6:35 PM
Aug 10, 2017
 |  BC Innovations  |  Tools & Techniques

K follows C in chemoproteomics

Chemoproteomic mapping of druggable lysines in the proteome

A year after publishing a map of druggable cysteines in the proteome, Ben Cravatt’s lab at Scripps has reported its chemoproteomics tools can also profile lysines. By identifying reactive lysines in dozens of proteins for which no chemical probes exist, let alone candidate molecules, the group continues to expand the universe of druggable targets.

Chemoproteomics was largely pioneered by Cravatt, a professor of chemical physiology at The Scripps Research Institute who has developed a range of chemical tools for identifying specific types of reactive sites on proteins.

Cravatt has founded three drug discovery companies based on the tools: ActivX Biosciences Inc., now a subsidiary of Kyorin Pharmaceutical Co. Ltd., Abide Therapeutics Inc. and Vividion Therapeutics Inc.

The idea is to use chemical fragments to covalently bind and tag a reactive target site, then recover labeled proteins and identify them using mass spectrometry. Cravatt’s team has shown the technology can work in cell extracts and living tissues (see “Ligating Lysines”).

Figure: Ligating lysines

In a Nature Chemistry paper published in July, researchers at The Scripps Research Institute used chemoproteomics to identify reactive lysines in the proteome that could be druggable.

The approach takes advantage of the nucleophilic properties of certain amino acids to fish them out of cell lysates via covalent interactions with electrophilic probe molecules, typically compound fragments.

In the study, the group treated human cell lysates with an STP alkyne probe that bound to the lysine’s amine side chain with high selectivity. The lysates were incubated with either low (0.1 mM) or high (1.0 mM) concentrations of the probe.

The tagged proteins in the low concentration group were then labeled with a heavy biotin tag and those in the high concentration group with a light biotin tag, and the tagged proteins were captured on streptavidin-conjugated beads (blue spheres).

The captured proteins were proteolytically digested and the resulting peptide fragments were characterized by mass spectrometry (MS).

A reactivity ratio (R value) was calculated for each lysine based on the relative labeling of the site under high versus low probe concentrations. An R value of 1.0 means that the lysine is highly reactive, as it indicates the residue was as likely to bind low concentrations of the probe as high. Higher values indicate lower reactivity.

Last year in Nature, his team used a covalent electrophilic probe to fish out 637 reactive cysteine-containing proteins from cells. Only 14% of the proteins already had drugs against them listed in the DrugBank database.

Now, his team has published a study in Nature Chemistry using the same principle to find reactive lysines, replacing the earlier probe with one highly selective for the nucleophilic amine side chain...

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