Glycosylated neurotensin analogues exhibit sub-picomolar anticonvulsant potency in a pharmacoresistant model of epilepsy - PubMed (original) (raw)

Glycosylated neurotensin analogues exhibit sub-picomolar anticonvulsant potency in a pharmacoresistant model of epilepsy

Hee-Kyoung Lee et al. ChemMedChem. 2009 Mar.

Abstract

Neurotensin (NT) is an endogenous neuropeptide involved in a variety of central and peripheral neuromodulatory effects. Herein we show the effects of site-specific glycosylation on the in vitro and in vivo pharmacological properties of this neuropeptide. NT analogues containing O-linked disaccharides (beta-melibiose and alpha-TF antigen) or beta-lactose units linked by a PEG(3) spacer were designed and chemically synthesized using Fmoc chemistry. For the latter analogue, Fmoc-Glu-(beta-Lac-PEG(3)-amide) was prepared. Our results indicate that the addition of the disaccharides does not negatively affect the sub-nanomolar affinity or the low-nanomolar agonist potency for the neurotensin receptor subtype 1 (NTS1). Interestingly, three glycosylated analogues exhibited sub-picomolar potency in the 6 Hz limbic seizure mouse model of pharmacoresistant epilepsy following intracerebroventricular administration. Our results suggest for the first time that chemically modified NT analogues may lead to novel antiepileptic therapies.

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Figures

Figure 1

Figure 1. The structure of glycoamino acids introduced to NT and the C-terminal hexapeptide, NT(8–13)

Figure 2

Figure 2. Synthesis of Fmoc-Glu-(β-Lac-PEG3-amide), 4, the intermediate compound for the preparation of NT(8–13)-PEG3-Lac

a) Pentafluorophenol (PFP), DCC, CH2Cl2, rt, 72%; b) DIPEA, CH2Cl2, 57%; c) H2, 10% Pd/C, EtOAc, 70%.

Figure 3

Figure 3. HPLC profile of NT-TF before deacetylation (A) and after deacetylation (B)

The retention time changed from 19.5 min to 16.3 min during deacetylation reaction. The analogs were purified by RP-HPLC using a semi-preparative C18 column in a linear gradient (5–65%) of Buffer B (90% acetonitrile, 0.1% TFA). The molecular weight of the analogs was confirmed by MALDI-TOF mass spectrometry.

Figure 4

Figure 4. Receptor binding curve for NT analogs (top) and the agonist activity from functional assay (bottom)

The binding affinity of NT and NT-TF for NTS1 remains same, 0.5 nM. EC50 values in intracellular calcium mobilization assay for NT and NT-TF are 1.1 nM and 0.9 nM, respectively.

Figure 5

Figure 5. Dose response curves of NT and a representative glycosylated NT analog, NT-TF, in the 6 Hz (32 mA) anticonvulsant assay in mice following the icv administration

The log values of dose were plotted against the percentage of mice protected from seizures. The ED50 values for NT and NT-TF were 1.0 pmol and 0.4 pmol, respectively.

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