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.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Amino Acid Sequence
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Animals
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Binding Sites
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Blood-Brain Barrier
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Crystallography, X-Ray
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Disks Large Homolog 4 Protein
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Drug Design
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Drug Evaluation, Preclinical
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Guanylate Kinases / antagonists & inhibitors
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Humans
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Infarction, Middle Cerebral Artery / complications
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Infarction, Middle Cerebral Artery / drug therapy*
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Infarction, Middle Cerebral Artery / pathology
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Intracellular Signaling Peptides and Proteins / antagonists & inhibitors*
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Learning Disabilities / etiology
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Learning Disabilities / prevention & control
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Male
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Membrane Proteins / antagonists & inhibitors*
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Mice
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Mice, Inbred C57BL
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Models, Molecular
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Molecular Sequence Data
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Molecular Targeted Therapy
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Movement Disorders / etiology
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Movement Disorders / prevention & control
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Neuroprotective Agents / chemical synthesis
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Neuroprotective Agents / pharmacology
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Neuroprotective Agents / therapeutic use*
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Nuclear Magnetic Resonance, Biomolecular
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PDZ Domains / drug effects
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Peptides / chemical synthesis
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Peptides / pharmacology
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Peptides / therapeutic use*
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Postural Balance
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Protein Conformation
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Sensation Disorders / etiology
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Sensation Disorders / prevention & control
Substances
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DLG4 protein, human
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Disks Large Homolog 4 Protein
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Dlg4 protein, mouse
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Intracellular Signaling Peptides and Proteins
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Membrane Proteins
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Neuroprotective Agents
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Peptides
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Tat-NPEG4(IETDV)2
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Guanylate Kinases