Photo-Cross-Linking Beads

In the course of our phenotypic screening from a chemical library in NPDepo and microbial metabolites, we identified several hundreds of compounds that induced unique cell-shape changes in src -NRK and HeLa cells. Among them, NPD6689, NPD8617, and NPD8969 were very potent, as evidenced by the nanomolar ranges of their effective doses. 

BNS-22 is a chemically synthesized derivative of the natural plant product GUT-70. GUT-70 has been reported to inhibit leukemic cell growth and induce caspase-mediated apoptosis [17]. Among more than 60 derivatives of GUT-70, BNS-22 was selected as the compound with the most robust biological activity. However, its molecular target and mechanism of action remained unknown. 

Using ChemProteoBase, we analyzed HeLa cells treated with BNS-22 [18]. We compared proteomic profiles of HeLa cell treated with BNS-22 to a dataset of 42 well-characterized anticancer drugs and found that the profile of BNS-22 was similar to that of ICRF-193, a DNA topoisomerase II (TOP2) catalytic inhibitor. BNS-22 was shown to inhibit TOP2 and TOP2 poison-mediated DNA damage. 

Harding, M.W., Galat, A, Uehling, D.E., and Schreiber, S.L. (1989) A receptor for the immunosuppressant FK506 is a cis-trans peptidyl-prolyl isomerase. Nature, 341, 758-760. 

Schreiber, S.L. (1991) Chemistry and biology of the immunophilins and their immunosuppressive ligands. Science, 

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A Target Identification System Based on MorphoBase, ChemProteoBase, and Photo-Cross-Linking Beads... 

Figure 11.3 Identification of protein targets using photo-cross-linking beads, (a) Upon UV Irradiation, aryl-diazirine groups covalently Introduced onto a solid matrix (e.g., agarose beads) are transformed into highly reactive carbenes, which in turn bind to or insert irreversibly into a proximal small molecule in a functionality independent manner, (b) Pull-down assays are performed by mixing...

Photo-Cross-Linking of Cyciosporin A on PALC- and CPALC Beads... 

Fig. 2. Detection of cleaved photo-cross-linked products from cyclosporin A-immobilized CPALC agarose beads, (a) Expected cleaved photo-cross-linked products and their molecular weights, (b) ESI-MS spectrum of the cleaved sample. Sample injection volume 20 pL Mobile phase MeOH-HjO (9 1, v/v) containing 0.1 % formic acid Flow rate 1 mlimin Ionization mode ESI No separation column used.

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