Modulation of the antagonistic properties of an insulin mimetic peptide by disulfide bridge modifications

Abstract

Insulin is a peptide responsible for regulating the metabolic homeostasis of the organism; it elicits its effects through binding to the transmembrane insulin receptor (IR). Insulin mimetics with agonistic or antagonistic effects toward the receptor are an exciting field of research and could find applications in treating diabetes or malignant diseases. We prepared five variants of a previously reported 20-amino acid insulin-mimicking peptide. These peptides differ from each other by the structure of the covalent bridge connecting positions 11 and 18. In addition to the peptide with a disulfide bridge, a derivative with a dicarba bridge and three derivatives with a 1,2,3-triazole differing from each other by the presence of sulfur or oxygen in their staples were prepared. The strongest binding to IR was exhibited by the peptide with a disulfide bridge. All other derivatives only weakly bound to IR, and a relationship between increasing bridge length and lower binding affinity can be inferred. Despite their nanomolar affinities, none of the prepared peptide mimetics was able to activate the insulin receptor even at high concentrations, but all mimetics were able to inhibit insulin-induced receptor activation. However, the receptor remained approximately 30% active even at the highest concentration of the agents; thus, the agents behave as partial antagonists. An interesting observation is that these mimetic peptides do not antagonize insulin action in proportion to their binding affinities. The compounds characterized in this study show that it is possible to modulate the functional properties of insulin receptor peptide ligands using disulfide mimetics.

Keywords: antagonism; dicarba; disulfide mimetics; insulin mimetic peptide; insulin receptor; staple; triazole.

FIGURE 1

Simplified structures of peptides 1–5. The parts by which the peptides differ are shown in blue. Black dots indicate C^α atoms at positions 11 and 18.

SCHEME 1

Synthesis of alkyne amino acids 7 and 11. Reagents, conditions, and yields: (a) NaH, DMF, propargyl bromide, 0°C to room temperature overnight; (b) TFA, DCM from 0°C to room temperature for 2 h; (c) Fmoc‐OSu, NaHCO₃, water, and dioxane, 1 h at 0°C, then overnight at rt (53% yield after three steps); (d) Na, NH₃(l); (e) propargyl bromide; (f) Fmoc‐OSu, NaHCO₃, water, and dioxane, 1 h at 0°C, then overnight at rt (58% yield after three steps); (g) propargyl bromide, NH₄OH overnight at rt (56% yield); (h) Fmoc‐OSu, NaHCO₃, water, and dioxane, 1 h at 0°C, then overnight at rt (89% yield); (i) propargyl bromide, NaHCO₃, TBAB, water, and ethyl acetate, 4 days at rt (77% yield)

SCHEME 2

Synthesis of azido amino acid 23. Reagents, conditions, and yields: O‐tert‐butyl‐N,N′‐diisopropyl isourea, DCM, overnight at rt (72% yield); (b) CBr₄, PPh₃, DCM, 1 h at 0°C, then overnight at rt (66% yield); (c) HOCH₂CH₂SH, K₂CO₃, AcCN, overnight at rt (61% yield); (d) CBr₄, PPh₃, DCM, 1 h at 0°C, then overnight at rt (68% yield); (e) NaN₃, DMSO, 70°C overnight (90% yiled); (f) MsCl, TEA, DCM, 1 h at 0°C; (g) NaN₃, DMSO, 70°C overnight (80% yield for two steps); (h) Na, MeOH, 1 h at rt then BrCH₂CH₂OH overnight; (i) Boc₂O, Na₂CO₃, water and dioxane, 0.5 h at 0°C, then overnight at rt (76% yield for two steps); (j) O‐tert‐butyl‐N,N′‐diisopropyl isourea, DCM, overnight at rt (55% yield); (k) TFA, TIPS, DCM, 1 h at rt; (l) Fmoc‐OSu, NaHCO₃, water, dioxane, 1 h at 0°C, then overnight at rt; (m) Na, MeOH, 0.5 h at rt then BrCH₂CH₂OH 3 h; (n) Fmoc‐OSu, NaHCO₃, water, and dioxane, 1 h at 0°C, then overnight at rt (73% yield for two steps); (o) O‐tert‐butyl‐N,N′‐diisopropyl isourea, THF, overnight at rt (77% yield); (p) HN (Boc)₂, PPh₃, DIAD, THF overnight at rt; (q) 24, KOH, MeOH, and THF overnight at rt; (r) Fmoc‐OSu, NaHCO₃, water and dioxane, 0°C then overnight at rt; (s) TFA, TIPS, DCM, 2 h at rt; (t) TfN₃, NaHCO₃, methanol, DCM and water, overnight at rt (50% yield for four steps).

FIGURE 2

Relative abilities of peptides 1–5 to antagonize insulin‐stimulated receptor phosphorylation (full lines). IR‐A transfected cells were stimulated with 0.0001 μM to 50 μM peptides in the presence of 10 nM insulin. Dashed lines show stimulation of IR‐A with insulin and selected peptides 1, 2, and 4 alone. The data are expressed as the contribution of phosphorylation relative to the signal of human insulin at 10 nM and are shown with S.D. The details are provided in Section 2.