The cation-independent mannose-6-phosphate/IGF2 receptor (CI-M6P/IGF2R) plays a crucial role in transporting lysosomal enzymes and other ligands. In this study, we designed and synthesized novel stable mannose-6-phosphate (M6P) derivatives to enhance their affinity for CI-M6P/IGF2R. To evaluate the binding potency, we employed a sensitive and cost-effective fluorescence polarization assay, enabling rapid quantification of receptor-ligand interactions in solution. The tested compounds included di-, tri-, and penta-M6P peptides along with various M6P-derived small molecules featuring phosphate isosteres or other functional modifications. Our findings indicate that ligands bearing multiple M6P moieties exhibit significantly higher receptor affinities than monomeric compounds and that phosphonate groups may serve as a more stable and potent alternative to native M6P. Computational modeling of ligand interactions with the CI-M6P/IGF2R domains further elucidated the binding mechanisms, offering new directions for the development of more effective ligands. This study advances the design of therapeutic strategies that leverage CI-M6P/IGF2R for targeted biomolecule delivery to lysosomes, thereby opening new possibilities for biomedical applications.

• Klíčová slova
• IGF2, Mannose‐6‐phosphate, fluorescence polarization assay, ligand binding, receptor,
• MeSH
• fluorescenční polarizace MeSH
• lidé MeSH
• ligandy MeSH
• lyzozomy metabolismus   MeSH
• mannosafosfáty * chemie   metabolismus   chemická syntéza   MeSH
• racionální návrh léčiv MeSH
• receptor IGF typ 2 * metabolismus   chemie   MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• IGF2R protein, human MeSH Prohlížeč
• ligandy MeSH
• mannosafosfáty * MeSH
• mannose-6-phosphate MeSH Prohlížeč
• receptor IGF typ 2 * MeSH

The preparation of specifically iodine-125 (125I)-labeled peptides of high purity and specific activity represents a key tool for the detailed characterization of their binding properties in interaction with their binding partners. Early synthetic methods for the incorporation of iodine faced challenges such as harsh reaction conditions, the use of strong oxidants and low reproducibility. Herein, we review well-established radiolabeling strategies available to incorporate radionuclide into a protein of interest, and our long-term experience with a mild, simple and generally applicable technique of 125I late-stage-labeling of biomolecules using the Pierce iodination reagent for the direct solid-phase oxidation of radioactive iodide. General recommendations, tips, and details of optimized chromatographic conditions to isolate pure, specifically 125I-mono-labeled biomolecules are illustrated on a diverse series of (poly)peptides, ranging up to 7.6 kDa and 67 amino acids (aa). These series include peptides that contain at least one tyrosine or histidine residue, along with those featuring disulfide crosslinking or lipophilic derivatization. This mild and straightforward late-stage-labeling technique is easily applicable to longer and more sensitive proteins, as demonstrated in the cases of the insulin-like growth factor binding protein-3 (IGF-BP-3) (29 kDa and 264 aa) and the acid-labile subunit (ALS) (93 kDa and 578 aa).

• Klíčová slova
• 125I-labeling of peptides, High specific activity, Intramolecular effect of 125I decay, Late-stage peptide labeling, Radiochemical stability, Radiohalogenated prosthetic groups, Site-specific labeling,
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

Preptin, a 34-amino acid peptide derived from pro-IGF2, is believed to influence various physiological processes, including insulin secretion and the regulation of bone metabolism. Despite its recognized involvement, the precise physiological role of preptin remains enigmatic. To address this knowledge gap, we synthesized 16 analogs of preptin, spanning a spectrum from full-length forms to fragments, and conducted comprehensive comparative activity evaluations alongside native human, mouse and rat preptin. Our study aimed to elucidate the physiological role of preptin. Contrary to previous indications of broad biological activity, our thorough analyses across diverse cell types revealed no significant biological activity associated with preptin or its analogs. This suggests that the associations of preptin with various diseases or tissue-specific abundance fluctuations may be influenced by factors beyond preptin itself, such as higher levels of IGF2 or IGF2 proforms present in tissues. In conclusion, our findings challenge the conventional notion of preptin as an isolated biologically active molecule and underscore the complexity of its interactions within biological systems. Rather than acting independently, the observed effects of preptin may arise from experimental conditions, elevated preptin concentrations, or interactions with related molecules such as IGF2.

• MeSH
• insulinu podobný růstový faktor II * metabolismus   MeSH
• inzulin metabolismus   MeSH
• krysa rodu Rattus MeSH
• lidé MeSH
• myši MeSH
• peptidové fragmenty metabolismus   MeSH
• proteinové prekurzory metabolismus   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• IGF2 protein, human MeSH Prohlížeč
• insulinu podobný růstový faktor II * MeSH
• inzulin MeSH
• peptidové fragmenty MeSH
• preptin MeSH Prohlížeč
• proteinové prekurzory MeSH