Development of a Potent Cyclic Peptide Inhibitor of the nNOS/PSD-95 Interaction.

The complex between the N-methyl-d-aspartate receptor (NMDAR), neuronal nitric oxide synthase (nNOS), and the postsynaptic density protein-95 (PSD-95) is an attractive therapeutic target for the treatment of acute ischemic stroke. The complex is formed via the PDZ protein domains of PSD-95, and efforts to disrupt the complex have generally been based on C-terminal peptides derived from the NMDAR. However, nNOS binds PSD-95 through a ß-hairpin motif, providing an alternative starting point for developing PSD-95 inhibitors. Here, we designed a cyclic nNOS ß-hairpin mimetic peptide and generated cyclic nNOS ß-hairpin peptide arrays with natural and unnatural amino acids (AAs), which provided molecular insights into this interaction. We then optimized cyclic peptides and identified a potent inhibitor of the nNOS/PSD-95 interaction, with the highest affinity reported thus far for a peptide macrocycle inhibitor of PDZ domains, which serves as a template for the development of treatment for acute ischemic stroke.

Targeting the APP-Mint2 Protein-Protein Interaction with a Peptide-Based Inhibitor Reduces Amyloid-ß Formation.

There is an urgent need for novel therapeutic approaches to treat Alzheimer's disease (AD) with the ability to both alleviate the clinical symptoms and halt the progression of the disease. AD is characterized by the accumulation of amyloid-ß (Aß) peptides which are generated through the sequential proteolytic cleavage of the amyloid precursor protein (APP). Previous studies reported that Mint2, a neuronal adaptor protein binding both APP and the γ-secretase complex, affects APP processing and formation of pathogenic Aß. However, there have been contradicting results concerning whether Mint2 has a facilitative or suppressive effect on Aß generation. Herein, we deciphered the APP-Mint2 protein-protein interaction (PPI) via extensive probing of both backbone H-bond and side-chain interactions. We also developed a proteolytically stable, high-affinity peptide targeting the APP-Mint2 interaction. We found that both an APP binding-deficient Mint2 variant and a cell-permeable PPI inhibitor significantly reduced Aß42 levels in a neuronal in vitro model of AD. Together, these findings demonstrate a facilitative role of Mint2 in Aß formation, and the combination of genetic and pharmacological approaches suggests that targeting Mint2 is a promising therapeutic strategy to reduce pathogenic Aß levels.

Site-Specific Phosphorylation of PSD-95 PDZ Domains Reveals Fine-Tuned Regulation of Protein-Protein Interactions.

The postsynaptic density protein of 95 kDa (PSD-95) is a key scaffolding protein that controls signaling at synapses in the brain through interactions of its PDZ domains with the C-termini of receptors, ion channels, and enzymes. PSD-95 is highly regulated by phosphorylation. To explore the effect of phosphorylation on PSD-95, we used semisynthetic strategies to introduce phosphorylated amino acids at four positions within the PDZ domains and examined the effects on interactions with a large set of binding partners. We observed complex effects on affinity. Most notably, phosphorylation at Y397 induced a significant increase in affinity for stargazin, as confirmed by NMR and single molecule FRET. Additionally, we compared the effects of phosphorylation to phosphomimetic mutations, which revealed that phosphomimetics are ineffective substitutes for tyrosine phosphorylation. Our strategy to generate site-specifically phosphorylated PDZ domains provides a detailed understanding of the role of phosphorylation in the regulation of PSD-95 interactions.

Importance of a Conserved Lys/Arg Residue for Ligand/PDZ Domain Interactions as Examined by Protein Semisynthesis.

PDZ domains are ubiquitous small protein domains that are mediators of numerous protein-protein interactions, and play a pivotal role in protein trafficking, synaptic transmission, and the assembly of signaling-transduction complexes. In recent years, PDZ domains have emerged as novel and exciting drug targets for diseases (in the brain in particular), so understanding the molecular details of PDZ domain interactions is of fundamental importance. PDZ domains bind to a protein partner at either a C-terminal peptide or internal peptide motifs. Here, we examined the importance of a conserved Lys/Arg residue in the ligand-binding site of the second PDZ domain of PSD-95, by employing a semisynthetic approach. We generated six semisynthetic PDZ domains comprising different proteogenic and nonproteogenic amino acids representing subtle changes of the conserved Lys/Arg residue. These were tested with four peptide interaction partners, representing the two different binding modes. The results highlight the role of a positively charged amino acid in the ß1-ß2 loop of PDZ domains, and show subtle differences for canonical and noncanonical interaction partners, thus providing additional insight into the mechanism of PDZ/ligand interaction.

Ligand binding to the PDZ domains of postsynaptic density protein 95.

Cellular scaffolding and signalling is generally governed by multidomain proteins, where each domain has a particular function. Postsynaptic density protein 95 (PSD-95) is involved in synapse formation and is a typical example of such a multidomain protein. Protein-protein interactions of PSD-95 are well studied and include the following three protein ligands: (i)N-methyl-d-aspartate-type ionotropic glutamate receptor subunit GluN2B, (ii) neuronal nitric oxide synthase and (iii) cysteine-rich protein (CRIPT), all of which bind to one or more of the three PDZ domains in PSD-95. While interactions for individual PDZ domains of PSD-95 have been well studied, less is known about the influence of neighbouring domains on the function of the respective individual domain. We therefore performed a systematic study on the ligand-binding kinetics of PSD-95 using constructs of different size for PSD-95 and its ligands. Regarding the canonical peptide-binding pocket and relatively short peptides (up to 15-mer), the PDZ domains in PSD-95 by and large work as individual binding modules. However, in agreement with previous studies, residues outside of the canonical binding pocket modulate the affinity of the ligands. In particular, the dissociation of the 101 amino acid CRIPT from PSD-95 is slowed down at least 10-fold for full-length PSD-95 when compared with the individual PDZ3 domain.

Biochemical investigations of the mechanism of action of small molecules ZL006 and IC87201 as potential inhibitors of the nNOS-PDZ/PSD-95-PDZ interactions.

ZL006 and IC87201 have been presented as efficient inhibitors of the nNOS/PSD-95 protein-protein interaction and shown great promise in cellular experiments and animal models of ischemic stroke and pain. Here, we investigate the proposed mechanism of action of ZL006 and IC87201 using biochemical and biophysical methods, such as fluorescence polarization (FP), isothermal titration calorimetry (ITC), and (1)H-(15)N HSQC NMR. Our data show that under the applied in vitro conditions, ZL006 and IC87201 do not interact with the PDZ domains of nNOS or PSD-95, nor inhibit the nNOS-PDZ/PSD-95-PDZ interface by interacting with the ß-finger of nNOS-PDZ. Our findings have implications for further medicinal chemistry efforts of ZL006, IC87201 and analogues, and challenge the general and widespread view on their mechanism of action.

The role of backbone hydrogen bonds in the transition state for protein folding of a PDZ domain.

Backbone hydrogen bonds are important for the structure and stability of proteins. However, since conventional site-directed mutagenesis cannot be applied to perturb the backbone, the contribution of these hydrogen bonds in protein folding and stability has been assessed only for a very limited set of small proteins. We have here investigated effects of five amide-to-ester mutations in the backbone of a PDZ domain, a 90-residue globular protein domain, to probe the influence of hydrogen bonds in a ß-sheet for folding and stability. The amide-to-ester mutation removes NH-mediated hydrogen bonds and destabilizes hydrogen bonds formed by the carbonyl oxygen. The overall stability of the PDZ domain generally decreased for all amide-to-ester mutants due to an increase in the unfolding rate constant. For this particular region of the PDZ domain, it is therefore clear that native hydrogen bonds are formed after crossing of the rate-limiting barrier for folding. Moreover, three of the five amide-to-ester mutants displayed an increase in the folding rate constant suggesting that the hydrogen bonds are involved in non-native interactions in the transition state for folding.

Probing backbone hydrogen bonding in PDZ/ligand interactions by protein amide-to-ester mutations.

PDZ domains are scaffolding modules in protein-protein interactions that mediate numerous physiological functions by interacting canonically with the C-terminus or non-canonically with an internal motif of protein ligands. A conserved carboxylate-binding site in the PDZ domain facilitates binding via backbone hydrogen bonds; however, little is known about the role of these hydrogen bonds due to experimental challenges with backbone mutations. Here we address this interaction by generating semisynthetic PDZ domains containing backbone amide-to-ester mutations and evaluating the importance of individual hydrogen bonds for ligand binding. We observe substantial and differential effects upon amide-to-ester mutation in PDZ2 of postsynaptic density protein 95 and other PDZ domains, suggesting that hydrogen bonding at the carboxylate-binding site contributes to both affinity and selectivity. In particular, the hydrogen-bonding pattern is surprisingly different between the non-canonical and canonical interaction. Our data provide a detailed understanding of the role of hydrogen bonds in protein-protein interactions.

Synthesis of peptides using tert-butyloxycarbonyl (Boc) as the α-amino protection group.

The use of the tert-butyloxycarbonyl (Boc) as the Nα-amino protecting group in peptide synthesis can be advantageous in several cases, such as synthesis of hydrophobic peptides and peptides containing ester and thioester moieties. The primary challenge of using Boc SPPS is the need for treatment of the resin-bound peptide with hazardous hydrogen fluoride (HF), which requires special equipment.

An expanded view of the protein folding landscape of PDZ domains.

Most protein domains fold in an apparently co-operative and two-state manner with only the native and denatured states significantly populated at any experimental condition. However, the protein folding energy landscape is often rugged and different transition states may be rate limiting for the folding reaction under different conditions, as seen for the PDZ protein domain family. We have here analyzed the folding kinetics of two PDZ domains and found that a previously undetected third transition state is rate limiting under conditions that stabilize the native state relative to the denatured state. In light of these results, we have re-analyzed previous folding data on PDZ domains and present a unified folding mechanism with three distinct transition states separated by two high-energy intermediates. Our data show that sequence composition tunes the relative stabilities of folding transition states within the PDZ family, while the overall mechanism is determined by topology. This model captures the kinetic folding mechanism of all PDZ domains studied to date.

Cell-permeable and plasma-stable peptidomimetic inhibitors of the postsynaptic density-95/N-methyl-D-aspartate receptor interaction.

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.

Modified peptides as potent inhibitors of the postsynaptic density-95/N-methyl-D-aspartate receptor interaction.

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.