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- Screening of novel cyclic depsipeptide library to identify erythropoietin ligands.
- Integrated experimental-computational study of erythropoietin-peptide interactions.
- Hydrophobicity and histidine govern erythropoietin binding, elution, respectively.
- Lead cyclic peptide ligand FSLLSH shows highest erythropoietin bind-elute yield.
- Lead cyclic peptide binds erythropoietin three times tighter than linear variant.
This work presents the selection and characterization of erythropoietin (EPO)-binding cyclic peptide ligands. The sequences were selected by screening a focused library of cyclic depsipeptides cyclo[(NÎ±-Ac)Dap(A)-X1-X6-AE], whose structure and amino acid compositions were tailored to mimic the EPO receptor. The sequences identified through library screening were synthesized on chromatographic resin and characterized via binding-and-elution studies against EPO to select a pool of candidate ligands. Sequences with higher hydrophobicity consistently showed stronger binding to EPO, with the exception of FSLLSH, which was noted for its lower hydrophobicity and high EPO binding. Mutagenesis studies performed on FSLLSH with natural and non-natural amino acid substitutions led to the identification of critical EPO-binding determinants, and the discovery of new peptide ligands. In particular, histidine-scanning mutagenesis performed on three lead sequences yielded the discovery of variants whose EPO-binding is more pH-sensitive, which facilitates EPO recovery. Selected ligands were studied to correlate the elution yield to the salinity of the binding buffer and the elution pH. Elution yields were consistently higher when EPO binding was performed at low ionic strength. The crystal structures of lead cyclic peptides were docked in silico against EPO to estimate the binding affinity in solution. Isotherm adsorption studies performed on FSLLSH indicated that the cyclic version of the ligand (KDÂ =Â 0.46Â Î¼M) has a higher affinity for EPO than its corresponding linear variant (KDÂ =Â 1.44Â Î¼M). Collectively, these studies set the stage for use of the cyclic peptide ligands as EPO purification and detection tools.
Journal: Journal of Chromatography A - Volume 1500, 2 June 2017, Pages 105-120