Protein-protein interactions (PPIs) are essential to vital cellular processes, and are involved in numerous pathophysiological states where they serve as potential targets for therapeutic intervention. PPIs have generally been perceived as difficult to target with small organic molecules, since they are often characterized by large, flat, and hydrophobic interfaces.
A class of PPIs is one involving PDZ domains [PDZ is an abbreviation for postsynaptic density protein-95 (PSD-95), Drosophila homologue discs large tumor suppressor (DlgA) and zonula occludens-1 protein (ZO-1)]. PDZ domains often function as modules in scaffolding proteins that are involved in assembling large protein complexes in the cell, and are highly abundant in eukaryotic organisms. PDZ domains comprise about 90 amino acids and generally interact with only a few amino acids of the C-terminal part of the interacting protein. PDZ domains are typically divided into three classes according to the sequence of their ligands. PSD-95, contains three PDZ domains, PDZ1-3, which bind peptide ligands with the consensus sequence Glu/Gln-Ser/Thr-X-Val-COOH, thus being designated class I PDZ domains.
The structural basis for the interaction of PDZ domains with C-terminal peptides was first elucidated by an X-ray crystallographic structure of PDZ3 of PSD-95 complexed with a native peptide ligand, CRIPT. PDZ3 contains six antiparallel β-strands (βA-βF) and two α-helices (αA and αB), and the C-terminal peptide ligand binds as an additional anti-parallel β-strand into a groove between the βB strand and αB helix. Two residues in the peptide ligand are considered particularly important for affinity and specificity, the first (P0) and the third (P−2) amino acids (counting from the C-terminal). The side chain of the amino acid in P0 position projects into a hydrophobic pocket and an amino acid with an aliphatic side chains (Val, Ile and Leu) is required. In the PDZ3-CRIPT structure, the hydroxyl oxygen of Thr (P−2) forms a hydrogen bond with the nitrogen of an imidazole side chain of His372, which is a highly conserved residue in class I PDZ domains. A conserved Gly-Leu-Gly-Phe (position 322-325 in PDZ3) motif and a positively charged residue (Arg318 in PDZ3) of PDZ domains mediate binding to the C-terminal carboxylate group.
The PDZ1 and PDZ2 domains of PSD-95 interact with a number of proteins including a group of ionotropic glutamate receptors, the N-methyl- D-aspartate (NMDA) receptor. This receptor is a hetero tetrameric ion channel generally formed by the two subunits, NR1 and NR2, and gated by glutamate and glycine. The NMDA receptor (NMDAR) plays a key role in several diseases in the CNS brain, but development of drugs that directly interact with the NMDA receptor has been difficult. Therefore, there is a need for alternative approaches to modulate the NMDA receptor activity; one such approach is perturbation of the PSD-95/NMDA receptor interaction. PSD-95 simultaneously binds the NMDA receptor, primarily NR2A and NR2B subunits, and the enzyme neuronal nitric oxide synthase (nNOS) through PDZ1 or PDZ2 (FIG. 1). Activation of the NMDA receptor causes an influx of Ca2+, which activates nNOS thereby leading to nitric oxide (NO) generation. Thus, PSD-95 mediates a specific association between NMDA receptor activation and NO production, which can be detrimental for the cells if sustained for a longer period (FIG. 1).
Inhibition of the PSD-95/NMDA receptor interaction is known to prevent ischemic brain damage in mice, presumably by impairing the functional link between Ca2+ entry and NO production, while the physiological function of the NMDA receptor remains intact.1 Uncoupling of PSD-95 from the NR2B subunit was achieved by a nonapeptide, corresponding to the C-terminal of NR2B, fused to HIV-1 Tat peptide, known for its ability to facilitate membrane permeability. This 20-mer peptide (Tat-NR2B, 3, Table 1) is currently in clinical trials as a potential drug for the treatment of cerebrovascular ischemia, as seen in stroke.2-4 However, peptides are generally not attractive drug candidates due to their poor bioavailability, instability in vivo and low patient tolerance due to development of “immunogenicity” to the administered peptide.
The binding pocket of PDZ domains, which embeds a small, linear peptide motif, has a relatively small surface area and a non-favourable geometry, which makes PDZ domains difficult to target with small molecules. These difficulties are reflected by the very low number of small molecule inhibitors of PDZ domain interactions.