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-NPEG4(IETDV)(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-N-dimer is a highly efficacious neuroprotective agent with therapeutic potential in stroke.

Fig. 1.

Dimeric PSD-95 inhibitors. (A) PSD-95 inhibitors targeting PDZ1-2 of PSD-95 block the formation of the ternary nNOS/PSD-95/NMDA receptor complex and uncouple the link between NMDA receptor activity and nitric oxide production, whereby neuroprotection against excitotoxicity is achieved. (B) Structures of O-dimer and N-dimer; and Tat-NPEG4(IETDV)₂ and Retroinverso-d-Tat-NPEG4(IETDV)₂, designated Tat-N-dimer and ReTat-N-dimer, respectively. Capital letters indicate l-amino acids, except for “N” (nitrogen), “O” (oxygen), “X” (N or O), and “R” (Tat or ReTat sequence), and lower case letters indicate d-amino acids.

Fig. 2.

Affinity, stability, and blood-brain barrier permeability. (A) Affinity towards PDZ1-2 of PSD-95 for Tat-N-dimer, ReTat-N-dimer and Tat-NR2B9c as measured by fluorescence polarization (Left); and stability in human blood plasma in vitro at 37 °C (Right). Data are shown as mean ± SEM, N≥3. Mean affinity inhibition constants (K_i) and half-lives (T_1/2) are shown in the table below. (B) Blood-brain barrier permeability of fluorescent analogues in nonmanipulated mice. The bar graph shows the mean fluorescence intensity of coronal brain sections of mice one hour after intravenous injection of F-N-dimer (p = 0.22), F-Tat-N-dimer (p = 0.0002), F-ReTat-N-dimer (p = 0.0008), F-Tat-NR2B9c (p = 0.011) compared to saline. Representative pictures of the cortical area of the coronal brain sections are shown. A nuclei-DAPI staining (blue) is included for anatomical reference showing layers I-VI of the frontal cortex. F: 5-FAM; Data are presented as mean ± SEM; ∗ / ∗∗ / ∗∗ ∗: p < 0.05/0.01/0.001 (nonparametric Mann-Whitney). Scale bar: 100 μm.

Fig. 3.

Biophysical characterization of PDZ1-2 from PSD-95 and interaction with inhibitors. (A) X-ray crystal structure of apo PDZ1-2 (PDB 3ZRT). Two conserved histidine residues (His130 and His225) important for ligand binding and with a relative distance of 42 Å are highlighted. (B) ¹H-¹⁵N correlation spectra of free [¹⁵N, ¹³C]-O-dimer (black contours) and [¹⁵N, ¹³C]-O-dimer in complex with PDZ1-2 from PSD-95 (red contours). Amino acid assignment is indicated with capital letters. (C) Secondary structure propensities (SSP) of unbound [¹⁵N, ¹³C]-O-dimer (black bars) and [¹⁵N, ¹³C]-O-dimer bound to PDZ1-2 (red bars) as calculated using the program SSP. A value of one indicates a fully formed α-helix and a value of minus one indicates a fully extended structure whereas a value around zero is indicative of a random coil. (D) Pair distance distribution functions derived from Fourier transformation of the SAXS data presented as the relative occurrence of distances (arbitrary scale) against distances in nm. Data from apo PDZ1-2 (green), PDZ1-2 with monomeric ligand (IETAV) (blue), and PDZ1-2 with dimeric ligand (O-dimer) (gray) are shown. The average radius of the two PDZ domains is visible as the first major peak (around 2 nm, PDZ1 and PDZ2 have similar radii), and the distance between the center of mass of PDZ1 and PDZ2 is seen as the second peak. (E) Surface representations of PDZ1-2 bound to IETAV (blue) or O-dimer (gray), showing that PDZ1-2 adopts a more compact conformation when bound to O-dimer compared to IETAV. (F) Comparison of the high-resolution O-dimer/PDZ1-2 model (black) and the experimental SAXS scattering data (red). X = 1.15. (G) High-resolution model of PDZ1-2 in complex with O-dimer.

Fig. 4.

Neuroprotection of Tat-N-dimer, 6 and 48 h after pMCAO in mice. (A) Compounds were administered 30 min postsurgery, followed by a survival period of 5.5 or 47.5 h. (B) Mean infarct volumes 6 h after pMCAO of mice treated with Tat-N-dimer (N = 17), Saline (N = 16), or Tat-NR2B9c (N = 18). (C) Immunofluorescent staining of cells containing Tat-based compounds, Tat-N-dimer and Tat-NR2B9c, 6 h after pMCAO, colabelled with nuclei-DAPI staining (blue). Sections from both saline-injected mice, and Tat-N-dimer-treated mice where the antibody was substituted with isotype control, were devoid of signal. Scale bar: 20 μm (Upper), 40 μm (Lower). (D) Toluidine blue staining showing the ischemic brain damage 48 h after pMCAO. IF: Infarct; Ctx: Cortex; Str: Striatum. Scale bar: 1 mm. (E) Mean infarct volumes 48 h after pMCAO of mice treated with Tat-N-dimer (N = 17), Saline (N = 16), or Tat-NR2B9c (N = 17). (F-H) Motor function assessment of mice with 48 h postsurgical survival. (F) Mean total grip strength of both front paws before (baseline) and after pMCAO. P-values: < 0.0001 (Saline), 0.13 (Tat-N-dimer), 0.0011 (Tat-NR2B9c). (G) Ischemia-induced grip strength asymmetry in the front paws. P-values: 0.014 (Saline), 0.11 (Tat-N-dimer), 0.013 (Tat-NR2B9c). (H) Rotarod performance test over four trials (T1-T4). A learning component is observed along the trials in all groups, but treatment with Tat-N-dimer gave a more pronounced improvement (cf. T2) compared to saline, and increased endurance compared to Tat-NR2B9c (see text). (B, E–H) Data are shown as mean ± SEM; ∗ / ∗∗ / ∗∗ ∗: p < 0.05/0.01/0.001; (B and E) Nonparametric Mann-Whitney test; (F–G) Paired Student’s t test; (H) Wilcoxon matched pairs test.