Selective binding and oligomerization of the murine granulocyte colony-stimulating factor receptor by a low molecular weight, nonpeptidyl ligand.
Granulocyte colony-stimulating factor regulates neutrophil production by binding to a specific receptor, the granulocyte colony-stimulating factor receptor, expressed on cells of the granulocytic lineage. Recombinant forms of granulocyte colony-stimulating factor are used clinically to treat neutropenias. As part of an effort to develop granulocyte colony-stimulating factor mimics with the potential for oral bioavailability, we previously identified a nonpeptidyl small molecule (SB-247464) that selectively activates murine granulocyte colony-stimulating factor signal transduction pathways and promotes neutrophil formation in vivo. To elucidate the mechanism of action of SB-247464, a series of cell-based and biochemical assays were performed. The activity of SB-247464 is strictly dependent on the presence of zinc ions. Titration microcalorimetry experiments using a soluble murine granulocyte colony-stimulating factor receptor construct show that SB-247464 binds to the extracellular domain of the receptor in a zinc ion-dependent manner. Analytical ultracentrifugation studies demonstrate that SB-247464 induces self-association of the N-terminal three-domain fragment in a manner that is consistent with dimerization. SB-247464 induces internalization of granulocyte colony-stimulating factor receptor on intact cells, consistent with a mechanism involving receptor oligomerization. These data show that small nonpeptidyl compounds are capable of selectively binding and inducing productive oligomerization of cytokine receptors.
Doyle
Michael L
ML
Department of Medicinal Chemistry, GlaxoSmithKline, Collegeville, Pennsylvania 19426, USA.
Tian
Shin-Shay
SS
Miller
Stephen G
SG
Kessler
Linda
L
Baker
Audrey E
AE
Brigham-Burke
Michael R
MR
Dillon
Susan B
SB
Duffy
Kevin J
KJ
Keenan
Richard M
RM
Lehr
Ruth
R
Rosen
Jon
J
Schneeweis
Lumelle A
LA
Trill
John
J
Young
Peter R
PR
Luengo
Juan I
JI
Lamb
Peter
P
D000818:Animals; D001562:Benzimidazoles / Q000494:pharmacology; D001854:Bone Marrow Cells / Q000378:metabolism; D002151:Calorimetry; D002460:Cell Line; D002942:Circular Dichroism; D016207:Cytokines / Q000378:metabolism; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D004492:Edetic Acid / Q000494:pharmacology; D006146:Guanidines / Q000494:pharmacology; D007477:Ions; D008024:Ligands; D051379:Mice; D008956:Models, Chemical; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D016188:Receptors, Granulocyte Colony-Stimulating Factor / Q000737:chemistry* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction; D014162:Transfection; D014461:Ultracentrifugation; D015032:Zinc
D001562:Benzimidazoles; D016207:Cytokines; D006146:Guanidines; D007477:Ions; D008024:Ligands; D010455:Peptides; D016188:Receptors, Granulocyte Colony-Stimulating Factor; D011993:Recombinant Fusion Proteins; C113274:SB 247464; D004492:Edetic Acid; D015032:Zinc