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Antimicrobial Agents and Chemotherapy, August 2009, p. 3478-3486, Vol. 53, No. 8
0066-4804/09/$08.00+0 doi:10.1128/AAC.00141-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Identification and Biochemical Characterization of Small-Molecule Inhibitors of Clostridium botulinum Neurotoxin Serotype A
,
Virginia Roxas-Duncan,1
Istvan Enyedy,2,
Vicki A. Montgomery,1
Vanessa S. Eccard,1
Marco A. Carrington,1
Huiguo Lai,3
Nizamettin Gul,1,
David C. H. Yang,3 and
Leonard A. Smith1*
Integrated Toxicology Division, U.S. Army Medical Research Institute of Infectious Diseases, Fort Detrick, Maryland 21702,1 Lombardi Cancer Center, Georgetown University, Washington, D.C. 20057,2 Department of Chemistry, Georgetown University, Washington, D.C. 200573
Received 30 January 2009/ Returned for modification 1 March 2009/ Accepted 2 June 2009
An integrated strategy that combined in silico screening and tiered biochemical assays (enzymatic, in vitro, and ex vivo) was used to identify and characterize effective small-molecule inhibitors of Clostridium botulinum neurotoxin serotype A (BoNT/A). Virtual screening was initially performed by computationally docking compounds of the National Cancer Institute (NCI) database into the active site of BoNT/A light chain (LC). A total of 100 high-scoring compounds were evaluated in a high-performance liquid chromatography (HPLC)-based protease assay using recombinant full-length BoNT/A LC. Seven compounds that significantly inhibited the BoNT/A protease activity were selected. Database search queries of the best candidate hit [7-((4-nitro-anilino)(phenyl)methyl)-8-quinolinol (NSC 1010)] were performed to mine its nontoxic analogs. Fifty-five analogs of NSC 1010 were synthesized and examined by the HPLC-based assay. Of these, five quinolinol derivatives that potently inhibited both full-length BoNT/A LC and truncated BoNT/A LC (residues 1 to 425) were selected for further inhibition studies in neuroblastoma (N2a) cell-based and tissue-based mouse phrenic nerve hemidiaphragm assays. Consistent with enzymatic assays, in vitro and ex vivo studies revealed that these five quinolinol-based analogs effectively neutralized BoNT/A toxicity, with CB 7969312 exhibiting ex vivo protection at 0.5 µM. To date, this is the most potent BoNT/A small-molecule inhibitor that showed activity in an ex vivo assay. The reduced toxicity and high potency demonstrated by these five compounds at the biochemical, cellular, and tissue levels are distinctive among the BoNT/A small-molecule inhibitors reported thus far. This study demonstrates the utility of a multidisciplinary approach (in silico screening coupled with biochemical testing) for identifying promising small-molecule BoNT/A inhibitors.
* Corresponding author. Mailing address: Integrated Toxicology Division, U.S. Army Medical Research Institute of Infectious Diseases, 1425 Porter St., Fort Detrick, MD 21702. Phone: (301) 619-4238. Fax: (301) 619-2348. E-mail: leonard.smith{at}amedd.army.mil
Published ahead of print on 15 June 2009.
The authors have paid a fee to allow immediate free access to this article.
Present address: Biogen Idec, Cambridge, MA 02142.
Present Address: Chemical Biological Medical Systems-Joint Product Management Office for Medical Identification and Treatment Systems (CBMS-JPMO MITS), 64 Thomas Johnson Dr., Frederick, MD 21702.
Antimicrobial Agents and Chemotherapy, August 2009, p. 3478-3486, Vol. 53, No. 8
0066-4804/09/$08.00+0 doi:10.1128/AAC.00141-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
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