Identification of nonabsorbable inhibitors of the scavenger receptor-BI (SR-BI) for tissue-specific administration.

The identification of a low-permeability scavenger receptor BI (SR-BI) inhibitor starting from the ITX-5061 template is described. Structure-activity and structure-permeability relationships were assessed for analogs leading to the identification of compound 8 as a potent and nonabsorbable SR-BI inhibitor.

Inhibition of colony-stimulating-factor-1 signaling in vivo with the orally bioavailable cFMS kinase inhibitor GW2580.

Colony-stimulating-factor-1 (CSF-1) signaling through cFMS receptor kinase is increased in several diseases. To help investigate the role of cFMS kinase in disease, we identified GW2580, an orally bioavailable inhibitor of cFMS kinase. GW2580 completely inhibited human cFMS kinase in vitro at 0.06 microM and was inactive against 26 other kinases. GW2580 at 1 microM completely inhibited CSF-1-induced growth of mouse M-NFS-60 myeloid cells and human monocytes and completely inhibited bone degradation in cultures of human osteoclasts, rat calvaria, and rat fetal long bone. In contrast, GW2580 did not affect the growth of mouse NS0 lymphoblastoid cells, human endothelial cells, human fibroblasts, or five human tumor cell lines. GW2580 also did not affect lipopolysaccharide (LPS)-induced TNF, IL-6, and prostaglandin E2 production in freshly isolated human monocytes and mouse macrophages. After oral administration, GW2580 blocked the ability of exogenous CSF-1 to increase LPS-induced IL-6 production in mice, inhibited the growth of CSF-1-dependent M-NFS-60 tumor cells in the peritoneal cavity, and diminished the accumulation of macrophages in the peritoneal cavity after thioglycolate injection. Unexpectedly, GW2580 inhibited LPS-induced TNF production in mice, in contrast to effects on monocytes and macrophages in vitro. In conclusion, GW2580's selective inhibition of monocyte growth and bone degradation is consistent with cFMS kinase inhibition. The ability of GW2580 to chronically inhibit CSF-1 signaling through cFMS kinase in normal and tumor cells in vivo makes GW2580 a useful tool in assessing the role of cFMS kinase in normal and disease processes.

The application of BacMam technology in nuclear receptor drug discovery.

The nuclear receptor (NR) superfamily represents a major class of drug targets for the pharmaceutical industry. Strategies for the development of novel, more selective and safer compounds aimed at these receptors are now emerging. Reporter assays have been used routinely for the identification and characterisation of NR ligands. As the NR drug development process evolves, the increase in screening demand in terms of both capacity and complexity has necessitated the development of novel assay formats with increased throughput and flexibility. BacMam technology, a modified baculovirus system for over-expressing genes of interest in mammalian cells has helped answer this requirement. BacMam has many advantages over traditional gene delivery systems including high transduction efficiencies, broad cell host range, speed, cost and ease of generation and use. As outlined in this review, the technology has shown itself to be robust and efficient in various NR assay formats including transactivation (ER alpha/beta, MR, PR and PXR) and transrepression (GR-NFkappaB). In addition, the flexibility of this system will allow greater multiplexing of receptor, reporter, and cell host combinations as NR assays become more complex in order to relate better to relevant cellular and biological systems.

Evaluation of cell-based assays for steroid nuclear receptors delivered by recombinant baculoviruses.

The authors describe the use of modified baculoviruses containing mammalian expression cassettes (BacMam technology) in steroid nuclear receptor reporter assays designed for screening and profiling agonist and antagonist compounds. Baculo-viruses were constructed that express full-length human genes for mineralocorticoid receptor (MR), glucocorticoid receptor (GR), progesterone receptor A (PR-A), and progesterone receptor B (PR-B) from the cytomegalovirus immediate early promoter. A virus carrying the mouse mammary tumor virus-firefly luciferase (MMTV-Luc) cassette was generated to provide a suitable reporter construct. Feasibility studies with BacMam-MR in single-dose tests of 1000 compounds showed high correlation to the standard transfection-based assay results. Likewise, in dose-response experiments, BacMam-based assays for GR and PR-B produced potency and efficacy values similar to transfection assay results. At various receptor/reporter ratios, the BacMam assays showed good flexibility, demonstrating consistent signal-to-background (S/B) ratios and compound potencies. Increasing transduction time from 24 to 48 h provided no benefit, actually reducing overall assay performance as measured by S/B and Z' values. The BacMam technology was applied in studies of isoforms PR-A and PR-B, which showed similar responses to a series of agonists. Taken together, the results demonstrate the utility of steroid nuclear receptor BacMam constructs for compound screening procedures with high reproducibility, reduced turnaround time, and lower cost.

A cell-based time-resolved fluorescence assay for selection of antibody reagents for G protein-coupled receptor immunohistochemistry.

A cell-based time-resolved fluorescence (celTRF) immunoassay is described for pre-screening antibodies to G protein-coupled receptor (GPCR) peptides that predicts suitability for immunohistochemistry (IHC). Rat GPCRs were expressed in Saos-2 human osteosarcoma cells via recombinant baculoviruses designed for mammalian cell expression, i.e., the transduced cells were used as a "screening lawn". The lawn was fixed and permeabilized similarly to IHC tissue. The celTRF, a dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA), employed Eu-labelled goat anti-rabbit IgG. It exhibited a broad dynamic range upon which enzyme-linked immunosorbant assay (ELISA)-positive affinity-purified anti-peptide antibody reagents were examined for specificity and potency. Over 150 anti-peptide reagents to 27 GPCRs were characterized. All celTRF-positive antibodies were found to be suitable for IHC, whereas ELISA alone did not predict IHC utility. Examples are illustrated with five rabbit anti-neuropeptide FF receptor 1 (NPFF1) antibodies, where a strong correlation between celTRF potency and IHC utility was observed in both applications. In contrast, two high anti-peptide ELISA titer but celTRF-negative antibodies failed to recognize the NPFF1 receptor in IHC. The celTRF assay was performed manually and in an automated fashion, in our case, using a Biomek FX station and Sami scheduling software. The celTRF is the first in vitro automated assay that offers confident pre-selection of antibodies for IHC and the versatility to accommodate the rapid screening of large numbers of GPCRs. The celTRF is readily applicable to other protein target classes.

In vivo expression of a GST-fusion protein mediates the rapid generation of affinity matured monoclonal antibodies using DNA-based immunizations.

Previously we demonstrated the rapid generation of affinity matured monoclonal antibody (MAb) producing cell lines following gene gun delivery of DNA using a mammalian expression vector (pAlpha/hFc), which enables the expression of human Fc-chimera proteins in vivo. Here we compare the pAlpha/hFc vector to modified vectors that replace human IgG(1) with either a Glutathione-S-Transferase (GST) fusion protein or a mouse IgG(2c) (mFc) fusion protein. We report that in vivo expression of a GST-chimera results in the rapid generation of affinity matured MAbs, comparable with antibodies raised using the pAlpha/hFc vector, that were reactive with annexin V. The mFc vector failed to induce early antigen-specific B-cell responses suitable for MAb development.