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Applied and Environmental Microbiology, February 2008, p. 653-659, Vol. 74, No. 3
0099-2240/08/$08.00+0     doi:10.1128/AEM.01690-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Use of a Recombinant Fluorescent Substrate with Cleavage Sites for All Botulinum Neurotoxins in High-Throughput Screening of Natural Product Extracts for Inhibitors of Serotypes A, B, and E{triangledown} ,{dagger}

Harry B. Hines,1 Alexander D. Kim,1,{ddagger} Robert G. Stafford,1 Shirin S. Badie,1 Ernst E. Brueggeman,1 David J. Newman,2 and James J. Schmidt1*

Department of Cell Biology and Biochemistry, Integrated Toxicology Division, United States Army Medical Research Institute of Infectious Diseases, Frederick, Maryland 21702-5011,1 Natural Products Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute, Frederick, Maryland 21701-12012

Received 23 July 2007/ Accepted 26 November 2007

The seven serotypes of botulinum neurotoxin (BoNTs) are zinc metalloproteases that cleave and inactivate proteins critical for neurotransmission. The synaptosomal protein of 25 kDa (SNAP-25) is cleaved by BoNTs A, C, and E, while vesicle-associated membrane protein (VAMP) is the substrate for BoNTs B, D, F, and G. BoNTs not only are medically useful drugs but also are potential bioterrorist and biowarfare threat agents. Because BoNT protease activity is required for toxicity, inhibitors of that activity might be effective for antibotulinum therapy. To expedite inhibitor discovery, we constructed a hybrid gene encoding (from the N terminus to the C terminus, with respect to the expressed product) green fluorescent protein, then a SNAP-25 fragment encompassing residues Met-127 to Gly-206, and then VAMP residues Met-1 to Lys-94. Cysteine was added as the C terminus. The expressed product, which contained the protease cleavage sites for all seven botulinum serotypes, was purified and coupled covalently through the C-terminal sulfhydryl group to maleimide-activated 96-well plates. The substrate was readily cleaved by BoNTs A, B, D, E, and F. Using this assay and an automated 96-well pipettor, we screened 528 natural product extracts for inhibitors of BoNT A, B, and E protease activities. Serotype-specific inhibition was found in 30 extracts, while 5 others inhibited two serotypes.

* Corresponding author. Mailing address: Department of Cell Biology and Biochemistry, Integrated Toxicology Division, USAMRIID, Frederick, MD 21702-5011. Phone: (301) 619-4840. Fax: (301) 619-2348. E-mail: james.schmidt{at}amedd.army.mil

{triangledown} Published ahead of print on 14 December 2007.

{dagger} Supplemental material for this article may be found at http://aem.asm.org/.

{ddagger} Present address: U.S. Patent and Trademark Office, 400 Dulany Street, Alexandria, VA 22314.

Applied and Environmental Microbiology, February 2008, p. 653-659, Vol. 74, No. 3
0099-2240/08/$08.00+0     doi:10.1128/AEM.01690-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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