Chemical array screening for small molecule inhibitors.


We have developed a unique photo-cross-linking approach for immobilizing a variety of small molecules in a functional-group-independent manner. Our approach depends on the reactivity of the carbene species generated from trifluoromethyldazirine upon UV irradiation. It was demonstrated in model experiments that the photogenerated carbenes were able to react with every small molecule tested, and they produced multiple conjugates in most cases. It was also found in on-array immobilization experiments that various small molecules were immobilized, and the immobilized small molecules retained their ability to interact with their binding proteins. With this approach, photo-cross linked microarrays of 2000 natural products and drugs were constructed. This photo-cross-linked microarray format was found to be useful not merely for ligand screening but also to study the structure-activity relationship, that is, the relationship between the structural motif (or pharmacophore) found in small molecules and its binding affinity toward a protein, by taking advantage of the nonselective nature of the photo-cross-linking process.

Overview of Systematic Chemical Array Screening Method.
Human genes of interest are cloned and restriction enzyme-digested fragments are inserted into pDsRed-express and sequenced. GLORIA contains 100 genes, which can be expressed as RFP-fused proteins in mammalian cells. HEK 293T cells are transfected with vectors encoding RFP fused-proteins or RFP for 24 h. After transfection, cell lysates are prepared for incubation with glass slides, where compound from NPDepo are immobilized in duplicate by a photo-crosslinking technique. Detection of "hit ligands" is done by merged display analysis. Spots that interact with RFP or RFP-fused proteins are designated green and red respectively. After two slides are merged, true hit compounds are detected as red signals, while false positive signals are yellow.(Ref: 1)


  1. I. Miyazaki, S. Simizu, H. Ichimiya, M. Kawatani, H. Osada.
    Robust and systematic drug screening method using chemical arrays and protein library: Identification of novel inhibitors of carbonic anhydrase II.
    Biosci. Biotechnol. Biochem., 72, 2739-2749 (2008).

  2. N. Kanoh, A. Asami, M. Kawatani, K. Homda, S. Kumashiro, H. Takayama, S. Shimizu, T. Amemiya, Y. Kondoh, S. Hatakeyama, K. Tsuganezawa, R. Utata, A. Tanaka, S. Yokoyama, H. Tashiro, H. Osada.
    Photo-cross-linked small-molecule microarrays as chemical genomic tools for dissecting protein-ligand interactions.
    Chem.Asian J., 1, 789-797 (2006).

  3. N. Kanoh, S. Kumashiro, S. Simizu, Y. Kondoh, S. Hatakeyama, H. Tashiro, H. Osada.
    Immobilization of natural products on glass slides by using a photoaffinity reaction and the detection of protein-small-molecule interactions.
    Angew. Chem. Int. Ed., 42, 5584-5587 (2003).

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