A Selective Irreversible Inhibitor Targeting a PDZ Protein Interaction Domain

Abstract

Irreversible inhibitors of proteases have proven themselves useful tools for determining which proteases are active under given conditions in tissues or cells and for studying the functional role that a protease plays in physiological processes. The application of such techniques to the study of the activity and function of protein-protein interactions has been hindered by the lack of guiding principles for the mechanistic design of irreversible inhibitors targeting the "active site" of a protein interaction. We report herein the first example of a mechanism-based irreversible inhibitor of a protein interaction that has been specifically targeted to one member of the PDZ family of protein interaction domains: the second PDZ domain of the membrane-associated guanylate kinase MAGI3. This inhibitor was designed using rationally directed computational evaluation to take advantage of a conserved histidine in the PDZ domain by introducing an ionizable group that will be held in close proximity to that nucleophile during binding. The novel compound exhibits all of the characteristics of an irreversible inhibitor of the interaction of the tumor suppressor PTEN with MAGI3 in in vitro models. In cells, the inhibitor can be shown to release PTEN from sequestration by MAGI3 and consequently upregulate the PKB signaling pathway.

An Antagonist of Dishevelled Protein-Protein Interaction Suppresses β-Catenin–Dependent Tumor Cell Growth

“…We confirmed that pretreatment with FJ9 for 30 min does not reduce the maximum signal intensity generated by the PDZ domain bound to Frz7 peptide. In addition, whereas the MAGI3 PDZ2 domain treated with the indole-3-carbinol compound shows a clear shift in mass spectrometry (29), the DVL3 PDZ domain exposed to FJ9 under identical conditions does not (data not shown). These data strongly suggest that FJ9 does not covalently bind to the DVL PDZ, at least within the period of observation.…”

“…Our aim in this study was to design a proof-ofprinciple small-molecule inhibitor to block the interaction between the PDZ domain of DVL and the COOH-terminal region of Frz receptor and to test whether it inhibits down-stream Wnt signaling and suppresses cancer cell growth. We reported the structure-based design of an indole-3-carbinol compound that mimics the tetrapeptide sequence binding to a PDZ domain (29). This compound formed a covalent adduct by electrophilic alkylation of a histidine residue in the PDZ domain and therefore had limited therapeutic potential because electrophilic inhibitors often lack specificity in targeting biomolecules.…”

Identification of a small-molecule inhibitor of the PICK1 PDZ domain that inhibits hippocampal LTP and LTD

“…Only a few small-molecule PDZ domain inhibitors have been described in the literature (10,11,16,17,35,36). Most of these have been designed on the basis of selected scaffolds rather than high-throughput screening of compound libraries including inhibitors of, e.g., dishevelled [affinity up to~10 μM (10, 36)] and NHERF1 (affinity~15 μM) (35).…”

“…However, only a few compounds have been identified, and in general they display low affinities for their target (>100 μM) (10,11,(16)(17)(18). Here we report the identification of a nonpeptide small-molecule inhibitor (FSC231) of the PICK1 PDZ domain.…”

From Laptop to Benchtop to Bedside: Structure-based Drug Design on Protein Targets

“…The core scaffolds that target Bcl-X L -Bad, gp41, Mdm2-p53, XIAP-caspase-9, CD4-MHC II [337] and IL-2-IL-2R interactions [338,339] are overwhelmingly hydrophobic. In contrast, such interactions as Ras-effector and PTEN-MAGI3 [340,341] are mainly mediated by -strands hydrogen bonds and electrostatic attractions, and those targets have unavoidably experienced obstacles to achieve low IC 50 . These facts imply the poorly-understood but pivotal roles of entropy and desolvation effects that trigger protein-protein association.…”