By David N. Leff

When a child born in the millennial year reaches its 25th birthday, the number of people in the world suffering from diabetes will have reached 300 million.

In the U.S. alone, there are 14 million patients with adult-onset Type II diabetes, of whom nearly 200,000 die of the disease every year. Their death doesn¿t happen all at once. Like the Chinese water torture, the disease destroys one organ or body part at a time. Thus, it causes 86,000 limb amputations a year, inflicts blindness on 24,000, kidney failure on 28,000 and fatal heart failure on 77,000.

Type I ¿juvenile-onset¿ diabetics can¿t produce enough insulin to convert their food ¿ critically glucose ¿ into energy. They must inject, swallow or inhale commercial insulin every day for life.

The far more common Type II patients produce enough insulin, but the cells that normally respond to the hormone fail to do so. Insulin works by stimulating the uptake of glucose into the body¿s cells, while tamping down glucose output by the liver.

Type II diabetes is usually diagnosed in people over 45 with a tendency to obesity and a sedentary lifestyle. Diet and exercise are the therapies of choice, together with drugs to control hyperglycemia (excess glucose in the blood). Often, they too must take exogenous insulin.

Constantly ongoing efforts to design new and better drug therapies against diabetes must confront the many components of how insulin¿s metabolic pathway works. Protein kinases transfer phosphate from the high-energy molecule ATP (adenosine triphosphate) to a particular amino acid on target proteins ¿ such as glucose. That phosphorylation step is switched off by phosphatases ¿ enzymes that remove phosphate from those target proteins. Protein kinases and phosphatases are both potential targets for therapeutic intervention.

Meet Glycogen Synthase Kinase 3 beta

One such candidate drug is a protein kinase called GSK3 beta, which is active in every cell of the human body. When insulin is activated by binding to its receptor, GSK3 beta switches off. This turns on glycogen synthase, an enzyme that converts glucose into glycogen, which enables the body to store energy. Modulation of GSK3 beta activity by design of a small molecule might activate glycogen synthase in patients with Type II diabetes, who lack adequate insulin activity to control blood glucose. (GSK3 beta owes its acronym to glycogen synthase kinase, its main substrate.)

GSK3 beta is under study by Vertex Pharmaceuticals Inc., of Cambridge, Mass. ¿We are now optimizing specific small-molecule inhibitors,¿ said Vertex crystallographer Ernst ter Haar, ¿based on our knowledge of the subtle differences between GSK3 beta and other closely related kinases.¿

Ter Haar is senior author of a progress report in Nature Structural Biology for July 2001. Its title: ¿Structure of GSK3b reveals a primed phosphorylation mechanism.¿

¿We started out by cloning the GSK3 beta gene,¿ ter Haar told BioWorld Today, ¿and expressed it in a baculovirus expression system.¿ With that expression, he said, ¿we obtained a lot of protein material, which we purified. Then we grew crystals, from which we collected refraction data.

¿We were able to grow the crystals in a slightly novel way,¿ ter Haar added, ¿because one of the major difficulties in X-ray crystallography is to obtain the actual crystals. However, we were able to increase our rate of success by using a novel microbatch crystallization robot we had obtained. With this robot we used about 10 times less material, and were able to test a lot of small, different positions. And because of that we could grow those crystals in less that one day.¿

He noted that the ATP binding site ¿has global similarity to other kinases, but when we looked more closely at the full-length, three-dimensional crystal structure of glycogen synthase kinase 3beta ¿ when we had its atomic coordinates ¿ we could actually identify some minor differences. We took advantage of those differences to design our unique GSK3 beta inhibitor.

¿It¿s a late-stage research program at Vertex,¿ he observed, ¿and we believe that we will have a clinical candidate coming pretty soon ¿ for preclinical testing, probably within the next 12 months. It will be an orally available compound, to help Type II patients better control their blood glucose level, as indefinite therapeutic maintenance.¿

Novartis Enlisted For Impending Clinical Trials

¿We would like to test the compound in the human body as soon as possible, and demonstrate a pharmacological impact in human patients,¿ ter Haar said. ¿Last year we entered a collaboration with Novartis to develop eight kinase inhibitors,¿ he added, ¿and I think the Phase I drug development will be closely coordinated with their scientists to take it into clinical trials.

¿The inhibitor has an interesting ability in terms of controlling blood glucose levels,¿ ter Haar noted. ¿It has a fast-acting activity in lowering glucose ¿ better than any mouse or rat model that we have seen.

¿Knowing those structures,¿ he said, ¿allows us two things: One, from the kinase research standpoint, those kinases are very similar to each other in terms of active sites. So there was a concern about how one can design a selective inhibitor. With the molecular detail of information in the structure that we have been able to solve, we were able to engineer selectivity into the clinical candidate that we are working on now.¿

He made a separate point: ¿I just mention the homology between GSK3 beta, which is a target for diabetes therapy, and another potential kinase target for cancer and proliferative diseases. Also p38,¿ he added, ¿which is yet another protein target actively pursued by Vertex. We have p38 inhibitors in clinical development for inflammatory disease.¿

To which ter Haar added two other potential disease targets: ¿GSK3 beta is known to phosphorylate the tau protein, which has been implicated in the neurofibrillary tangles of Alzheimer¿s disease. Another possibility would be looking at the role of GSK3 beta in a NFkappaB signaling event. That would have some implication potentially for a GSK3 beta inhibitor to treat cardiovascular disease.

¿We still need to fully evaluate our inhibitory compound to see its potential applications. Over the next 12 months,¿ ter Haar concluded, ¿we will be doing further tests on our lead molecules, optimizing them and selecting the best ones for development.¿

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