The protein--protein interaction between the NMDA receptor and its intracellular scaffolding protein, PSD-95, is a potential target for treating ischemic brain diseases, neuropathic pain, and Alzheimer's disease. We have previously demonstrated that N-alkylated tetrapeptides are potent inhibitors of this interaction, and here, this template is exploited for the development of blood plasma-stable and cell-permeable inhibitors. Initially, we explored both the amino acid sequence of the tetrapeptide and the nature of the N-alkyl groups, which consolidated N-cyclohexylethyl-ETAV (1) as the most potent and selective compound. Next, the amide moieties of N-methylated ETAV were systematically replaced with thioamides, demonstrating that one of three amide bonds could be replaced without compromising the affinity. Subsequent optimization of the N-alkyl groups and evaluation of cell permeability led to identification of N-cyclohexylethyl-ETA(S)V (54) as the most potent, plasma-stable and cell-permeable inhibitor, which is a promising tool in unraveling the therapeutic potential of the PSD-95/NMDA receptor interaction.

Dimeric ligands can be potent inhibitors of protein-protein or enzyme-substrate interactions. They have increased affinity and specificity toward their targets due to their ability to bind two binding sites simultaneously and are therefore attractive in drug design. However, few studies have addressed the kinetic mechanism of interaction of such bivalent ligands. We have investigated the binding interaction of a recently identified potent plasma-stable dimeric pentapeptide and PDZ1-2 of postsynaptic density protein-95 (PSD-95) using protein engineering in combination with fluorescence polarization, isothermal titration calorimetry, and stopped-flow fluorimetry. We demonstrate that binding occurs via a two-step process, where an initial binding to either one of the two PDZ domains is followed by an intramolecular step, which produces the bidentate complex. We have determined all rate constants involved in the binding reaction and found evidence for a conformational transition of the complex. Our data demonstrate the importance of a slow dissociation for a successful dimeric ligand but also highlight the possibility of optimizing the intramolecular association rate. The results may therefore aid the design of dimeric inhibitors in general.Biological molecules that comprise two or more binding units enable di-or multivalent binding to their protein partners. This concept is well known in nature as a way to increase affinity and selectivity, such as in virus-cell and antibody-antigen recognition (1). Consequently, linking two ligands together can be a strategy to enhance binding of drug candidates to therapeutically relevant proteins by exploiting a bivalent binding site (2-5). It is proposed that such dimeric inhibitors will foster a more potent response by increasing the affinity toward their targets by some hundred-fold (6, 7). Indeed, in vitro binding studies have shown improved affinities of dimeric inhibitors toward their targets as compared with their monomeric counterparts (5, 8 -11). It is complex to predict the overall affinity enhancement by linking two ligands because the observed binding energy is not a direct summation of the binding energies of individual components, and the entropy and enthalpy compensation are difficult to estimate (6,7,12). Therefore, experimental determination of the binding mechanism of dimeric ligands is useful for future design of dimeric ligands. However, there are only a few cases where in solution methods have been used to determine the mechanism of interaction of such ligands (5,7,10). One class of proteins where dimeric ligands have been exploited in an attempt to develop potential inhibitors for therapeutically relevant interactions in the cell is the PDZ (PSD-95/Dlg/Zonula occludens-1) domain family of proteins (5, 8). PDZ domains constitute a class of protein-protein interacting modules that functions as scaffolds and adapters in signaling cascades, and they are found in a few hundred proteins in the human genome (13). PDZ domains generally bind to the C termini of thei...

The PDZ domain mediated interaction between the NMDA receptor and its intracellular scaffolding protein, PSD-95, is a potential target for treatment of ischemic brain diseases. We have recently developed a number of peptide analogues with improved affinity for the PDZ domains of PSD-95 compared to the endogenous C-terminal peptide of the NMDA receptor, as evaluated by a cell-free protein-protein interaction assay. However, it is important to address both membrane permeability and effect in living cells. Therefore a bioluminescence resonance energy transfer (BRET) assay was established, where the C-terminal of the NMDA receptor and PDZ2 of PSD-95 were fused to green fluorescent protein (GFP) and Renilla luciferase (Rluc) and expressed in COS7 cells. A robust and specific BRET signal was obtained by expression of the appropriate partner proteins and subsequently, the assay was used to evaluate a Tat-conjugated peptide for its ability to disrupt the PSD-95/NMDA receptor interaction in living cells.