여름정기학술대회
2022여름초록
발표자 및 발표 내용
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| Brief Oral Presentation 발표신청 |
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주저자
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공동저자
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접수자
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We developed the LC-MS/MS method to rapidly screen the covalent binding of inhibitors to proteins. This high-throughput screening platform can be used to accurately
identify and quantify the formation of covalent adducts between electrophilic
inhibitors and nucleophilic residues such as cysteine in a protein. MDM2, E3 ubiquitin-protein ligase that mediates
ubiquitination of p53/TP53, is our target protein. To make covalent bond
with the electrophilic inhibitors, cysteine was introduced in the druggable
pocket of MDM2 (Mouse double minute 2 homolog). From a result of previous
experiment, 30 electrophilic inhibitors with acrylamide and chloride warhead
group were selected from a cysteine-focused inhibitors library, where they were
identified to be bound to the target cycstein residue of MDM2. We divided 30 electrophilic
inhibitors into 3 groups containing 10 different ones. 10 electrophilic inhibitors
in a group were incubated with cyctein introduced MDM2(M62C) at the same time
and digested with trypsin followed by LC-MS/MS analysis. To list the degree of binding affinity of 30
electrophilic inhibitors to the druggable pocket of
the protein, we calculated the relative
intensity ratios of the inhibitor-bound target peptides to their corresponding
internal standard peptide, and identified the inhibitors with significantly higher the ratio than others. Finally,
interlabaratory experiment was performed to evaluate whether this LC-MS/MS based high
throughput screening platform could be used universally. Using the same manual,
seven laboratories treated MDM2(M62C)
incubated with electrophilic inhibitors and performed
LC-MS/MS analysis. By comparing the results from each laboratory, we will
discuss the usefulness of this LC-MS/MS based high
throughput screening platform.
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