Lawnmower-like Enzyme Rewinds Decades of Molecular Aging in Human Tissue

Imagine a steak sizzling in a pan. As it cooks, it undergoes non-enzymatic browning called the Maillard reaction. This chemical process between amino acids and reducing sugars creates flavor compounds, a savory aroma, and a nice, seared meat. But this reaction is not exclusive to food, it also occurs in humans.

“Instead of cooking at 400 degrees for 30 minutes, we are cooking at 98 degrees for 50–70 years, and we build up some of the same products [as food]…and the body has no way to get rid of them,” said Aaron Cravens, the chief executive officer of Revel Pharmaceuticals, a company that develops therapeutic enzymes. People accumulate advanced glycation end products (AGEs) in long-lived proteins, such as collagen, elastin, and eye lens proteins. The resulting protein modifications are harmful and can lead to accelerated biological aging through cellular damage and chronic inflammation; worse yet, this damage is thought to be irreversible.

Determined to reverse this damage, Cravens and his colleagues engineered an enzyme that targets the most abundant AGE in the human body. Their findings, published in Nature Communications, demonstrated that the enzyme oxidized the target AGE and thus repaired harmful modifications, bringing damage levels of a 75-year-old tissue donor down to those seen in a 31-year-old.¹ This work is a proof of concept that protein damage can be reversed thanks to enzymatic repair. The researchers hope this work provides a foundation for developing interventions, particularly for patient populations that accumulate AGEs at a faster rate, like those with type 2 diabetes.

Previous groups have tried to develop enzymes to target and cleave AGEs; however, their attempts were unsuccessful. According to Cravens, one of the biggest challenges is that there’s no obvious naturally occurring enzyme that does this reaction, so how do researchers even begin to find it? “You’re looking for a needle in a haystack and then once you find that needle, you have to engineer it because it’s super dull…you have to sharpen [it] to actually turn it into a useful tool.”

In their search, the researchers targeted Nε-carboxymethyl-lysine (CML), the most abundant AGE formed on lysine residues on proteins.² CML can then act as a ligand by binding to the receptor for AGE; this promotes cell signaling that drives inflammation, oxidative stress, and disease progression.³ So, the team’s goal was to find an enzyme that could bind to CML and also reverse this harmful process. “We believe you can remove [CML damage] enzymatically, by going in and developing these lawnmower enzymes that can just cut and clip these changes off of the proteins,” explained Cravens.

Next, the team screened large DNA libraries to find an enzyme with CML-cleaving activity either in a free state or peptidyl-CML, which is when CML is incorporated in a larger protein like collagen. In their search, the researchers found glycine oxidase from the bacterium Calidithermus roseus (CrGO) to be a potential candidate. Then, the researchers used directed evolution to enhance the enzyme’s oxidase activity, which would restore the native lysine and reverse CML modifications on long-lived proteins.

The researchers assessed the activity of their newly sharpened enzyme, the variant CrGO-897 (CMLase), on a panel of physiologically relevant proteins including casein, hemoglobin, collagen, and retinal total protein extract. They induced CML modifications of similar degrees by treating the proteins with glycolic acid. Then, the team measured CML levels pre- and post-enzyme treatment and found that CMLase could remove the majority of CML damage on the proteins.

Because CML accumulation typically occurs in long-lived organisms, short-lived animal models are ill-suited. “If you’re 50, 60, or 70, you’ve had a totally different life experience than a mouse that’s lived for two years. And so, your tissues have had a very different set of damage built up,” explained Cravens. So, the team tested CMLase on human tissue, where CML has had decades to accumulate.

The researchers tested CMLase on aorta, skin, and lens tissue—places of CML accumulation— from donors aged 20 to 25 and compared them to a 75-year-old donor. They then looked for reduction or presence of CML. The results surprised Cravens. “We were pretty floored because we were expecting a 20 percent reduction,” he said. “But after these studies, the post-enzyme treatment showed that we were reducing the levels back to that of what we’d see in like a 30-year-old’s tissue.”

“I thought it was pretty bold to even try and tackle [this] problem,” said James Galligan, a pharmacologist and toxicologist at the University of Arizona, who studies protein glycation and how it impacts health and disease and was not involved in the study. “If you look at the post-translational modifications (PTMs), they’re rock solid. They’re not going to go anywhere…these have long been thought to be irreversible.” However, he added, “[Here], they’re able to develop something by the metrics they’re using that is selective to remove these modifications.”

Based on these findings, the researchers’ work is a proof of concept for reversing CML-related damage to aging tissues. Galligan added, “[This work] paves the way to at least iron out the biology of what these PTMs actually do.” Cravens remarked his excitement for potentially using this treatment as a “one-and-done” approach, where patients can clean up and remove decades of damage. Reducing the burden of CML on long-lived proteins may be a viable strategy in lessening disease burden in people with diabetes and other age-related diseases.