ABSTRACT
Store-operated calcium (SOC) channels are vital for activation of the immune cells, and mutations in the channel result in severe combined immunodeficiency in human patients. In lymphocytes, SOC entry is mediated by the Orai1 channel, which is activated by direct binding of STIM1. Here we describe an alternative approach for identifying inhibitors of SOC entry using minimal functional domains of STIM1 and Orai1 to screen a small-molecule microarray. This screen identified AnCoA4, which inhibits SOC entry at submicromolar concentrations and blocks T cell activation in vitro and in vivo. Biophysical studies revealed that AnCoA4 binds to the C terminus of Orai1, directly inhibiting calcium influx through the channel and also reducing binding of STIM1. AnCoA4, unlike other reported SOC inhibitors, is a molecule with a known binding site and mechanism of action. These studies also provide proof of principle for an approach to ion channel drug discovery.
Subject(s)
Benzodioxoles/therapeutic use , Chromones/therapeutic use , Drosophila Proteins/metabolism , Membrane Proteins/metabolism , Small Molecule Libraries/chemistry , Animals , Benzodioxoles/chemistry , Benzodioxoles/pharmacology , Chromones/chemistry , Chromones/pharmacology , Disease Models, Animal , Drosophila , Drosophila Proteins/antagonists & inhibitors , Drosophila Proteins/genetics , Fura-2/chemistry , Gene Expression/drug effects , HEK293 Cells , Humans , Hypersensitivity, Delayed/drug therapy , Hypersensitivity, Delayed/metabolism , Hypersensitivity, Delayed/pathology , Immunosuppressive Agents/chemistry , Immunosuppressive Agents/pharmacology , Immunosuppressive Agents/therapeutic use , Lymphocyte Activation/drug effects , Membrane Proteins/antagonists & inhibitors , Membrane Proteins/chemistry , Membrane Proteins/genetics , Mice , Mice, Inbred BALB C , Molecular Docking Simulation , Neoplasm Proteins/chemistry , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , ORAI1 Protein , Protein Binding , Protein Structure, Tertiary , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Small Molecule Libraries/metabolism , Small Molecule Libraries/pharmacology , Stromal Interaction Molecule 1 , T-Lymphocytes/drug effects , T-Lymphocytes/immunology , T-Lymphocytes/metabolismSubject(s)
Biological Factors , Protein Array Analysis/methods , Recombinant Fusion Proteins/metabolism , Streptomyces/metabolism , Tacrolimus Binding Protein 1A/metabolism , Biological Factors/isolation & purification , Biological Factors/pharmacology , Protein Binding , Sirolimus/isolation & purification , Sirolimus/pharmacologyABSTRACT
Herein we report the expanded functional group compatibility of small-molecule microarrays to include immobilization of primary alcohols, secondary alcohols, phenols, carboxylic acids, hydroxamic acids, thiols, and amines on a single slide surface. Small-molecule "diversity microarrays" containing nearly 10,000 known bioactive small molecules, natural products, and small molecules originating from several diversity-oriented syntheses were produced by using an isocyanate-mediated covalent capture strategy. Selected printed bioactive compounds were detected with antibodies against compounds of interest. The new surface of the diversity microarrays is highly compatible with approaches involving cellular lysates. This feature has enabled a robust, optimized screening methodology using cellular lysates, allowing the detection of specific interactions with a broad range of binding affinity by using epitope-tagged or chimeric fluorescent proteins without prior purification. We believe that this expanded research capability has considerable promise in biology and medicine.
Subject(s)
Oligonucleotide Array Sequence Analysis , Cell Line , Fluorescence , Humans , Tacrolimus Binding Protein 1A/chemistryABSTRACT
This protocol describes a robust method for the covalent capture of small molecules with diverse reactive functional groups in microarray format, and outlines a procedure for probing small-molecule microarrays (SMMs) with proteins of interest. A vapor-catalyzed, isocyanate-mediated surface immobilization scheme is used to attach bioactive small molecules, natural products and small molecules derived from diversity-oriented synthesis pathways. Additionally, an optimized methodology for screening SMMs with purified proteins and cellular lysates is described. Finally, a suggested model for data analysis that is compatible with commercially available software is provided. These procedures enable a platform capability for discovering novel interactions with potential applications to immunoglobulin profiling, comparative analysis of cellular states and ligand discovery. With the appropriate materials and experimental setup, the printing of SMMs can be completed in 14 hours over 3 days. Screening and data analysis requires 2 days. A detailed timeline is provided.
