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Infection and Immunity, November 2005, p. 7548-7557, Vol. 73, No. 11
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.11.7548-7557.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Metalloproteinase Inhibitors, Nonantimicrobial Chemically Modified Tetracyclines, and Ilomastat Block Bacillus anthracis Lethal Factor Activity in Viable Cells

Salih S. Kocer,1 Stephen G. Walker,2 Brad Zerler,6 Lorne M. Golub,2 and Sanford R. Simon1,3,4,5*

Program in Cellular and Molecular Biology,1 Department of Oral Biology & Pathology,2 Department of Biochemistry,3 Department of Pathology,4 Institute for Cell and Developmental Biology, State University of New York at Stony Brook, Stony Brook, New York 11794,5 CollaGenex Pharmaceuticals, Inc., Newtown, Pennsylvania 189406

Received 22 March 2005/ Returned for modification 25 April 2005/ Accepted 10 July 2005

Lethal toxin, produced by the bacterium Bacillus anthracis, is a major contributor to morbidity and mortality in animals and humans who have contracted anthrax. One component of this toxin, lethal factor (LF), proteolytically inactivates members of the mitogen-activated protein kinase kinase (MAPKK or MEK) family. In this study we show that CMT-300, CMT-308, and Ilomastat, agents initially characterized as matrix metalloproteinase inhibitors which are in early stages of development as pharmaceuticals, effectively inhibit the zinc metalloproteinase activity of LF. All three inhibitors, CMT-300, CMT-308, and Ilomastat, inhibit LF-mediated cleavage of a synthetic peptide substrate based on the N-terminal domain of MEKs. Inhibition of LF-mediated MEK proteolysis by all three agents was also achieved using lysates of the human monocytoid line MonoMac 6 as sources of MAPKKs and visualization of the extent of cleavage after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by detection by Western blotting. Finally, we have demonstrated inhibition of intracellular MEKs in viable human monocytes and MonoMac 6 cells by these agents after incubation of the cells with a reconstituted preparation of recombinant lethal toxin. All three agents are effective inhibitors when incubated with LF prior to exposure to cells, while the CMTs, but not Ilomastat, are also effective when added after LF has already entered the viable cell targets. These results offer promise for strategies to combat effects of the lethal toxin of B. anthracis.

* Corresponding author. Mailing address: Department of Pathology, BST-9 Room 148, State University of NY at Stony Brook, Stony Brook, NY 11794-8691. Phone: (631) 444-3007. Fax: (631) 444-7507. E-mail: ssimon{at}notes.cc.sunysb.edu.

Editor: J. T. Barbieri

Infection and Immunity, November 2005, p. 7548-7557, Vol. 73, No. 11
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.11.7548-7557.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.





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