Bivalent diketopiperazine-based tropomysin receptor kinase C (TrkC) antagonists

Abstract

Bivalent molecules containing two beta-turn mimics with side chains that correspond to hot-spots on the neurotrophin NT-3 were prepared. Binding assays showed the mimetics to be selective TrkC ligands, and biological assays showed one mimetic to be an antagonist of the TrkC receptor.

Figure 1

a. Key distance of Cβ-separations of the i + 1 to i + 2 residues of a type I β-turn and of the monovalent turn mimics A and B featured here; b. comparative overlay of the two types of 2,5-DKP mimics (colored) onto a type I β-turn (blue).

Figure 2

Direct binding of biotin-peptidomimetic 6dh to TrkC-expressing cells. Cells expressing the indicated receptor were first bound at 4 °C with test ligand (50 μM), followed by fluorescein-avidin. After washing, cells were analyzed by FACScan/CellQuest. The background mean channel fluorescence (MCF) of NIH-IGF-1R were subtracted to analyze the specific MCF binding to test cells. Results shown are fluorescence ± standard error of mean (SEM), n =3 independent experiments.

Figure 3

Mimetic 6dh antagonizes NT-3 action in cell survival assays. NIH-3T3 cells expressing either TrkA or TrkC or IGF-1R were cultured in SFM supplemented with optimal (a, 10 nM) or sub-optimal (b, 0.2 nM) concentrations of the appropriate growth factor (GF) ± peptidomimetic (20 μM). Survival was measured in MTT assays, and was calculated relative to optimal growth factor-mediated survival (100%). Results shown are average ± sd.

Figure 4

Effect of 6dh on TrkC receptor phosphorylation and MAPK activation in NIH3T3 TrkC cells. Cells were exposed to the indicated peptidomimetic (20 μM) and growth factor (2 nM) for 20 min. Detergent lysates were analyzed by Western blotting with anti-pTyr monoclonal antibody (mAb) 4G10 or anti-phospho-MAPK. Membranes were re-blotted with anti-actin to verify protein loading. Blots were quantified by densitometry.

Scheme 1

Solution phase synthesis of monomeric DKP-based peptidomimetics. Parts a and b refer to syntheses of the monovalent scaffolds, and parts c deals with functionalization of these with linker fragments. Reagents and conditions: (a) 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), 1-hydroxybenzotriazole (HOBt), N-methylmorpholine (NMM), CH₂Cl₂, 25 °C, 12 h; (b) trifluoroacetic acid (TFA)/CH₂Cl₂, 0 – 25 °C, 2 h; (c) bromoacetyl bromide, 0.5 M K₂CO₃ (aq), CH₂Cl₂, 0 °C, 2 h; (d) ⁱ 50 % NaOH(aq), CH₂Cl₂, 25 °C, 12 h; (e) TFA, Pr₃SiH, CH₂Cl₂, 25 °C, 10 h. Reagents and conditions: (a) EDCI, HOBt, NMM, CH₂Cl₂, 25 °C, 12 h; (b) H₂, Pd/C, MeOH, 25 °C, 12 h; (c) bromoacetyl bromide, 0.5 M K₂CO₃ (aq), CH₂Cl₂, 0 °C, 2 h; (d) 50 % NaOH (aq), CH₂Cl₂, 25 °C, 12 h; (e) Me₃SnOH, 1,2-dichloroethane, 80 °C, 6 h. Reagents and conditions: For 4a and 4e: 3a, (a) (i) short or long linker, EDCI, HOBt, NMM, CH₂Cl₂, 25 °C, 12 h; (ii) H₂, Pd/C, MeOH, 25 °C, 16 h; (iii) Boc anhydride, Et₃N, CH₂Cl₂, 25 °C, 12 h; For 4b and 4f: 3b, (b) (i) short or long linker, EDCI, HOBt, NMM, CH₂Cl₂, 25 °C, 12 h; (ii) H₂, Pd/C, MeOH, 25 °C, 16 h; For 4c, d and 4g, h: 3c or 3d, (c) short or long linker, EDCI, HOBt, NMM, CH₂Cl₂, 25 °C, 12 h.

Scheme 1

Solution phase synthesis of monomeric DKP-based peptidomimetics. Parts a and b refer to syntheses of the monovalent scaffolds, and parts c deals with functionalization of these with linker fragments. Reagents and conditions: (a) 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), 1-hydroxybenzotriazole (HOBt), N-methylmorpholine (NMM), CH₂Cl₂, 25 °C, 12 h; (b) trifluoroacetic acid (TFA)/CH₂Cl₂, 0 – 25 °C, 2 h; (c) bromoacetyl bromide, 0.5 M K₂CO₃ (aq), CH₂Cl₂, 0 °C, 2 h; (d) ⁱ 50 % NaOH(aq), CH₂Cl₂, 25 °C, 12 h; (e) TFA, Pr₃SiH, CH₂Cl₂, 25 °C, 10 h. Reagents and conditions: (a) EDCI, HOBt, NMM, CH₂Cl₂, 25 °C, 12 h; (b) H₂, Pd/C, MeOH, 25 °C, 12 h; (c) bromoacetyl bromide, 0.5 M K₂CO₃ (aq), CH₂Cl₂, 0 °C, 2 h; (d) 50 % NaOH (aq), CH₂Cl₂, 25 °C, 12 h; (e) Me₃SnOH, 1,2-dichloroethane, 80 °C, 6 h. Reagents and conditions: For 4a and 4e: 3a, (a) (i) short or long linker, EDCI, HOBt, NMM, CH₂Cl₂, 25 °C, 12 h; (ii) H₂, Pd/C, MeOH, 25 °C, 16 h; (iii) Boc anhydride, Et₃N, CH₂Cl₂, 25 °C, 12 h; For 4b and 4f: 3b, (b) (i) short or long linker, EDCI, HOBt, NMM, CH₂Cl₂, 25 °C, 12 h; (ii) H₂, Pd/C, MeOH, 25 °C, 16 h; For 4c, d and 4g, h: 3c or 3d, (c) short or long linker, EDCI, HOBt, NMM, CH₂Cl₂, 25 °C, 12 h.

Scheme 1

Solution phase synthesis of monomeric DKP-based peptidomimetics. Parts a and b refer to syntheses of the monovalent scaffolds, and parts c deals with functionalization of these with linker fragments. Reagents and conditions: (a) 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), 1-hydroxybenzotriazole (HOBt), N-methylmorpholine (NMM), CH₂Cl₂, 25 °C, 12 h; (b) trifluoroacetic acid (TFA)/CH₂Cl₂, 0 – 25 °C, 2 h; (c) bromoacetyl bromide, 0.5 M K₂CO₃ (aq), CH₂Cl₂, 0 °C, 2 h; (d) ⁱ 50 % NaOH(aq), CH₂Cl₂, 25 °C, 12 h; (e) TFA, Pr₃SiH, CH₂Cl₂, 25 °C, 10 h. Reagents and conditions: (a) EDCI, HOBt, NMM, CH₂Cl₂, 25 °C, 12 h; (b) H₂, Pd/C, MeOH, 25 °C, 12 h; (c) bromoacetyl bromide, 0.5 M K₂CO₃ (aq), CH₂Cl₂, 0 °C, 2 h; (d) 50 % NaOH (aq), CH₂Cl₂, 25 °C, 12 h; (e) Me₃SnOH, 1,2-dichloroethane, 80 °C, 6 h. Reagents and conditions: For 4a and 4e: 3a, (a) (i) short or long linker, EDCI, HOBt, NMM, CH₂Cl₂, 25 °C, 12 h; (ii) H₂, Pd/C, MeOH, 25 °C, 16 h; (iii) Boc anhydride, Et₃N, CH₂Cl₂, 25 °C, 12 h; For 4b and 4f: 3b, (b) (i) short or long linker, EDCI, HOBt, NMM, CH₂Cl₂, 25 °C, 12 h; (ii) H₂, Pd/C, MeOH, 25 °C, 16 h; For 4c, d and 4g, h: 3c or 3d, (c) short or long linker, EDCI, HOBt, NMM, CH₂Cl₂, 25 °C, 12 h.

Scheme 2

Solution phase synthesis of biotin-labeled bivalent peptidomimetic library. Reagents and conditions: (a) 1:1 TFA/CH₂Cl₂, 25 °C, 10 h; (b) (i) 1:1 TFA/CH₂Cl₂, 25 °C, 10 h; (ii) DTAB, K₂CO₃, DMSO, 25 °C, 2 h; (c) K₂CO₃, DMSO, 25 °C, 5–7 d.