Potent Agonists of a Hematopoietic Stem Cell Cytokine Receptor, c‐Mpl

Abstract

Several growth factors feature prominently in the control of hematopoiesis. Thrombopoietin, a class I hematopoietic cytokine, plays critical roles in regulating hematopoietic stem cell numbers and also stimulates the production and differentiation of megakaryocytes, the bone marrow cells that ultimately produce platelets. Thrombopoietin interacts with the c-Mpl cell-surface receptor. Recently, several peptide and small-molecule 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 doubled the mean blood platelet counts when injected into mice.

Immobilisation of a thrombopoietin peptidic mimic by self-assembled monolayers for culture of CD34+ cells

“…1). Previously reported peptide compound 4 (RILL) [26] was prepared by automated solid-phase synthesis by GenScript Corporation (Piscataway, NJ, USA). Fmoc (9-fluorenylmethoxycarbonyl)-amino acids and Rink amide-AM resin were purchased from GL Biochem (Shanghai, China).…”

“…The TPO mimetic peptide RILL (4), a potent c-Mpl agonist with a half-maximal effective concentration (EC 50 ) of 4 nM, was developed in our laboratory [26]. As suggested by its name, Retro Inverso Linear Linear (RILL), the peptide sequence 4 consists of two retroinversed linear sequences linked by lysine in the centre.…”

“…Mixed monolayers of OEG-and oxyamine-terminated alkanethiol were added with aldehyde-or ketone-functionalised ligand molecules for immobilisation to form covalently linked oxime products. We immobilised a thrombopoietin (TPO) mimetic peptide, RILL (name derived from the nature of the peptide sequence "Retro Inverso Linear Linear"), on a SAM surface for use in HSC culture [26].…”

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“…These methods have been used to design and optimise green pesticides [16][17][18][19][20] in collaboration with Du Pont and Schering Plough, have discovered new peptides and small molecules to control stem cells and cancers, [21][22][23][24][25][26][27] are accelerating the development of biomaterials for implantation and stem cell culture, [28][29][30][31][32][33] have provided new scientific insight into the potential adverse properties of nanomaterials, [34][35][36][37][38] have yielded clinical candidates for Australian SMEs and international companies, and were instrumental in the discovery of antibiotics [39,40] with a novel mode of action for the biotechnology spin off company, Betabiotics. Some of this research is discussed in more detail in the following sections.…”

“…We identified a key binding motif, RQW, in these peptide libraries and used this to develop potent small molecule and peptide agonists and antagonists of the TPO receptor. [22,23] The agonists were capable of potently stimulating platelet counts in animals, and also increased megakaryocyte ploidy. Peptide TPO mimetics could replace native TPO in the expansion of CD34 þ CD38 À primary cells up to 8 days.…”