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Carbohydrate-based medicines – more than a sugarcoating
8 March 2012
A recent breakthrough in sugar chemistry could open the door to a new generation of synthetic medicines and diagnostic tools.
BBSRC-funded researchers at the Universities of Oxford and York have found compelling evidence that almost half of the enzymes that attach sugar molecules to proteins might do so using a very rare type of chemical reaction. The finding could provide a route to designing new protein- and inhibitor-based molecules with a wide variety of clinical uses.
Carbohydrates have been described as the 'next frontier' in biomedicine (ref 1). But despite their central role in biology (combining with proteins and fats on cell surfaces to bring about cellular signalling and recognition processes, the functioning of the immune system, the ability of various infectious agents to make us ill, the progression of cancer and even determining our blood groups) there are surprisingly few drugs on the market that target carbohydrate chemistry.
According to Professor Benjamin Davis from the University of Oxford's Department of Chemistry, the main barrier to drug development in this area has been a lack of understanding of the complex mechanisms by which sugars are metabolised.
Since the Nobel prize-winning work in the 1950s of the Argentinean chemist Luis Leloir, who identified that highly energised forms of sugar molecules called sugar nucleotides were fundamental to carbohydrate metabolism, relatively little progress has been made in understanding exactly how some sugars attach to other molecules – a process known as glycosylation.
"Leloir's work revealed that sugar nucleotides act as substrates for key enzymes in the glycosylation process – the glycosyltransferases," says Davis. "While more recent studies have suggested a catalytic mechanism used by many of these enzymes, so-called inverting glycosyltransferases, the reactions of almost half, the retaining glycosyltransferases, remain unclear.
"It is critical that we fully understand these important enzymes in order to aid the design of potent inhibitor molecules, and any associated therapeutic strategies."
Learning from mimics
Observed electron density for a transition-state mimic binding synergistically with UDP in the active centre of OtsA.
Copyright Gideon Davies, University of York
As part of a recent BBSRC-funded study, Davis's team, together with Professor Gideon Davies and colleagues at the University of York have found the first compelling evidence that retaining glycosyltransferases use a mechanism called internal nucleophilic substitution or SNi to link sugar nucleotides to other molecules. Their findings were published in Nature Chemical Biology.
Their breakthrough came by mimicking the transition state – the point in a chemical reaction at which reactant molecules will always go on to form products.
The team had previously described the X-ray structure of a so-called ternary complex of the glycosyltransferase enzyme OtsA bound to two other molecules, validoxylamine A 6'-O-phosphate (VA6P) and uridine diphosphate (UDP). Together these two molecules mimic the normal 'donor' and 'acceptor' substrates of OtsA – the sugar nucleotide uridine diphosphate glucose (UDP-Glc) and glucose-6-phosphate respectively.
"In our previous study we showed that VA6P was a competitive inhibitor with regard to the donor substrate UDP-Glc. The potency of VA6P as an inhibitor increased in the presence of UDP," Davis explains.
But while this 3D structure of their ternary complex was suggestive of a transition state operating via an SNi mechanism, the team needed more experimental data to test their hypothesis. In their latest research, Davis and colleagues have uncovered new evidence, based on studies of so-called linear free-energy relationships (LFER) and kinetic isotope effects (KIE), that their ternary complex is indeed a true reflection of the transition state.
Davis explains, "By measuring KIEs in labelled substrates, we determined that a carbon-oxygen bond in the natural donor substrate, UDP-Glc, breaks during the transition state generating an oxygen leaving group. We also showed that the natural acceptor substrate, glucose-6-phosphate, interacts with this oxygen and is involved in the transition state, forming several hydrogen bonds with the enzyme's active site.
"These findings, together with the LFERs, supported our earlier observations that the 3D structure of our ternary complex is likely to reflect the transition state of a retaining glycosyltransferase," says Davis.
He goes on to say, "While the free-energy relationship data alone could also be interpreted as consistent with other mechanisms, as a whole, the complete data presented in our latest paper, combined with our earlier structural data provide a more definitive set of conclusions, which are consistent with an SNi mechanism.
"This was a really interesting discovery as SNi is found very rarely in organic chemistry.
"The only way to demonstrate this was to bring together all the parts of the puzzle, which was made possible through this latest BBSRC-funded project."
"What is additionally exciting is how the work dovetails with computational approaches and work on non-enzymatic reactions published by others subsequently; these are all pointing to a consistent, but highly unusual, mechanism," says Professor Gideon Davies FRS, head of structural enzymology and carbohydrate chemistry at the University of York (ref 2 and ref 3 respectively).
The discovery should prompt the useful reassessment of many biocatalysts, their substrates and inhibitors, which Davis believes could revolutionise medicine. For example in the design of synthetic inhibitor molecules that can prevent cancers from spreading.
Directing drug development
As well as their fundamental studies of the glycosylation process, Davis and his team are also investigating the production of glycosylated proteins.
"There are many synthetic small molecules that are used as medicines. But imagine completely re-building an entire protein, says Davis. "We're not just talking about modifying natural proteins but building synthetic proteins at will."
In 2003 Davis co-founded the spin-out company Glycoform, applying the patented methods for glycosylating proteins that he and his team had developed to direct the production of new compounds in a targeted manner.
His second co-founded spin-out, Oxford Contrast is close to securing first round investment. This new company will focus on the application of patented technology developed by Davis and others at Oxford for in vivo diagnostics and monitoring of important diseases such as Multiple Sclerosis.
It was this innovative approach that led to Davis's nomination as a finalist in the 2010 BBSRC innovator of the year competition.
Davis concludes, "Sugars always seem to have been the 'Cinderella' biomolecule, neglected perhaps because of their apparent complexity. Yet, they allow fascinating and useful forms of control in biology that, through chemistry, might allow us to both map and treat disease."
Mechanistic evidence for a front-side, SNi-type reaction in a retaining glycosyltranferase. Nature Chemical Biology DOI:10.1038/NCHEMBIO.628
- Carbohydrates as the next frontier in pharmaceutical research, Chemistry – a European Journal (2005) DOI: 1002/chem.200500025
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