| METHOD |
These days, the search for clinically useful enzyme inhibitors relies on one of two strategies: Either huge libraries are screened for small molecules that bind tightly to a given target or X-ray crystal structures guide the design of inhibitors. Authors thinks there's a better way. The key to designing better drugs, he says, is to figure out what the enzyme's transition state looks like. Mimics of the substrate in the transition state make powerful enzyme inhibitors, because the enzyme binds the transition state more tightly than either substrate or product. "But transition states live for only one-tenth of a picosecond, so no direct method is available to 'see' their structures," he adds. This lack of structural information makes designing transition-state mimics challenging. To get a picture of an enzyme's fleeting transition-state structure, authors measures how isotopic substitutions in the substrate affect enzyme catalysis. He uses these kinetic isotope effects as boundaries for computational analysis of detailed transition-state structures. "This transition-state structure becomes a blueprint for chemical synthesis of a small number of closely related, chemically stable molecules. Author reported his method's first clinical success story--immucillin-H, now in human clinical trials for the treatment of T-cell leukemia--at the 18th biannual Enzyme Mechanisms Conference, held last month in Galveston, Texas. Authors began investigating PNP's transition-state structure in the early 1990s. The pair systematically replaced the atoms in one of the enzyme's natural substrates, inosine, with the corresponding heavier isotope. By assessing how each change affected the rate of the enzyme-catalyzed reaction, they established bond lengths and angles in the transition state. Semiempirical and quantum computational methods were used to convert these constraints into an electrostatic and geometric picture of PNP's transition state. Authors set out to build a molecule that resembled the altered substrate in their experimental transition-state structure. Immucillin-H's ribose ring nitrogen--which is positively charged when bound to the enzyme--simulates the oxycarbenium character of the transition state's ribose ring. In addition, its protonated purine ring looks similar to that of the transition state. At first author's proposed inhibitor "looked way too complicated. So he spent quite some time synthesizing simpler variants. "They were uniformly useless. "So eventually, I bit the bullet and synthesized immucillin-H." Author's team has since worked out a more practical convergent route to the inhibitor and related compounds. But the hard work paid off. Immucillin-H binds PNP a million times more tightly than substrates do. And as would be expected, its affinity for PNP (23 pM) exceeds by orders of magnitude those of all other known inhibitors. This dramatic improvement should mean that dosages can be kept low to reduce side effects. |
| UPDATE | 02.03 |
| AUTHOR | This data is not available for free |
| LITERATURE REF. | This data is not available for free |
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