Blacksburg, Va., June 21, 2004 -- A multi-university research team led by Virginia Tech University Distinguished Professor of Chemistry David G. I. Kingston and his collaborators Professor Susan Bane at the State University of New York (SUNY) Binghamton and Professor James P. Snyder at Emory University has succeeded in enhancing the structure of paclitaxel (TaxolTM) to make it more effective in killing cancer cells. They did so by rational design, based on a hypothesis developed by Snyder that paclitaxel adopts a "T-shaped" conformation in its binding pocket on beta-tubulin.
This work is described in the Proceedings of the National Academy of Sciences (PNAS) online early edition the week of June 21 (www.pnas.org), in the article "The bioactive Taxol conformation on (beta)-tubulin: Experimental evidence from highly active constrained analogs," by Research Scientist Thota Ganesh, Graduate Student Rebecca C. Guza and David Kingston, all of Virginia Tech; Research Scientist Rudravajhala Ravindra, Graduate Student Natasha Shanker, and Susan Bane, all of SUNY Binghamton; and Graduate Student Ami Lakdawala and James P. Snyder of Emory, all with the chemistry departments of their respective institutions.
Paclitaxel, a natural compound from yew trees, is the world's best-selling anticancer drug. Several analogs are in clinical trial, with others in preclinical development. One problem that paclitaxel and all its analogs share, however, is that they are all highly complex compounds, and cannot be prepared synthetically in a commercially viable way. "The Holy Grail of paclitaxel research would be to find a compound that had the same biological effect as paclitaxel, but could be prepared synthetically in a few steps," said Kingston. The discovery by the cross-university team marks a significant step along the road to this objective.
Kingston explains that paclitaxel binds to tubulin, a protein molecule that forms the backbone of microtubules. Microtubules are a cell component whose duties include allowing chromosomes to move into the correct position for the cell to divide into two daughter cells. "When paclitaxel binds to tubulin, it stabilizes the microtubules and messes up the equilibrium between tubulin and microtubule," said Kingston. "A cell with stable microtubules proceeds to programmed cell death without dividing."
How does paclitaxel bind to tubulin? There is a binding pocket in the protein into which part of the paclitaxel molecule fits. This binding pocket has been visualized by some elegant electron crystallography experiments carried out by scientists at the Lawrence Berkeley National Laboratory (Nogales, Wolff Downing, Nature 1998, 39, 199-203). Paclitaxel consists of a rigid ring system attached to a flexible side chain, but the exact arrangement of the side chain in space is not known. "The issue has been, what is the shape or orientation of the side chain when paclitaxel is sitting on the microtubule? If we could figure that out, we could design a molecule that would plug in better than paclitaxel for better binding and possibly better activity against cancer," said Kingston.
What is the right conformation of the side chain? The key to answering this question came from a hypothesis put forward by Snyder. Based on a computer model of the paclitaxel binding site, he and his colleagues proposed a particular orientation of the side chain, known as "T-taxol" (Snyder et al., Proc. Natl. Acad. Sci. USA, 2001, 98, 5312-5316). Kingston, his colleagues, and Snyder then designed molecules with bridges between a ring and a short side chain. "The strategy is a perfect example of structure based molecular design. The unique T-conformation of Taxol invites construction of a tether between two atoms in the 3-D structure of the molecule that appear distant when drawn in 2-D on paper," said Snyder.
"We thought that if we linked the bridge in the right position, maybe it would hold the side chain in the right place," Kingston says.
Bridging per se wasn't a new idea. The teams of Gunda Georg of the University of Kansas, Iwao Ojima of SUNY Stony Brook, and JoŽlle Dubois of CNRS in France all made bridged paclitaxel derivatives, using a different hypothesis to guide them, but these were all less effective than natural paclitaxel.
Kingston collaborated with Snyder to design a paclitaxel analog that linked in a new way, and Bane tested it in her assays. "We synthesized a number of the compounds and refined the details of how the link is formed until, last summer, we were able to get activity as good as paclitaxel." Presently, their best compound is about 20 times more active than paclitaxel in one assay, or biological test measuring the analog's ability to kill cancer cells. In another assay, the compound is one and a half times more active; and in a third assay, it is three times more deadly to cancer cells. "In measurements of interaction with tubulin, it is two to three times more active than paclitaxel," Kingston says.
The research is significant because it has validated Snyder's model, provided a more exact picture of the 3-D form that paclitaxel takes in order to bind to tubulin, and "it offers the exciting possibility that now that we know that shape, we can design simpler molecules with a similar shape, which is what we are doing now," Kingston said. The team is optimistic that simpler molecules can be designed as future medicines.
The Virginia Tech graduate student on the project, Becky Guza of Ivanhoe, Minn., received her B.A. in chemistry and biology from the College of Saint Benedict in St. Joseph, Minn. in 2002, graduated with a master's degree in chemistry from Virginia Tech this spring, and will be a Ph.D. candidate in biochemistry at the University of Minnesota.
The Emory graduate student on the project, Ami Lakdawala, is now principal scientist at Glaxo-Smith-Kline.
Source: Eurekalert & othersLast reviewed: By John M. Grohol, Psy.D. on 21 Feb 2009
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