ABSTRACT
BACKGROUND: The technetium 99 m (99mTc)-radiolabeled, leukocyte-avid peptide-glycoseaminoglycan complex, [99mTc]P1827DS, has been synthesized as an improved infection/inflammation imaging agent to [99mTc]P483H (LeukoTect, Diatide). In a phase I/II clinical trail, [99mTc]P483H images were equivalent to those obtained with 111In ex vivo labeled leukocytes. However, there was physiologic accumulation of radioactivity in the body that could hamper interpretation of the images. In this study, the potential of [99mTc]P1827DS for infection imaging was assessed in comparison with [99mTc]P483H and the well-described imaging agent [99mTc] hydrazinonicotinamide (HYNIC)-interleukin 8 (IL-8). METHODS: The binding of [99mTc]P1827DS to human blood cell was studied in vitro. A rabbit Escherichia coli infection model was used to perform the biodistribution and imaging studies with [99mTc]P1827DS, [99mTc]P483H and [99mTc]HYNIC-IL-8. RESULTS: [99mTc]P1827DS binds to leukocytes but not to erythrocytes. The leukocyte binding was not saturable up to an investigated concentration of 10 microM. The accumulation of [99mTc]P1827/DS at the infection site strongly depends on the P1827/DS ratio and was optimal at a molar ratio of 10:1. [99mTc]P1827DS shows improved biodistribution over [99mTc]P483H with similar uptake at the infection site. Abscess uptake of [99mTc]HYNIC-IL-8 was approximately three times higher than that of [99mTc]P1827DS. [99mTc]HYNIC-IL-8 showed high accumulation in the kidneys, whereas [99mTc]P1827DS showed high lung uptake and slightly higher accumulation in the liver and spleen. CONCLUSION: [99mTc]P1827DS is a potential new inflammation imaging agent, which clearly visualized the abscess in the rabbit E. coli infection model and showed improved biodistribution compared to [99mTc]P483H. However, the infection uptake and biodistribution of [99mTc]P1827DS is not superior to that of [99mTc]HYNIC-IL-8 in this animal model.
Subject(s)
Escherichia coli Infections/diagnostic imaging , Escherichia coli Infections/metabolism , Glycosaminoglycans/pharmacokinetics , Interleukin-8/pharmacokinetics , Organotechnetium Compounds/pharmacokinetics , Animals , Drug Evaluation, Preclinical , Metabolic Clearance Rate , Organ Specificity , Rabbits , Radionuclide Imaging , Radiopharmaceuticals/pharmacokinetics , Tissue DistributionABSTRACT
The members of the pentameric ligand-gated receptor channel family are involved in information transfer in synapses and the neuromuscular junction. They often contain several copies of the same subunit isoform. Here, we present a method to functionally dissect the role of individual subunits that occur in multiple copies in these receptors. Opening of the inherent chloride channel in the GABA(A) receptor is achieved through the binding of two agonist molecules; however, it has been difficult to obtain information on the contribution of the two individual binding sites. The sites are both located at beta+/alpha- subunit interfaces, suggesting similar properties. One pair of subunits is flanked by gamma and beta (site 1) and the other by alpha and gamma (site 2), the different environment possibly affecting the binding sites. Here, we used concatenated subunits and two point mutations of amino acid residues, each in alpha and beta subunits, both located in the agonist binding pocket, to investigate the properties of these two sites. The sites were individually mutated, and consequences of these mutations on GABA and muscimol-induced channel opening and its competitive inhibition by bicuculline were studied. A model predicts that opening also occurs for receptors occupied with a single agonist molecule but is promoted approximately 60-fold in those occupied by two agonists and that site 2 has an approximately threefold higher affinity for GABA than site 1, whereas muscimol and bicuculline show some preference for site 1.
Subject(s)
GABA Agonists/metabolism , Receptors, GABA-A/genetics , Receptors, GABA-A/metabolism , Animals , Binding Sites/genetics , Binding Sites/physiology , Binding, Competitive/drug effects , Binding, Competitive/genetics , Blotting, Western , Dose-Response Relationship, Drug , GABA Agonists/pharmacology , GABA Antagonists/pharmacology , Mutagenesis, Site-Directed , Oocytes/metabolism , Patch-Clamp Techniques , Point Mutation , Receptors, GABA-A/drug effects , Structure-Activity Relationship , XenopusABSTRACT
In this study, we explored the application of a yeast three-hybrid (Y3H)-based compound/protein display system to scanning the proteome for targets of kinase inhibitors. Various known cyclin-dependent kinase (CDK) inhibitors, including purine and indenopyrazole analogs, were displayed in the form of methotrexate-based hybrid ligands and deployed in cDNA library or yeast cell array-based screening formats. For all inhibitors, known cell cycle CDKs as well as novel candidate CDK-like and/or CDK-unrelated kinase targets could be identified, many of which were independently confirmed using secondary enzyme assays and affinity chromatography. The Y3H system described here may prove generally useful in the discovery of candidate drug targets.
Subject(s)
Adenine/analogs & derivatives , Cyclin-Dependent Kinases/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Two-Hybrid System Techniques , Adenine/chemistry , Adenine/metabolism , Animals , Binding Sites , CDC2 Protein Kinase/antagonists & inhibitors , CDC2 Protein Kinase/chemistry , CDC2 Protein Kinase/metabolism , Cyclin-Dependent Kinases/chemistry , Cyclin-Dependent Kinases/metabolism , Enzyme Inhibitors/chemistry , Ligands , Protein Array Analysis , Proteome/genetics , Saccharomyces/geneticsABSTRACT
The major isoform of the gamma-aminobutyric acid type A (GABA(A)) receptor is thought to be composed of 2alpha(1), 2beta(2), and 1gamma(2) subunit(s), which surround the ion pore. Definite evidence for the subunit arrangement is lacking. We show here that GABA(A) receptor subunits can be concatenated to a trimer that can be functionally expressed upon combination with a dimer. Many combinations did not result in the functional expression. In contrast, four different combinations of triple subunits with dual subunit constructs, all resulting in the identical pentameric receptor gamma(2)beta(2)alpha(1)beta(2)alpha(1), could be successfully expressed in Xenopus oocytes. We characterized the functional properties of these receptors in respect to agonist, competitive antagonist, and diazepam sensitivity. All properties were similar to those of wild type alpha(1)beta(2)gamma(2) GABA(A) receptors. Thus, together with information on the crystal structure of the homologous acetylcholine-binding protein (Brejc, K., van Dijk, W. J., Klaassen, R. V., Schuurmans, M., van Der Oost, J., Smit, A. B., and Sixma, T. K., (2001) Nature 411, 269-276, we provide evidence for an arrangement gamma(2)beta(2)alpha(1)beta(2)alpha(1), counterclockwise when viewed from the synaptic cleft. Forced subunit assembly will also allow receptors containing different subunit isoforms or mutant subunits to be expressed, each in a desired position. The methods established here should be applicable to the entire ion channel family comprising nicotinic acetylcholine, glycine, and 5HT(3) receptors.