RESUMEN
Toxoplasma gondii is unable to synthesize purines de novo, instead salvages them from its environment, inside the host cell, for which they need high affinity carriers. Here, we report the expression of a T. gondii Equilibrative Nucleoside Transporter, Tg244440, in a Trypanosoma brucei strain from which nucleobase transporters have been deleted. Tg244440 transported hypoxanthine and guanine with similar affinity (Km ~1 µM), while inosine and guanosine displayed Ki values of 4.05 and 3.30 µM, respectively. Low affinity was observed for adenosine, adenine, and pyrimidines, classifying Tg244440 as a high affinity oxopurine transporter. Purine analogues were used to probe the substrate-transporter binding interactions, culminating in quantitative models showing different binding modes for oxopurine bases, oxopurine nucleosides, and adenosine. Hypoxanthine and guanine interacted through protonated N1 and N9, and through unprotonated N3 and N7 of the purine ring, whereas inosine and guanosine mostly employed the ribose hydroxy groups for binding, in addition to N1H of the nucleobase. Conversely, the ribose moiety of adenosine barely made any contribution to binding. Tg244440 is the first gene identified to encode a high affinity oxopurine transporter in T. gondii and, to the best of our knowledge, the first purine transporter to employ different binding modes for nucleosides and nucleobases.
Asunto(s)
Proteínas de Transporte de Nucleósidos/metabolismo , Nucleósidos/metabolismo , Purinonas/metabolismo , Toxoplasma/fisiología , Toxoplasmosis/parasitología , Fibroblastos , Técnicas de Silenciamiento del Gen , Humanos , Proteínas de Transporte de Nucleósidos/genética , Nucleósidos/química , Filogenia , Unión Proteica , Purinonas/química , Toxoplasma/clasificaciónRESUMEN
Dopamine replacement represents the standard therapy for Parkinson's disease (PD), a common, chronic, and incurable neurological disorder; however, this approach only treats the symptoms of this devastating disease. In the search for novel disease-modifying therapies that target other relevant molecular and cellular mechanisms, Drosophila has emerged as a valuable tool to study neurodegenerative diseases due to the presence of a complex central nervous system, the blood-brain barrier, and a similar neurotransmitter profile to humans. Human PD-related genes also display conservation in flies; DJ-1ß is the fly ortholog of DJ-1, a gene for which mutations prompt early-onset recessive PD. Interestingly, flies mutant for DJ-1ß exhibit PD-related phenotypes, including motor defects, high oxidative stress (OS) levels and metabolic alterations. To identify novel therapies for PD, we performed an in vivo high-throughput screening assay using DJ-1ß mutant flies and compounds from the Prestwick® chemical library. Drugs that improved motor performance in DJ-1ß mutant flies were validated in DJ-1-deficient human neural-like cells, revealing that zaprinast displayed the most significant ability to suppress OS-induced cell death. Zaprinast inhibits phosphodiesterases and activates GPR35, an orphan G-protein-coupled receptor not previously associated with PD. We found that zaprinast exerts its beneficial effect in both fly and human PD models through several disease-modifying mechanisms, including reduced OS levels, attenuated apoptosis, increased mitochondrial viability, and enhanced glycolysis. Therefore, our results support zaprinast as a potential therapeutic for PD in future clinical trials.
Asunto(s)
Enfermedad de Parkinson , Animales , Drosophila/genética , Drosophila/metabolismo , Estrés Oxidativo , Enfermedad de Parkinson/tratamiento farmacológico , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismo , Proteína Desglicasa DJ-1/genética , Proteína Desglicasa DJ-1/metabolismo , Purinonas/metabolismo , Purinonas/farmacología , Purinonas/uso terapéuticoAsunto(s)
Osteoporosis/tratamiento farmacológico , Purinonas/metabolismo , Pirimidinas/metabolismo , Animales , Densidad Ósea/efectos de los fármacos , Remodelación Ósea/efectos de los fármacos , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 5/metabolismo , Dexametasona/farmacología , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Glucocorticoides , Masculino , Osteoporosis/inducido químicamente , Oxidación-Reducción/efectos de los fármacos , Estrés Oxidativo , Inhibidores de Fosfodiesterasa 5/metabolismo , RatasRESUMEN
Due to toxicity and compliance issues and the emergence of resistance to current medications new drugs for the treatment of Human African Trypanosomiasis are needed. A potential approach to developing novel anti-trypanosomal drugs is by inhibition of the 6-oxopurine salvage pathways which synthesise the nucleoside monophosphates required for DNA/RNA production. This is in view of the fact that trypanosomes lack the machinery for de novo synthesis of the purine ring. To provide validation for this approach as a drug target, we have RNAi silenced the three 6-oxopurine phosphoribosyltransferase (PRTase) isoforms in the infectious stage of Trypanosoma brucei demonstrating that the combined activity of these enzymes is critical for the parasites' viability. Furthermore, we have determined crystal structures of two of these isoforms in complex with several acyclic nucleoside phosphonates (ANPs), a class of compound previously shown to inhibit 6-oxopurine PRTases from several species including Plasmodium falciparum. The most potent of these compounds have Ki values as low as 60 nM, and IC50 values in cell based assays as low as 4 µM. This data provides a solid platform for further investigations into the use of this pathway as a target for anti-trypanosomal drug discovery.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Redes y Vías Metabólicas/efectos de los fármacos , Purinonas/metabolismo , Tripanocidas/farmacología , Trypanosoma brucei brucei/metabolismo , Dominio Catalítico , Descubrimiento de Drogas , Inhibidores Enzimáticos/química , Humanos , Hipoxantina Fosforribosiltransferasa/antagonistas & inhibidores , Hipoxantina Fosforribosiltransferasa/química , Hipoxantina Fosforribosiltransferasa/genética , Hipoxantina Fosforribosiltransferasa/metabolismo , Modelos Moleculares , Pentosiltransferasa/antagonistas & inhibidores , Pentosiltransferasa/química , Pentosiltransferasa/genética , Pentosiltransferasa/metabolismo , Interferencia de ARN , Tripanocidas/química , Trypanosoma brucei brucei/efectos de los fármacos , Trypanosoma brucei brucei/enzimología , Trypanosoma brucei brucei/genéticaRESUMEN
Ligands of the translocator protein (18 kDa) (TSPO) have demonstrated rapid anxiolytic efficacy in stress responses and stress-related disorders. This protein is involved in the synthesis of endogenous neurosteroids including pregnenolone, dehydroepiandrosterone, and progesterone. These neurosteroids promote γ-aminobutyric acid-mediated neurotransmission in the central neural system (CNS). A TSPO ligand, N-benzyl-N-ethyl-2-(7,8-dihydro-7-benzyl-8-oxo-2-phenyl-9H-purin-9-yl) acetamide (ZBD-2) was recently synthesized. The purpose of the present study was to investigate the neuroprotective effects of ZBD-2 and. In cultured cortical neurons, treatment with ZBD-2 attenuated excitotoxicity induced by N-methyl-d-aspartate (NMDA) exposure. It significantly decreased the number of apoptotic cells by downregulating GluN2B-containing NMDA receptors (NMDARs), the ratio of Bax/Bcl-2, and levels of pro-caspase-3. Systemic treatment of ZBD-2 provided significant neuroprotection in mice subjected to middle cerebral artery occlusion. These findings provide direct evidence that neuroprotection by ZBD-2 is partially mediated by inhibiting GluN2B-containing NMDA receptor-mediated excitotoxicity.
Asunto(s)
Acetamidas/farmacología , Isquemia Encefálica/prevención & control , N-Metilaspartato/toxicidad , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Purinonas/farmacología , Receptores de GABA/metabolismo , Acetamidas/metabolismo , Animales , Apoptosis/efectos de los fármacos , Encéfalo/citología , Isquemia Encefálica/patología , Caspasa 3/metabolismo , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Ligandos , Masculino , Ratones , Neuronas/citología , Neuronas/metabolismo , Fármacos Neuroprotectores/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Purinonas/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Proteína X Asociada a bcl-2/metabolismoRESUMEN
The activation of Translocator protein (18 kDa) (TSPO) has been demonstrated to mediate rapid anxiolytic efficacy in stress response and stress-related disorders. This protein is involved in the synthesis of endogenous neurosteroids that promote γ-aminobutyric acid (GABA)-mediated neurotransmission in the central neural system. However, little is known about the functions and the underlying mechanisms of TSPO in chronic pain-induced anxiety-like behaviors. The novel TSPO ligand N-benzyl-N-ethyl-2-(7,8-dihydro-7-benzyl-8-oxo-2-phenyl-9H-purin-9-yl) acetamide (ZBD-2) was used in the present study. We found that ZBD-2 (0.15 or 1.5 mg/kg) significantly attenuated anxiety-like behaviors in mice with chronic inflammatory pain induced by hindpaw injection of complete Freund's adjuvant (CFA). However, the treatment did not alter the nociceptive threshold or inflammation in the hindpaw. Hindpaw injection of CFA induced the upregulation of TSPO, GluR1-containing α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, and NR2B-containing N-methyl-D-aspartate (NMDA) receptors in the basolateral amygdala (BLA). ZBD-2 administration reversed the alterations of the abovementioned proteins in the BLA of the CFA-injected mice. Electrophysiological recording revealed that ZBD-2 could prevent an imbalance between excitatory and inhibitory transmissions in the BLA synapses of CFA-injected mice. Therefore, as the novel ligand of TSPO, ZBD-2 induced anxiolytic effects, but did not affect the nociceptive threshold of mice under chronic pain. The anxiolytic effects of ZBD-2 were related to the regulation of the balance between excitatory and inhibitory transmissions in the BLA.
Asunto(s)
Acetamidas/metabolismo , Ansiolíticos/farmacología , Dolor Crónico/tratamiento farmacológico , Purinonas/metabolismo , Receptores de GABA/metabolismo , Sinapsis/metabolismo , Animales , Ansiedad/tratamiento farmacológico , Modelos Animales de Enfermedad , Adyuvante de Freund/uso terapéutico , Masculino , Ratones Endogámicos C57BL , Transmisión SinápticaRESUMEN
Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) is a key enzyme of the purine recycling pathway that catalyzes the conversion of 5-phospho-ribosyl-α-1-pyrophosphate and guanine or hypoxanthine to guanosine monophosphate (GMP) or inosine monophosphate (IMP), respectively, and pyrophosphate (PPi). We report the first crystal structure of a fungal 6-oxopurine phosphoribosyltransferase, the Saccharomyces cerevisiae HGPRT (Sc-HGPRT) in complex with GMP. The crystal structures of full length protein with (WT1) or without (WT2) sulfate that mimics the phosphate group in the PPi binding site were solved by molecular replacement using the structure of a truncated version (Δ7) solved beforehand by multiwavelength anomalous diffusion. Sc-HGPRT is a dimer and adopts the overall structure of class I phosphoribosyltransferases (PRTs) with a smaller hood domain and a short two-stranded parallel ß-sheet linking the N- to the C-terminal end. The catalytic loops in WT1 and WT2 are in an open form while in Δ7, due to an inter-subunit disulfide bridge, the catalytic loop is in either an open or closed form. The closure is concomitant with a peptide plane flipping in the PPi binding loop. Moreover, owing the flexibility of a GGGG motif conserved in fungi, all the peptide bonds of the phosphate binding loop are in trans conformation whereas in nonfungal 6-oxopurine PRTs, one cis-peptide bond is required for phosphate binding. Mutations affecting the enzyme activity or the previously characterized feedback inhibition by GMP are located at the nucleotide binding site and the dimer interface.
Asunto(s)
Glicina/química , Guanosina Monofosfato/metabolismo , Hipoxantina Fosforribosiltransferasa/química , Hipoxantina Fosforribosiltransferasa/metabolismo , Saccharomyces cerevisiae/enzimología , Sitios de Unión , Dominio Catalítico , Cristalografía por Rayos X , Glicina/metabolismo , Modelos Moleculares , Conformación Proteica , Multimerización de Proteína , Purinonas/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismoRESUMEN
DNA is constantly exposed to agents that induce structural damage, from sources both internal and external to an organism. Endogenous species, such as oxidizing chemicals, and exogenous agents, such as ultraviolet rays in sunlight, together produce more than 70 distinct chemical modifications of native nucleotides. Of these, about 15 of the lesions have been detected in cellular DNA. This kind of structural DNA damage can be cytotoxic, carcinogenic, or both and is being linked to an increasingly lengthy list of diseases. The formamidopyrimidine (Fapy) lesions are a family of DNA lesions that result after purines undergo oxidative stress. The Fapy lesions are produced in yields comparable to the 8-oxopurines, which, owing in part to a perception of mutagenicity in some quarters, have been subjected to intense research scrutiny. But despite the comparable abundance of the formamidopyrimidines and the 8-oxopurines, until recently very little was known about the effects of Fapy lesions on biochemical processes involving DNA or on the structure and stability of the genomic material. In this Account, we discuss the detection of Fapy lesions in DNA and the mechanism proposed for their formation. We also describe methods for the chemical synthesis of oligonucleotides containing Fapy·dA or Fapy·dG and the outcomes of chemical and biochemical studies utilizing these compounds. These experiments reveal that the formamidopyrimidines decrease the fidelity of polymerases and are substrates for DNA repair enzymes. The mutation frequency of Fapy·dG in mammals is even greater than that of 8-oxodGuo (8-oxo-7,8-dihydro-2'-deoxyguanosine, one of the 8-oxopurines), suggesting that this lesion could be a useful biomarker and biologically significant. Despite clear similarities, the formamidopyrimidines have lived in the shadow of the corresponding 8-oxopurine lesions. But the recent development of methods for synthesizing oligonucleotides containing Fapy·dA or Fapy·dG has accelerated research on these lesions, revealing that the formamidopyrimidines are repaired as efficiently and, in some cases, more rapidly than the 8-oxopurines. Fapy·dG appears to be a lesion of biochemical consequence, and further study of its mutagenicity, repair, and interactions with DNA structure will better define the cellular details involving this important product of DNA stress.
Asunto(s)
Daño del ADN , Estrés Oxidativo , Purinonas/metabolismo , Pirimidinas/metabolismo , Reparación del ADN , ADN Polimerasa Dirigida por ADN/metabolismo , Humanos , Purinonas/química , Pirimidinas/químicaRESUMEN
The poorly characterized G-protein-coupled receptor GPR35 has been suggested as a potential exploratory target for the treatment of both metabolic disorders and hypertension. It has also been indicated to play an important role in immune modulation. A major impediment to validation of these concepts and further study of the role of this receptor has been a paucity of pharmacological tools that interact with GPR35. Using a receptor-ß-arrestin-2 interaction assay with both human and rat orthologues of GPR35, we identified a number of compounds possessing agonist activity. These included the previously described ligand zaprinast. Although a number of active compounds, including cromolyn disodium and dicumarol, displayed similar potency at both orthologues of GPR35, a number of ligands, including pamoate and niflumic acid, had detectable activity only at human GPR35 whereas others, including zaprinast and luteolin, were markedly selective for the rat orthologue. Previous studies have demonstrated activation of Gα13 by GPR35. A Saccharomyces cerevisiae-based assay employing a chimaeric Gpa1-Gα13 G-protein confirmed that all of the compounds active at human GPR35 in the ß-arrestin-2 interaction assay were also able to promote cell growth via Gα13. Each of these ligands also promoted binding of [35S]GTP[S] (guanosine 5'-[γ-[35S]thio]triphosphate) to an epitope-tagged form of Gα13 in a GPR35-dependent manner. The ligands identified in these studies will be useful in interrogating the biological actions of GPR35, but appreciation of the species selectivity of ligands at this receptor will be vital to correctly attribute function.
Asunto(s)
Arrestinas/metabolismo , Descubrimiento de Drogas , Subunidades alfa de la Proteína de Unión al GTP G12-G13/metabolismo , Receptores Acoplados a Proteínas G/agonistas , Animales , Proliferación Celular/efectos de los fármacos , Cromolin Sódico/metabolismo , Dicumarol/metabolismo , Subunidades alfa de la Proteína de Unión al GTP G12-G13/genética , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/genética , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Genes Reporteros , Células HEK293 , Humanos , Ligandos , Concentración Osmolar , Purinonas/metabolismo , Ratas , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Recombinantes de Fusión/agonistas , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Bibliotecas de Moléculas Pequeñas , Especificidad de la Especie , Arrestina beta 2 , beta-ArrestinasRESUMEN
Trypanosomes are purine-auxotrophic parasites that depend upon nucleoside hydrolase (NH) activity to salvage nitrogenous bases necessary for nucleic acid and cofactor synthesis. Nonspecific and purine-specific NHs have been widely studied, yet little is known about the 6-oxopurine-specific isozymes, although they are thought to play a primary role in the catabolism of exogenously derived nucleosides. Here, we report the first functional and structural characterization of the inosine-guanosine-specific NH from Trypanosoma brucei brucei. The enzyme shows near diffusion-limited efficiency coupled with a clear specificity for 6-oxopurine nucleosides achieved through a catalytic selection of these substrates. Pre-steady-state kinetic analysis reveals ordered product release, and a rate-limiting structural rearrangement that is associated with the release of the product, ribose. The crystal structure of this trypanosomal NH determined to 2.5 Å resolution reveals distinctive features compared to those of both purine- and pyrimidine-specific isozymes in the framework of the conserved and versatile NH fold. Nanomolar iminoribitol-based inhibitors identified in this study represent important lead compounds for the development of novel therapeutic strategies against trypanosomal diseases.
Asunto(s)
N-Glicosil Hidrolasas/química , Nucleósidos/química , Proteínas Protozoarias/química , Purinonas/química , Trypanosoma brucei brucei/enzimología , Animales , Cristalografía por Rayos X , Cinética , N-Glicosil Hidrolasas/metabolismo , Nucleósidos/metabolismo , Proteínas Protozoarias/metabolismo , Purinonas/metabolismo , Relación Estructura-ActividadRESUMEN
The ATP-sensitive potassium (K(ATP)) channel couples intracellular metabolic state to membrane excitability. Recently, we demonstrated that neuronal K(ATP) channels are functionally enhanced by activation of a nitric oxide (NO)/cGMP/cGMP-dependent protein kinase (PKG) signaling cascade. In this study, we further investigated the intracellular mechanism underlying PKG stimulation of neuronal K(ATP) channels. By performing single-channel recordings in transfected HEK293 and neuroblastoma SH-SY5Y cells, we found that the increase of Kir6.2/SUR1 (i.e., the neuronal-type K(ATP)) channel currents by PKG activation in cell-attached patches was diminished by 5-hydroxydecanoate (5-HD), an inhibitor of the putative mitochondrial K(ATP) channel; N-(2-mercaptopropionyl)glycine, a reactive oxygen species (ROS) scavenger, and catalase, a hydrogen peroxide (H(2)O(2))-decomposing enzyme. These reagents also ablated NO-induced K(ATP) channel stimulation and prevented the shifts in the single-channel open- and closed-time distributions resulting from PKG activation and NO induction. Bath application of H(2)O(2) reproduced PKG stimulation of Kir6.2/SUR1 but did not activate tetrameric Kir6.2LRKR368/369/370/371AAAA channels. Moreover, neither the PKG activator nor exogenous H(2)O(2) was able to enhance the function of K(ATP) channels in the presence of Ca(2+) chelators and calmodulin antagonists, whereas the stimulatory effect of H(2)O(2) was unaffected by 5-HD. Altogether, in this report we provide novel evidence that activation of PKG stimulates neuronal K(ATP) channels by modulating intrinsic channel gating via a 5-HD-sensitive factor(s)/ROS/Ca(2+)/calmodulin signaling pathway that requires the presence of the SUR1 subunit. This signaling pathway may contribute to neuroprotection against ischemic injury and regulation of neuronal excitability and neurotransmitter release by modulating the function of neuronal K(ATP) channels.
Asunto(s)
Antiarrítmicos/metabolismo , Ácidos Decanoicos/metabolismo , Hidroxiácidos/metabolismo , Canales KATP/metabolismo , Neuronas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/fisiología , Animales , Catalasa/metabolismo , Línea Celular , Cricetinae , Cricetulus , Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , Activación Enzimática , Flufenazina/análogos & derivados , Flufenazina/metabolismo , Glicina/análogos & derivados , Glicina/metabolismo , Humanos , Peróxido de Hidrógeno/metabolismo , Canales KATP/antagonistas & inhibidores , Ratones , Neuronas/citología , Donantes de Óxido Nítrico/metabolismo , Oxidantes/metabolismo , Técnicas de Placa-Clamp , Inhibidores de Fosfodiesterasa/metabolismo , Canales de Potasio de Rectificación Interna/genética , Canales de Potasio de Rectificación Interna/metabolismo , Purinonas/metabolismo , Compuestos de Sulfhidrilo/metabolismoRESUMEN
Leishmania possess distinct xanthine phosphoribosyltransferase and hypoxanthine-guanine phosphoribosyltransferase enzymes that mediate purine salvage, an obligatory nutritional function for these pathogenic parasites. The xanthine phosphoribosyltransferase preferentially uses xanthine as a substrate, while the hypoxanthine-guanine phosphoribosyltransferase phosphoribosylates only hypoxanthine and guanine. These related phosphoribosyltransferases were used as model system to investigate the molecular determinants regulating the 6-oxopurine specificity of these enzymes. Analysis of the purine binding domains showed two conserved acidic amino acids; glutamate residues in the xanthine phosphoribosyltransferase (E198 and E215) and aspartate residues in the hypoxanthine-guanine phosphoribosyltransferase (D168 and D185). Genetic and biochemical analysis established that the single E198D and E215D mutations increased the turnover rates of the xanthine phosphoribosyltransferase without altering purine nucleobase specificity. However, the E215Q and E198,215D mutations converted the Leishmania xanthine phosphoribosyltransferase into a broad-specificity enzyme capable of utilizing guanine, hypoxanthine, and xanthine as substrates. Similarly, the D168,185E double mutation transformed the Leishmania hypoxanthine-guanine phosphoribosyltransferase into a mutant enzyme capable phosphoribosylating only xanthine, albeit with a much lower catalytic efficiency. These studies established that these conserved acidic residues play an important role in governing the nucleobase selectivity of the Leishmania 6-oxopurine phosphoribosyltransferases.
Asunto(s)
Leishmania donovani/enzimología , Pentosiltransferasa/química , Pentosiltransferasa/metabolismo , Purinonas/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Biocatálisis/efectos de los fármacos , Guanosina Monofosfato/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Hipoxantina Fosforribosiltransferasa/química , Hipoxantina Fosforribosiltransferasa/genética , Hipoxantina Fosforribosiltransferasa/metabolismo , Cinética , Magnesio/farmacología , Manganeso/farmacología , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Pentosiltransferasa/genética , Conformación Proteica , Alineación de Secuencia , Especificidad por SustratoRESUMEN
The purine salvage enzyme hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRT) is essential for purine nucleotide and hence nucleic acid synthesis in the malaria parasite, Plasmodium falciparum. Acyclic nucleoside phosphonates (ANPs) are analogues of the nucleotide product of the reaction, comprising a purine base joined by a linker to a phosphonate moiety. K(i) values for 19 ANPs were determined for Pf HGXPRT and the corresponding human enzyme, HGPRT. Values for Pf HGXPRT were as low as 100 nM, with selectivity for the parasite enzyme of up to 58. Structures of human HGPRT in complex with three ANPs are reported. On binding, a large mobile loop in the free enzyme moves to partly cover the active site. For three ANPs, the IC(50) values for Pf grown in cell culture were 1, 14, and 46 microM, while the cytotoxic concentration for the first compound was 489 microM. These results provide a basis for the design of potent and selective ANP inhibitors of Pf HGXPRT as antimalarial drug leads.
Asunto(s)
Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Hipoxantina Fosforribosiltransferasa/antagonistas & inhibidores , Nucleósidos/química , Organofosfonatos/química , Organofosfonatos/farmacología , Animales , Antimaláricos/síntesis química , Antimaláricos/química , Antimaláricos/farmacología , Antimaláricos/toxicidad , Dominio Catalítico , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Cristalografía por Rayos X , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/toxicidad , Eritrocitos/efectos de los fármacos , Eritrocitos/parasitología , Humanos , Hipoxantina Fosforribosiltransferasa/química , Concentración 50 Inhibidora , Modelos Moleculares , Organofosfonatos/síntesis química , Organofosfonatos/toxicidad , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/enzimología , Plasmodium falciparum/crecimiento & desarrollo , Purinonas/metabolismo , Especificidad por SustratoRESUMEN
Using a mechanically grinded pyrolytic graphite electrode in edge orientation, a sensitive electrochemical method was developed for simultaneous determination of uric acid (UA), xanthine (XAN), hypoxanthine (HYP) (products of purine catabolism in human), allopurinol (ALO), and oxypurinol (OXY) (a drug used in treatment of purine catabolism disorders and its metabolite, respectively). It is demonstrated that differential pulse voltammetry in connection with this electrode can serve as a simple and efficient tool for monitoring transformation of purine catabolites (HYP --> XAN --> UA) catalyzed by xanthine oxidase (XO) as well as inhibition of this pathway by ALO being enzymatically converted to OXY. Our protocol is based on direct electrochemical measurement of oxidation peaks for each of the substances during in vitro reactions in a single detection step by the same electrode system. In addition, we show that the proposed electrochemical technique can be applied to parallel detection of metabolites involved in the XO pathway excreted in urine without any pretreatment of the clinical samples.
Asunto(s)
Alopurinol/análisis , Técnicas Electroquímicas/métodos , Oxipurinol/análisis , Purinonas/análisis , Purinonas/metabolismo , Xantina Oxidasa/metabolismo , Técnicas Biosensibles/economía , Técnicas Biosensibles/métodos , Carbono/química , Técnicas Electroquímicas/economía , Electrodos , Inhibidores Enzimáticos/análisis , Humanos , Hipoxantina/análisis , Hipoxantina/metabolismo , Hipoxantina/orina , Purinonas/orina , Sensibilidad y Especificidad , Ácido Úrico/análisis , Ácido Úrico/metabolismo , Ácido Úrico/orina , Xantina/análisis , Xantina/metabolismo , Xantina/orina , Xantina Oxidasa/antagonistas & inhibidoresRESUMEN
A series of trisubstituted purinones was synthesized and evaluated as A(2A) receptor antagonists. The A(2A) structure-activity relationships at the three substituted positions were studied and selectivity against the A(1) receptor was investigated. One antagonist 12o exhibits a K(i) of 9nM in an A(2A) binding assay, a K(b) of 18nM in an A(2A) cAMP functional assay, and is 220-fold selective over the A(1) receptor.
Asunto(s)
Antagonistas del Receptor de Adenosina A2 , Purinonas/síntesis química , Animales , Humanos , Unión Proteica/efectos de los fármacos , Unión Proteica/fisiología , Purinonas/metabolismo , Purinonas/farmacología , Ratas , Receptor de Adenosina A2A/metabolismo , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/metabolismo , Relación Estructura-ActividadRESUMEN
Nitric oxide (NO) is an inhibitory signalling molecule in the gastrointestinal (GI) tract that is released from neurons and from leucocytes during inflammation. NO stimulates soluble guanylate cyclase (sGC), elevates cyclic guanosine 3',5'-monophospate (cGMP), and subsequently activates cGMP-dependent protein kinase (PKG). Targets for NO in the guinea pig caecum were investigated by characterizing the cellular distribution of sGC, cGMP and PKG. Immunoreactivity for both isoforms of sGC, sGCalpha1 and sGCbeta1, was observed in the interstitial cells of Cajal (ICC) and enteric neurons in the tunica muscularis. Double labelling with anti-Kit and anti-sGC antibodies showed sGCalpha1 and sGCbeta1-like immunoreactivity (LI) in almost all intramuscular (IM) and myenteric ICC. Neuronal processes with neuronal NO synthase were closely apposed to ICC expressing sGC-LI. Cells with sGC-LI possessed ultrastructural features of ICC-IM: caveolae, close association with nerve bundles and contacts with smooth muscle cells (SMC). Sodium nitroprusside, added with the phosphodiesterase inhibitors (3-isobutyl-1-methylxanthine and zaprinast), enhanced cGMP-LI in almost all ICC and in some enteric neurons. Nerve stimulation also increased cGMP-LI in ICC and enteric neurons. In contrast, no resolvable increase in cGMP-LI was observed in any cells when the sGC inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one was present. ICC and SMC also expressed PKG type I-LI. These data show that ICC express the downstream signalling molecules necessary to transduce nitrergic signals and activate inhibitory pathways and thus are primary targets for NO released from neurons and other cells in the GI tract.
Asunto(s)
Ciego , Óxido Nítrico/metabolismo , Transducción de Señal/fisiología , Animales , Ciego/fisiología , Ciego/ultraestructura , GMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , Activación Enzimática , Femenino , Guanilato Ciclasa/metabolismo , Cobayas , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Ratones , Ratones Endogámicos BALB C , Óxido Nítrico Sintasa de Tipo I/metabolismo , Nitroprusiato/metabolismo , Inhibidores de Fosfodiesterasa/metabolismo , Purinonas/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Guanilil Ciclasa Soluble , Factor de Células Madre/metabolismoRESUMEN
Adenosine triphosphate (ATP)-sensitive potassium (K(ATP)) channels couple cellular metabolic status to membrane electrical activity. In this study, we performed patch-clamp recordings to investigate how cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG) regulates the function of K(ATP) channels, using both transfected human SH-SY5Y neuroblastoma cells and embryonic kidney (HEK) 293 cells. In intact SH-SY5Y cells, the single-channel currents of Kir6.2/sulfonylurea receptor (SUR) 1 channels, a neuronal-type K(ATP) isoform, were enhanced by zaprinast, a cGMP-specific phosphodiesterase inhibitor; this enhancement was abolished by inhibition of PKG, suggesting a stimulatory role of cGMP/PKG signaling in regulating the function of neuronal K(ATP) channels. Similar effects of cGMP accumulation were confirmed in intact HEK293 cells expressing Kir6.2/SUR1 channels. In contrast, direct application of purified PKG suppressed rather than activated Kir6.2/SUR1 channels in excised, inside-out patches, while tetrameric Kir6.2LRKR368/369/370/371AAAA channels expressed without the SUR subunit were not modulated by zaprinast or purified PKG. Lastly, reconstitution of the soluble guanylyl cyclase/cGMP/PKG signaling pathway by generation of nitric oxide led to Kir6.2/SUR1 channel activation in both cell types. Taken together, here, we report novel findings that PKG exerts dual functional regulation of neuronal K(ATP) channels in a SUR subunit-dependent manner, which may provide new means of therapeutic intervention for manipulating neuronal excitability and/or survival.
Asunto(s)
Adenosina Trifosfato/metabolismo , Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , Canales KATP/metabolismo , Animales , Carbazoles/metabolismo , Línea Celular , Inhibidores Enzimáticos/metabolismo , Humanos , Neuronas/citología , Neuronas/metabolismo , Óxido Nítrico/metabolismo , Donantes de Óxido Nítrico/metabolismo , Compuestos Nitrosos/metabolismo , Técnicas de Placa-Clamp , Canales de Potasio de Rectificación Interna/metabolismo , Purinonas/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal/fisiologíaRESUMEN
Zaprinast is a phosphodiesterase inhibitor that is active in various models of pain when administered locally. In addition, the antinociception of zaprinast is involved in the nitric oxide (NO)-cGMP pathway. However, the effect of zaprinast administered spinally has not been examined. Therefore, this study examined the effect of zaprinast on the formalin-induced nociception at the spinal level. Next, the role of the NO-cGMP-potassium channel pathway on the effect of zaprinast was further clarified. Catheters were inserted into the intrathecal space of male Sprague-Dawley (SD) rats. Pain was induced by applying 50 microl of a 5% formalin solution to the hindpaw. The change in the zaprinast-induced effect was examined after an intrathecal pretreatment with a NO synthase inhibitor (l-NMMA), a guanylyl cyclase inhibitor (ODQ) or a potassium channel blocker (glibenclamide). Zaprinast produced an antinociceptive effect during phase 1 and phase 2 in the formalin test. Intrathecal l-NMMA, ODQ and glibenclamide did not reverse the antinociception of zaprinast in either phase of the formalin test. These results suggest that zaprinast is effective against both acute pain and the facilitated pain state at the spinal level. However, the NO-sensitive cGMP-potassium channel pathway is not contributable to the antinociceptive mechanism of zaprinast in the spinal cord.
Asunto(s)
Analgésicos/farmacología , GMP Cíclico/metabolismo , Óxido Nítrico/metabolismo , Dimensión del Dolor/efectos de los fármacos , Canales de Potasio/metabolismo , Purinonas/farmacología , Transducción de Señal/fisiología , 3',5'-GMP Cíclico Fosfodiesterasas/antagonistas & inhibidores , Analgésicos/administración & dosificación , Analgésicos/metabolismo , Animales , Formaldehído/efectos adversos , Gliburida/metabolismo , Inyecciones Espinales , Masculino , Oxadiazoles/metabolismo , Dolor/inducido químicamente , Inhibidores de Fosfodiesterasa/administración & dosificación , Inhibidores de Fosfodiesterasa/metabolismo , Inhibidores de Fosfodiesterasa/farmacología , Purinonas/administración & dosificación , Purinonas/metabolismo , Quinoxalinas/metabolismo , Ratas , Ratas Sprague-Dawley , omega-N-Metilarginina/metabolismoRESUMEN
Derivatives of the herpes simplex thymidine kinase inhibitor HBPG [2-phenylamino-9-(4-hydroxybutyl)-6-oxopurine] have been synthesized and tested for inhibitory activity against recombinant enzymes (TK) from herpes simplex types 1 and 2 (HSV-1, HSV-2). The compounds inhibited phosphorylation of [3H]thymidine by both enzymes, but potencies differed quantitatively from those of HBPG and were generally greater for HSV-2 than HSV-1 TKs. Changes in inhibitory potency were generally consistent with the inhibitor/substrate binding site structure based on published X-ray structures of HSV-1 TK. In particular, several 9-(4-aminobutyl) analogues with bulky tertiary amino substituents were among the most potent inhibitors. Variable substrate assays showed that the most potent compound, 2-phenylamino-9-[4-(1-decahydroquinolyl)butyl]-6-oxopurine, was a competitive inhibitor, with Ki values of 0.03 and 0.005 microM against HSV-1 and HSV-2 TKs, respectively. The parent compound HBPG was uniquely active in viral infection models in mice, both against ocular HSV-2 reactivation and against HSV-1 and HSV-2 encephalitis. In assays lacking [3H]thymidine, HBPG was found to be an efficient substrate for the enzymes. The ability of the TKs to phosphorylate HBPG may relate to its antiherpetic activity in vivo.
Asunto(s)
Antivirales/síntesis química , Guanina/análogos & derivados , Guanina/síntesis química , Herpesvirus Humano 1/enzimología , Herpesvirus Humano 2/enzimología , Purinonas/síntesis química , Timidina Quinasa/antagonistas & inhibidores , Animales , Antivirales/metabolismo , Antivirales/farmacología , Clonación Molecular , Encefalitis por Herpes Simple/tratamiento farmacológico , Encefalitis por Herpes Simple/virología , Infecciones Virales del Ojo/tratamiento farmacológico , Infecciones Virales del Ojo/virología , Guanina/química , Guanina/farmacología , Herpesvirus Humano 1/efectos de los fármacos , Herpesvirus Humano 2/efectos de los fármacos , Ratones , Fosforilación , Purinonas/metabolismo , Purinonas/farmacología , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Relación Estructura-Actividad , Timidina Quinasa/biosíntesis , Timidina Quinasa/aislamiento & purificación , Activación Viral/efectos de los fármacosRESUMEN
In the starfish, Asterias amurensis, the cooperation of three components of the egg jelly, i.e. ARIS (acrosome reaction-inducing substance), Co-ARIS and asterosap, is responsible for inducing the acrosome reaction. Experimentally, ARIS and asterosap are sufficient for the induction. However, when sperm are treated only with asterosap, they become unresponsive to the egg jelly to undergo the reaction. In this study, we analysed the mechanism of the acrosome reaction, using sperm inactivation by asterosap as a clue. Asterosap causes a rapid and transient increase in intracellular cGMP through the activation of the asterosap receptor, a guanylyl cyclase, and causes an increase in intracellular Ca(2+). When sperm were pretreated with asterosap, the guanylyl cyclase seemed to be inactivated irreversibly by dephosphorylation. They were still responsive to ARIS but no longer to asterosap. However, in the presence of IBMX or zaprinast, inhibitors against phosphodiesterases (PDEs), they retained their capacity to undergo the acrosome reaction in response to the egg jelly or ARIS alone. IBMX and zaprinast suppressed the intracellular catabolism of cGMP, but not of cAMP. These results suggest that guanylyl cyclase and cGMP-specific, IBMX- and zaprinast-susceptible PDEs are involved in the regulation of the acrosome reaction.