ABSTRACT
(1,1-Dioxo-2H-[1,2,4]benzothiadiazin-3-yl) azolo[1,5-a]pyridine and azolo[1,5-a]pyrimidine derivatives have been investigated as potential anti-HCV drugs. Their synthesis, HCV NS5B polymerase inhibition, and replicon activity are discussed.
Subject(s)
Antiviral Agents/chemical synthesis , Azoles/chemical synthesis , Benzothiadiazines/chemical synthesis , Enzyme Inhibitors/chemical synthesis , Hepatitis C/drug therapy , Pyridines/chemical synthesis , Pyrimidines/chemical synthesis , Viral Nonstructural Proteins/antagonists & inhibitors , Animals , Antiviral Agents/pharmacology , Azoles/pharmacology , Benzothiadiazines/pharmacology , Chemistry, Pharmaceutical/methods , Drug Design , Enzyme Inhibitors/pharmacology , Hepacivirus/metabolism , In Vitro Techniques , Inhibitory Concentration 50 , Magnetic Resonance Spectroscopy , Models, Chemical , Molecular Structure , Pyridines/pharmacology , Pyrimidines/pharmacology , Structure-Activity Relationship , Viral Nonstructural Proteins/chemistryABSTRACT
Alpha(1)-acid glycoprotein (AGP) is an important drug-binding protein in human plasma and, as an acute-phase protein, it has a strong influence on pharmacokinetics and pharmacodynamics of many pharmaceuticals. We report the crystal structure of the recombinant unglycosylated human AGP at 1.8 A resolution, which was solved using the new method of UV-radiation-damage-induced phasing (UV RIP). AGP reveals a typical lipocalin fold comprising an eight-stranded beta-barrel. Of the four loops that form the entrance to the ligand-binding site, loop 1, which connects beta-strands A and B, is among the longest observed so far and exhibits two full turns of an alpha-helix. Furthermore, it carries one of the five N-linked glycosylation sites, while a second one occurs underneath the tip of loop 2. The branched, partly hydrophobic, and partly acidic cavity, together with the presumably flexible loop 1 and the two sugar side chains at its entrance, explains the diverse ligand spectrum of AGP, which is known to vary with changes in glycosylation pattern.
Subject(s)
Diazepam/metabolism , Lipocalins/blood , Molecular Biology/methods , Orosomucoid/chemistry , Orosomucoid/metabolism , Ultraviolet Rays , Amino Acid Sequence , Binding Sites , Crystallography, X-Ray , Diazepam/chemistry , Histidine/chemistry , Humans , Ligands , Lipocalins/chemistry , Models, Molecular , Molecular Sequence Data , Progesterone/chemistry , Progesterone/metabolism , Protein Structure, Secondary , Protein Structure, Tertiary , Sequence Alignment , Structural Homology, Protein , Surface PropertiesABSTRACT
At least ten different lipocalins occur in the human body: retinol-binding protein (RBP), alpha1-acid glycoprotein, alpha1-microglobulin, apolipoprotein D, beta-trace protein, complement component 8gamma, glycodelin, neutrophil gelatinase-associated lipocalin, odorant-binding protein, and tear lipocalin. Although many of these lipocalins seem to play an important physiological role, their precise biological function is not always clear. Especially the interpretation of their diverse ligand-binding activities has been hampered by the fact that the natural lipocalins were prepared from different sources and with varying purity. Here we present a generic expression and purification strategy for the recombinant lipocalins, which is based on secretion into the periplasm of E. coli, where disulphide bonds are readily formed, followed by affinity purification via the Strep-tag II and gel filtration. The ten human lipocalins were successfully prepared and their ligand-binding activities were compared via fluorescence titration with a set of typical ligands: retinol, retinoic acid (RA), 11-(5-(dimethylamino)-1-naphthalene-sulfonylamino)undecanoic acid (DAUDA), and 8-anilino-1-naphtalene-sulfonic acid (ANS). As result, merely two lipocalins, RBP and beta-trace, revealed high affinities both for retinol and for RA, which probably reflects a specialized physiological function in retinoid complexation. Surprisingly, the strongest retinol affinity was detected for apolipoprotein D, whereas this lipocalin exhibits much weaker binding activity for retinoic acid. Binding studies with the two spectroscopic probes DAUDA and ANS revealed mixed patterns, which demonstrates that the affinity for lipophilic substances varies considerably among human lipocalins. Notably, RBP with its perfectly moulded retinol-binding site did not show any detectable binding activity for both compounds. Hence, our recombinant expression and purification system should be useful for further structural and functional studies of lipocalins from human origin and beyond.
Subject(s)
Carrier Proteins/chemistry , Carrier Proteins/metabolism , Blood Proteins/biosynthesis , Blood Proteins/chemistry , Blood Proteins/metabolism , Carrier Proteins/biosynthesis , Chromatography, Gel , Dansyl Compounds/chemistry , Escherichia coli/metabolism , Fatty Acids/chemistry , Humans , Ligands , Protein Structure, Tertiary , Recombinant Proteins/biosynthesis , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Tretinoin/chemistry , Vitamin A/chemistryABSTRACT
A polarimetric assay has been developed for the identification of alpha-amino acid racemase activity. The setup consists of a microcuvette polarimeter (40 microL volume) connected to a pipetting robot for microtiter plates, a pump, and data processing. It could be demonstrated for a glutamate racemase from Lactobacillus fermentii, expressed in Escherichia coli, serving as model enzyme, that its activity can be determined from the time-dependent change of the optical rotation using l-glutamate as substrate. Thus, the specific activity was determined to 111.4 mdeg/min which corresponds to 45.7 micromol/min per mg purified enzyme. Moreover, a protocol was developed that allows the measurement of racemase activity from 96-well microtiter plates using purified enzymes. Thus, the method described can be used to determine racemase activity in an automatic manner. It should be also applicable for the screening of enzyme libraries created by directed evolution.