The use of Bacillus anthracis in acts of terrorism and/or biological warfare is a demonstrated threat to U.S. security. The long-term objective of this proposal is to develop inhibitors against a new drug target, the anthracis enzyme nicotinamide adenine dinucleotide (NAD) synthetase, that are effective therapeutic agents for preventing and/or treating infections caused by B. anthracis. NAD synthetase catalyzes the last step in both the de novo and salvage pathways for the biosynthesis of NAD, an essential cofactor in energy metabolism. Since exogenous NAD cannot support bacterial growth due to insufficient cell membrane permeability/transport, inhibitors of NAD biosynthesis should be bacteriostatic and/or bacteriocidal. We have identified the first low micromolar inhibitors of Bacillus NAD synthetase, and these effectively inhibit the vegetative growth of Bacillus anthracis, and are bacteriocidal, at concentrations around 1 (mu/g/mL. We will now perform reiterative design, chemical synthesis, and in vitro analysis to develop mature lead compounds.
The Specific Aims are: (1) Using parallel, solution phase synthetic chemistry, we will optimize inhibitory activities for existing classes of lead structures until low nanomolar inhibitors are obtained. (2) Determine the molecular mechanism by which existing lead synthetic compounds inhibit NAD synthetase. In addition to protein crystallography, we will: (a) Measure the Ki and evaluate the type of inhibition for synthetic inhibitors of NAD synthetase. (b) Characterize the enzyme homodimer/monomer equilibrium and modulation by inhibitors. (c) Perform photoaffinity labeling experiments using inhibitors containing a photoreactive alkylating group (azide). (3) Alternative structural templates will be developed as new NAD synthetase inhibitors. All synthetic compounds will be evaluated in high throughput screens as enzyme inhibitors (IC50 and/or Ki) and antibacterials (MIC). Selectivity for the inhibition of Bacillus NAD synthetase over the human (erythrocyte) enzyme will also be evaluated.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project--Cooperative Agreements (U01)
Project #
5U01AI056477-03
Application #
6860067
Study Section
Special Emphasis Panel (ZAI1-ALR-M (M4))
Program Officer
Baker, Phillip J
Project Start
2003-08-01
Project End
2008-01-31
Budget Start
2005-02-01
Budget End
2006-01-31
Support Year
3
Fiscal Year
2005
Total Cost
$362,500
Indirect Cost
Name
University of Alabama Birmingham
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
063690705
City
Birmingham
State
AL
Country
United States
Zip Code
35294
Moro, Whitney Beysselance; Yang, Zhengrong; Kane, Tasha A et al. (2009) SAR studies for a new class of antibacterial NAD biosynthesis inhibitors. J Comb Chem 11:617-25
Moro, Whitney Beysselance; Yang, Zhengrong; Kane, Tasha A et al. (2009) Virtual screening to identify lead inhibitors for bacterial NAD synthetase (NADs). Bioorg Med Chem Lett 19:2001-5
McDonald, Heather M; Pruett, Pamela S; Deivanayagam, Champion et al. (2007) Structural adaptation of an interacting non-native C-terminal helical extension revealed in the crystal structure of NAD+ synthetase from Bacillus anthracis. Acta Crystallogr D Biol Crystallogr 63:891-905
Velu, Sadanandan E; Luan, Chi-Hao; Delucas, Lawrence J et al. (2005) Tethered dimer inhibitors of NAD synthetase: parallel synthesis of an aryl-substituted SAR library. J Comb Chem 7:898-904
Yang, Zhengrong W; Tendian, Susan W; Carson, W Michael et al. (2004) Dimethyl sulfoxide at 2.5% (v/v) alters the structural cooperativity and unfolding mechanism of dimeric bacterial NAD+ synthetase. Protein Sci 13:830-41