title: High-throughput screen for small molecule inhibitors of Mint1-PDZ domains.
abstract: Several hundred PDZ (postsynaptic density-95, Drosophila disks-large, ZO-1) domain-containing proteins have been identified in the human genome. PDZ domains play a critical role in organization and function of cellular signaling pathways. Thus, small molecule inhibitors of PDZ domain association with their targets have wide potential applications as research and therapeutic agents. PDZ domains typically bind to a carboxyl-terminal tail of the target protein. Here we describe a high-throughput screening (HTS) assay for small molecule inhibitors of association between Mint1-PDZ domains and N-type Ca2+ channel carboxyl-terminal peptide (NC peptide). The performance of a homogeneous time-resolved fluorescence resonance energy transfer (HTRF) and an amplified luminescent proximity homogeneous assay (ALPHA) were systematically compared in parallel pilot HTS experiments with glutathione S-transferase-Mint1-PDZ1/2 protein and biotinylated NC peptide. Both of the two assays showed similar sensitivities in our target protein assay. Using HTRF-based assay we screened a library of 100,000 small molecule compounds and identified a number of potential "hits." The activity of isolated "hits" was confirmed by ALPHA assay. However, further evaluation revealed that isolated "hits" most likely act as "promiscuous binders," not as specific Mint-PDZ inhibitors, and that additional screening will be required to identify the true Mint-PDZ inhibitors. The assays described provided an example of HTS for a small molecule inhibitor of Mint-PDZ domain that can be easily adapted to other PDZ domain-mediated interactions.
authors: Department of Physiology, University of Texas Southwestern Medical Center at Dallas, Dallas, TX 75390-9040, USA.
authors: Xuesong
authors: X
authors: Chen
authors: Jamie C
authors: JC
authors: Longgood
authors: Carolyn
authors: C
authors: Michnoff
authors: Shuguang
authors: S
authors: Wei
authors: Doug E
authors: DE
authors: Frantz
authors: Llya
authors: L
authors: Bezprozvanny
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000037:antagonists & inhibitors*; D000595:Amino Acid Sequence; D000818:Animals; D004353:Drug Evaluation, Preclinical; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology; D031541:Fluorescence Resonance Energy Transfer; D005982:Glutathione Transferase / Q000037:antagonists & inhibitors*; D007202:Indicators and Reagents; D008027:Light; D049449:Luminescence; D008565:Membrane Proteins / Q000037:antagonists & inhibitors*; D010100:Oxygen / Q000737:chemistry / Q000528:radiation effects; D054731:PDZ Domains / Q000187:drug effects*; D051381:Rats; D017382:Reactive Oxygen Species / Q000737:chemistry / Q000528:radiation effects; D011994:Recombinant Proteins / Q000737:chemistry; D019809:Streptavidin / Q000737:chemistry; D013379:Substrate Specificity
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C513071:Apba1 protein, rat; D004791:Enzyme Inhibitors; D007202:Indicators and Reagents; D008565:Membrane Proteins; D017382:Reactive Oxygen Species; D011994:Recombinant Proteins; D019809:Streptavidin; D005982:Glutathione Transferase; D010100:Oxygen