ABSTRACT
FKBP12 is best known as the target of the widely used immunosuppressive drug FK506 but may also play a role in neuronal survival. Nonimmunosuppressive ligands of FKBP12 have been shown to have neuroprotective and neuroregenerative activity both in vitro and in vivo, stimulating interest in the development of high-throughput screens to rapidly identify novel ligands. FKBP12 was expressed as a His(6)-fusion in bacteria and purified by metal ion affinity and gel filtration chromatography. A high-throughput fluorescence polarization assay was developed to identify novel ligands of FKBP12. Dissociation constant values of known FKBP12 ligands measured by the new method agreed closely with K(i) values obtained by assaying inhibition of the rotamase activity of the enzyme. The fluorescence polarization assay is rapid, robust, and inexpensive and does not generate radioactive waste. It is very well suited for high-throughput screening efforts.
Subject(s)
Fluorescence Polarization/methods , Ligands , Tacrolimus Binding Protein 1A/metabolism , Drug Evaluation, Preclinical/methods , Histidine/genetics , Humans , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Reproducibility of Results , Tacrolimus Binding Protein 1A/genetics , Tacrolimus Binding Protein 1A/isolation & purificationABSTRACT
Four human 5-HT receptor subtypes (5-HT1A, 5-HT1D alpha, 5-HT1D beta and 5-HT1E) have been expressed in Sf9 insect cells. All four human 5-hydroxytryptamine receptors produced by Sf9 cells had the expected pharmacological properties. Surprisingly, levels of expression of these receptors were relatively low (1-5 pmol/mg protein). High affinity agonist binding to the four 5-hydroxytryptamine receptors was reduced to different extents by guanine nucleotides and/or NaCl. This suggests that the nature of receptor-G protein coupling and/or the predominant conformational state of the receptors in Sf9 cell membranes varies among the different receptors. Activation of all four receptors inhibited forskolin-stimulated cAMP formation in intact Sf9 cells. Expression of 5-hydroxytryptamine receptors in Sf9 cells should be useful for purification of these receptors, for studies of post-translational modification and for pharmaceutical screening.
Subject(s)
Receptors, Serotonin/metabolism , Amino Acid Sequence , Animals , Baculoviridae , Cell Line , Cell Membrane/metabolism , Cloning, Molecular , Colforsin/pharmacology , Cyclic AMP/metabolism , GTP-Binding Proteins/metabolism , Guanine Nucleotides/metabolism , Humans , Molecular Sequence Data , Moths , Radioligand Assay , Receptors, Serotonin/chemistry , Receptors, Serotonin/genetics , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Serotonin Receptor Agonists/metabolism , Sodium Chloride/metabolismABSTRACT
The blood clearance kinetics of five gadolinium complexes, Gd(L), were determined in rats and the results interpreted in terms of an open two-compartment pharmacokinetic model. The complexes were tested in vitro for stability in serum and in aqueous solutions of ions that they might encounter in vivo and that might be expected to react with the Gd(L) complexes to produce uncomplexed gadolinium. Reaction with serum was observed in two instances. Chemical structural differences among the chelating ligands appear to govern the overall reactivity of their Gd(L) complexes. It may be inferred from the results that a preferred structural feature of the ligand is the presence of a 12-membered 1,4,7,10-tetraaza macrocycle.
