Background: The 18 kDa translocator protein (TSPO), previously known as the peripheral-
type benzodiazepine receptor, plays a key role for the synthesis of neurosteroids by promoting
transport of cholesterol from the outer to the inner mitochondrial membrane, which is the ratelimiting
step in neurosteroid biosynthesis. Neurosteroids interact with nonbenzodiazepine site of
GABAa receptor causing an anxiolytic effect without the side effects.
Methods: Using the original peptide drug-based design strategy, the first putative dipeptide ligand
of the TSPO N-carbobenzoxy-L-tryptophanyl-L-isoleucine amide (GD-23) was obtained. Molecular
docking of GD-23 in the active pocket of the TSPO receptor using Glide software was carried
out. The lead compounds GD-23 and its analogues were synthesized using activated succinimide
esters coupling method. The anxiolytic activity of GD-23 and its analogues was investigated in
vivo, using two validated behavioral tests, illuminated open field and elevated plus-maze.
Results: The in vivo studies revealed that the following parameters are necessary for the
manifestation of anxiolytic activity of new compounds: the L-configuration of tryptophan, the
presence of an amide group at the C-terminus, the specific size of the N-acyl substituent at the Nterminus.
Compound GD-23 (N-carbobenzoxy-L-tryptophanyl-L-isoleucine amide) demonstrated
a high anxiolytic-like effect in the doses of 0.05–1.0 mg/kg i.p. comparable with that of diazepam.
Compound GD-23 was also active in the open field test when was administered orally in the doses
of 0.1-5.0 mg/kg. The involvement of TSPO receptor in the mechanism of anxiolytic-like activity
of new compounds was proved by the antagonism of compound GD-23 with TSPO selective inhibitor
PK11195 as well as with inhibitors of enzymes which are involved in the biosynthesis of
neurosteroids, trilostane and finasteride.
Conclusion: A series of N-acyl-tryptophanyl-containing dipeptides were designed and synthesized
as 18 kDa translocator protein (TSPO) ligands. Using a drug-based peptide design method a series
of the first dipeptide TSPO ligands have been designed and synthesized and their anxiolytic activity
has been evaluated. In general, some of the compounds displayed a high level of anxiolytic efficacy
comparable with that of diazepam. The involvement of TSPO receptor in the mechanism of
anxiolytic activity of new compounds was proved using two methods. On this basis, the N-acyl-Ltryptophanyl-
isoleucine amides could potentially be a novel class of TSPO ligands with anxiolytic