A Potential Approach to Tackling CEL and CML Advanced Glycation End Products

Advanced glycation end-products (AGEs) form in tissues as a side-effect of the normal operation of cellular metabolism where it touches on the processing of sugars. There are many types of AGEs, most short-lived, but some persistent and challenging for our biochemistry to break down. These persistent AGEs lead to cross-links, binding together molecules in the extracellular matrix and thereby altering the structural properties of tissues. This is perhaps most harmful where it reduces tissue elasticity, and is thus an important contributing cause of skin and vascular aging.

While sugars are involved, it is much debated as to whether the contents of diet, either fully formed AGEs from certain cooked and processed foods, or precursors in the form of excessive amounts of sugar, has much influence at all over the generation of the types of AGE involved in aging. As mentioned, there are many types of AGE. One of the big questions in the small research community focused on AGEs is whether or not glucosepane AGEs are the only target worthy of attention in the matter of aging. There is certainly good evidence for cross-links in humans to be overwhelmingly made of glucosepane, but equally there is a faction who argue that the research community does not yet have sufficiently robust data to be able to ignore AGEs such as carboxymethyl-lysine (CML).

The challenge inherent to all work on AGEs, and why this part of the larger field has been a comparative backwater for decades despite its great importance to aging, is that the usual tools for cell, tissue, and molecular biochemistry work just don't exist. AGEs are hard to work with. The usual recipes for making the molecule of interest, the standardized tests for assessing its presence, and so forth, just don't exist or didn't exist until comparatively recently. Most research groups take a look at this desert of tooling and move on to something easier - it is a self-reinforcing problem. This was the case until the SENS Research Foundation and allied philanthropists turned up to try to solve the missing tools problem. Those efforts have led to significant progress in the past five years or so, but there is still a fair way to go yet. Today's paper is of interest for showing progress towards tooling for CML, rather than for glucosepane. It is not open access, but sufficiently interesting to note nonetheless.

Biocatalytic Reversal of Advanced Glycation End Product Modification

Advanced glycation end products (AGEs) are non-enzymatic post-translational modifications of proteins derived from the condensation of reducing sugars and nucleophilic amino acid residues, such as lysine and arginine. Although AGEs are formed in the body as a part of normal metabolism, they can accumulate to high concentrations and contribute to the progressive decline of multiple organ systems. This process is accelerated in diabetics, owing to their hyperglycemic conditions. In addition to causing spontaneous damage by altering protein structure and function, AGEs also interact with the receptor for AGEs (RAGE), eliciting oxidative stress and activating the transcription factor NF-κB thought to be a major contributor of AGE-associated chronic inflammation and cellular damage.

Elevated levels of AGEs are linked to the pathology of many metabolic and degenerative diseases of aging, such as diabetic complications, atherosclerosis, and Alzheimer's disease. This association is manifested by age-dependent increases in cross-linking, browning, fluorescence, and AGE content in long-lived proteins such as collagens and lens crystallins. Structural characterization and synthesis of some of the more prevalent AGEs (e.g., glucosepane) have allowed more focused investigations into their individual chemical properties and formation. Indeed, chemical studies have shown strong correlations between specific AGEs and the development of age-related illnesses; however, it has been difficult to unequivocally demonstrate that any AGEs are direct causal factors largely due to the lack of tools for investigating the reversal of mature AGE modifications at the molecular level.

Here, we show that MnmC, an enzyme involved in a bacterial tRNA-modification pathway, is capable of reversing the AGEs carboxyethyl-lysine (CEL) and carboxymethyl-lysine (CML) back to their native lysine structure. Combining structural homology analysis, site-directed mutagenesis, and protein domain dissection studies, we generated a variant of MnmC with improved catalytic properties against CEL in free amino acid form. We show that this enzyme variant is also active on a CEL-modified peptidomimetic and an AGE-containing peptide that has been established as an authentic ligand of the receptor for AGEs (RAGE).

To the best of our knowledge, this is the first biochemical demonstration of an enzyme that can reverse a mature AGE-functionalized peptide. While the kinetic parameters, which are similar to known Amadoriases, could be substantially improved, C-MnmC variants represent lead catalysts for further directed evolution and development. As MnmC natively acts on nucleic acids, glycated DNA (e.g., carboxyethyl/carboxymethyl-deoxyguanosine) may also be suitable substrates to test in future studies. Such improved AGE-reversal tools could in principle enable a better understanding of the biology of AGEs at the molecular level, elucidate their direct roles in the pathogenesis of age-related diseases, and serve as leads for recombinant enzyme therapies.

Comments

I often hear about cross linking being a particularly hard problem to fix, but often enough I also read about how, after senescent cells, it is likely to be the next domino to fall. What is the reason for this optimism, and how soon do people predict something like this might be in the clinic? Given an effective cross link breaker has the potential to make people look younger, this seems especially important. A world in which people can restore the appearance of youth is a world in which chronological age and biological age are no longer synonymous in people's minds.

Posted by: Ben at May 2nd, 2019 12:22 AM

@Ben
>Given an effective cross link breaker has the potential to make people look younger, this seems especially importan...

It is not only looks. The vascular system, and probably, other tissues will be better, reverted to Las aged level, do effectively rejuvenated...

Posted by: Cuberat at May 2nd, 2019 6:16 AM

Yes, I know, but once people's appearance may be rejuvenated every person who makes use of such an intervention would become a walking billboard for SENS. The pro-aging trance relies upon the fallacy that chronological and biological age are the same thing, and that age related damage is not a problem to be solved, but an aspect of identity. Smash the fallacy and you wake people from the trance.

Posted by: Ben at May 2nd, 2019 1:28 PM

Its a long process and cross-link breakers will probably emerge treating something like stiffness in the arteries. We are probably talking a 15-20 years process once a drugable target is identified to the approval by the FDA of a really good drug.

It seems overwhelming the whole process, but the other side is that a drug like that with a huge potential market, would be a multi-billion a year drug. For the sake of argument, say such a drug earned $35 billion over the course of its on-patent life.

Let us say it could be quite lucrative if research & development costs amounted to $6 billion, once there is the cost of capital and so forth. If you employ someone for their entire career of ~40 years at $150,000 a year, that works out to $6 million dollars career cost.

So for $6 billion you could employ 1,000 of those people for their entire careers. I am showing these numbers to give you guys a feeling of the scale of labor involved in these projects.

Posted by: aa3 at May 2nd, 2019 7:15 PM

When is Revel LLC ever going to launch and get their glucosepane breaker into clinical trials?. It seems like Aubrey de Grey has been talking about it for 5 years.

Posted by: jimofoz at May 5th, 2019 1:03 AM

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