A Small, Nonpeptidyl Mimic of Granulocyte-Colony- Stimulating Factor

Abstract

A nonpeptidyl small molecule SB 247464, capable of activating granulocyte-colony-stimulating factor (G-CSF) signal transduction pathways, was identified in a high-throughput assay in cultured cells. Like G-CSF, SB 247464 induced tyrosine phosphorylation of multiple signaling proteins and stimulated primary murine bone marrow cells to form granulocytic colonies in vitro. It also elevated peripheral blood neutrophil counts in mice. The extracellular domain of the murine G-CSF receptor was required for the activity of SB 247464, suggesting that the compound acts by oligomerizing receptor chains. The results indicate that a small molecule can activate a receptor that normally binds a relatively large protein ligand.

Pharmaceutical intervention in the JAK/STAT signaling pathway

“…Validation experiments run in high throughput format showed that this assay has a stable baseline and can detect compounds with e cacies as low as 10% that of G-CSF. This cell line was used to screen *110 000 individual compounds, and a single compound that triggered an increase in luciferase activity was studied further (Tian et al, 1998). This compound, termed SB 247464, has a molecular weight of 526 and is symmetrical in structure (Figure 4).…”

Selective Binding and Oligomerization of the Murine Granulocyte Colony-stimulating Factor Receptor by a Low Molecular Weight, Nonpeptidyl Ligand

“…Luciferase Assay-Cytokine-independent NFS60 cells containing a stably integrated STAT-responsive luciferase reporter gene that is induced by G-CSF have been described previously (23). The cells were grown in RPMI containing 10% FBS and split to a density of 2 ϫ 10 5 /ml 2 days before the assay.…”

“…Transient Transfection Assays-The human and mouse G-CSF receptor expression constructs have been described previously (23). The chimeric receptors encode the following amino acids: M/H1, mouse amino acids 1-612, human amino acids 612-707; M/H2, mouse amino acids 1-601, human amino acids 601-707; M/H3, mouse amino acids 1-594, human amino acids 592-707.…”

“…HepG2 cells were maintained in Dulbecco's modified Eagle's medium containing 10% FBS. Cells were co-transfected with the indicated G-CSF receptor expression construct and a luciferase reporter under the control of a multimerized STATbinding element, 4ϫIRFtkluc (23). Cells were transfected in triplicate in 24-well plates using Superfect (Qiagen Inc., Chatsworth, CA), as instructed by the manufacturer.…”

“…We have previously described the discovery and initial characterization of a nonpeptidyl small molecule, SB-247464, that acts as a mimic of G-CSF (23). This compound was discovered as part of a high throughput screen designed to detect compounds that activate G-CSF signal transduction pathways in a murine cell line.…”