| Over the past several years, the field of activity-based protein profiling (ABPP) has enjoyed tremendous growth in terms of both technology development and biological applications. These advances can be traced not only to a rich history of research on mechanism-based enzyme inhibitors, but also to the success of genome sequencing projects and innovations in mass spectrometry. Nevertheless, as a chemical technology, further advances in the scope and application of ABPP requires the development of novel chemical probes. Toward this end, this thesis outlines our efforts to develop and apply a ∼50 member library of natural product-like activity based probes.; The library was modeled and prepared around a structural motif that appears in several reported protein-reactive natural products, and at the time, was unexplored in functional proteomics studies. With these chemical tools in hand, we incorporated the library into a cell-based screening platform, from which we discovered a bioactive compound, termed MJE3. Further characterization of the target repertoire of MJE3 suggested that this compound exerted its anti-proliferation effects by alkylating and inhibiting brain-type phosphoglycerate mutase (PGAM1) in human breast cancer cell lines. Complementing this work, we studied the target repertoire of other library members using the techniques associated with non-directed ABPP. In multiple human cancer cell lines, we identified and characterized a protein almost exclusively labeled by MJE24, and that is upregulated in aggressive human cancer cell lines. We have assembled evidence that this protein is annexin A1, and ongoing work is dedicated to understanding the properties and functional consequences of this small-molecule-protein interaction. In summary, our efforts to expand the scope and application of ABPP and its techniques has culminated in the development of a novel set of chemical tools for functional proteomics, and their application has led to the characterization of two unique small molecule-protein interactions in cancer proteomes. |
