RESUMEN
Human paraoxonase 1 (PON1) is a high-density lipoprotein (HDL)-associated serum enzyme that exhibits a broad substrate specificity. In addition to protecting against exposure to some organophosphorus (OP) pesticides by hydrolyzing their toxic oxon metabolites, PON1 is important in protecting against vascular disease by metabolizing oxidized lipids. Recently, PON1 has also been shown to play a role in inactivating the quorum sensing factor N-(3-oxododecanoyl)-l-homoserine lactone (3OC12-HSL) of Pseudomonas aeruginosa. Native, untagged engineered recombinant human PON1 (rHuPON1) expressed in Escherichia coli and purified by conventional column chromatographic purification is stable, active, and capable of protecting PON1 knockout mice (PON1(-/-)) from exposure to high levels of the OP compound diazoxon. The bacterially derived rHuPON1 can be produced in large quantities and lacks the glycosylation of eukaryotic systems that can produce immunogenic complications when inappropriately glycosylated recombinant proteins are used as therapeutics. Previous studies have shown that the determination of PON1 status, which reveals both PON1(192) functional genotype and serum enzyme activity level, is required for a meaningful evaluation of PON1's role in risk of disease or exposure. We have developed a new two-substrate assay/analysis protocol that provides PON1 status without use of toxic OP substrates, allowing for use of this protocol in non-specialized laboratories. Factors were also determined for inter-converting rates of hydrolysis of different substrates. PON1 status also plays an important role in revealing changes in HDL-associated PON1 activities in male patients with Parkinson disease (PD). Immunolocalization studies of PONs 1, 2 and 3 in nearly all mouse tissues suggest that the functions of PONs 1 and 3 extend beyond the plasma and the HDL particle.
Asunto(s)
Arildialquilfosfatasa/metabolismo , Enfermedad , Exposición a Riesgos Ambientales/efectos adversos , Intoxicación por Organofosfatos , Animales , Arildialquilfosfatasa/genética , Arildialquilfosfatasa/uso terapéutico , Biomarcadores/metabolismo , Humanos , RiesgoRESUMEN
The crystal structure of Sr(OH)2*8H2O has been determined at 20, 100 and 200 K from neutron diffraction data. The structure consists of double layers of H2O and OH- ions separated by Sr2+ along the c axis. The Sr2+ ions are eight-coordinated by water O atoms in a square antiprism configuration. Each H2O molecule is engaged in three hydrogen bonds. The OH- ions form chains of acceptor and donor bonds along the fourfold axis with O atoms engaged in four bonds with H2O molecules, such that both non-equivalent O atoms have square-pyramidal environments of five H atoms and the overall bonding configurations of distorted octahedra.
RESUMEN
A worldwide initiative in structural genomics aims to capitalize on the recent successes of the genome projects. Substantial new investments in structural genomics in the past 2 years indicate the high level of support for these international efforts. Already, enormous progress has been made on high-throughput methodologies and technologies that will speed up macromolecular structure determinations. Recent international meetings have resulted in the formation of an International Structural Genomics Organization to formulate policy and foster cooperation between the public and private efforts.
Asunto(s)
Biología Computacional , Genómica , Conformación Proteica , Proteínas/química , Proteoma , Animales , Congresos como Asunto , Costos y Análisis de Costo , Cristalografía por Rayos X , Bases de Datos Factuales , Guías como Asunto , Humanos , Gestión de la Información , Servicios de Información , Cooperación Internacional , Internet , Resonancia Magnética Nuclear Biomolecular , Patentes como Asunto , Sector Privado , Pliegue de Proteína , Sector Público , Edición , Transferencia de TecnologíaRESUMEN
In an effort to gain greater insight into the evolution of the redox active, catalytic antibody 28B4, the germline genes used by the mouse to generate this antibody were cloned and expressed, and the X-ray crystal structures of the unliganded and hapten-bound germline Fab of antibody 28B4 were determined. Comparison with the previously determined structures of the unliganded and hapten-bound affinity-matured Fab [Hsieh-Wilson, L. C., Schultz, P. G., and Stevens, R. C. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 5363] shows that the germline antibody binds the p-nitrophenyl ring of hapten 3 in an orientation significantly different from that seen in the affinity-matured antibody, whereas the phosphonate moiety is bound in a similar mode by both antibodies. The affinity-matured antibody 28B4 has more electrostatic and hydrophobic interactions with hapten 3 than the germline antibody and binds the hapten in a lock-and-key fashion. In contrast, significant conformational changes occur in the loops of CDR H3 and CDR L1 upon hapten binding to the germline antibody, consistent with the notion of structural plasticity in the germline antibody-combining site [Wedemayer, G. J., Patten, P. A., Wang, L. H., Schultz, P. G., and Stevens, R. C. (1997) Science 276, 1665]. The structural differences are reflected in the differential binding affinities of the germline Fab (K(d) = 25 microM) and 28B4 Fab (K(d) = 37 nM) to hapten 3. Nine replacement mutations were found to accumulate in the affinity-matured antibody 28B4 compared to its germline precursor. The effects of each mutation on the binding affinity of the antibody to hapten 3 were characterized in detail in the contexts of both the germline and the affinity-matured antibodies. One of the mutations, Asp95(H)Trp, leads to a change in the orientation of the bound hapten, and its presence is a prerequisite for other somatic mutations to enhance the binding affinity of the germline antibody for hapten 3. Thus, the germline antibody of 28B4 acquired functionally important mutations in a stepwise manner, which fits into a multicycle mutation, affinity selection, and clonal expansion model for germline antibody evolution. Two other antibodies, 20-1 and NZA6, with very different antigen specificities were found to be highly homologous to the germline antibody of 28B4, consistent with the notion that certain germline variable-region gene combinations can give rise to polyspecific hapten binding sites [Romesberg, F. E., Spiller, B., Schultz, P. G., and Stevens, R. C. (1998) Science 279, 1929]. The ultimate specificity of the polyspecific germline antibody appears to be defined by CDR H3 variability and subsequent somatic mutation. Insights into the evolution of antibody-combining sites provided by this and other structural studies are discussed.
Asunto(s)
Anticuerpos Catalíticos/química , Fragmentos Fab de Inmunoglobulinas/química , Región Variable de Inmunoglobulina/química , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Afinidad de Anticuerpos , Clonación Molecular , Cristalografía por Rayos X , Evolución Molecular , Haptenos , Cadenas Pesadas de Inmunoglobulina/química , Cadenas Ligeras de Inmunoglobulina/química , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Estructura Secundaria de Proteína , Complejo Receptor-CD3 del Antígeno de Linfocito T/inmunología , Proteínas Recombinantes/químicaAsunto(s)
Biotecnología , Cristalografía por Rayos X , Conformación Proteica , Proteínas/química , Automatización , Computadores , Costos y Análisis de Costo , Cristalización , Miniaturización , Resonancia Magnética Nuclear Biomolecular , Proteínas/genética , Proteínas/aislamiento & purificación , RobóticaRESUMEN
Design and characterization of helical ribbon assemblies of a bolaamphiphilic conjugated polymer and their color-coded transformation into nanofibers are described. An L-glutamic acid modified bolaamphiphilic diacetylene lipid was synthesized and self-assembled into right-handed helical ribbons with micron scale length and nano scale thickness under mild conditions. The ribbon structures were further stabilized by polymerizing well-aligned diacetylene units to form bisfunctional polydiacetylenes (PDAs). Transitions from flat sheets to helical ribbons and tubes were observed by transmission electron microscopy. The helical ribbons appear to originate from the rupture of flat sheets along domain edges and the peeling off between stacked lipid layers. These results point to the applicability of chiral packing theory in bolaamphiphilic supramolecular assemblies. Contact mode atomic force microscopy observations revealed that high order existed in the surface packing arrangement. Hexagonal and pseudorectangular packings were observed in flat and twisted regions of the ribbons, respectively, suggesting a correlation between microscopic morphologies and nanoscopic packing arrangements. The tricarboxylate functionalities of the bolaamphiphilic lipid provide a handle for the manipulation of the bisfunctional PDAs' morphology. Increasing solution pH caused the fraying of helical ribbons into nanofibers accompanied by a sharp blue-to-red chromatic transition. A dramatic change in circular dichroism spectra was observed during this process, suggesting the loss of chirality in packing. A model is proposed to account for the pH-induced morphological change and chromatic transition. The color-coded transition between two distinct microstructures would be useful in the design of sensors and other "smart" nanomaterials requiring defined molecular templates.
Asunto(s)
Acetileno/análogos & derivados , Acetileno/química , Ácido Glutámico/análogos & derivados , Lípidos/química , Membranas Artificiales , Polímeros/química , Acetileno/síntesis química , Ácido Glutámico/síntesis química , Ácido Glutámico/química , Concentración de Iones de Hidrógeno , Lípidos/síntesis química , Modelos Químicos , Conformación Molecular , Imitación Molecular , Polímero Poliacetilénico , Polímeros/síntesis química , PoliinosRESUMEN
Deficiencies in the human enzyme phenylalanine hydroxylase (PAH) due to mutations in the PAH gene (PAH) result in the inborn error of metabolism phenylketonuria (PKU). The clinical symptom of this disease is an elevated concentration of L-phenylalanine (L-Phe) in blood serum. To prevent mental retardation due to the buildup of neurotoxic metabolites of L-Phe, patients with severe PKU must be treated with a low-L-Phe diet starting early in their life. Owing to extensive newborn screening programmes and genotyping efforts, more than 400 different mutations have been identified in the PAH gene. Recently, there have been several reports of PKU patients showing a normalization of their L-Phe concentrations upon oral administration of the natural cofactor to PAH, (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4). In an attempt to correlate the clinical responsiveness to BH4 administration with PKU genotype, we propose specific structural consequences for this subset of PAH mutations. Based on the location and proximity of this subset of mutations to the cofactor-binding site in the three-dimensional structure of PAH, a hypothesis for BH4 responsiveness in PKU patients is presented. It is believed that some of these mutations result in expressed mutant enzymes that are Km variants (with a lower binding affinity for BH4) of the standard PAH enzyme phenotype. Oral administration of excess BH4 thus makes it possible for these mutant enzymes to suppress their low binding affinity for BH4, enabling this subset of PAH mutations to perform the L-Phe hydroxylation reaction. Most of the BH4-responsive PAH mutations map to the catalytic domain of PAH in either of two categories. Residues are located in cofactor-binding regions or in regions that interact with the secondary structural elements involved in cofactor binding. Based on the series of known mutations that have been found to be responsive to BH4, we propose that other subsets of PAH mutations will have a high likelihood of being responsive to oral BH4 administration.
Asunto(s)
Errores Innatos del Metabolismo de los Aminoácidos/tratamiento farmacológico , Errores Innatos del Metabolismo de los Aminoácidos/metabolismo , Biopterinas/análogos & derivados , Biopterinas/uso terapéutico , Fenilalanina/metabolismo , Fenilcetonurias/tratamiento farmacológico , Fenilcetonurias/metabolismo , Errores Innatos del Metabolismo de los Aminoácidos/genética , Secuencia de Aminoácidos , Animales , Biopterinas/química , Humanos , Datos de Secuencia Molecular , Fenilalanina Hidroxilasa/química , Fenilcetonurias/genética , Conformación ProteicaRESUMEN
Tetanus neurotoxin (TeNT) blocks neurotransmitter release by cleaving VAMP/synaptobrevin, a membrane associated protein involved in synaptic vesicle fusion. Such activity is exerted by the N-terminal 50kDa domain of TeNT which is a zinc-dependent endopeptidase (TeNT-L-chain). Based on the three-dimensional structure of botulinum neurotoxin serotype A (BoNT/A) and serotype B (BoNT/B), two proteins closely related to TeNT, and on X-ray scattering studies of TeNT, we have designed mutations at two active site residues to probe their involvement in activity. The active site of metalloproteases is composed of a primary sphere of residues co-ordinating the zinc atom, and a secondary sphere of residues that determines proteolytic specificity and activity. Glu-261 and Glu-267 directly co-ordinates the zinc atom in BoNT/A and BoNT/B respectively and the corresponding residue of TeNT was replaced by Asp or by the non conservative residue Ala. Tyr-365 is 4.3A away from zinc in BoNT/A, and the corresponding residue of TeNT was replaced by Phe or by Ala. The purified mutants had CD, fluorescence and UV spectra closely similar to those of the wild-type molecule. The proteolytic activity of TeNT-Asp-271 (E271D) is similar to that of the native molecule, whereas that of TeNT-Phe-375 (Y375F) is lower than the control. Interestingly, the two Ala mutants are completely devoid of enzymatic activity. These results demonstrate that both Glu-271 and Tyr-375 are essential for the proteolytic activity of TeNT.
Asunto(s)
Metaloendopeptidasas/química , Toxina Tetánica/química , Secuencia de Aminoácidos , Toxinas Botulínicas Tipo A/química , Proteínas de la Membrana/metabolismo , Metaloendopeptidasas/metabolismo , Datos de Secuencia Molecular , Mutagénesis , Proteínas R-SNARE , Relación Estructura-Actividad , Toxina Tetánica/metabolismo , Zinc/metabolismoRESUMEN
The synthesis of the first examples of stilbene-tethered hydrophobic C-nucleosides is described. Compounds of this type are targeted for use with our recently reported "blue-fluorescent antibodies" with the aim of probing native and nonnatural DNA. The nucleophilic addition of aryl Grignard reagents to either a protected 2'-deoxy-1'-chloro-ribofuranose or a protected 2'-deoxy-ribonolactone was the key synthetic step and afforded C-nucleosides in good yields. Both routes resulted in a final product that was >/=90% of the beta-anomer. Amide- and ether-based linkers for attachment of trans-stilbene to the nucleobase were assessed for utility during synthesis and in binding of the ligands to a blue-fluorescent monoclonal antibody. X-ray structures of each complex were obtained and serve as a guideline for second-generation stilbene-tethered C-nucleosides. The development of these hydrophobic nucleosides will be useful in current native and nonnatural DNA studies and invaluable for investigations regarding novel, nonnatural genomes in the future.
Asunto(s)
Anticuerpos/química , Nucleósidos/síntesis química , Estilbenos/química , Cristalografía por Rayos X , Fluorescencia , Estructura Molecular , Resonancia Magnética Nuclear BiomolecularRESUMEN
Directed evolution can be a powerful tool to predict antibiotic resistance. Resistance involves the accumulation of mutations beneficial to the pathogen while maintaining residue interactions and core packing that are critical for preserving function. The constraint of maintaining stability, while increasing activity, drastically reduces the number of possible mutational combination pathways. To test this theory, TEM-1 beta-lactamase was evolved using a hypermutator E. coli-based directed evolution technique with cefotaxime selection. The selected mutants were compared to two previous directed evolution studies and a database of clinical isolates. In all cases, evolution resulted in the generation of the E104K/M182T/G238S combination of mutations ( approximately 500-fold increased resistance), which is equivalent to clinical isolate TEM-52. The structure of TEM-52 was determined to 2.4 A. G238S widens access to the active site by 2.8 A whereas E104K stabilizes the reorganized topology. The M182T mutation is located 17 A from the active site and appears to be a global suppressor mutation that acts to stabilize the new enzyme structure. Our results demonstrate that directed evolution coupled with structural analysis can be used to predict future mutations that lead to increased antibiotic resistance.
Asunto(s)
Evolución Molecular Dirigida , Farmacorresistencia Microbiana/genética , beta-Lactamasas/química , beta-Lactamasas/genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos/genética , Sitios de Unión , Cefotaxima/farmacología , Cristalografía por Rayos X , Bases de Datos como Asunto , Estabilidad de Enzimas/genética , Escherichia coli/genética , Evolución Molecular , Enlace de Hidrógeno , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis/genética , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Selección Genética , Alineación de Secuencia , Relación Estructura-Actividad , Supresión Genética/genética , beta-Lactamasas/metabolismoRESUMEN
alpha-Conotoxins are small disulfide-constrained peptide toxins which act as antagonists at specific subtypes of nicotinic acetylcholine receptors (nACh receptors). In this study, we analyzed the structures and activities of three mutants of alpha-conotoxin ImI, a 12 amino acid peptide active at alpha7 nACh receptors, in order to gain insight into the primary and tertiary structural requirements of neuronal alpha-conotoxin specificity. NMR solution structures were determined for mutants R11E, R7L, and D5N, resulting in representative ensembles of 20 conformers with average pairwise RMSD values of 0.46, 0.52, and 0.62 A from their mean structures, respectively, for the backbone atoms N, C(alpha), and C' of residues 2-11. The R11E mutant was found to have activity near that of wild-type ImI, while R7L and D5N demonstrated activities reduced by at least two orders of magnitude. Comparison of the structures reveals a common two-loop architecture, with variations observed in backbone and side-chain dihedral angles as well as surface electrostatic potentials upon mutation. Correlation of these structures and activities with those from previously published studies emphasizes that existing hypotheses regarding the molecular determinants of alpha-conotoxin specificity are not adequate for explaining peptide activity, and suggests that more subtle features, visualized here at the atomic level, are important for receptor binding. These data, in conjunction with reported characterizations of the acetylcholine binding site, support a model of toxin activity in which a single solvent-accessible toxin side-chain anchors the complex, with supporting weak interactions determining both the efficacy and the subtype specificity of the inhibitory activity.
Asunto(s)
Conotoxinas/química , Conotoxinas/farmacología , Antagonistas Nicotínicos/química , Antagonistas Nicotínicos/farmacología , Receptores Nicotínicos/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Conotoxinas/genética , Relación Dosis-Respuesta a Droga , Electrofisiología , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Mutación/genética , Resonancia Magnética Nuclear Biomolecular , Unión Proteica , Estructura Secundaria de Proteína , Subunidades de Proteína , Receptores Nicotínicos/química , Alineación de Secuencia , Electricidad Estática , Relación Estructura-Actividad , Especificidad por Sustrato , Receptor Nicotínico de Acetilcolina alfa 7RESUMEN
Structure-based biological discovery is entering a new era with the development of industrialized macromolecular structure determination pipelines. Intense, highly focused X-rays from integrated synchrotron radiation beam lines combined with significant advances in protein expression, purification, and micro-crystallization automation allow for the full streamlining of the traditionally tedious and time consuming process of determining the three dimensional structures of macromolecules.
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Automatización/métodos , Cristalografía por Rayos X/métodos , Proteínas/química , Cristalización , Conformación Proteica , Proteínas/metabolismo , Manejo de Especímenes/métodos , SincrotronesRESUMEN
An important goal of structural genomics is to complete the structural analysis of all the enzymes in metabolic pathways and to understand the structural similarities and differences. A preliminary glimpse of this type of analysis was achieved before structural genomics efforts with the glycolytic pathway and efforts are underway for many other pathways, including that of catecholamine metabolism. Structural enzymology necessitates a complete structural characterization, even for highly homologous proteins (greater than 80% sequence homology), as every active site has distinct structural features and it is these active site differences that distinguish one enzyme from another. Short cuts with homology modeling cannot be taken with our current knowledge base. Each enzyme structure in a pathway needs to be determined, including structures containing bound substrates, cofactors, products and transition state analogs, in order to obtain a complete structural and functional understanding of pathway-related enzymes.
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Enzimas/metabolismo , Genoma , Sitios de Unión , Catecolaminas/metabolismo , Enzimas/química , Glucólisis , Conformación ProteicaRESUMEN
A novel family of glycopolythiophenes containing sialic acid or mannose ligands were prepared and evaluated for their ability to bind lectins, virus, and bacteria. For the set of glycopolythiophenes studied, the spacer-length between the polymer backbone and the ligand was varied to optimize binding interactions. The glycopolymers were blue-shifted (absorbance of ca. 400 nm) relative to the corresponding homo-polythiophenes (absorbance ca. 440 nm), suggesting a twisted conformation for the glycopolymers. The altered conformation is likely due to electrostatic or H-bonding interactions between the polymer chains, arising from the carbohydrate ligand. Further conformational changes in the polythiophene backbone were detected by the binding of specific receptors; lectins (wheat germ agglutinin, concanavalin A), Influenza virus, and Escherichia coli. The binding interactions result in an unusual red-shift in the visible absorption of the polymer backbone, suggesting a lengthening of the effective conjugated length upon interaction of the ligand with its congnate receptor. These conjugated glycopolymeric systems offer a potentially new platform for the detection of molecular binding interactions.
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Colorimetría/métodos , Escherichia coli/aislamiento & purificación , Orthomyxoviridae/aislamiento & purificación , Polímeros/química , Tiofenos/química , Espectroscopía de Resonancia Magnética , Espectrometría de Masas/métodos , Estructura MolecularRESUMEN
The combinatorial chemistry industry has made major advances in the handling and mixing of small volumes, and in the development of robust liquid-handling systems. In addition, developments have been made in the area of material handling for the high-throughput drug screening and combinatorial chemistry fields. Lastly, improvements in beamline optics at synchrotron sources have enabled the use of flash-frozen micron-sized (10-50 microm) crystals. The combination of these and other recent advances will make high-throughput protein crystallography possible. Further advances in high-throughput methods of protein crystallography will require application of the above developments and the accumulation of success/failure data in a more systematic manner. Major changes in crystallography technology will emerge based on the data collected by first-generation high-throughput systems.
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Proteínas/química , Automatización , CristalizaciónRESUMEN
The forte of catalytic antibodies has resided in the control of the ground-state reaction coordinate. A principle and method are now described in which antibodies can direct the outcome of photophysical and photochemical events that take place on excited-state potential energy surfaces. The key component is a chemically reactive optical sensor that provides a direct report of the dynamic interplay between protein and ligand at the active site. To illustrate the concept, we used a trans-stilbene hapten to elicit a panel of monoclonal antibodies that displayed a range of fluorescent spectral behavior when bound to a trans-stilbene substrate. Several antibodies yielded a blue fluorescence indicative of an excited-state complex or "exciplex" between trans-stilbene and the antibody. The antibodies controlled the isomerization coordinate of trans-stilbene and dynamically coupled this manifold with an active-site residue. A step was taken toward the use of antibody-based photochemical sensors for diagnostic and clinical applications.
Asunto(s)
Anticuerpos Catalíticos/química , Anticuerpos Monoclonales/química , Fluorescencia , Estilbenos/química , Estilbenos/inmunología , Sitios de Unión , Sitios de Unión de Anticuerpos , Fenómenos Químicos , Química Física , Cristalografía por Rayos X , Haptenos , Ligandos , Microscopía Fluorescente , Modelos Químicos , Modelos Moleculares , Fotoquímica , Espectrometría de Fluorescencia , Estereoisomerismo , Temperatura , Rayos UltravioletaRESUMEN
Botulinum neurotoxin serotype B is a zinc protease that disrupts neurotransmitter release by cleaving synaptobrevin-II (Sb2), one of three SNARE proteins involved in neuronal synaptic vesicle fusion. The three-dimensional crystal structure of the apo botulinum neurotoxin serotype B catalytic domain (BoNT/B-LC) has been determined to 2.2 A resolution, and the complex of cleaved Sb2 with the catalytic domain (Sb2-BoNT/B-LC) has been determined to 2.0 A resolution. A comparison of the holotoxin catalytic domain and the isolated BoNT/B-LC structure shows a rearrangement of three active site loops. This rearrangement exposes the BoNT/B active site. The Sb2-BoNT/B-LC structure illustrates two distinct binding regions, which explains the specificity of each botulinum neurotoxin for its synaptic vesicle protein. This observation provides an explanation for the proposed cooperativity between binding of full-length substrate and catalysis and suggest a mechanism of synaptobrevin proteolysis employed by the clostridial neurotoxins.
Asunto(s)
Toxinas Botulínicas/química , Toxinas Botulínicas/metabolismo , Clostridium botulinum/química , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Proteínas de Transporte Vesicular , Secuencia de Aminoácidos , Apoproteínas/química , Apoproteínas/metabolismo , Sitios de Unión , Toxinas Botulínicas Tipo A , Catálisis , Dominio Catalítico , Cristalografía por Rayos X , Modelos Químicos , Modelos Moleculares , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Unión Proteica , Conformación Proteica , Proteínas R-SNARE , Proteínas SNARE , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
The effect of food on didanosine bioavailability and interpatient pharmacokinetic variability was examined in children infected with human immunodeficiency virus type 1 (HIV-1). Didanosine pharmacokinetics were determined during fasting and fed conditions in HIV-infected children enrolled in the Pediatric AIDS Clinical Trials Group Protocol 144 randomized to receive didanosine at 50 mg/m2 or 150 mg/m2 orally every 12 hr. Pharmacokinetic parameters from patients in the low (n = 39) and high (n = 38) dosing groups were not significantly different, but intersubject variability was substantial. The fraction absorbed was higher while fasting than with food (0.27+/-0.13 versus 0.19+/-0.09, p < 0.0001); the zero order absorption rate was faster (0.48+/-0.31 versus 0.76+/-0.72 hr, p = 0.003); and the plasma half-life was shorter (0.93+/-0.43 versus 1.39+/-0.65 hr, p < 0.0001). The lower fraction absorbed with food was offset by the absorption rate becoming rate limiting for elimination, resulting in similar areas under the concentration-time curves (normalized to 100 mg/m2) when fasted (853.9+/-465.8 microg/liter-hr) versus fed (796.3+/-367.5 microg/liter x hr). Oral clearances during fasting (152.5+/-81.7 liters/hr/m2) and fed states (163.6+/-99.3 liters/hr/m2) were similar, but these values in children are substantially higher than previously reported for adults. The systemic exposure (i.e., AUC) of didanosine was highly variable in children but similar in the presence and absence of food. Administration of didanosine with food in children may be permissible if total systemic exposure rather than maximum plasma concentration is sufficient for antiretroviral activity. The higher oral clearance and substantial pharmacokinetic variability suggest the need to reexamine current didanosine dose recommendations for HIV-infected children.