Modified Peptides as Potent Inhibitors of the Postsynaptic Density-95/N-Methyl-d-Aspartate Receptor Interaction

Abstract

The protein-protein interaction between the NMDA receptor and its intracellular scaffolding protein, PSD-95, is a potential target for treatment of ischemic brain diseases. An undecapeptide corresponding to the C-terminal of the NMDA was used as a template for finding lead candidates for the inhibition of the PSD-95/NMDA receptor interaction. Initially, truncation and alanine scan studies were carried out, which resulted in a pentapeptide with wild-type affinity, as examined in a fluorescence polarization assay. Further examination was performed by systematic substitutions with natural and unnatural amino acids, which disclosed a tripeptide with micromolar affinity and N-methylated tetrapeptides with improved affinities. Molecular modeling studies guided further N-terminal modifications and introduction of a range of N-terminal substitutions dramatically improved affinity. The best compound, N-cyclohexylethyl-ETAV (56), demonstrated up to 19-fold lower K i value ( K i = 0.94 and 0.45 microM against PDZ1 and PDZ2 of PSD-95, respectively) compared to wild-type values, providing the most potent inhibitors of this interaction reported so far. These novel and potent inhibitors provide an important basis for development of small molecule inhibitors of the PSD-95/NMDA receptor interaction.

Identification of a small-molecule inhibitor of the PICK1 PDZ domain that inhibits hippocampal LTP and LTD

“…Given that screening of phage libraries has identified short peptides with affinities in this range, e.g., for the Erbin PDZ domain (38), it is not unlikely that the nonpeptide small-molecule ligands can achieve such high affinity. Moreover, it is of interest that peptidomimetics have been developed against the PDZ domain of α1-syntrophin and PDZ1 and PDZ2 of PSD-95 with affinities of ≈0.5-1 μM (39,40). PDZ domain selectivity also may be of some concern in the design of inhibitors, given the structural conservation and the fact that the human genome encodes up to 540 different PDZ domains.…”

“…The molecular biology procedures and constructs are described in SI Methods. GST-PICK1 and N-terminally polyhistidine tagged PSD-95 PDZ domains were expressed in Escherichia coli BL21(DE3) pLysS (Novagen) and purified as described (19,39,40) (SI Methods).…”

Molecular basis of the alternative recruitment of GABAA versus glycine receptors through gephyrin

“…This principle has been demonstrated for the interaction between the N-methyl-D-aspartate-type glutamate receptors and the scaffolding postsynaptic density protein 95 (PSD-95), which has been targeted by peptide-based inhibitors [47][48][49][50][51][52] . This approach has shown great promise both as a pharmacological tool 51,53 and, in particular, in the development of therapeutically relevant compounds 54,55 .…”

Targeting the cyclin-binding groove site to inhibit the catalytic activity of CDK2/cyclin A complex using p27KIP1-derived peptidomimetic inhibitors

“…This method allows extension of the peptides with pharmacologically acceptable fragment hits. Unnatural amino acid derivatives such as 2-amino-3-phenylpropanoic acid, 2-amino butyric acid, chlorophenyl, and different derivatives were added at the N-terminal end of the LFG residues to mimic the amide bond of the peptide [7]. ChemDraw Ultra 11.0 suite was employed to draw the 2D structure of the LFG sequence and a number of modifications.…”

“…Now, the belief has changed and it is comprehended that not all the substrate protein residues are essential for interaction and only few are crucial for binding in the active site of protein [7]. Earlier in vitro and in vivo studies proved that cyclin-dependent kinase inhibitor (CDKI)-derived peptides specifically bind to CBG and induce antitumor effects [23].…”