DOI: 10.1002/cmdc.201300089 Potent Agonists of a Hematopoietic Stem Cell Cytokine Receptor, c-Mpl Anna Tarasova, [a, c] David N. Haylock, [a, d] Laurence Meagher, [a, c] Cheang Ly Be, [a] Jacinta White, [a, c] Susan K. Nilsson, [a, d] Jessica Andrade, [a] Kellie Cartledge, [a] and David A. Winkler* [a, b, c] Introduction Thrombopoietin (TPO) is a potent primary regulator of mega- karyocytopoiesis, a cellular developmental process leading to platelet production. [1–5] As shown in Figure 1, TPO is an essen- tial factor that drives all stages of thrombopoiesis. This cyto- kine also supports the survival and proliferation of megakaryo- cyte progenitors and affects committed erythroid progenitors, hematopoietic progenitors, and leukemic cells. [2] TPO interacts synergistically with cytokines such as stem cell factor, [6] inter- leukin-3, [6] or Fms-related tyrosine kinase 3 ligand [7] to stimulate the proliferation of hematopoiet- ic stem cells (HSC) and progeni- tor cells in vitro. [8] TPO has been shown to maintain HSC quies- cence in vivo and therefore reg- ulate the size of the HSC com- partment. [9, 10] Human TPO is a 332-residue protein containing two domains. The N-terminal domain compris- es the first 153 residues, and the 179-residue C-terminal domain contains multiple potential N- linked glycosylation sites. [11] A number of studies have shown that the N-terminal domain is sufficient for receptor binding, signaling and stimulation of megakaryocytopoiesis both in vivo [1] and in vitro. [12, 13] This domain is arranged into a four- a-helix bundle with an up-up-down-down topology with the four a-helices located at residues 9–29 (helix A), 56–77 (helix B), 85–107 (helix C), and 127–147 (helix D). In addition, the region contains two intramolecular disulfide bonds, and from sequence analysis has shown moderate sequence similari- ty to erythropoietin (EPO; ~ 23 % amino acid identity), another member of the four-helix bundle cytokine family. The X-ray crystal structure of the human TPO functional domain com- plexed to a neutralizing antibody TN1 Fab was published by Feese et al., [14] confirming the topology of the cytokine. TPO binds to its receptor, myeloproliferative leukemia virus oncogene (c-Mpl, also known as TPO receptor TPO-R), with a K d value of 100–200 pm. Binding induces oligomerization of independent receptor chains and transphosphorylation of the Several growth factors feature prominently in the control of hematopoiesis. Thrombopoietin, a class I hematopoietic cyto- kine, plays critical roles in regulating hematopoietic stem cell numbers and also stimulates the production and differentia- tion of megakaryocytes, the bone marrow cells that ultimately produce platelets. Thrombopoietin interacts with the c-Mpl cell-surface receptor. Recently, several peptide and small-mole- cule agonists and antagonists of c-Mpl have been reported. We conducted a bioinformatics and molecular modeling study aimed at understanding the agonist activities of peptides that bind to c-Mpl, and developed new potent peptide agonists with low nanomolar activity. These agonists also show very high activity in human CD34 + primary cell cultures, and dou- bled the mean blood platelet counts when injected into mice. Figure 1. Stages in megakaryocytopoiesis driven by thrombopoietin. [a] Dr. A. Tarasova, Prof. D. N. Haylock, Dr. L. Meagher, C. L. Be, J. White, Prof. S. K. Nilsson, Dr. J. Andrade, K. Cartledge, Prof. D. A. Winkler CSIRO Materials Science & Engineering Bag 10, Clayton South MDC 3169 (Australia) E-mail : dave.winkler@csiro.au [b] Prof. D. A. Winkler Monash Institute of Pharmaceutical Sciences 381 Royal Parade, Parkville 3052 (Australia) [c] Dr. A. Tarasova, Dr. L. Meagher, J. White, Prof. D. A. Winkler Cooperative Research Centre for Polymers 8 Redwood Drive, Notting Hill 3168 (Australia) [d] Prof. D. N. Haylock, Prof. S. K. Nilsson Department of Anatomy and Cell Biology Monash University, Clayton 3168 (Australia) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cmdc.201300089.  2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemMedChem 2013, 8, 763 – 771 763 CHEMMEDCHEM FULL PAPERS