title: Targeting protein-protein interactions with trimeric ligands: high affinity inhibitors of the MAGUK protein family.
abstract: PDZ domains in general, and those of PSD-95 in particular, are emerging as promising drug targets for diseases such as ischemic stroke. We have previously shown that dimeric ligands that simultaneously target PDZ1 and PDZ2 of PSD-95 are highly potent inhibitors of PSD-95. However, PSD-95 and the related MAGUK proteins contain three consecutive PDZ domains, hence we envisioned that targeting all three PDZ domains simultaneously would lead to more potent and potentially more specific interactions with the MAGUK proteins. Here we describe the design, synthesis and characterization of a series of trimeric ligands targeting all three PDZ domains of PSD-95 and the related MAGUK proteins, PSD-93, SAP-97 and SAP-102. Using our dimeric ligands targeting the PDZ1-2 tandem as starting point, we designed novel trimeric ligands by introducing a PDZ3-binding peptide moiety via a cysteine-derivatized NPEG linker. The trimeric ligands generally displayed increased affinities compared to the dimeric ligands in fluorescence polarization binding experiments and optimized trimeric ligands showed low nanomolar inhibition towards the four MAGUK proteins, thus being the most potent inhibitors described. Kinetic experiments using stopped-flow spectrometry showed that the increase in affinity is caused by a decrease in the dissociation rate of the trimeric ligand as compared to the dimeric ligands, likely reflecting the lower probability of simultaneous dissociation of all three PDZ ligands. Thus, we have provided novel inhibitors of the MAGUK proteins with exceptionally high affinity, which can be used to further elucidate the therapeutic potential of these proteins.
authors: Department of Drug Design and Pharmacology, University of Copenhagen, Universitetsparken 2, Copenhagen, Denmark.
authors: Klaus B
authors: KB
authors: Nissen
authors: Department of Drug Design and Pharmacology, University of Copenhagen, Universitetsparken 2, Copenhagen, Denmark.
authors: Linda M
authors: LM
authors: Haugaard-Kedström
authors: Department of Drug Design and Pharmacology, University of Copenhagen, Universitetsparken 2, Copenhagen, Denmark.
authors: Theis S
authors: TS
authors: Wilbek
authors: Department of Drug Design and Pharmacology, University of Copenhagen, Universitetsparken 2, Copenhagen, Denmark.
authors: Line S
authors: LS
authors: Nielsen
authors: Department of Medical Biochemistry and Microbiology, Uppsala University, Biomedical Centre, Uppsala, Sweden.
authors: Emma
authors: E
authors: Åberg
authors: Department of Drug Design and Pharmacology, University of Copenhagen, Universitetsparken 2, Copenhagen, Denmark.
authors: Anders S
authors: AS
authors: Kristensen
authors: Department of Drug Design and Pharmacology, University of Copenhagen, Universitetsparken 2, Copenhagen, Denmark.
authors: Anders
authors: A
authors: Bach
authors: Department of Medical Biochemistry and Microbiology, Uppsala University, Biomedical Centre, Uppsala, Sweden.
authors: Per
authors: P
authors: Jemth
authors: Department of Drug Design and Pharmacology, University of Copenhagen, Universitetsparken 2, Copenhagen, Denmark.
authors: Kristian
authors: K
authors: Strømgaard
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D001667:Binding, Competitive; D000074288:Discs Large Homolog 1 Protein; D000076122:Disks Large Homolog 4 Protein; D005454:Fluorescence Polarization; D051528:Guanylate Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D016296:Mutagenesis; D009687:Nuclear Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D054731:PDZ Domains; D010455:Peptides / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D011485:Protein Binding; D055503:Protein Multimerization; D014157:Transcription Factors / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D025521:Tumor Suppressor Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C513314:DLG1 protein, human; C116869:DLG3 protein, human; C501444:DLG4 protein, human; D000074288:Discs Large Homolog 1 Protein; D000076122:Disks Large Homolog 4 Protein; D047908:Intracellular Signaling Peptides and Proteins; D008024:Ligands; D008565:Membrane Proteins; D009687:Nuclear Proteins; D010455:Peptides; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; C496468:DLG2 protein, human; D051528:Guanylate Kinases