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Drug Development

Drug developers look to lysine on disease-linked proteins

Covalent drugs gained steam by targeting cysteine on disease-related proteins. Now a second amino acid is in drug developers’ sights

by Shi En Kim
August 28, 2022 | A version of this story appeared in Volume 100, Issue 30


Protein structure of the AURKA protein, which contains a lysine residue. A small molecule carrying an aldehyde group forms a covalent bond with the lysine on this protein.
Credit: Jack Taunton
A small molecule carrying an aldehyde group covalently binds to a lysine (yellow segment on the blue ribbon) on a kinase protein.

Misbehaving proteins are behind many diseases. One way drugmakers incapacitate these bad actors is to deploy molecules that bind to them. But finding solid footholds on the proteins to block their action isn’t always easy.

Drugs that form covalent bonds with proteins latch irreversibly onto their targets. This means they have a longer action time and are effective in smaller doses than noncovalent drugs, which can come and go from the proteins they attach to. In recent years, covalent molecules have become increasingly popular among drug developers for their potency and their ability, if designed properly, to bind selectively to their intended targets and shut them down for good.

Covalent drugs usually zero in on reactive amino acid side chains that jut from the surface or nooks of a disease-causing protein. For the past few decades, efforts to develop covalent drugs have targeted cysteine. But now another amino acid promises to vastly expand the space of druggable targets: lysine.

An alternative to cysteine

Cysteine as a covalent binding site is a double-edged sword. On the one hand, it offers some degree of selectivity: the fraction of cysteine-containing proteins in the human body is small, so drug molecules have fewer opportunities to bind to the wrong place. On the other hand, many proteins lack a cysteine, which puts them out of contention as potential covalent drug targets.

Lysine promises to make many more human proteins available to target with covalent drugs. This amino acid residue is nearly three times as abundant as cysteine in the body: an average of 32 lysines dot every protein.

There is tremendous utility in being able to expand the covalent alphabet to lysine.
Peter Thompson, CEO and cofounder, Terremoto Biosciences

Research on cysteine-targeting covalent drugs has also found that some cancer-related proteins, such as the epidermal growth factor receptor, can mutate to swap out their cysteine residue for another amino acid, rendering the once-druggable protein impervious to attack. Homing in on lysine promises a lower probability of proteins picking up mutations that lead to drug resistance. That’s because, for certain large classes of proteins such as in kinases, the lysine is critical to the protein’s function and can’t be substituted for another amino acid. As a drug target, lysine isn’t just versatile—it’s also reliable.

Lysine has already drawn interest from the pharmaceutical industry. Several companies developing covalent drugs are looking to this amino acid after focusing on the well-established cysteine.

“There is tremendous utility in being able to expand the covalent alphabet to lysine,” says Peter Thompson, CEO and cofounder of Terremoto Biosciences, a San Francisco–based company that recently emerged to develop lysine-based covalent drugs. The company, which has not disclosed details about diseases it is targeting, expects competition in the future: “There will probably be many companies that will pursue this,” Thompson says.

Experts figure that lysine is more than a decade behind cysteine as a covalent drug target. There are no lysine-targeting drugs available yet, while cysteine drug developers boast several game-changing medicines, such as the anticancer drugs ibrutinib and osimertinib. But the field of lysine covalency is “being developed as we speak,” Thompson says. “We’re moving very rapidly—faster than I thought.”

Lysine’s potential

Among all the amino acids, cysteine is drugmakers’ top choice for a covalent target because it’s the most nucleophilic and hence reactive. That means it’s the most eager among the amino acids to share its electrons with an electrophile to make a bond. Lysine is slightly less nucleophilic, so it’s a logical next covalent target.

Despite their prevalence, not all lysines are options as drug-docking sites. When exposed to an acidic or even mildly basic environment, such as at the physiological pH of 7.4, lysine can become protonated and will no longer make a covalent bond. So drug discovery efforts typically go after lysines nestled within the inner folds of a protein, where they are protected from protonation.

To explore the extent of lysine’s potential, researchers recently charted the landscape of lysines in human proteins to look for druggable sites. Their effort showed plenty of opportunities in this relatively unexplored space. Chemist Mikail Abbasov of Cornell University and colleagues combed through the proteins in human immune cells and cancer cells for lysines that could be targeted given their protected location in the protein (Nat. Chem. 2021, DOI: 10.1038/s41557-021-00765-4). The researchers mapped more than 14,000 lysines; at least 3,000 were potentially druggable with a covalent molecule.

The group also performed experiments to evaluate various reactive groups’ efficiency at binding to the lysines at different sites. Understanding their binding behavior could provide a starting point for covalent drug design. “This was the first work that not only assessed the reactivity of a large variety of small molecules,” Abbasov says, “but also screened in cancer and immune systems” for lysines that small molecules could bind to. The landscape is vast for covalent drug candidates that seek out lysine. “There are a lot of things to explore,” he says.

“There’s been a lot of work in the lysine-targeting field, especially in the last 5 years,” says Katya Vinogradova, a chemical biologist who studies immunology and proteomics at the Rockefeller University. “The [lysine covalent] platform is very powerful.”

To bind or not to bind

Lysine’s lower nucleophilicity compared with cysteine requires that researchers use more reactive electrophiles as drug candidates. But that reactivity may make the electrophiles less stable, causing them to potentially react with water, enzymes, or other compounds in the bloodstream. Drugmakers need to balance reactivity with stability in electrophile design.

Scientists led by Maurizio Pellecchia of the University of California, Riverside, have approached this balance from both directions: in one case they started with a highly reactive sulfonyl fluoride and reined it in by tacking on the right electron-donating groups (J. Med. Chem. 2021, DOI: 10.1021/acs.jmedchem.1c01459). In another case, the researchers designed a workaround to coax reluctant fluorosulfates to react. They made a molecular scaffold to hold the fluorosulfate group across from the lysine of interest long enough for the two to form a covalent bond (J. Med. Chem. 2019, DOI: 10.1021/acs.jmedchem.9b01108).

Chemical structure of the amino acid residues lysine and cysteine.

Pellecchia is marching these custom-made fluorosulfate molecules toward commercialization. He is the president and founder of Armida Labs, which is seeking seed funding as it moves to test its best candidate, called CovaLys, in mouse models to treat cancer.

One of the key components of a covalent drug molecule is, ironically, its noncovalent portion: the ligand that’s attached to the electrophile. While the electrophile forms the covalent bond to the amino acid, the ligand strikes up noncovalent interactions with the protein frame surrounding the correct amino acid target, thereby guiding the drug to the right spot. The ligand reinforces the attraction to the site of interest, locking the electrophile in place so that a covalent bond with the right amino acid can form.

Cysteine-selective drugs already use this trick, but it’s even more critical when abundant lysine is the target.

Scientists at Kyoto University, for example, employed a ligand to guide highly reactive sulfonamide groups onto a lysine on Hsp90, a protein typically found in cancer cells (Nat. Commun. 2018, DOI: 10.1038/s41467-018-04343-0). Their molecule was able to tamp down the expression of the protein and suppress cancer cell growth.

Without the bulky ligand, the antsy sulfonamide would stray to other lysines and even water molecules in the protein environment, says Itaru Hamachi, the chemical biologist who helmed the research. “The ligand [gave] us binding and selectivity.”

Making and breaking covalent bonds

Another emerging strategy to achieve selectivity is counterintuitive: some researchers are moving from fully covalent drugs to explore molecules that bind to proteins covalently but reversibly. In this case, the irreversible bond that first sold scientists on covalent drugs is now deliberately engineered to be breakable.

“I see it as having a compound with the best of the two worlds—the covalent and noncovalent,” Abbasov says.

Engineering reversibility into the bond contributes to the drug candidate’s selectivity by giving it room for self-correction. The electrophiles are still reactive enough to form strong covalent bonds, but they will detach from off targets until they find the lysine that they have the most affinity with, which is, by design, on the target protein.

Irreversible electrophiles’ selectivity depends only on one rate constant—a factor called the on rate, which is the rate the molecule attaches to the right lysine site, according to Jack Taunton, a chemist at the University of California, San Francisco. “With reversible electrophiles, you also have an off rate, and so it just gives you an extra dimension of selectivity.”

The molecular structure of the drug candidate CovaLys, which is covalently bound to a lysine amino acid on the protein structure of the cancer-linked XIAP.
Credit: Udompholkul P., et al., Pegan S., and Pellecchia M. of UC Riverside. The image was produced using Chimera from the Computer Graphics Laboratory, UCSF (supported by NIH P41 RR-01081).
The drug candidate CovaLys binds covalently to a lysine in the cancer-linked protein XIAP.

By tinkering with this off rate of covalent bond formation, Taunton’s team of researchers demonstrated that its aldehyde-based covalent inhibitors eventually landed on the desired proteins and didn’t stay put on the wrong targets, despite the high on rate. The team showed that a collection of these molecules could selectively seek over 200 different protein targets in human cell lines and mice (Nat. Chem. Biol. 2022, 10.1038/s41589-022-01019-1).

Taunton has another reason to bet on aldehydes: there’s a precedent for drugs that rely on these functional groups, suggesting they can be safe in the human body. In 2019, the US Food and Drug Administration approved Global Blood Therapeutics’ voxelotor to treat sickle cell disease. The compound relies on an aldehyde to bind to an N-terminal amine on hemoglobin.

And although Novartis decided for business reasons to abandon it, the company took the reversible covalent inhibitor roblitinib, a cysteine-seeking aldehyde, through Phase 2 clinical trials as a potential therapy for liver cancer. Neither of these molecules targets lysine, but Taunton thinks a lysine-based covalent drug with an aldehyde handle has good odds of being safe.

Taunton hopes engineering delicately balanced, reversible agents will eventually allow him to hit a loftier goal: molecules that anchor on surface lysines just outside the protective inner pockets of proteins. These targets are swarmed with water molecules that weaken the lysine’s nucleophilicity, so the optimal trade-off between the reactivity of the electrophile and the stability of the reversible interactions is tricky to achieve.

“It’s going to require really paying close attention to the chemistry,” Taunton says.

Lots to do for lysine

Fashioning effective covalent drugs requires putting all the pieces together. “Everything matters—the scaffold, the [electrophile] reactivity, and the local environment surrounding the lysine residue,” Abbasov says. There is no one electrophile candidate that stands above the rest; the more researchers can devise, the more options will be available to tailor a drug to a specific lysine on a candidate protein.

Despite its abundance, lysine isn’t meant to replace cysteine as the covalent target of choice. Instead, researchers hope that cysteine and lysine will complement each other to widen the overall druggable space. Researchers are already exploring ways to covalently snare other amino acids, such as tyrosine and serine. Each brings its own challenges but has the potential to expand drug developers’ disease-fighting repertoire.

A breadth of options will be a win beyond drug development: researchers can use the same chemistry to tag amino acids with covalent molecular probes that can discover a protein’s secrets. Having more sites available to bind to allows scientists to poke, prod, and modify a protein to better understand its structure and function.

“There’s a lot to do in this field,” Abbasov says. “This is just the beginning of what we can envision for the future.”


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