Pin the ligand on the target!

Covalent drugs, therapeutic agents that permanently attach to their target, have been around since ancient times, ever since people figured out that something from a bark of a willow tree (salicylic acid) helped their headache, or fever, or rash get better. Salicylic acid and its more recent man-made derivative, aspirin, are one example of the reactive type of a molecule that exerts its pharmacological bioactivity by reacting with the target. Because they are reactive, and can potentially go off and react with everything at hand, drugs like this have not been trusted by the drug discovery and development communities, or regulators.

And yet, contrary to the expected free-for-all reactivity, what we have been seeing is that covalent drugs, both old (like aspirin and penicillin) and new ones (like recently FDA-approved kinase inhibitors afatinib, osimertinib and ibrutinib), are effective and not overtly toxic. Plus: they come with some advantages over their non-covalent cousins which make them attractive as therapeutic agents. For example, they have improved potency - they bind to the target and stay there, exerting more lasting effects. They often react with unique residues found only on the target (or a small number of related proteins), resulting in improved selectivity. Because irreversible covalent inhibitors permanently disarm the target, only de novo biosynthesis of the target protein can restore its function, which means improved pharmacodynamics and decreased frequency of dosing. Given these advantages, and despite the concerns about indiscriminate reactivity and toxicity, the area of targeted covalent inhibitors has been booming.

We published a review on recent advances in selective covalent ligand development and validation last week. In that review we offered a perspective that seems to be missing in the current literature: a chemical biology one! Our main motivation for taking this more basic science perspective on the area of such a large drug development interest was our desire to highlight the value of covalent ligand as research tools and to emphasize the importance of robust validation. We look forward to receiving feedback on our proposed workflow for covalent ligand validation from the community and invite commentary and discussion.

One big question that was left out of this review is: How many targets for covalent ligands are there?

In the review I mentioned above we highlighted that there is a great deal yet to be discovered. Some things that need to be discovered that will lead us to new and/or different targets are up to chemists to tackle. We definitely, and perhaps even desperately, need new covalent warheads. In this area, I am especially excited about what covalent inhibitor developers can learn from efforts in bioconjugate chemistry. Some of the most creative blending of these two areas have recently emerged from the lab of Chris Chang on targeting histidines, the work his lab did in collaboration with F. Dean Toste on developing Redox Activated Chemical Tagging (ReACT) for methionines, and the work from Itaru Hamachi and colleagues on ligand-directed chemistry. But more needs to be done and only chemists can do it, as whimsically, yet powerfully illustrated by Phil Baran, Justine deGruyter and Lara Malins in a must-read perspective that comes with this genius "Amino Acid Side-Chain Modification Report Card" that everyone in the game of developing covalent ligands should immediately print out and stick to the wall as one of those motivational posters!

But, I would like to argue that we shouldn't limit ourselves to the side chains of unmodified amino acids. When thinking about covalent targeting we have to start seriously considering posttranslationally modified forms of the proteins, aka proteoforms, as targets. Each posttranslational modification (PTM), more than 400 discrete types of them to be more precise, diversifies our proteome in a way that may be exploitable through chemical ingenuity. I don't think we can ignore the potential targeting opportunities that this might open - and we have a precedent to consider as an example in this area. Kate Carroll's lab has shown that cysteine residues oxidized into sulfenic acid, a reversible PTM, can be targeted using not the traditional electrophilic warheads, but nucleophilic ones.

In my view this is just a tip of a very large iceberg. A recent perspective provided a provocative estimate that if we take all the PTMs and all the isoforms, splice and proteolytic variants into account the size of the proteome is likely to exceed the ~20,000 human gene-encoded protein number by at least an two orders of magnitude. This is a fascinating new ocean where chemical biologists ought to go fishing, perhaps by starting to think beyond unmodified proteins as only types of targetable entities.

So, this should make us sit up and pay attention to target opportunities beyond the 20,000 that we seem to have been fixated on, not in the very least because we only have chemical tools and/or drugs and/or drug leads for a less than, a very depressing, 10% of those. I hope that my musings here will stimulate discussions. Even if what I elicit are mostly bursts of disagreement, I will be OK with that. We need to talk and we need to talk now, and I happen to think that expanding the range of targets for covalent and other ligands to include a range of different proteoforms, and a range of different chemistries makes a lot of sense!