Feature: Chemistry

David Vocadlo’s insights into glycans may lead to therapeutics for cancer and Alzheimer’s disease.

By Tyler Irving

While the study of DNA and proteins has become almost routine, another important class of biomolecules — glycans — is just beginning to give up its secrets. David Vocadlo of Simon Fraser University in Burnaby, British Columbia is a researcher at the frontiers of chemical glycobiology. ACCN spoke to Vocadlo to find out how the study of glycans is changing our view of biochemical processes and why they may be key to treating diseases like diabetes, Alzheimer’s and cancer.

ACCN What is a glycan?

DV Glycans are one of the key classes of biological molecules. You have nucleic acids like DNA and RNA, proteins, lipids and carbohydrates, which are also called glycans. The other biomolecules are quite heavily studied, but glycans are more difficult to study for a number of different reasons, so the field hasn’t advanced as far. Simply put, glycans are an assembly of different sugar units that form very specific structures. When someone ingests some sugar such as glucose, it gets converted within cells into other types of sugar building blocks. In humans there are essentially 10 different types of sugar units that are used to build up glycans. Glucose is just one of them, there are others such as mannose and galactose. In principle,the diversity of different structures is tremendous, there was recently a paper conservatively estimating that mammals have 7,000 different types of glycans.

ACCN How do you study them?

DV Our laboratory is mostly interested in chemical glycobiology. We study enzymes and other proteins that interact with carbohydrates and then develop chemical tools with which we can study those biological processes as well as the enzymes themselves. Ideally, we use these tools to look at them in cells or even in vivo to try to discern what their function is. It’s exciting because it’s a frontier area; there’s a lot to discover in the field of glycobiology. And it’s turning out that these glycans are very important in a number of different diverse biological processes.

ACCN Can you give some examples?

DV Every animal cell has a carbohydrate coating around it, known as the glycocalyx. The glycocalyx extends out from the cell surface and is the first line of contact between the cell and other cells or, alternatively, viruses or bacteria. It’s what the other cells see and they use it to communicate with each other. Some beautiful work has shown how some glycans on the cell surface are essential for organisms to develop in an appropriate way.

An older example that people can relate to is the ABO blood group antigens, which are glycans. The differences between them are simply the presence or absence of different sugar units within the glycan structure. For example, the O-type glycan has one less sugar unit than the A-type or the B-type. These blood group antigens are interesting: we know that they exist and we know that they’re important for transfusion, but it’s not known what their precise biological functions actually are or why there are different types. We speculate that this diversity has been important historically to make sure that populations are resistant to epidemics. Blood group antigens have a long history in Canada; Ray Lemieux, of the University of Alberta, was the first to synthesize them chemically, a fantastic feat at the time.

ACCN Much of your work revolves around the O-GlcNAc post-translational modification. What is this?

DV Proteins can be modified in many ways: they can be phosphorylated, acetylated, methylated and of course glycosylated. O-GlcNAc is a form of glycosylation, a modification of proteins found inside the cell. It’s just a single saccharide unit, O-linked N-acetylglucosamine (O-GlcNAc) that gets added on to proteins. There are two enzymes involved: one called O-GlcNAc transferase (OGT) that puts O-GlcNAc on the proteins and one that removes it, called O-GlcNAcase (OGA).

We started off trying to understand how these enzymes (OGA and OGT) work at a mechanistic level and how they catalyze the reactions that they do. We then developed inhibitors that would target them in a specific way. We’ve developed very good inhibitors of both of them, so we’re now using those to understand what some of the biological roles of the O-GlcNAc modification are. The modification is interesting since it’s been implicated in both diabetes and Alzheimer’s disease.

 
In the body, proteins (represented in red) are converted between their native form (left) and glycosylated form (right). The enzyme OGT adds the glycan O-GlcNAc (in black) onto the protein while the enzyme OGA removes it. David Vocadlo’s team has developed robust inhibitors of both OGA and OGT to probe the role of this modification in such biological processes as diabetes, cancer and Alzheimer’s disease.

ACCN How so?

DV It’s an elegant hypothesis. When glucose is taken up by cells, a certain percentage of it is shunted into a biosynthetic pathway, the end result of which is the substrate for OGT. OGT takes that substrate, adds it on to proteins and the net result is increased O-GlcNAc levels. The second theory is that O-GlcNAc glycosylation and phosphorylation of proteins are sometimes reciprocal. This is important because insulin signaling depends on phorphorylation pathways. When you take those two ideas together, what you can see is that with increased blood glucose levels — one of the symptoms of diabetes — proteins within cells are going to have increased O-GlcNAc levels. If O-GlcNAc levels on proteins go up, they might compete with and impair phosphorylation and that might attenuate insulin signaling. It becomes a vicious cycle where increased blood glucose has toxic effects and impairs insulin signaling, which results in even higher blood glucose and more damage. So we started to develop selective compounds that would target OGA, the enzyme that removes the sugar, in order to determine the involvement of O-GlcNAc in insulin resistance. What we found out by doing this was that you could increase O-GlcNAc levels dramatically, but it didn’t actually result in insulin resistance.

ACCN Were you surprised that the results didn’t fit with the hypothesis?

DV For us it was a very surprising observation. But that is the nature of science and what makes it continually interesting to me — there are always surprises. In any case, the experiments we did suggest that the current proposal is not as simple as thought and offer some new approaches and ideas as to how the model might be refined. I think in the end our findings turned out to be quite important for other reasons as well. They show you can inhibit the enzyme and not cause serious problems like insulin resistance. Actually, that is what prompted us to consider looking at Alzheimer’s disease and the potential benefit of increasing O-GlcNAc levels; you obviously don’t want to cause insulin resistance while trying to improve Alzheimer’s.

ACCN How does Alzheimer’s come into it?

DV One of the hallmarks of Alzheimer’s are these neurofibrillary tangles in the brain, which contain a hyperphosphorylated form of a protein called Tau. A lot of people are interested in blocking the aggregation of Tau and its toxicity by blocking its phosphorylation. In addition to that, it’s known that in Alzheimer’s disease, one of the early symptoms is an inability of neurons to appropriately use glucose. So the idea there is that if you get decreased levels of O-GlcNAc within cells, this might enable hyperphosphorylation of Tau, which leads to its aggregation and downstream toxicity.

We published a paper on using our inhibitors to block OGA, the enzyme that removes O-GlcNAc. It showed that if you increase O-GlcNAc levels dramatically with this inhibitor, you see decreases in phosphorylation of Tau at certain specific sites that are pathologically relevant.

ACCN Are there other diseases where O-Glc-NAc is thought to play a role?

DV Recently there have been a few papers published implicating O-GlcNAc in cancer and there’s some fairly good data supporting that idea. The theory in that particular case is that somehow O-GlcNAc is involved in the ability of cancer cells to survive and proliferate. If you can decrease levels of O-GlcNAc in cells, you may be able to prevent the progression of cancer. That’s one of the really nice things about inhibitors — you can quickly deploy them in different experimental models.

ACCN Tell us about your company, Alectos Therapeutics, formed in 2007 to commercialize these discoveries.

DV A lot of the large pharmaceutical companies are very interested in Alzheimer’s disease because there’s no therapeutic right now that will block its progression. Alectos Therapeutics contacted Merck about the possibility of establishing a research-collaboration and licensing agreement and they were very interested in the target as well as the technology. So Alectos is focusing on developing inhibitors of OGA for Alzheimer’s disease. The ones we have are a starting point; although they’re quite good in terms of specificity, there are always things that may need to be tweaked to generate compounds that have the most desirable properties in the body. Right now we’re looking at a number of different issues that have to be addressed for the program to progress.

ACCN Why is it so much harder to study glycans than DNA or proteins?

DV It’s been difficult to dissect the critical functions of glycans in biology because, in many cases, if you just knock out an enzyme that’s involved in building up a glycan, you can have developmental effects in the organism that you’re studying. It’s not comparable to changing the levels of glycans in an adult organism. Also, knocking out an enzyme is not the same thing as the enzyme being inhibited; if you knock out the enzyme the protein is gone and that can complicate issues. That’s why we’ve gone with an inhibitor-based approach.

Another factor is that glycans are difficult to synthesize. If you want to synthesize a nucleic acid, you can use a nucleic acid synthesizer — you can just call up a company and get it delivered. For glycans that’s very difficult; it’s a tremendously laborious process. You have to use traditional chemical synthesis. It’s a really time-consuming process and there are people who specialize only in synthesizing different glycans. To make a glycan with just six units can take from six months to one year.

ACCN Have there been any major advances in the past few years?

DV There’s been some really tremendous efforts directed at solid-phase synthesis of carbohydrates and there are a number of groups around the world that are working on that. Basically you use polymer beads as an anchor, just like with peptide synthesis and then you add to the glycan chain while it’s on the beads and cleave it off at the end. It’s not in the mainstream yet but there are now carbohydrate synthesizers that have been developed and they’re being prototype tested. Researchers have also been advancing the use of enzymes to rapidly build glycans and this has shown a lot of promise as well.

ACCN What excites you about glycobiology?

DV When you teach science, often you’re teaching people about what is known, but the most exciting part of science is what’s not known. In the field of glycobiology and glycoscience, there’s a tremendous amount that is not known and that’s the stuff that is really exciting. It’s pushing that limit of what we know, seeing what lies just over there. What will this study show about the role glycans are playing in biology? Can we manipulate this system for potentially therapeutic benefit? I think those are some of the really interesting questions in the field.

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