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IN SILICO SCREENING AND IN VIVO EVALUATION OF POTENTIAL ANALGESIC EFFECT OF NEW NEUROTENSIN (NT8-13) PEPTIDE MIMETICS

SILVIA MIHAYLOVA #*, TAMARA PAIPANOVA ²#, KALOYAN GEORGIEV #

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This study aimed to optimise the analgesic effects of neurotensin, which activates NTS1 and NTS2 receptors. Modifications were made to enhance selectivity towards NTS2 and prevent proteolytic degradation of the active neurotensin (8-13) sequence. Six peptides (T1 - T6) were synthesised via solid - phase peptide synthesis (SPPS) using a 2 - chlorotrityl chloride resin and the Fmoc/tBu strategy. HOBt/DIPEA served as the coupling mixture in a plastic syringe. Lipinski's rule of five (Ro5) parameters were determined using Molinspiration Cheminformatics software, assessing topological polar surface area (TPSA) and percentage of absorption (%ABS) in silico. Antinociceptive activity was evaluated in male ICR mice using the acetic acid writhing test (antipyretic type analgesia) and the hot plate test (narcotic type analgesia) after intraperitoneal administration (5 mg/kg bw). The native neurotensin fragment significantly reduced pain sensitivity. Peptide T2 showed a mild analgesic effect, while peptides T1, T4, and T5 had minimal impact. Notably, peptide T3 significantly reduced abdominal contractions in the peripheral pain model. Preparative high - performance liquid chromatography (HPLC) purification yielded the synthesised neurotensin analogues (T1 - T6) with yields of 26 - 59% and purity > 95%. Their molecular weights (Mw) fell within the extended Ro5 limits (Mw ≤ 1000 Da), and their miLogP values indicated hydrophilic properties. This study provides insights into modifying short - chain peptides, aiding future research in neurotensin mimetics for targeted pain modulation therapeutics.