Peptide Agonist of the Thrombopoietin Receptor as Potent as the Natural Cytokine

Abstract

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.

Peptide immobilized on gold particles enhances cell growth

“…Cwirla et al (1997) reported that a type of dimer of AF12505, AF13948, had high activity, which was close to that of rhTPO.…”

“…rhTPO had the highest activity. The peptide sequence of TPO-P is the same as that of AF12505, reported by Cwirla et al (1997). They reported that the activity of AF12505 was about 10 -4 times as high as that of rhTPO, calculated from the proliferative response of Ba/F3 hTPOR cells.…”

“…Several small peptides of 14-16 amino acid residues and their dimers have been reported as potent agonists of the TPO receptor c-Mpl (Cwirla et al 1997), with in vivo efficacy in mice. Recently, nonpeptide synthetic compounds, such as a benzodiazepine derivative, hydrazinonaphthalenes (Kimura et al 1998), azonaphthalenes (Duffy et al 2002), and a substituted thiazole (Inagaki et al 2004), have been reported to have TPO-like activity.…”

Peptide agonists of the thrombopoietin receptor

“…The library design was based on the linear sequence of AF12434 (ICS0 = 20 nM) where the 2B core sequence (GPTLRQWL) was fixed, and flanking parental residues were either deleted, substituted with alanine, or conserved. The oligonucleotides for this library were synthesized by an automated, codon-based, split and pool procedure [4]. As with the previous mutagenesis step, clones isolated from this library were ranked by their signal strength in the MBP ELISA, as shown in Figure 5.…”

Identifizierung und Strukturbestimmung eines niedermolekularen Aktivators der LFA‐1/ICAM‐1‐Bindung

“…[8,9] Obwohl die Inhibierung von Protein-Protein-Interaktionen (PPIs) mittels kleiner Moleküle lange Zeit als die ultimative Herausforderung in der Wirkstoffentwicklung galt, wurden nur sehr wenige Beispiele für solche PPI-Inhibitoren beschrieben. [10][11][12][13] Demgegenüber gibt es klare mechanistischkonzeptionelle Argumente dafür, dass PPI-Stabilisatoren großes Potential haben kçnnten. [14] LFA-1-Aktivatoren wären ein vielversprechender Ansatz für die Behandlung einer seltenen Erbkrankheit, bekannt als Leukozytenadhäsi-onsdeffizienz (LAD), oder als mçgliche Verstärker in der Tumorimmuntherapie.…”