Synthesis and In Vitro Neuroprotective Activity of Analogs with Various Spacer Lengths of a Dimeric Dipeptide Mimetic (GK-2) of Nerve Growth Factor

Dimeric dipeptide mimetic GK-2 or hexamethylenediamide bis-(N-monosuccinyl-L-glutamyl-L-lysine), which activated TrkA receptors and exhibited neuroprotective activity in vitro (10^–5 – 10^–9 M) and in vivo (0.05 – 5 mg/kg i.p.), was designed by us earlier based on the beta-turn of the nerve growth factor (NGF) fourth loop. The effect of dimerizing spacer length on the manifestation of neuroprotective activity was studied by synthesizing GK-2 (11 σ-bonds) analogs tetramethylenediamide bis-(N-monosuccinyl-L-glutamyl-Llysine) (GK-2c); pentamethylenediamide bis-(N-monosuccinyl-L-glutamyl-L-lysine) (GK-2b); and trimethylenediamide bis-(N-monosuccinyl-L-glutamyl-L-lysyl-6-aminohexanoic acid) (GK-2a) containing 9, 10, and 22 σ-bonds, respectively, between the lysine C_α-atoms of the dipeptide fragments. Neuroprotective activity was investigated in vitro using HT-22 mouse hippocampal neuronal cell culture under H₂O₂-induced oxidative stress and showed that the neuroprotective activity as measured by the minimum effective concentration was 104 times less if the distance between the dipeptide fragments increased from 11 to 22 σ-bonds. The neuroprotective activity decreased insignificantly if this distance was decreased from 11 to 10 σ bonds (GK-2b) and disappeared with 9 σ -bonds (GK-2c). Therefore, the hexamethylene spacer was optimal although the distance between the corresponding amino-acid moieties of natural NGF from an x-ray structure analysis was considerably greater at 33 σ-bonds. This contradiction could have arisen because two ionically bound mimetic molecules interacted with TrkA receptors. Another possible explanation was that TrkA molecules could approach closer in the presence of bis-dipeptide GK-2 than NGF.