A high-affinity, dimeric inhibitor of PSD-95bivalently interacts with PDZ1-2 and protectsagainst ischemic brain damageAnders Bacha,1, Bettina H. Clausenb, Magda Møllera, Bente Vestergaarda, Celestine N. Chic, Adam Roundd,Pernille L. Sørensena, Klaus B. Nissena, Jette S. Kastrupa, Michael Gajhedea, Per Jemthc, Anders S. Kristensena,Patrik Lundströme, Kate L. Lambertsenb, and Kristian Strømgaarda,1

aDepartment of Medicinal Chemistry, University of Copenhagen, DK-2100 Copenhagen, Denmark; bDepartment of Neurobiology Research, Institute ofMolecular Medicine, University of Southern Denmark, DK-5000 Odense, Denmark; cDepartment of Medical Biochemistry and Microbiology, UppsalaUniversity, BMC, SE-75123 Uppsala, Sweden; dEuropean Molecular Biology Laboratory, Grenoble Outstation, 38042 Grenoble, France; and eDepartmentof Physics, Chemistry and Biology, Division of Molecular Biotechnology, Linköping University, SE-58183, Linköping, Sweden

Edited by James A. Wells, University of California, San Francisco, San Francisco, CA, and approved January 4, 2012 (received for review August 23, 2011)

Inhibition of the ternary protein complex of the synaptic scaffold-ing protein postsynaptic density protein-95 (PSD-95), neuronalnitric 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 designand synthesis of a novel dimeric inhibitor, Tat-NPEG4ðIETDVÞ2(Tat-N-dimer), which binds the tandem PDZ1-2 domain of PSD-95with an unprecedented high affinity of 4.6 nM, and displays exten-sive protease-resistance as evaluated in vitro by stability-measure-ments in human blood plasma. X-ray crystallography, NMR, andsmall-angle X-ray scattering (SAXS) deduced a true bivalent inter-action between dimeric inhibitor and PDZ1-2, and also provided adynamic model of the conformational changes of PDZ1-2 inducedby the dimeric inhibitor. A single intravenous injection of Tat-N-di-mer (3 nmol∕g) to mice subjected to focal cerebral ischemia reducesinfarct volume with 40% and restores motor functions. Thus, Tat-N-dimer is a highly efficacious neuroprotective agent with therapeuticpotential in stroke.

drug discovery ∣ ischemic stroke ∣ protein-protein interactions

Protein-protein interactions mediated by postsynaptic densityprotein-95 (PSD-95)/Discs-large/ZO-1 (PDZ) domains areimportant for intracellular signaling events, and several PDZdomains are potential drug targets for neuronal diseases andcancer (1, 2). The postsynaptic scaffolding protein PSD-95 simul-taneously binds the N-methyl-D-aspartate (NMDA)-type of iono-tropic glutamate receptors and the enzyme neuronal nitric oxidesynthase (nNOS) through its PDZ1 and PDZ2 domains (3). Acti-vation of the NMDA receptor causes influx of Ca2þ, which acti-vates nNOS thereby leading to nitric oxide generation (4), a keyfacilitator of glutamate-mediated excitotoxicity (5, 6). Ligands thatbind to the first two PDZ domains of PSD-95 inhibit the formationof the ternary nNOS/PSD-95/NMDA receptor complex and un-couple the harmful production of nitric oxide from NMDA recep-tor activity (Fig. 1A). As PSD-95 inhibition does not affect ion-flux(7) or prosurvival signaling pathways (8) mediated by the NMDAreceptor, it is believed that compounds targeting PDZ1 and PDZ2of PSD-95 can provide an efficient and safe treatment of ischemicbrain damage (9), where excitotoxicity is known to dominate in theacute poststroke period, as well as other NMDA receptor-relateddisorders such as chronic pain and Alzheimer’s disease (10–14).

The shallow and elongated binding pocket of PDZ domainsgenerally favor binding of peptides or peptide analogues and so farno drug-like small-molecule inhibitors of PDZ domains with affi-nities below 5 μM have been identified (15). Accordingly, the mostadvanced PSD-95 inhibitor is a 20-mer peptide, Tat-NR2B9c (7, 8,16), composed of nine amino acids corresponding to the C-term-inal of the GluN2B subunit of the NMDA receptor, fused to theHIV-1 Tat peptide (17). This peptide has shown promising effects

against ischemic brain damage in rats (7, 8, 16), and is currentlyinvestigated in clinical trials (18). However, Tat-NR2B9c suffersfrom low affinity to PDZ1-2 of PSD-95 (19), which might limitits biological effectiveness and clinical potential. Here we presentdimeric inhibitors that evade these shortcomings by exploiting thatPSD-95 possesses two neighboring PDZ domains (Fig. 1A) andshow up to 1,000 fold improved affinity compared to Tat-NR2B9cas well as improved blood plasma stability. Our dimeric inhibitorspermeate the blood-brain barrier and demonstrate substantialneuroprotective properties in mice subjected to focal ischemicbrain damage. Biophysical studies demonstrate that the dimericdesign results in a bivalent binding mode, where the two ligandmoieties bind PDZ1 and PDZ2 simultaneously, hence explainingthe high affinity.

ResultsDesign and Activity of Novel Dimeric Inhibitors. We have previouslydemonstrated that the five C-terminal amino acids of GluN2Bare sufficient for maintaining wild-type binding affinity towardsthe individual PDZ1 (Ki ¼ 14 μM) and PDZ2 (Ki ¼ 3 μM) do-mains (19). Thus, by linking two pentapeptides (IETAV) with amonodisperse polyethylene glycol (PEG) linker of optimal length(PEG4), a high-affinity dimeric ligand, PEG4ðIETAVÞ2 (O-di-mer) (Fig. 1B), was obtained with Kd of 10 nM towards tandemPDZ1-2 of PSD-95 (20, 21). We here redesigned the PEG linker,and synthesized a unique PEG-based linker, termed NPEG(Scheme S1A), where the central oxygen of the PEG4 linker isreplaced by nitrogen, which upon linkage with two IETAV moi-eties provides NPEG4ðIETAVÞ2 (N-dimer) (Fig. 1B). In order toimprove permeability across the blood-brain barrier, we attachedthe cell-penetrating peptide sequences, Tat and its inverse D-ami-no acid-containing version Retroinverso-D-Tat (22), to the linkernitrogen, and used IETDVas ligand moiety, which is more selec-tive than IETAV towards PDZ1 and PDZ2 over PDZ3 (20). Thisresulted in dimeric ligands Tat-NPEG4ðIETDVÞ2 (Tat-N-dimer)and Retroinverso-D-Tat-NPEG4ðIETDVÞ2 (ReTat-N-dimer)(Fig. 1B, Scheme S1B, and Fig. S1).

Author contributions: A.B., B.H.C., B.V., C.N.C., M.G., P.J., A.S.K., K.L.L., and K.S. designedresearch; A.B., B.H.C., M.M., C.N.C., A.R., P.L.S., K.B.N., P.L., and K.L.L. performedresearch; A.B., B.H.C., B.V., C.N.C., J.S.K., M.G., P.L., K.L.L., and K.S. analyzed data; andA.B. and K.S. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Data deposition: Coordinates and structure factors for the PDZ1-2 crystal structure havebeen deposited in the Protein Data Bank, www.pdb.org (PDB ID code 3ZRT).1To whom correspondence may be addressed. E-mail: anba@farma.ku.dk or krst@farma.ku.dk.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1113761109/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1113761109 PNAS ∣ February 28, 2012 ∣ vol. 109 ∣ no. 9 ∣ 3317–3322

BIOCH

EMISTR

Y

Dow

nloa

ded

by g

uest

on

May

30,

202

1

http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1113761109/-/DCSupplemental/pnas.1113761109_SI.pdf?targetid=STXThttp://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1113761109/-/DCSupplemental/pnas.1113761109_SI.pdf?targetid=STXThttp://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1113761109/-/DCSupplemental/pnas.1113761109_SI.pdf?targetid=SF1www.pdb.orgwww.pdb.orgwww.pdb.orghttp://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1113761109/-/DCSupplementalhttp://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1113761109/-/DCSupplementalhttp://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1113761109/-/DCSupplementalhttp://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1113761109/-/DCSupplementalhttp://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1113761109/-/DCSupplementalhttp://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1113761109/-/DCSupplemental

For in vitro affinity evaluation we developed a fluorescencepolarization assay, where we prepared fluorescently (5-FAM, F)labeled N-dimer (F-N-dimer, Fig. S1) and used it as a high-affi-nity probe (Kd; mean� SEM: 7.8� 0.1 nM). This allowed us to

determine accurate Ki values for high-affinity ligands in a simpleand fast format, as compared to previous studies based on iso-thermal titration calorimetry (20, 23). Tat-N-dimer and ReTat-N-dimer displayed affinities (Ki; mean� SEM) of 4.6� 0.3 and5.1� 0.4 nM, respectively, against PDZ1-2 of PSD-95, whereasN-dimer bound PDZ1-2 with an affinity (Ki; mean� SEM) of9.3� 1 nM (Fig. 2A). Tat-N-dimer is thereby by far the most po-tent PDZ domain inhibitor described (15, 24–26), and provides a1,000-fold increase in affinity relative to monomeric Tat-NR2B9c(Ki; mean� SEM: 4;600� 300 nM, Fig. 2A).

A general concern of peptide-based compounds is their sus-ceptibility to enzymatic cleavage in biological fluids and tissues.Monomeric pentapeptide IETDV and Tat-NR2B9c showedhalf-lives (T1∕2; mean� SEM) of 37� 6 and 1;100� 300 min inhuman blood plasma in vitro, respectively, while Tat-N-dimer hada T1∕2 (mean� SEM) of 4;900� 100 min (Fig. 2A), thus having>100-fold improved stability compared to IETDV. Hence, thecombination of dimerization and attachment of cell-penetratingpeptides has a strikingly positive effect on protease stability.Furthermore, no detectable degradation was observed for ReTat-N-dimer within the period of measurements, demonstrating theeffect of incorporating D-amino acids into the cell-penetratingpeptide sequence (Fig. 2A).

To test the effect of introducing cell-penetrating peptideson the permeability of the blood-brain barrier, the ligands werelabeled with 5-FAM (F) and injected intravenously in mice(3 nmol∕g) 1 h prior to termination of the animals. Fluorescencemicroscopy of coronal brain sections revealed that F-Tat-N-dimer, F-ReTat-N-dimer, and F-Tat-NR2B9c resulted in a stronguniform fluorescence throughout the brain including layer I-VI ofthe frontal cortex, as compared to sections from F-N-dimer andsaline-treated mice (Fig. 2B). Thus blood-brain barrier perme-ability of the dimeric compounds is obtained by introductionof Tat or ReTat (7).

Fig. 1. Dimeric PSD-95 inhibitors. (A) PSD-95 inhibitors targeting PDZ1-2 ofPSD-95 block the formation of the ternary nNOS/PSD-95/NMDA receptor com-plex and uncouple the link between NMDA receptor activity and nitric oxideproduction, whereby neuroprotection against excitotoxicity is achieved. (B)Structures of O-dimer and N-dimer; and Tat-NPEG4ðIETDVÞ2 and Retroinver-so-D-Tat-NPEG4ðIETDVÞ2, designated Tat-N-dimer and ReTat-N-dimer, respec-tively. 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 lettersindicate 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 mea-sured 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 affinityinhibition constants (Ki) and half-lives (T1∕2) are shown in the table below. (B) Blood-brain barrier permeability of fluorescent analogues in nonmanipulatedmice. The bar graph shows themean fluorescence intensity of coronal brain sections of mice one hour after intravenous injection of F-N-dimer (p ¼ 0.22), F-Tat-N-dimer (p ¼ 0.0002), F-ReTat-N-dimer (p ¼ 0.0008), F-Tat-NR2B9c (p ¼ 0.011) compared to saline. Representative pictures of the cortical area of the coronalbrain 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 arepresented as mean� SEM; �∕ � �∕ � ��: p < 0.05∕0.01∕0.001 (nonparametric Mann-Whitney). Scale bar: 100 μm.

3318 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1113761109 Bach et al.

Dow

nloa

ded

by g

uest

on

May

30,

202

1

http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1113761109/-/DCSupplemental/pnas.1113761109_SI.pdf?targetid=SF1http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1113761109/-/DCSupplemental/pnas.1113761109_SI.pdf?targetid=SF1

Dimeric Inhibitor Bivalently Interacts with PDZ1-2, Leading to a Flex-ible and Extended Conformation. Previous kinetic and mutationalstudies have suggested that a dimeric ligand binds to PDZ1-2 ina two-step process, where initial binding to either of the two PDZdomains is followed by an intramolecular step leading to thebidentate complex (21). In order to provide insight into the struc-tural and dynamic properties of the complex between PDZ1-2and dimeric ligand, and to ascertain that both PDZ domains areoccupied simultaneously by peptide moieties from the same di-meric ligand, we elucidated the molecular mode of action of ourdimeric ligands. First, the domain orientation of the two PDZdomains in apo PDZ1-2 was determined by X-ray crystallogra-phy, which, however, showed that the two binding sites faceapproximately 180° away from each other in an extended confor-mation that cannot accommodate the dimeric ligands (Fig. 3Aand Table S1). Therefore, we prepared a labeled dimeric ligand,[15N, 13C]-O-dimer, and subjected it to structural NMR studies.In a heteronuclear single quantum correlation (HSQC) spectrumof the dimeric, symmetrical ligand alone, five peaks were ob-served, whereas ten different peaks appeared when the ligandwas incubated with PDZ1-2 (Fig. 3B). From secondary structuralcalculations based on the chemical shifts for the bound andunbound labeled dimeric ligand, we observed that the unboundligand exhibits random coil character, while the bound ligandadopts a β-stranded structure (Fig. 3C). Thus, the two peptidemoieties of the dimeric ligand face different protein environ-ments (PDZ1 and PDZ2) and β-strand character of the peptidemoieties is imposed by binding to PDZ1-2 similar to a canonicalPDZ-peptide binding mode (27).

The dynamic properties of PDZ1-2 upon ligand binding wererevealed by calculating correlation times for molecular tumblingof PDZ1 and PDZ2 in PDZ1-2 in complex withO-dimer based onR1 and R2 relaxation rate measurements. This gave correlationtimes slightly larger and with less deviation (14.6 and 13.6 ns forPDZ1 and PDZ2, respectively, Table S2) than those reported forPDZ1-2 bound to monomeric ligand (14.4 and 12.5 ns) (28), but

very different from values for unbound PDZ1-2 (18.3 and 18.2 ns,Table S2). Hence, these values suggest that the interdomainmotion of PDZ1-2 is only slightly more restricted in complex withdimeric ligand than with a monomeric ligand, while unboundPDZ1-2 is essentially rigid.

To further examine the structural changes in PDZ1-2 uponligand binding, we performed small-angle X-ray scattering(SAXS) studies of PDZ1-2. In apo PDZ1-2, a distinct distance ofapproximately 3.3 nm is observed (Fig. 3D), which likely repre-sents the average distance between PDZ1 and PDZ2 when noligand is bound to the protein. Interestingly, this distance in-creases upon binding of O-dimer (3.6 nm) and even more uponbinding of monomeric ligand IETAV (3.9 nm) (Fig. 3D andTable S3). This demonstrates that the closed and compact apoPDZ1-2 becomes more extended when the dimeric ligand binds,and even further extended by the monomeric ligand, as visualizedby low-resolution ab initio models of the O-dimer and IETAVPDZ1-2 complexes (Fig. 3E). The two PDZ domains can belocated within theO-dimer/PDZ1-2 ab initio model, which differsfrom other reported PDZ1-2 conformations (Fig. S2), by energyminimization so that O-dimer bivalently binds to PDZ1-2, andthe peptide moieties of O-dimer interact with each PDZ domainin a canonical manner (Fig. 3 F–G). In conclusion, the SAXSand NMR studies unambiguously demonstrate that the dimericligand spans the distance between individual domains, interactswith the PDZ domains in a canonical binding mode, and inducesa conformation of PDZ1-2 that is more extended and flexiblethan unbound PDZ1-2, but more compact and restricted com-pared to PDZ1-2 in complex with monomeric ligand.

Tat-N-dimer Protects Against Ischemic Brain Damage in Mice. Encour-aged by the remarkably high affinity of Tat-N-dimer towardsPSD-95 and its stability in blood plasma, we examined Tat-N-dimeras a potential in vivo active neuroprotectant. We tested Tat-N-dimer as well as Tat-NR2B9c and O-dimer in the permanent mid-dle cerebral artery occlusion (pMCAO) model of focal ischemia

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 conservedhistidine 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 capitalletters. (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 calculatedusing 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 zerois indicative of a random coil. (D) Pair distance distribution functions derived from Fourier transformation of the SAXS data presented as the relative occurrenceof distances (arbitrary scale) against distances in nm. Data from apo PDZ1-2 (green), PDZ1-2 with monomeric ligand (IETAV) (blue), and PDZ1-2 with dimericligand (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) orO-dimer (gray), showing that PDZ1-2 adopts a more compact conformation when bound toO-dimer compared to IETAV. (F) Comparison of the high-resolutionO-dimer/PDZ1-2 model (black) and the experimental SAXS scattering data (red). X ¼ 1.15. (G) High-resolution model of PDZ1-2 in complex with O-dimer.

Bach et al. PNAS ∣ February 28, 2012 ∣ vol. 109 ∣ no. 9 ∣ 3319

BIOCH

EMISTR

Y

Dow

nloa

ded

by g

uest

on

May

30,

202

1

http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1113761109/-/DCSupplemental/pnas.1113761109_SI.pdf?targetid=ST1http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1113761109/-/DCSupplemental/pnas.1113761109_SI.pdf?targetid=ST2http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1113761109/-/DCSupplemental/pnas.1113761109_SI.pdf?targetid=ST2http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1113761109/-/DCSupplemental/pnas.1113761109_SI.pdf?targetid=ST3http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1113761109/-/DCSupplemental/pnas.1113761109_SI.pdf?targetid=SF2

in adult mice (29). Compounds were injected (3 nmol∕g) once, in-travenously, 30 min after the pMCAO insult, followed by a 6-hpostsurgical survival period (Fig. 4A). Animals receiving Tat-N-dimer showed a 40% reduction in ischemic tissue damage com-pared to saline-treated animals (p ¼ 0.0081) (Fig. 4B), whereastreatment with O-dimer gave no neuroprotective effect (p ¼ 0.62,N ¼ 19), indicating the necessity for introducing cell-penetratingpeptides into the dimeric ligands to obtain effects in vivo. Treat-ment with Tat-NR2B9c did not provide a statistically significantreduction in infarct volumes (p ¼ 0.22) (Fig. 4B), although weverified by immunofluorescence using an anti-Tat antibody thatboth Tat-N-dimer and Tat-NR2B9c were located inside neuron-likecells (30) 6 h after pMCAO (Fig. 4C).

To evaluate prolonged effects of Tat-N-dimer, we performed thepMCAO experiment with a 48 h postsurgical survival period(Fig. 4A) and observed a 37% reduction in infarct size compared tosaline-treated mice (p ¼ 0.0042) (Fig. 4 D–E). Again, Tat-NR2B9cgave no statistically significant infarct reductions (p ¼ 0.89)(Fig. 4D-E), but treating animals with ReTat-N-dimer under similarconditions led to a 34% reduction in infarct volumes (p ¼ 0.0094)(Fig. S3). Body temperature, weight, and blood gas parameters weremonitored during the pMCAO experiment and no differences wereobserved among the treated groups (Fig. S4 and Table S4).

Focal cerebral ischemia induced by pMCAO in mice affectscortical brain areas controlling the contralateral front- and hind-limb including the paws (31). Thus, we included behavioral teststhat challenged the motor function of mice subjected to pMCAO.In accordance with reduced infarct volumes, the total grip strengthof both paws was unaffected for animals treated with Tat-N-dimer,but reduced for those given saline or Tat-NR2B9c (Fig. 4F). Simi-

larly, there was no difference in grip strength between the right andleft front paw for Tat-N-dimer-treated animals in contrast to treat-ment with saline or Tat-NR2B9c (Fig. 4G). Finally, in the rotarodperformance test we observed that Tat-N-dimer treatment resultedin a more pronounced short-term learning skill improvement com-pared to mice treated with saline (Fig. 4H), and the total timespent on the rod was significantly longer (83.5� 4.1 s; averageof trial 1–4� SEM) than for mice treated with Tat-NR2B9c(65.7� 3.7 s; average of trial 1-4� SEM) (P < 0.001). The effectson motor control of ReTat-N-dimer were similar to those observedfor Tat-N-dimer (Fig. S3).

DiscussionPSD-95 is a potential target for protection against ischemic braindamage (9), and recent studies demonstrate promising effectsof PSD-95 inhibitors in animal models of pain (10–12) and Alz-heimer’s disease (13). Despite this intriguing role of PSD-95,there is a lack of inhibitors against PSD-95, and PDZ domains ingeneral, with reasonably affinity and in vivo activity, which im-pede the possibilities for exploiting PDZ domains as drug targets.Here we have advanced the concept of dimeric inhibitors againstthe tandem PDZ1-2 domain of PSD-95 in order to developpotent in vivo active ligands. Previously, two decapeptides target-ing PDZ1 and PDZ2 have been linked via disulphide bondsfrom N-terminal cysteine residues, which resulted in a 5–20-foldincrease in affinity relative to the monomeric ligand (24), and arelated design with peptide sequences of 15 amino acids resultedin a similar 8–25-fold increase in affinity (25). In the presentwork, the dimeric ligands provide a remarkable 400-fold increasein affinity compared to the monomeric peptide. This has been

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 periodof 5.5 or 47.5 h. (B) Mean infarct volumes 6 h after pMCAO of mice treated with Tat-N-dimer (N ¼ 17), Saline (N ¼ 16), or Tat-NR2B9c (N ¼ 18). (C) Immuno-fluorescent 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. Scalebar: 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. Scalebar: 1 mm. (E) Mean infarct volumes 48 h after pMCAO of mice treated with Tat-N-dimer (N ¼ 17), Saline (N ¼ 16), or Tat-NR2B9c (N ¼ 17). (F-H) Motor functionassessment of mice with 48 h postsurgical survival. (F) Mean total grip strength of both front paws before (baseline) and after pMCAO. P-values:

achieved by optimizing peptide length and sequence as well asthe linker length, and in particular, by the essential modificationof changing the PEG linker into NPEG, which allows cell-pene-trating peptides to be attached to the dimeric structure withoutcompromising binding affinities. Tat-N-dimer displays an unpre-cedented high affinity for any PDZ domain-mediated interactionwith a Ki value of 4.6 nM, which is a 1,000-fold improvementcompared to Tat-NR2B9c. In addition, our design provides asolution to the inherent problem of peptides being degraded inbiological fluids, as Tat-N-dimer, and in particular ReTat-N-dimer, demonstrates greatly enhanced stability in blood plasma.Moreover, both compounds cross the blood-brain barrier in miceand demonstrate significant in vivo neuroprotective properties,hence Tat-N-dimer reduces ischemic stroke damage in mice with40% and significantly improves motor functions.

We observe that the high-affinity compounds, Tat-N-dimer andReTat-N-dimer, are more efficient in vivo neuroprotectants in themouse pMCAO model compared to the low-affinity monomericinhibitor Tat-NR2B9c when these compounds are tested in par-allel and under the same conditions and dosages. Tat-NR2B9chas previously shown robust neuroprotective effects in both tran-sient and permanent focal ischemic stroke models in rats (7, 8,16), but these studies cannot be directly compared to the currentmouse study due to experimental differences (32). Therefore,whether our results represent generally improved neuroprotec-tive properties across species and types of ischemic stroke modelsof our compounds relative to Tat-NR2B9c needs confirmation byfuture studies. However, the permanent MCAO model induces asmaller ischemic penumbra than the transient MCAO model inthe acute phase after stroke (

14. Gardoni F (2008) MAGUK proteins: New targets for pharmacological intervention inthe glutamatergic synapse. Eur J Pharmacol 585:147–152.

15. Bach A, et al. (2011) Cell-permeable and plasma-stable peptidomimetic inhibitors ofthe postsynaptic density-95/N-methyl-D-aspartate receptor interaction. J Med Chem54:1333–1346.

16. Sun HS, et al. (2008) Effectiveness of PSD95 inhibitors in permanent and transient focalischemia in the rat. Stroke 39:2544–2553.

17. Schwarze SR, Ho A, Vocero-Akbani A, Dowdy SF (1999) In vivo protein transduction:Delivery of a biologically active protein into the mouse. Science 285:1569–1572.

18. , Tat-NR2B9c is found under the name NA-1 at www.clinicaltrials.gov.19. Bach A, et al. (2008) Modified peptides as potent inhibitors of the postsynaptic den-

sity-95/N-methyl-D-aspartate receptor interaction. J Med Chem 51:6450–6459.20. Bach A, et al. (2009) Design and synthesis of highly potent and plasma-stable

dimeric inhibitors of the PSD-95–NMDA receptor interaction. Angew Chem Int Ed48:9685–9689.

21. Chi CN, et al. (2010) Deciphering the kinetic binding mechanism of dimeric ligandsusing a potent plasma-stable dimeric inhibitor of postsynaptic density protein-95 asan example. J Biol Chem 285:28252–28260.

22. Wender PA, et al. (2000) The design, synthesis, and evaluation of molecules thatenable or enhance cellular uptake: Peptoid molecular transporters. Proc Natl AcadSci USA 97:13003–13008.

23. Huang X (2003) Fluorescence polarization competition assay: The range of resolvableinhibitor potency is limited by the affinity of the fluorescent ligand. J Biomol Screen8:34–38.

24. Long JF, et al. (2003) Supramodular structure and synergistic target binding of theN-terminal tandem PDZ domains of PSD-95. J Mol Biol 327:203–214.

25. Sainlos M, et al. (2011) Biomimetic divalent ligands for the acute disruption of synapticAMPAR stabilization. Nat Chem Biol 7:81–91.

26. Paduch M, et al. (2007) Bivalent peptides as models for multimeric targets of PDZdomains. Chembiochem 8:443–452.

27. Doyle DA, et al. (1996) Crystal structures of a complexed and peptide-free membraneprotein-binding domain: Molecular basis of peptide recognition by PDZ. Cell85:1067–1076.

28. Wang W, et al. (2009) Creating conformational entropy by increasing interdomainmobility in ligand binding regulation: A revisit to N-terminal tandem PDZ domainsof PSD-95. J Am Chem Soc 131:787–796.

29. Lambertsen KL, et al. (2009) Microglia protect neurons against ischemia by synthesis oftumor necrosis factor. J Neurosci 29:1319–1330.

30. Lyck L, Krøigård T, Finsen B (2007) Unbiased cell quantification reveals a continuedincrease in the number of neocortical neurones during early post-natal developmentin mice. Eur J Neurosci 26:1749–1764.

31. Pronichev I, Lenkov D (1998) Functional mapping of the motor cortex of the whitemouse by a microstimulation method. Neurosci Behav Physiol 28:80–85.

32. Ginsberg MD (2008) Neuroprotection for ischemic stroke: Past, present and future.Neuropharmacology 55:363–389.

33. Hata R, et al. (2000) Dynamics of regional brain metabolism and gene expression aftermiddle cerebral artery occlusion in mice. J Cereb Blood Flow Metab 20:306–315.

34. Hata R, et al. (2000) Evolution of brain infarction after transient focal cerebralischemia in mice. J Cereb Blood Flow Metab 20:937–946.

35. Nakagawa T, et al. (2004) Quaternary structure, protein dynamics, and synapticfunction of SAP97 controlled by l27 domain interactions. Neuron 44:453–467.

36. Feng W, Zhang M (2009) Organization and dynamics of PDZ-domain-related supra-modules in the postsynaptic density. Nat Rev Neurosci 10:87–99.

3322 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1113761109 Bach et al.

Dow

nloa

ded

by g

uest

on

May

30,

202

1

www.clinicaltrials.govwww.clinicaltrials.govwww.clinicaltrials.gov