The functional mimicry of a protein by an unrelated small molecule has been a formidable challenge. Now, however, the biological activity of a 166-residue hematopoietic growth hormone, erythropoietin (EPO), with its class 1 cytokine receptor has been mimicked by a 20-residue cyclic peptide unrelated in sequence to the natural ligand. The crystal structure at 2.8 A resolution of a complex of this agonist peptide with the extracellular domain of EPO receptor reveals that a peptide dimer induces an almost perfect twofold dimerization of the receptor. The dimer assembly differs from that of the human growth hormone (hGH) receptor complex and suggests that more than one mode of dimerization may be able to induce signal transduction and cell proliferation. The EPO receptor binding site, defined by peptide interaction, corresponds to the smaller functional epitope identified for hGH receptor. Similarly, the EPO mimetic peptide ligand can be considered as a minimal hormone, and suggests the design of nonpeptidic small molecule mimetics for EPO and other cytokines may indeed be achievable.

Random phage display peptide libraries and affinity selective methods were used to isolate small peptides that bind to and activate the receptor for the cytokine erythropoietin (EPO). In a panel of in vitro biological assays, the peptides act as full agonists and they can also stimulate erythropoiesis in mice. These agonists are represented by a 14- amino acid disulfide-bonded, cyclic peptide with the minimum consensus sequence YXCXXGPXTWXCXP, where X represents positions allowing occupation by several amino acids. The amino acid sequences of these peptides are not found in the primary sequence of EPO. The signaling pathways activated by these peptides appear to be identical to those induced by the natural ligand. This discovery may form the basis for the design of small molecule mimetics of EPO.

Two families of small peptides that bind to the human thrombopoietin receptor and compete with the binding of the natural ligand thrombopoietin (TPO) were identified from recombinant peptide libraries. The sequences of these peptides were not found in the primary sequence of TPO. Screening libraries of variants of one of these families under affinity-selective conditions yielded a 14-amino acid peptide (Ile-Glu-Gly-Pro-Thr-Leu-Arg-Gln-Trp-Leu-Ala-Ala-Arg-Ala) with high affinity (dissociation constant approximately 2 nanomolar) that stimulates the proliferation of a TPO-responsive Ba/F3 cell line with a median effective concentration (EC50) of 400 nanomolar. Dimerization of this peptide by a carboxyl-terminal linkage to a lysine branch produced a compound with an EC50 of 100 picomolar, which was equipotent to the 332-amino acid natural cytokine in cell-based assays. The peptide dimer also stimulated the in vitro proliferation and maturation of megakaryocytes from human bone marrow cells and promoted an increase in platelet count when administered to normal mice.

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The specific signals inducing the growth and maturation of thymocytes remain undefined. We show here that recombinant IL-7 induces growth of fetal and adult mouse CD4-8- thymocytes. IL-7 also induces a lower but significant response in CD4+8- and CD4-8+ thymocytes. Day 14 fetal thymic lobes cultured in IL-7 for 6 days show a 2-fold increase in cell number when compared to control cultures. The thymocyte subsets that proliferate in response to IL-7 can be maintained in culture for extended periods of time. CD4-8- thymocytes maintained in IL-7 did not change their phenotype with respect to CD4 and CD8 expression. In addition, we show that the combination of IL-7 plus IL-6 provides a potent growth stimulus for CD4+8- and CD4-8+ thymocytes. A cloned thymic epithelial cell, that can be induced to express MHC class II molecules, transcribes both IL-7 and IL-6 mRNA. A cloned thymic macrophage cell line produces IL-6 but no detectable IL-7 mRNA. The pattern of biological activities present in the supernatants of these cell lines is also presented. These observations suggest that the thymic epithelial and macrophage cell types may be an in vivo source of signals which mediate thymocyte development.