BioWorld International Correspondent

LONDON - Pharmacologists who want to design drugs to treat diabetes, by inhibiting an enzyme that damps down the release of insulin, now have access to this protein's 3-dimensional structure to help them.

Scientists working at Novo Nordisk A/S in Bagsvaerd, Denmark, have worked out the crystal structure of the enzyme known as dipeptidyl peptidase-IV (DPP-IV). Many teams are working hard to find inhibitors of DPP-IV, and some of these molecules are already in clinical trials for treating Type II diabetes.

The Novo Nordisk team reported its results in a paper in Nature Structural Biology (advance online publication Dec. 16, 2002) titled "Crystal structure of human dipeptidyl peptidase IV/CD26 in complex with a substrate analog."

DPP-IV is an exciting drug target because of the role it plays in regulating glucose levels. Mice that lack the gene for DPP-IV secrete higher than normal levels of insulin and reduce their blood glucose levels more rapidly than normal. That is partly because they have higher levels of two peptides - glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP). Both GLP-1 and GIP play important roles in stimulating insulin secretion, but both are inactivated by DPP-IV.

Hanne Rasmussen, research scientist at Novo Nordisk, told BioWorld International, "By inhibiting DPP-IV, the levels of intact GLP-1 in the human body are increased. In this way, we can get increased insulin release. Importantly, this strategy does not risk inducing hypoglycemia, because insulin release is also dependent on the glucose level. Once the blood glucose level falls, the release of insulin also decreases."

Rasmussen and her colleagues solved the structure of DPP-IV using crystallographic methods. Many previous investigators tackling this problem had been misled by assuming that the structure of DPP-IV was similar to that of a closely related enzyme, prolyl oligopeptidase (POP). This has two domains, including a "propeller domain" containing seven blades.

The study reported in Nature Structural Biology, however, shows that one of the two domains of DPP-IV is a "propeller domain" - but it has eight blades rather than seven. It also contains a large cavity, approximately 30 to 45 angstroms wide, with a small pocket inside the cavity that forms the active site.

Rasmussen and her co-authors said, "The large cavity is accessible via two openings. Substrates and products may pass either through a funnel in the center of the propeller domain or through a much bigger opening in the side."

An extended "arm" of the molecule looks as though it functions as a lid to the active site, because it is situated close to the active site and has a size that matches the side opening. This may explain why DPP-IV is enzymatically active as a dimer, they wrote: "If the dimer dissociates, this arm could move towards the cavity, thereby closing the side opening of the active site."

The group speculates that the entrance to the active site is via the large side opening, as that is the shortest and most easily accessible way to the active site. The negative charge in the cleft that forms the active site would attract the positively charged N terminus of the peptide substrate into the cavity, they suggest.

They note that only elongated peptides, or unfolded or partly unfolded protein fragments, can reach the active site. "This explains why most natural DPP-IV substrates are peptides <80 amino acids," they wrote. "However, larger proteins might be substrates as long as they have an unfolded N-terminal region."

Rasmussen said, "The great advantage, now that we have this structure, is that we can use it to design very specific inhibitors of DPP-IV. This will allow us to ensure that our drug candidates have an optimal fit with this enzyme."

She said she could not comment on what stage of evaluation candidate molecules had reached, but added: "This has given us new ideas, and allowed us to make shortcuts to arrive at the right decisions about what we should synthesize."

Apart from the search for the perfect inhibitor of DPP-IV, Rasmussen and her colleagues plan to carry out further studies of the biology of this enzyme.