GPCR screening via ERK 1/2: a novel platform for screening G protein-coupled receptors

Discovery of novel agonists and antagonists for G protein-coupled receptors (GPCRs) relies heavily on cell-based assays because determination of functional consequences of receptor engagement is often desirable. Currently, there are several key parameters measured to achieve this, including mobilization of intracellular Ca2+ and formation of cyclic adenosine monophosphate or inositol triphosphate. However, no single assay platform is suitable for all situations, and all of the assays have limitations. The authors have developed a new high-throughput homogeneous assay platform for GPCR discovery as an alternative to current assays, which employs detection of phosphorylation of the key signaling molecule p42/44 MAP kinase (ERK 1/2). The authors show that ERK 1/2 is consistently activated in cells stimulated by Gq-coupled GPCRs and provides a new high-throughput platform for screening GPCR drug candidates. The activation of ERK 1/2 in Gq-coupled GPCR systems generates comparable pharmacological data for receptor agonist and antagonist data obtained by other GPCR activation measurement techniques.     

Total and semisynthesis and in vitro studies of both enantiomers of 20-fluorocamptothecin

Both enantiomers of 20-fluorocamptothecin and the racemate have been prepared by total synthesis. The (R)-enantiomer is essentially inactive in a topoisomerase-I/DNA assay, while the (S)-enantiomer is much less active than (20S)-camptothecin. The lactone ring of 20-fluorocamptothecin hydrolyzes more rapidly than that of camptothecin in PBS. The results provide insight into the role of the 20-hydroxy group in the binding of camptothecin to topoisomerase-I and DNA.     

A proteomic analysis of murine bone marrow and its response to ionizing radiation

To characterize the mouse bone marrow tissue proteome and investigate the response to radiation damage we took bone marrow before and after 4-Gy gamma-irradiation from mouse strains (C57BL/6 and CBA/Ca) that differ in their short-term and long-term radiation responses and analyzed extracellular proteins by high-resolution 2-DE. Twenty proteins were identified from 71 protein spots in both C57BL/6 and CBA/Ca. We detected significant differences between control and irradiated bone marrow and between genotypes and identified many of the changed proteins by MS. In C57BL/6, 27 spots were significantly different between control and irradiated samples. In CBA/Ca, 18 spots showed significant changes following irradiation. Proteins such as serum albumin, apolipoprotein A-I, ferritin, haptoglobin (Hp) and alpha-1-antitrypsin were changed in irradiated bone marrow of both mouse strains, reflecting an ongoing acute-phase reaction. Several other proteins including serotransferrin, neutrophil collagenase, peroxiredoxin 2 and creatine kinase M chain were changed specifically in an individual mouse strain. The proteomic approach makes an important contribution to characterizing bone marrow proteome and investigating the tissue response of bone marrow to radiation, assists in identifying genotype-dependent responses and provides support for the importance of microenvironmental factors contributing to the overall response.     

Evidence for the presence of three distinct binding sites for the thioflavin T class of Alzheimer's disease PET imaging agents on beta-amyloid peptide fibrils

Imaging the progression of Alzheimer's disease would greatly facilitate the discovery of therapeutics, and a wide range of ligands are currently under development for the detection of beta-amyloid peptide (Abeta)-containing plaques by using positron emission tomography. Here we report an in-depth characterization of the binding of seven previously described ligands to in vitro generated Abeta-(1-40) polymers. All of the compounds were derived from the benzothiazole compound thioflavin T and include 2-[4'-(methylamino)phenyl]benzothiazole and 2-(4'-dimethylamino-)phenyl-imidazo[1,2-a]-pyridine derivatives, 2-[4'-(dimethylamino)phenyl]-6-iodobenzothiazole and 2-[4'-(4'-methylpiperazin-1-yl)phenyl]-6-iodobenzothiazole, and a benzofuran compound (5-bromo-2-(4-dimethylaminophenyl)benzofuran). By using a range of fluorescent and radioligand binding assays, we find that these compounds display a more complex binding pattern than described previously and are consistent with three classes of binding sites on the Abeta fibrils. All of the compounds bound with very high affinity (low nm K(d)) to a low capacity site (BS3) (1 ligand-binding site per approximately 300 Abeta-(1-40) monomers) consistent with the previously recognized binding site for these compounds on the fibrils. However, the compounds also bound with high affinity (K(d) approximately 100 nm) to either one of two additional binding sites on the Abeta-(1-40) polymer. The properties of these sites, BS1 and BS2, suggest they are adjacent or partially overlapping and have a higher capacity than BS3, occurring every approximately 35 or every approximately 4 monomers of Abeta-(1-40)-peptide, respectively. Compounds appear to display selectivity for BS2 based on the presence of a halogen substitution (2-[4'-(dimethylamino)phenyl]-6-iodobenzothiazole, 2-[4'-(4'-methylpiperazin-1-yl)phenyl]-6-iodobenzothiazole, and 5-bromo-2-(4-dimethylaminophenyl)benzofuran) on their aromatic ring system. The presence of additional ligand-binding sites presents potential new targets for ligand development and may allow a more complete modeling of the current positron emission tomography data.     

Delineation of positron emission tomography imaging agent binding sites on beta-amyloid peptide fibrils

A range of imaging agents for use in the positron emission tomography of Alzheimer's disease is currently under development. Each of the main compound classes, derived from thioflavin T (PIB), Congo Red (BSB), and aminonaphthalene (FDDNP) are believed to bind to mutually exclusive sites on the beta-amyloid (Abeta) peptide fibrils. We recently reported the presence of three classes of binding sites (BS1, BS2, BS3) on the Abeta fibrils for thioflavin T derivatives and now extend these findings to demonstrate that these sites are also able to accommodate ligands from the other chemotype classes. The results from competition assays using [3H]Me-BTA-1 (BS3 probe) indicated that both PIB and FDDNP were able to displace the radioligand with Ki values of 25 and 42 nM, respectively. BSB was unable to displace the radioligand tracer from the Abeta fibrils. In contrast, each of the compounds examined were able to displace thioflavin T (BS1 probe) from the Abeta fibrils when evaluated in a fluorescence competition assay with Ki values for PIB, FDDNP, and BSB of 1865, 335, and 600 nM, respectively. Finally, the Kd values for FDDNP and BSB binding to Abeta fibrils were directly determined by monitoring the increases in the ligand intrinsic fluorescence, which were 290 and 104 nM, respectively. The results from these assays indicate that (i) the three classes of thioflavin T binding sites are able to accommodate a wide range of chemotype structures, (ii) BSB binds to two sites on the Abeta fibrils, one of which is BS2, and the other is distinct from the thioflavin T derivative binding sites, and (iii) there is no independent binding site on the fibrils for FDDNP, and the ligand binds to both the BS1 and BS3 sites with significantly lower affinities than previously reported.     

The catalytically active serine protease domain of human complement factor I

Factor I (fI) is a major regulator of complement. As a protease it has very restricted specificity, cleaving only C3b or C4b in the presence of a cofactor such as factor H (fH). Cleavage of C3b by fI yields iC3b, a major opsonin. The cleavage occurs through the formation of a ternary complex between the enzyme, the substrate, and the cofactor. The catalytic subunit of fI, the SP domain, accommodates substrate recognition and cleavage. The role of the fI heavy chain within the catalysis complex is unknown. Using partial proteolysis and affinity chromatography an intact form of the SP domain was generated and isolated from fI in high yield. fI and the SP domain were found to have similar amidolytic activities but strikingly different proteolytic activities on C3(NH(3)). fI did not cleave C3(NH(3)) in the absence of fH, while in its presence it cleaved C3(NH(3)) rapidly at two sites. The SP domain, however, slowly cleaved C3(NH(3)) in the absence of fH, at more than two sites. Cleavage by the SP domain was inhibited, not stimulated, by fH. Pefabloc SC and antipain inhibited the proteolytic activity of both fI and the SP domain, but suramin inhibited only fI and not the SP domain. The contrast in the proteolytic activities suggests that the heavy chain domains and the cofactor must have roles in orienting the natural substrates and restricting cleavage to the two sites which yield iC3b through a highly specific catalysis.     

IFN-alpha beta secreted during infection is necessary but not sufficient for negative feedback regulation of IFN-alpha beta signaling by Mycobacterium tuberculosis

IFN-alphabeta functions in the transition from innate to adaptive immunity and may impinge on the interaction of Mycobacterium tuberculosis with its host. Infection by M. tuberculosis causes IFN-alphabeta secretion and down-regulation of IFN-alphabeta signaling in human APC and the human monocytic cell line THP-1, which provides a model for these studies. Neutralization of secreted IFN-alphabeta prevents inhibition of IFN-alpha signaling during infection, but several lines of evidence distinguish inhibition due to infection from a negative feedback response to only IFN-alphabeta. First, greater inhibition of IFN-alpha-stimulated STAT-1 tyrosine phosphorylation occurs 3 days postinfection than 1 or 3 days after IFN-alphabeta pretreatment. Second, LPS also induces IFN-alphabeta secretion and causes IFN-alphabeta-dependent down-regulation of IFN-alpha signaling, yet the inhibition differs from that caused by infection. Third, IFN-alpha signaling is inhibited when cells are grown in conditioned medium collected from infected cells 1 day postinfection, but not if it is collected 3 days postinfection. Because IFN-alphabeta is stable, the results with conditioned medium suggest the involvement of an additional, labile substance during infection. Further characterizing signaling for effects of infection, we found that cell surface IFN-alphabeta receptor is not reduced by infection, but that infection increases association of protein tyrosine phosphatase 1c with the receptor and with tyrosine kinase 2. Concomitantly, IFN-alpha stimulation of tyrosine kinase 2 tyrosine phosphorylation and kinase activity decreases in infected cells. Moreover, infection reduces the abundance of JAK-1 and tyrosine-phosphorylated JAK-1. Thus, the distinctive down-regulation of IFN-alpha signaling by M. tuberculosis occurs together with a previously undescribed combination of inhibitory intracellular events.     

Long-term continuous monitoring of dissolved oxygen in cell culture medium for perfused bioreactors using optical oxygen sensors

For long-term growth of mammalian cells in perfused bioreactors, it is essential to monitor the concentration of dissolved oxygen (DO) present in the culture medium to ascertain the health of the cells. An optical oxygen sensor based on dynamic fluorescent quenching was developed for long-term continuous measurement of DO for NASA-designed rotating perfused bioreactors. Tris(4,7-diphenyl-1,10-phenanthroline) ruthenium(II) chloride is employed as the fluorescent dye indicator. A pulsed, blue LED was chosen as the excitation light source. The sensor can be sterilized using an autoclave. The sensors were tested in a perfused rotating bioreactor supporting a BHK-21 (baby hamster kidney) cell culture over one 28-day, one 43-day, and one 180-day cell runs. The sensors were initially calibrated in sterile phosphate-buffered saline (PBS) against a blood-gas analyzer (BGA), and then used continuously during the entire cell culture without recalibration. In the 180-day cell run, two oxygen sensors were employed; one interfaced at the outlet of the bioreactor and the other at the inlet of the bioreactor. The DO concentrations determined by both sensors were compared with those sampled and measured regularly with the BGA reference. The sensor outputs were found to correlate well with the BGA data throughout the experiment using a single calibration, where the DO of the culture medium varied between 25 and 60 mm Hg at the bioreactor outlet and 80-116 mm Hg at the bioreactor inlet. During all 180 days of culture, the precision and the bias were +/-5.1 mm Hg and -3.8 mm Hg at the bioreactor outlet, and +/- 19 mm Hg and -18 mm Hg at inlet. The sensor dynamic range is between 0 and 200 mm Hg and the response time is less than 1 minute. The resolution of the sensor is 0.1 mm Hg at 50 mm Hg, and 0.25 mm Hg at 130 mm Hg.