This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Small organic molecules and peptides have long been exploited to modulate the activity of a given target enzyme. Important drugs are usually discovered by screening large libraries of natural products or chemically synthesized compounds using a single biological assay. In most cases the targeted enzyme has been fully characterized and in many cases complete crystal structures are known. While this use for small molecules is certainly of great importance, the potential for use of the small molecule as a discovery tool is often overlooked. We propose to exploit small, electrophilic peptides which covalently tag a target in the process of inhibition. These compounds are likely to be extremely valuable reagents for the identification of novel nucleophilic enzymes involved in a wide variety of biological processes. We have found that C-terminal vinyl sulfone peptides represent a new class of suicide substrate inhibitors of the threonine protease known as the proteasome. Michael addition of the hydroxyl nucleophile to the ivnyl sulfone renders the protease inactive and simultaneously results in a covalent, irreversible modification of the active site. Attachment of a radio-label at the N-terminus of the peptide vinyl sulfone allows specific detection of the target protein by simple SDS-PAGE. This system provides a starting point for the development of additional peptide electrophiles designed to target proteases other than the proteasome. While radiolabeled forms of small molecule electrophiles provide a method of visualizing target proteins, they may also be equipped with a biotin moiety to aid in affinity purification. The designed affinity reagents will allow rapid isolation of small quantitites of proteins which must then be characterized using in-gel protease digestion followed by mass spectrometric sequencing of peptide fragments. Further, our lab will be focusing on synthesis of combinatorial libraries of electrophiles synthesized as complex mixtures. Since our assay is dependent only on a compound's ability to modify a protein target, we plan to screen libraries as mixtures of compounds, thus eliminating the need to deconvolute and isolate individual members. However, the synthesis of compounds as mixtures will prevent characterization of products by standard NMR analysis. Therefore, we will only be able to determine relative amounts and purity of each library component by LC-MS. The bulk of the small molecules that we synthesize, while often peptide in nature, require characterization by mass spectrometry. The success of the described projects therefore heavily depends on access to the Mass Spectrometry Facility at UCSF.
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