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J. Biol. Chem., Vol. 280, Issue 2, 1669-1677, January 14, 2005

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Determination of Selectivity and Efficacy of Fatty Acid Synthesis Inhibitors^*

Srinivas Kodali, Andrew Galgoci, Katherine Young, Ronald Painter, Lynn L. Silver, Kithsiri B. Herath, Sheo B. Singh, Doris Cully, John F. Barrett, Dennis Schmatz, and Jun Wang¶

From the Department of Human and Animal Infectious Disease, Merck Research Laboratories, Rahway, New Jersey 07065

Type II fatty acid synthesis (FASII) is essential to bacterial cell viability and is a promising target for the development of novel antibiotics. In the past decade, a few inhibitors have been identified for this pathway, but none of them lend themselves to drug development. To find better inhibitors that are potential drug candidates, we developed a high throughput assay that identifies inhibitors simultaneously against multiple targets within the FASII pathway of most bacterial pathogens. We demonstrated that the inverse t value of the FASII enzyme-catalyzed reaction gives a measure of FASII activity. The K_m values of octanoyl-CoA and lauroyl-CoA were determined to be 1.1 ± 0.3 and 10 ± 2.7 µM in Staphylococcus aureus and Bacillus subtilis, respectively. The effects of free metals and reducing agents on enzyme activity showed an inhibition hierarchy of Zn²⁺ > Ca²⁺ > Mn²⁺ > Mg²⁺; no inhibition was found with -mercaptoethanol or dithiothreitol. We used this assay to screen the natural product libraries and isolated an inhibitor, bischloroanthrabenzoxocinone (BABX) with a new structure. BABX showed IC₅₀ values of 11.4 and 35.3 µg/ml in the S. aureus and Escherichia coli FASII assays, respectively, and good antibacterial activities against S. aureus and permeable E. coli strains with minimum inhibitory concentrations ranging from 0.2 to 0.4 µg/ml. Furthermore, the effectiveness, selectivity, and the in vitro and in vivo correlations of BABX as well as other fatty acid inhibitors were elucidated, which will aid in future drug discovery.

Received for publication, June 18, 2004 , and in revised form, October 15, 2004.

^* 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 Figs. S1 and S2.

Both authors contributed equally to this work.

Current address: LL Silver Consulting, 3403 Park Place, Spring-field, NJ 07081.

^¶ To whom correspondence should be addressed: Merck & Co., Inc., P. O. Box 2000, R80Y-205; Rahway, NJ 07065. Tel.: 732-594-2776; Fax: 732-594-1399; E-mail: jun_wang2{at}merck.com.

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