Nucleophilic covalent fragments against cysteine (!)

Covalent fragment-based drug discovery continues to gain momentum, as evidenced by the number of talks at the CHI Drug Discovery Chemistry meeting in April. All of those involved nucleophilic residues on proteins, especially cysteine, reacting with electrophilic fragments. However, as we noted last year, it is possible to do the reverse. This is the topic of a new paper in Nat. Chem. Biol. by Jing Yang (Beijing Institute of Lifeomics), Kate Carroll (UF Scripps), and collaborators.

 

The reason most covalent fragments are not nucleophiles is that none of the twenty standard amino acids are particularly electrophilic. The work we mentioned last year focused on post-translational modifications introducing aldehydes or ketones into proteins. But it turns out that the thiol group of cysteine, which is normally nucleophilic, can be oxidized to a sulfenic acid, which is electrophilic. The Carroll group has been studying this “cysteine redoxome” for years and found that S-sulfenation can serve a regulatory function akin to phosphorylation.

 

To assess the reactivity of sulfenic acids across the proteome, the researchers synthesized cyanoacetamide and nitroacetamide derivatives of 3,5-bis(trifluoromethyl)aniline. The chloroacetamide and acrylamide derivatives of this fragment have previously been used in chemoproteomics experiments to probe for reactive cysteines. Cell lysates were treated with one of these four fragments, followed by treatment with a generic probe for sulfenic acids or thiols. If a cysteine residue is modified with the fragment, it will be unavailable to react with the second probe, and this loss in signal can be quantifiably detected using mass spectrometry.

 

For the thiol-reactive chloroacetamide and acrylamide, the researchers found that 25.2% and 11.0% of quantifiable cysteines in the proteome could form adducts. But only 24 cysteine residues formed adducts with the nitroacetamide (1.3% of the total sulfenic acids quantified), and none formed adducts with the cyanoacetamide.

 

Despite this lower hit rate, the researchers constructed a library of 65 cyanoacetamide- and nitroacetamide-containing fragments, which was similarly screened in cell lysates. Adding hydrogen peroxide to cell lysates to mimic oxidative stress increased the number of sulfenic acid sites. In total the researchers found 524 liganded sites across 441 proteins. As expected from the earlier experiments, nitroacetamides tended to bind to more sites than cyanoacetamides.

 

The researchers studied the functional effect of covalent modification for several proteins. The enzymes GAPDH, GSTO1, and ACAT1 all have active-site cysteine residues that can be reversibly modified by oxidation to the sulfenic acid. Reaction of this form of the recombinant proteins with one of the covalent fragments led to irreversible inhibition.

 

Similarly, the researchers demonstrated that a fragment which hits the enzyme PRXL2A activated MAPK signaling in cells, as expected. Importantly, this effect was not seen in cells containing PRXL2A with a cysteine to serine mutation. The non-enzyme proteins HDGF and BCCIP could also be functionally inhibited in cells with covalent fragments.

 

This “umpolung” approach to covalent ligand discovery is scientifically interesting, but how useful will it be? In most cases sulfenic acid formation is already deactivating, so targeting this form of the protein will simply keep it in the off-state. However, the researchers do note that sulfenic acid formation can be activating.

 

A second challenge is that sulfenic acid formation tends to be substoichiometric, with only a small percentage of cysteines existing predominantly in the sulfenic acid form. Thus, it will be difficult to achieve the near quantitative level of modification often required for biological effects. That said, there are cases where you want to tweak a pathway rather than shut it down entirely. Protein activation or the development of new PROTACs could also benefit from limited target protein engagement.

 

As for the covalent fragments themselves, nitroacetamides may be too reactive, but the cyanoacetamide moiety is actually found in a few approved drugs, such as the anti-inflammatory tofacitinib. And if compelling sulfenic acid targets are identified, chemists will likely develop additional nucleophilic probes suitable for dosing in humans.