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

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 109; no. 9; pp. 3317 - 3322
• Main Authors: Bach, Anders, Clausen, Bettina H, Møller, Magda, Vestergaard, Bente, Chi, Celestine N, Round, Adam, Sørensen, Pernille L, Nissen, Klaus B, Kastrup, Jette S, Gajhede, Michael, Jemth, Per, Kristensen, Anders S, Lundström, Patrik, Lambertsen, Kate L, Strømgaard, Kristian
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 28.02.2012; National Acad Sciences
• Subjects: Amino Acid Sequence; Animals; Behavioral neuroscience; Binding Sites; Biological Sciences; Blood; Blood brain barrier; blood plasma; Brain damage; Brain injury; Crystallography, X-Ray; Disks Large Homolog 4 Protein; Drug Design; drug discovery; Drug Evaluation, Preclinical; Drug interactions; dynamic models; enzyme inhibitors; Glutamic acid receptors; Glutamic acid receptors (ionotropic); Guanylate Kinases - antagonists & inhibitors; Humans; infarction; Infarction, Middle Cerebral Artery - complications; Infarction, Middle Cerebral Artery - drug therapy; Infarction, Middle Cerebral Artery - pathology; Intracellular Signaling Peptides and Proteins - antagonists & inhibitors; Intravenous administration; intravenous injection; Ischemia; ischemic stroke; Learning Disorders - etiology; Learning Disorders - prevention & control; Ligands; Male; Membrane Proteins - antagonists & inhibitors; Mice; Mice, Inbred C57BL; Models, Molecular; Molecular Sequence Data; Molecular Targeted Therapy; Movement Disorders - etiology; Movement Disorders - prevention & control; N-Methyl-D-aspartic acid receptors; N.M.R; Neuroprotective agents; Neuroprotective Agents - chemical synthesis; Neuroprotective Agents - pharmacology; Neuroprotective Agents - therapeutic use; neuroprotective effect; Nitric-oxide synthase; nuclear magnetic resonance spectroscopy; Nuclear Magnetic Resonance, Biomolecular; Oxides; PDZ Domains - drug effects; Peptides - chemical synthesis; Peptides - pharmacology; Peptides - therapeutic use; Plasma stability; postsynaptic density; Postsynaptic density proteins; Postural Balance; Protein Conformation; Protein folding; protein-protein interactions; Proteins; Receptors; Rodents; Sensation Disorders - etiology; Sensation Disorders - prevention & control; Stroke; Strokes; TECHNOLOGY; TEKNIKVETENSKAP; X-radiation; X-ray crystallography; X-ray diffraction; X-ray scattering
• ISSN: 0027-8424; 1091-6490; 1091-6490
• DOI: 10.1073/pnas.1113761109

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.