Rigid linkers for bioactive peptides

Abstract

Rigid linkers of variable length were used to connect two high-affinity Nle4-D-Phe7-alpha-melanocyte stimulating hormone (NDP-alpha-MSH) or two low-affinity MSH(4) ligands. The linked peptides were synthesized by solid-phase methods. Control experiments indicate there is little or no effect of these linkers on NDP-alpha-MSH or MSH(4) binding to the human melanocortin 4 receptor (hMC4R). Tethering two high-affinity ligands gave no binding enhancement, while tethering two low-affinity ligands resulted in binding enhancement that decreased with increased linker length. Furthermore, for the low-affinity ligands, the enhancement of affinity is inversely proportional to the estimated molecular moments of inertia. These results are consistent with a model wherein binding is enhanced when the rate of ligand reattachment to the receptor is fast relative to the rate of ligand diffusion.

Figure 1

Rigid amino acid linkers 1–4 and N-protected derivatives 5–8.

Figure 2

Dimeric and monomeric NDP-α-MSH compounds 32 and 33.

Figure 3

Dimeric and monomeric MSH(4) compounds 37–44.

Figure 4

Representative ligand binding curves generated through competitive ligand binding analysis compare the binding affinities of ligands 10 (□) and 13 (■). The calculated IC₅₀ for 10 is 8 nM with an R² value = 0.91 and for 13 10 nM with an R² value = 0.93.

Figure 5

Binding of Linked MSH Ligands to hMC4R. State A: L₁ is bound, L₂ is unavailable for binding. State B: L₁and L₂ are unbound, L₁ is positioned for binding. State C: L₁and L₂ are unbound, L₂ is positioned for binding. State D: L₂ is bound, L₁ is unavailable for binding. State E: Compound is dissociated from cell surface (not shown).

Figure 6

Inverse correlation of compound binding affinity and linker length.

Scheme 1. Solid-phase Synthesis of Compounds 9–11 Containing Linked NDP-α-MSH Ligands and Control Compounds 12–14

Reagents and conditions: (a) piperidine. (b) Fmoc/tBu solid-phase synthesis (Refs and 19). (c) N-protected linker (, , or 7), HOCt-DIC, DMF. (d) THF/MeOH/N₂H₄ (70/15/15). (e) pyridine/Ac₂O (50/50). (f) CF₃CO₂H/HSCH₂CH₂SH/PhSMe/H₂O (91/3/3/3).