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J. Biol. Chem., Vol. 282, Issue 7, 5004-5014, February 16, 2007

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Inhibition of Metalloprotease Botulinum Serotype A from a Pseudo-peptide Binding Mode to a Small Molecule That Is Active in Primary Neurons^*

James C. Burnett, Gordon Ruthel, Christian M. Stegmann^¶, Rekha G. Panchal, Tam L. Nguyen, Ann R. Hermone, Robert G. Stafford, Douglas J. Lane, Tara A. Kenny, Connor F. McGrath, Peter Wipf^||, Andrea M. Stahl, James J. Schmidt, Rick Gussio^**, Axel T. Brunger^¶1, and Sina Bavari²

From the Target Structure-based Drug Discovery Group, SAIC-Frederick, Inc., and the ^**Information Technology Branch, Developmental Therapeutics Program, National Cancer Institute-Frederick, Frederick, Maryland 21702, the United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, Maryland 21702, the ^¶Howard Hughes Medical Institute (HHMI) and Departments of Molecular and Cellular Physiology, Neurology and Neurological Sciences, and the Stanford Synchrotron Radiation Laboratory, Stanford University, School of Medicine, Stanford, California 94305, and the ^||Combinatorial Chemistry Center, University of Pittsburgh, Pittsburgh, Pennsylvania 15260

An efficient research strategy integrating empirically guided, structure-based modeling and chemoinformatics was used to discover potent small molecule inhibitors of the botulinum neurotoxin serotype A light chain. First, a modeled binding mode for inhibitor 2-mercapto-3-phenylpropionyl-RATKML (K_i = 330 nM) was generated, and required the use of a molecular dynamic conformer of the enzyme displaying the reorientation of surface loops bordering the substrate binding cleft. These flexible loops are conformationally variable in x-ray crystal structures, and the model predicted that they were pivotal for providing complementary binding surfaces and solvent shielding for the pseudo-peptide. The docked conformation of 2-mercapto-3-phenylpropionyl-RATKML was then used to refine our pharmacophore for botulinum serotype A light chain inhibition. Data base search queries derived from the pharmacophore were employed to mine small molecule (non-peptidic) inhibitors from the National Cancer Institute's Open Repository. Four of the inhibitors possess K_i values ranging from 3.0 to 10.0 µM. Of these, NSC 240898 is a promising lead for therapeutic development, as it readily enters neurons, exhibits no neuronal toxicity, and elicits dose-dependent protection of synaptosomal-associated protein (of 25 kDa) in a primary culture of embryonic chicken neurons. Isothermal titration calorimetry showed that the interaction between NSC 240898 and the botulinum A light chain is largely entropy-driven, and occurs with a 1:1 stoichiometry and a dissociation constant of 4.6 µM.

Received for publication, August 24, 2006 , and in revised form, October 20, 2006.

The atomic coordinates and structure factors (code 2ISE, 2ISG, and 2ISH) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* The work was supported by Defense Threat Reduction Agency Grant 3.10024_06_RD_B, 2S.Bavari, the United States Army Medical Research and Material Command, and IAA Y3 CM 100505 (MRMC and NCI, National Institutes of Health). This project has been funded in whole or in part with federal funds from the National Cancer Institute, National Institutes of Health, under Contract N01-CO-12400. This research was supported (in part) by the Developmental Therapeutics Program in the Division of Cancer Treatment and Diagnosis of the NCI. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains additional Methods, Results and Discussion, references, Figs. S1-S4, and Tables S1-S2.

¹ To whom correspondence may be addressed: HHMI and Depts. of Molecular and Cellular Physiology, Neurology and Neurological Sciences, and Stanford Synchrotron Radiation Laboratory, Stanford University, School of Medicine, 318 Campus Dr., Rm. E300, Stanford, CA 94305. Tel.: 650-736-1031; Fax: 650-736-1961; E-mail: brunger{at}stanford.edu.

² To whom correspondence may be addressed: United States Army Research Institute of Infectious Diseases, 1425 Porter St., Fort Detrick, Frederick, MD 21702. Tel.: 301-619-4246; Fax: 301-619-2348; E-mail: sina.bavari{at}us.army.mil.

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