A high-affinity, dimeric inhibitor of PSD-95 bivalently interacts with PDZ1-2 and protects against ischemic brain damage

Abstract

Inhibition of the ternary protein complex of the synaptic scaffolding protein postsynaptic density protein-95 (PSD-95), neuronal nitric oxide synthase (nNOS), and the N-methyl-D-aspartate (NMDA) receptor is a potential strategy for treating ischemic brain damage, but high-affinity inhibitors are lacking. Here we report the design and synthesis of a novel dimeric inhibitor, Tat-NPEG4ðIETDVÞ 2 (Tat-N-dimer), which binds the tandem PDZ1-2 domain of PSD-95 with an unprecedented high affinity of 4.6 nM, and displays extensive protease-resistance as evaluated in vitro by stability-measurements in human blood plasma. X-ray crystallography, NMR, and small-angle X-ray scattering (SAXS) deduced a true bivalent interaction between dimeric inhibitor and PDZ1-2, and also provided a dynamic model of the conformational changes of PDZ1-2 induced by the dimeric inhibitor. A single intravenous injection of Tat-N-dimer (3 nmol∕g) to mice subjected to focal cerebral ischemia reduces infarct volume with 40% and restores motor functions. Thus, Tat-Ndimer is a highly efficacious neuroprotective agent with therapeutic potential in stroke.drug discovery | ischemic stroke | protein-protein interactions P rotein-protein interactions mediated by postsynaptic density protein-95 (PSD-95)/Discs-large/ZO-1 (PDZ) domains are important for intracellular signaling events, and several PDZ domains are potential drug targets for neuronal diseases and cancer (1, 2). The postsynaptic scaffolding protein PSD-95 simultaneously binds the N-methyl-D-aspartate (NMDA)-type of ionotropic glutamate receptors and the enzyme neuronal nitric oxide synthase (nNOS) through its PDZ1 and PDZ2 domains (3). Activation of the NMDA receptor causes influx of Ca 2þ , which activates nNOS thereby leading to nitric oxide generation (4), a key facilitator of glutamate-mediated excitotoxicity (5, 6). Ligands that bind to the first two PDZ domains of PSD-95 inhibit the formation of the ternary nNOS/PSD-95/NMDA receptor complex and uncouple the harmful production of nitric oxide from NMDA receptor activity (Fig. 1A). As PSD-95 inhibition does not affect ion-flux (7) or prosurvival signaling pathways (8) mediated by the NMDA receptor, it is believed that compounds targeting PDZ1 and PDZ2 of PSD-95 can provide an efficient and safe treatment of ischemic brain damage (9), where excitotoxicity is known to dominate in the acute poststroke period, as well as other NMDA receptor-related disorders such as chronic pain and Alzheimer's disease (10-14).The shallow and elongated binding pocket of PDZ domains generally favor binding of peptides or peptide analogues and so far no drug-like small-molecule inhibitors of PDZ domains with affinities below 5 μM have been identified (15). Accordingly, the most advanced PSD-95 inhibitor is a 20-mer peptide, Tat-NR2B9c (7, 8, 16), composed of nine amino acids corresponding to the C-terminal of the GluN2B subunit of the NMDA receptor, fused to the HIV-1 Tat peptide (17). This peptide has shown promising effects against ischemic brain damage in rats (...

Efficacy of the PSD95 inhibitor Tat-NR2B9c in mice requires dose translation between species

“…Since the literature on dose translation between species suggests that mice require approximately double the rat dose for dose equivalence, [22][23][24] we examined the impact of Tat-NR2B9c at both the 3 nM/g dose previously reported to be ineffective in mice 19,20 as well a higher dose of 10 nM/g. These were tested in two separate studies that differed only in the duration of tMCAO.…”

“…Thus, it was surprising to us that two groups that tested Tat-NR2B9c in mice reported a lack of efficacy in their hands. 19,20 Both groups used a dose of 3 nMole/g, which is effective in rats. 2,4,10,[14][15][16] We had never validated Tat-NR2B9c in mice since our research did not require genetically modified animals and mouse models reportedly exhibit high variability between labs even using the same strain, duration of MCAo, and survival period.…”

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 .…”

Membrane-permeable C-terminal Dopamine Transporter Peptides Attenuate Amphetamine-evoked Dopamine Release*

“…Also, TAT fusion peptides with sequences from the C terminus of the N-methyl-D-aspartate (NMDA)-type ionotropic glutamate receptor were shown to perturb NMDA receptor-PSD-95 interaction and thereby attenuate ischemic brain damage (33). Targeting the PDZ domain(s) of PSD-95 represents a pharmacological strategy for treatment of acute stroke (33,36).…”

“…1A) (32,33). Previous reports have successfully used TAT-conjugated dominant-negative peptides to efficiently perturb particular protein-protein interactions (32)(33)(34)(35)(36). In addition to a TAT-conjugated WT peptide (TAT-C24WT), we generated an equivalent scrambled peptide (TAT-C24Scr) (Fig.…”