiomeric glycerol derivatives demonstrated a 5-fold stereoselectivity for the S-isomer. Stereoisomerically defined analogues of 7 containing a cyclopropyl group in place of the branched carbon were less potent than 7 as antagonists, with IC(50) values of 2-3 microM. No agonist activity was observed for these analogues. A new rhodopsin-based molecular model of the P2Y1 receptor indicated that the optimal docked orientation of the two monophosphate moieties relative to the adenine N(6) (compared to a rigid, bicyclic analogue) was consistent with the dependence of antagonist potency on chain length. The 3'-phosphate was predicted to occupy a restricted space, deeper in the binding cleft than the 5'-phosphate location. In summary, modification of the flexible spacer chain linking bisphosphate groups to the adenine moiety provided many moderately potent antagonists.
Assuntos
Difosfato de Adenosina/análogos & derivados , Difosfato de Adenosina/síntese química , Antagonistas do Receptor Purinérgico P2 , Difosfato de Adenosina/química , Difosfato de Adenosina/farmacologia , Animais , Cromatografia Líquida de Alta Pressão , Ativação Enzimática , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Membrana Eritrocítica/efeitos dos fármacos , Membrana Eritrocítica/metabolismo , Técnicas In Vitro , Fosfatos de Inositol/biossíntese , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Modelos Moleculares , Receptores Purinérgicos P2/química , Receptores Purinérgicos P2Y1 , Estereoisomerismo , Relação Estrutura-Atividade , Turquia , Fosfolipases Tipo C/antagonistas & inibidores , Fosfolipases Tipo C/metabolismoRESUMO
A novel nucleotide inhibitor (ADP-S-HBES-S-dGTP) of mouse ribonucleotide reductase was designed to span the active site and the allosteric specificity site of the enzyme. The inhibitor contains ADP and dGTP moieties which are linked by 1,6-hexane-(bis-ethylenesulfone), and has a Ki value of 12 microM.
Assuntos
Difosfato de Adenosina/análogos & derivados , Nucleotídeos de Desoxiguanina/síntese química , Inibidores Enzimáticos/síntese química , Ribonucleotídeo Redutases/antagonistas & inibidores , Difosfato de Adenosina/síntese química , Difosfato de Adenosina/química , Difosfato de Adenosina/farmacologia , Sítio Alostérico , Animais , Sítios de Ligação , Nucleotídeos de Desoxiguanina/química , Nucleotídeos de Desoxiguanina/farmacologia , Desenho de Fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Escherichia coli/enzimologia , Cinética , Camundongos , Modelos Moleculares , Conformação Proteica , Ribonucleotídeo Redutases/química , Relação Estrutura-AtividadeRESUMO
A novel photoactivatable radioactive ADP derivative, namely, 2-azido-3'-O-naphthoyl-[beta-(32)P]ADP (2-azido-N-[(32)P]ADP), was synthesized with the aim at mapping the substrate binding site(s) of the yeast mitochondrial ADP/ATP carrier. It was used with mitochondria isolated from genetically modified strains of Saccharomyces cerevisiae, producing the native or the His-tagged Anc2p isoform of the carrier. In darkness, 2-azido-N-[(32)P]ADP was reversibly bound to the carrier in mitochondria, without being transported. Upon photoirradiation, only the ADP/ATP carrier was covalently radiolabeled among all mitochondrial proteins. Specificity of labeling was demonstrated since carboxyatractyloside (CATR), a potent inhibitor of ADP/ATP transport, totally prevented the incorporation of the photoprobe. To localize the radioactive region(s), the purified photolabeled carrier was submitted to CNBr or hydroxylamine cleavage. The resulting fragments were characterized and identified by SDS-PAGE, Western blotting, amino acid sequencing, and MALDI-MS and ESI-MS analyses. Two short photolabeled distinct segments, eight and nine residues long, were identified: S183-R191, located in the central part of the ADP/ATP carrier; and I311-K318, belonging to its C-terminal end. Plausible models of organization of the nucleotide binding site(s) of the carrier involving the two regions specifically labeled by 2-azido-N-[(32)P]ADP are proposed.