GlycoSENS: Breaking extracellular crosslinks

 

Many of the major structural features of the body are built out of proteins that are laid down early in our life, and then more or less have to last for a lifetime: they are never recycled or replaced, or are only recycled over the course of many decades slowly. The healthy functioning of these tissues relies on these constituent proteins maintaining their proper structure. Such proteins are responsible for the elasticity of the artery wall, the transparency of the lens of the eye, and the high tensile strength of the ligaments, for example. But occasionally, blood sugar (and other molecules in the fluids in which these tissues are bathed) will react with these proteins, creating chemical bonds called crosslinks. Crosslinks act like molecular “handcuffs,” taking two neighboring proteins that were previously able to move independently of one another and binding them together, impairing their function in the same way that occurs to participants in a three-legged race. In the case of the artery wall, for instance, the crosslinking of strands of the protein collagen prevents them from spreading apart from one another to accommodate the surge of the pulse being driven forward by the pumping action of the heart. As more and more strands of collagen become crosslinked together over time, the blood vessels to become ever more rigid, leading to a gradual rise in systolic blood pressure with age. With the loss of the cushioning effect provided by free-moving collagen in the blood vessels, the force of the surge of blood that is driven into the arteries by the pumping action of the heart is carried directly to organs like the kidneys and the brain, damaging to the structures that filter our blood and that connect the functional regions of our brain, and putting us at risk of a stroke.
 
Stiffening by Other Means
In addition to the chemical damage that occurs to structural proteins, there is also mechanical damage: subtle (and not-so-subtle) wear and tear. One subtle form of mechanical damage that probably contributes to the stiffening of the large arteries and of other organs like the the lung over time is the fraying and breakage of structures made of the stretchy protein elastin. Whereas collagen fibrils accommodate the pulse flowing through the blood vessels by spreading outward from each other, elastin helps to cushion the pounding of the pulse by stretching forward and outward. But like an elastic band that is stretched and relaxed over and over again, undergoing 100,000 rounds of stretching and relaxing each day in response to the heartbeat gradually wears out the individual fibrils of elastin, causing them to fray and tear. Over time, the loss of functioning elastin structures in the arteries also leaves the kidneys and brain more vulnerable to the direct pounding of the pulse.
 
The Solution
Fortunately, the crosslinks that occur as chemical accidents in our structural tissues have very unusual chemical structures, which are not found in proteins or other molecules that the body makes on purpose. This should make it possible to identify or design drugs that can react with the crosslinks and sever them, without breaking apart  any essential structural bystanders. Indeed, several years ago a group of chemists seemed to have found such a molecule: a drug called alagebrium (or ALT-711), which substantially lowers blood pressure in aging or diabetic animals, in addition to several other beneficial effects. In humans, however, alabebrium’s effects were very minor and limited. This may be because the kind of crosslink that alagebrium severs is much rarer in humans than it is in rodents and other experimental animals, or perhaps because the drug has additional beneficial properties in animals that extend beyond breaking crosslinks that are less important in humans.
 
So the search is on now to develop new and more human-specific crosslink breakers. It’s now known that the single greatest contributor to total unintentional collagen crosslinking in humans is a very complex molecule called glucosepane; therefore, drugs that cleave this molecule are likely to have the strongest rejuvenative effect on tissue elasticity. But glucosepane may be too complex a molecule to be broken by the kind of small-sized molecules that are usually used as drugs. So some novel approaches deserve exploring – such as:
 
  • Finding or engineering enzymes, instead of drugs, to break the crosslinks. Enzymes are potentially more effective than drugs in this application because they can use cellular energy (ATP) to power the crosslink-cleaving reaction. There is very little ATP in the extracellular space, so such an enzyme might need to shuttle back and forth across the cell membrane to ‘recharge’ itself. Fortunately, while glucosepane crosslinks are the most common class of these stiffening links, their absolute number in aging tissues is still fairly low, and they form very slowly. This should mean that the amount of ATP that such an enzyme might need to “steal” from local cells in order to effect a real rejuvenation in tissue elasticity would not significantly affect cell function.
  • Developing "one-shot" proteins that would break the crosslink but would themselves be destroyed in the process. Such proteins are already known to exist for other purposes: for example, the DNA repair protein MGMT. The “disposable” nature of these proteins means that we will need to supply them in higher numbers than we would with an enzyme, but again, the very slow rate at which glucosepane crosslinks form means that we will only have to use a relatively small number of them in order to restore elasticity in our tissues.
 
On the other hand, it’s possible that the bulk of the glucosepane crosslinks in our arteries are ‘buried’ inside tightly-knit fibers collagen, so such protein-based therapeutics may be too big to reach their targets. Clearly, a range of possible approaches to AGE-breaking will need to be explored. 
 
An entirely different solution will be required to overcome the fraying of the elastin structures in the arteries and elsewhere in our bodies. This may involve transplantation of new elastin structures, or of wholesale replacement of parts of the large arteries. Preliminary experiments with tissue engineered patches for the pulmonary artery are a step in this direction.
 
* Unlike these long-lived structural proteins, the proteins inside our cells are continuously being broken down and rebuilt to replace damaged proteins and to match the available proteins with the metabolic needs of the cell. When the systems responsible for breaking down and recycling these proteins fail, they leave harmful waste material inside cells, but for the great majority of proteins, these systems operate quite well when the cell is youthful and healthy.
 
Research Funded by SENS Research Foundation 
Collaborative Project on Glucosepane Crosslink-Breakers
In late 2011, SENS Research Foundation and the Cambridge University Institute of Biotechnology finalized an agreement for the new SENS Research Foundation Laboratory at Cambridge. Subsequently, the Foundation recruited two post-doctoral candidates to carry out GlycoSENS research there. The initial goal of this project is to develop reagents that can rapidly and specifically detect glucosepane-linked proteins. The development of such reagents is an important enabling technology for the development and testing of potential glucosepane-breaking drugs. The overall GlycoSENS research program will be carried out as a collaboration between the Cambridge SENS Research Foundation Laboratory and Dr. David A. Spiegel’s group at Yale University, which has special expertise in making glycation crosslinks and has recently been studying the mechanisms and chemical vulnerabilities of precursors of glucosepane. In addition to being put to use in SENS Foundation-funded research, the labeling reagents that emerge from the first phase of this work will made available to other scientists, to accelerate research into the role of crosslinks in disease and aging, and into ways to combat them. Dr. Spiegel’s group will then move on to developing agents that may be able to cleave glucosepane, which can then be tested in vitro and in vivo at the Cambridge center.
 
Resources
For a more in-depth account of protein crosslinks and how to remove them, see the chapter “Breaking the Shackles of AGE” in  Ending Aging. The Afterword of the paperback edition contains additional information on the mechanical fatigue of elastin structures in the arteries.
 
Publications
Click here to view a list of SRF-funded academic publications relevant to GlycoSENS, or click here for a much more comprehensive reading list of third-party publications on this topic.