W0265

When Small Molecules Become Protein-sized...(and Proteins Diffract like Small Molecules). Ethan A Merritt, Misol Ahn, Zhongsheng Zhang, Erkang Fan, Wim GJ Hol, Biomolecular Structure Center, University of Washington, Seattle WA 98195.

Cholera Toxin presents some unusual opportunities for structure-based drug design. Because the toxin / receptor interaction occurs in the lumen of the intestine, an orally delivered antagonist to receptor binding can block the toxin's effect without the need for absorption into the bloodstream. In a strict topological sense a receptor antagonist for Cholera Toxin does not have to enter the body at all. In particular it need not cross any cell membranes, and this removes a severe restraint on ligand size that normally applies to the design of potential drugs. At the same time, the 5-fold symmetry of receptor binding sites on the toxin practically begs to be attacked by the design of a corresponding 5-fold symmetric ligand. We therefore have both the motive and the opportunity to explore the design and synthesis of large, multivalent, receptor blockers as potential drugs against Cholera Toxin and closely related toxins from E coli.

We have successfully synthesized and characterized a series of receptor blockers, ranging from simple sugars to large multivalent assemblies, and crystallized them in complex with the toxin B-pentamer. We are fortunate in that many of these complexes diffract to near-atomic resolution. The largest of these to date is a "Ten-fingered Pentapus" with formula C₄₇₅N₇₅O₁₉₅H₇₅₅. It consists of ten galactose moieties linked via branched chains to a pentacyclene core, and exhibits an apparent binding constant of 200nM. Amazingly, crystals of this 11kD inhibitor complexed with the 55kD Cholera Toxin B-pentamer diffract to 1.4Å. I will summarize how we deal with 'small' molecules which have grown to protein size, proteins that can be refined like small-molecules, and how this is contributing to structure-based inhibitor design targeting Cholera Toxin.