A better map of targets

LA JOLLA, Calif.—Even before the struggle of developing molecules that are safe and effective against therapeutic targets, drug developers face the issue of identifying those targets in the first place. There are about 25,000 proteins in the human body, but broad methods for determining which proteins are “druggable” are few. Researchers at The Scripps Research Institute (TSRI) have a new approach to fill that need, however, one they used to identify more than 100 human proteins that can likely be targeted by small-molecule drugs. The work appeared in Nature Chemistry in an article titled “Global profiling of lysine reactivity and ligandability in the human proteome.”

The team included researchers from the Department of Chemical Physiology and the Department of Integrative Structural and Computational Biology at the Scripps campus in La Jolla, and from the Laboratory of Protein Design & Immunoengineering, Ecole Polytechnique Fédérale de Lausanne. Members of the team, including senior investigator Benjamin F. Cravatt, have tackled the issue of finding drug targets before, having in the past developed techniques for identifying druggable sites based on cysteine or serine amino acids on proteins, methods that have revealed hundreds of targetable proteins, including ones thought to be un-druggable. This latest approach turns an eye toward lysine amino acids instead.

“We think this approach has the potential to substantially expand the map of the druggable human proteome,” said Cravatt, a professor of chemical biology at TSRI.

“Nucleophilic amino acids make important contributions to protein function, including performing key roles in catalysis and serving as sites for post-translational modification,” the authors explained in their abstract. “Electrophilic groups that target amino-acid nucleophiles have been used to create covalent ligands and drugs, but have, so far, been mainly limited to cysteine and serine.”

Lysine sites, the team found, sometimes present with a high degree of chemical reactivity, which increases the likelihood that they are involved in their protein's function, making them potential targets. The sites' reactivity also means certain compounds could make nearly irreversible bonds with them known as covalent bonds, in which atoms share electron pairs. This is promising given that theoretically, a drug that can bind covalently to its target protein will be more potent.

The scientists, which also included first authors Stephan M. Hacker and Dr. Keriann M. Backus (postdoctoral research associates at the time of the study, the latter of which is now a member of Cravatt's laboratory), began their work by finding a compound—pentynoic acid sulfotetrafluorophenyl ester—capable of selectively and covalently binding to reactive lysines on proteins. Using the compound as a probe, they were able to characterize the reactivity of more than 4,000 lysines.

Of those, 310 qualified as being abnormally reactive, and they were highly likely to be found at functional sites on proteins. The team also applied a library of small molecules to see if any could compete with their probe in binding covalently to the reactive lysines, and found 121 lysines in 113 proteins that were targetable with the small molecules (and, theoretically, with drug molecules).

“We have quantified, in total, more than 9,000 lysines in human cell proteomes and have identified several hundred residues with heightened reactivity that are enriched at protein functional sites and can frequently be targeted by electrophilic small molecules,” the paper's abstract reported. “We have also discovered lysine-reactive fragment electrophiles that inhibit enzymes by active site and allosteric mechanisms, as well as disrupt protein–protein interactions in transcriptional regulatory complexes, emphasizing the broad potential and diverse functional consequences of liganding lysine residues throughout the human proteome.”

The druggable proteins their work identified fell into a number of classes, such as “enzymes that catalyze biochemical reactions, scaffold proteins that act as hubs for other protein signaling complexes and transcription factors that regulate gene activity,” as noted in a TSRI press release. To back up their theory, the scientists chose several proteins and demonstrated that small molecules targeting the proteins also blocked their functions.

The next step for this discovery consists of a broader effort by the team to identify small molecules that can bind covalently to reactive lysines.

“This new protein profiling method allows us to expand the universe of proteins that can be targeted by small molecules, which in turn should encourage the development of new drugs as well as probes for studying protein biology,” Cravatt stated.

SOURCE: TSRI press release