Discovery and characterization of a selective, nonpeptidyl thrombopoietin receptor agonist.
OBJECTIVE
Peptide and other small molecule agonists have been described for several cytokines and growth factors. Hydrazone compounds described here as thrombopoietin receptor agonists were identified as activating STAT proteins in a Tpo responsive cell line.


METHODS
STAT activation and analysis of signal transduction pathways in cell lines and normal human platelets was elucidated by Western blot and electrophoretic mobility shift assays. Proliferation assays in cell types responsive to other cytokines determined specificity for Tpo receptor. Flow cytometry quantified differentiation of CD34(+) cells into CD41(+) megakaryocytes and platelet production in vitro.


RESULTS
Activation of STAT5, mitogen-activated protein kinase, p38, and early response genes by SB 394725 was similar to that induced by Tpo. SB 394725 induced a reporter gene response under a STAT activation promoter as well as the megakaryocyte-specific gpIIb promoter. The compound induced proliferation of Tpo responsive lines but demonstrated no activity in cell lines responding to other cytokines, i.e., erythropoietin, granulocyte-colony stimulating factor, interleukin-3, interferon-gamma. The response of normal human Tpo receptors was elucidated by measuring growth and differentiation of human bone marrow in vitro. Activation of endogenous Tpo receptors by SB 394725 was demonstrated in human and chimp platelets, but not in platelets of other species including mouse, dog, rabbit, or cynomolgus monkey.


CONCLUSIONS
SB 394725, a small molecule with a molecular weight of 452 Da, is capable of activating Tpo-specific signal transduction, proliferation, and differentiation responses similar to the responses and functions of the protein growth factor, Tpo.
Erickson-Miller
Connie L
CL
SmithKline Beecham Pharmaceuticals, Collegeville, Pa., USA. Connie_L_Erickson-Miller@GSK.com
DeLorme
Evelyne
E
Tian
Shin-Shay
SS
Hopson
Christopher B
CB
Stark
Kenneth
K
Giampa
Leslie
L
Valoret
Elizabeth I
EI
Duffy
Kevin J
KJ
Luengo
Juan L
JL
Rosen
Jon
J
Miller
Stephen G
SG
Dillon
Susan B
SB
Lamb
Peter
P
D000818:Animals; D001792:Blood Platelets; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D004268:DNA-Binding Proteins / Q000378:metabolism; D006412:Hematopoietic Stem Cells / Q000187:drug effects; D006801:Humans; D006835:Hydrazones / Q000494:pharmacology*; D008533:Megakaryocytes; D051379:Mice; D008894:Milk Proteins / Q000378:metabolism; D009363:Neoplasm Proteins / Q000819:agonists; D011518:Proto-Oncogene Proteins / Q000819:agonists; D018121:Receptors, Cytokine / Q000819:agonists*; D053628:Receptors, Thrombopoietin; D050799:STAT5 Transcription Factor; D015398:Signal Transduction / Q000187:drug effects; D013045:Species Specificity; D015534:Trans-Activators / Q000378:metabolism
D004268:DNA-Binding Proteins; D006835:Hydrazones; D008894:Milk Proteins; D009363:Neoplasm Proteins; D011518:Proto-Oncogene Proteins; D018121:Receptors, Cytokine; D053628:Receptors, Thrombopoietin; D050799:STAT5 Transcription Factor; D015534:Trans-Activators; C082218:MPL protein, human