Covalent inhibitors have numerous applications as drugs, tools for drug discovery and probes for chemical biology. This class of compounds is designed such that the initial reversible association is followed by the formation of a covalent bond between an electrophile on the ligand and a nucleophilic center in the biological target protein, typically solvent-exposed, noncatalytic cysteine. Although cysteine is the most reactive at physiological pH, it is also the least prevalent. Thus, novel methods of covalent inhibition are necessary to target a larger proportion of the proteome and increase the scope of therapeutic targets for covalent inhibitors.

Recently, Jack Taunton and colleagues from the University of California, San Francisco (UCSF) have developed salicylaldehyde-based chemical probes that reversibly and covalently modify the catalytic lysine of protein kinases, with sustained occupancy in cells and animals. They published their findings in the May 19, 2022, online edition of Nature Chemical Biology. Taunton is a professor of cellular and molecular pharmacology at UCSF where his laboratory develops chemical and biochemical tools to illuminate cellular processes relevant to cancer and autoimmune disease.

Using quantitative chemoproteomic methods, Taunton and his team estimated the residence times of the salicylaldehyde probes for endogenous kinases in cultured human cells, as well as in treated mice. According to the authors, the in vivo use of covalent inhibitors requires electrophiles with the proper balance of metabolic stability and reactivity for their targets.

Taunton and his team used the salicylaldehyde group as a reversible covalent electrophile to develop probes that react with the catalytic lysine in kinases. Using these probes, in combination with mass spectrometry, they found that approximately 50% of the kinome was able to react with the probes under equilibrium labeling conditions, including 80 kinases from living mice treated with the probes.

Despite this promiscuity, the team discovered that these probes exhibited remarkable kinetic selectivity for a small subset of kinases, with an apparent dissociation half-life of greater than 6 hours from the aforesaid kinases. Taunton's team found that the kinase selectivity could be improved further by linking the salicylaldehyde to a more selective noncovalent-recognition scaffold. The authors postulate that the salicylaldehyde probes occupy a 'sweet spot' in electrophile space, efficiently reacting with an appropriately positioned lysine, while reacting slowly with most off-target nucleophiles when added to cells at low micromolar concentration.

According to the authors, these are among the first lysine-reactive kinase probes to exhibit the requisite stability and reactivity for in vivo applications. These novel probes demonstrate how to achieve kinase selectivity, despite targeting the conserved, catalytically essential lysine residues.

In order to demonstrate the further utility of the clickable salicylaldehyde probe, Taunton's team performed a competitive labeling experiment in mice to quantify kinase engagement by PF-06873600 (ebvaciclib), a cyclin-dependent kinase 2/4/6 (CDK2/4/6) inhibitor, currently in phase II trials for metastatic breast and ovarian cancer.

The chemoproteomic results reported in the study demonstrated the feasibility of conducting competitive kinase engagement experiments in animals using a reversible, lysine-targeted salicylaldehyde probe.

The kinases that bind most tightly to the salicylaldehyde probes reported in the study play diverse biological and pathophysiological roles. The salicylaldehyde probes engaged four kinases (Aurora kinase A [AURKA], Aurora kinase B [AURKB], microtubule-associated serine/threonine kinase 3 [MAST3] and serum/glucocorticoid regulated kinase 3 [SGK3]) and two kinases (MAST3 and SGK3), respectively, in a sustained manner, with half-lives more than 6 hours. AURKA and AURKB have distinct functions in cell cycle progression and cancer. AURKA has been shown to drive neuroblastoma progression and its inhibition can overcome acquired or intrinsic resistance to PI3K inhibitors in preclinical breast cancer models. Both MAST3 and SGK3 can drive cancer progression and few selective inhibitors have been reported for either MAST3 or SGK3.

Taunton hopes that work validates the possibility of developing salicylaldehyde-based inhibitors with prolonged residence times for these and many other therapeutically relevant kinases, which lack a druggable cysteine.