Design of Potent Mannose-6-Phosphate Derivatives as Ligands for CI-M6P/IGF2R Using Fluorescence Polarization Assay

Abstract

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.

Keywords: IGF2; Mannose‐6‐phosphate; fluorescence polarization assay; ligand binding; receptor.

Figure 1

Model structure of the human CI‐M6P/IGF2 receptor (AlphaFold structure ID: AF‐P11717‐F1‐v4). The extracellular portion of the receptor consists of 15 homologous domains, with four domains identified as mannose 6‐phosphate (M6P) ligand‐binding sites. Domains 3 (D3) and 9 (D9) function as high‐affinity binding sites for M6P ligands, whereas domains 5 (D5) and 15 (D15) have been characterized as low‐affinity binding sites. Ligand binding induces extensive conformational changes in the receptor structure, which may influence the binding of additional ligands.^{[ 6 ]}

Figure 2

Target glycopeptides 1–6 bearing the 6‐phosphonomethyl mannose moiety (blue spheres). The fluorescein group is shown in yellow and N‐terminal acetylation is shown in green.

Scheme 1

Synthesis of mannose‐6‐phosphate (MP6) analogues 8–14. Reagents and conditions: i) (COCl)₂, DMSO, CH₂Cl₂, −78 °C, then Et₃N, −78 °C – r.t., 2.5 h, 80%; ii) tetraethyl methylenediphosphonate, n‐BuLi, THF, ‐78 °C – r.t., 3 h, 73%; iii) 10% Pd/C, H₂, EtOH, r.t., 24 h, 97%; iv) TMSBr, 2,6‐lutidine, CH₃CN, 20 h, 70%; v) I₂, Ph₃P, imidazole, 80 °C, 2 h, 86%; vi) P(OEt)₃, 140 °C, 120 h, 75%; vii) 10% Pd/C, H₂, EtOH, r.t., 24 h, 98%; viii) TMSBr, 2,6‐lutidine, CH₃CN, r.t., 3 h, 74%; ix) a) ethyl cyanoacetate, 60% NaH, DMF, 70 °C, 2 h, 65%; b) LiCl, DMSO/H₂O, 160 °C, 3 h, 76%; x) NaN₃, Et₃N•HCl, DMF, 90 °C, 180 h, 58%; xi) 10% Pd/C, H₂, EtOH, r.t., 24 h, 78%; xii) NaOH, EtOH/H₂O, 90 °C, 24 h, 96%; xiii) 10% Pd/C, H₂, EtOH, r.t., 24 h, 91%; xiv) KCN, DMF, 70 °C, 5 h, 98%; xv) NaN₃, Et₃N•HCl, DMF, 90 °C, 120 h, 75%; xvi) 10% Pd/C, H₂, EtOH, r.t., 24 h, 85%; xvii) Ac₂O, pyridine, r.t., 24 h, 87%; xviii) Ac₂O, H₂SO₄, AcOH, 40 °C, 1 h, 78%; xix) allylTMS, BF₃•Et₂O, TMSOTf, CH₃CN, r.t., 16 h, 78%; xx) 4‐iodophenol, Pd(OAc)₂, TBAB, NaHCO₃, DMF, 85 °C,16 h, 92%; xxi) a) TMSBr, pyridine, CH₂Cl₂, r.t., 4 h; b) NaOMe, MeOH, r.t., 4 h, 59%; xxii) 2‐bromoethanol, BF₃•Et₂O, 0 °C – r.t., 16 h, 78%; xxiii) a) NaN₃, TBAB, DMSO, r.t., 17 h, 84%; xxiv) TMSBr, pyridine, CH₃CN, r.t., 3 h, 96%. For abbreviations, see Supporting Information.

Figure 3

The reverse saturation plot, obtained from the binding of fluorescent glycopeptide 1 (10 nM) to soluble CI‐M6P/IGF2R:D1–D15, was determined by fluorescence polarization and Scatchard analysis (insert plot) after calculation of bound (B) and free (F) receptor concentrations. The values presented in this figure were obtained after subtracting the basal anisotropy values of peptide 1 measured in the absence of the receptor (at c = 0 [nM]). The original measured data are shown in Figure S20.

Figure 4

Representative titration curves of compounds for CI‐M6P/IGF2R:D1–D15. Panel A: peptide 2 (black), peptide 3 (red), peptide 4 (blue), peptide 5 (green), and peptide 6 (magenta); Panel B: peptide 2 (black), compound 7 (red), compound 8 (green), compound 9 (magenta), compound 11 (blue), and compound 13 (dark yellow).

Figure 5

Modeled structure of ligand 13 in CI‐M6P/IGF2R:D9. A: Overall view; B: Detailed zoomed view of the binding site with the interacting CI‐M6P/IGF2R residues marked.