The hemoregulatory
peptide (pGlu-
Glu-Asp-Cys-Lys,
pEEDCK) is a potent inhibitor of stem cell recruitment, which is a major source of hematological complications after
cytostatic tumor therapy. By preventing recruitment,
pEEDCK can keep hemopoietic stem cells in their normal nonproliferative state and in this way prevent damage by certain cell cycle-specific
cytostatic drugs.
pEEDCK could play a role as hemoprotector in
tumor chemotherapy. As a
thiol-containing
peptide,
pEEDCK is highly sensitive to oxidation, resulting in the formation of a dimer. Although monomeric
pEEDCK is a strong inhibitor of colony-forming units-granulocyte/macrophage (CFU-GM) clonal growth, the dimer was previously found to enhance
colony-stimulating factor-triggered CFU-GM colony formation. It seemed, thus, necessary to find methods that avoid undesired dimerization reactions. A solid phase strategy for
pEEDCK synthesis is presented. The primary synthetic product, S-tert-butyl-sulfenyl-
pEEDCK, was purified and stored with the
thiol-protecting group remaining attached. Conversion to active monomeric
pEEDCK was achieved by reductive treatment in situ before application and removal of tert-butyl-mercaptane in vacuo. The activation
reagent (
dithioerythritol) prevented reoxidation also in
culture media, where unprotected
peptide was oxidized rapidly (t1/2 less than 13 min). The purified synthetic
peptide was found to be a potent inhibitor of CFU-GM colony formation (IC50 = 1.1 x 10(-12) M) in vitro. It was also found to inhibit colony formation of some leukemic cell lines (HL-60, RAJI) although at much higher concentrations (10(-8) to 10(-9) M). Friend
leukemia cells were not inhibited in the dose range where CFU-GM were sensitive.