A method for the covalent capture and screening of diverse small molecules in a microarray format

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.

[1]  Stuart L Schreiber,et al.  A robust small-molecule microarray platform for screening cell lysates. , 2006, Chemistry & biology.

[2]  David G Myszka,et al.  Analyzing ligand and small molecule binding activity of solubilized GPCRs using biosensor technology. , 2006, Analytical biochemistry.

[3]  B. Zimmermann,et al.  Biomolecular interaction analysis in functional proteomics , 2006, Journal of Neural Transmission.

[4]  I. Shin,et al.  Fabrication of chemical microarrays by efficient immobilization of hydrazide-linked substances on epoxide-coated glass surfaces. , 2005, Angewandte Chemie.

[5]  Do-Hyoung Kim,et al.  Proteomimetic libraries: design, synthesis, and evaluation of p53-MDM2 interaction inhibitors. , 2006, Journal of combinatorial chemistry.

[6]  S. Schreiber,et al.  Expanding the functional group compatibility of small-molecule microarrays: discovery of novel calmodulin ligands. , 2003, Angewandte Chemie.

[7]  H. Osada,et al.  Immobilization of natural products on glass slides by using a photoaffinity reaction and the detection of protein-small-molecule interactions. , 2003, Angewandte Chemie.

[8]  Milan Mrksich,et al.  Carbohydrate arrays for the evaluation of protein binding and enzymatic modification. , 2002, Chemistry & biology.

[9]  Jonathan Bard,et al.  Evaluation of fluorescence-based thermal shift assays for hit identification in drug discovery. , 2004, Analytical biochemistry.

[10]  Stuart L. Schreiber,et al.  Small-Molecule Microarrays: Covalent Attachment and Screening of Alcohol-Containing Small Molecules on Glass Slides , 2000 .

[11]  Ruiwu Liu,et al.  Combinatorial chemistry identifies high-affinity peptidomimetics against α4β1 integrin for in vivo tumor imaging , 2006 .

[12]  Xuan Huang,et al.  Microarrays of tagged combinatorial triazine libraries in the discovery of small-molecule ligands of human IgG. , 2004, Journal of combinatorial chemistry.

[13]  John D Lambris,et al.  Hydrophobic effect and hydrogen bonds account for the improved activity of a complement inhibitor, compstatin. , 2006, Journal of medicinal chemistry.

[14]  Stuart L. Schreiber,et al.  Printing Small Molecules as Microarrays and Detecting Protein−Ligand Interactions en Masse , 1999 .

[15]  D. Holt,et al.  Synthesis and activity of bivalent FKBP12 ligands for the regulated dimerization of proteins. , 1998, Bioorganic & medicinal chemistry.

[16]  P. Clemons,et al.  Synthesis of calcineurin-resistant derivatives of FK506 and selection of compensatory receptors. , 2002, Chemistry & biology.

[17]  Herbert Waldmann,et al.  Staudinger ligation: a new immobilization strategy for the preparation of small-molecule arrays. , 2003, Angewandte Chemie.