Bacillus anthracis, the causative agent of anthrax, has become a serious bioterrorism threat. Due to the highly virulent nature of this microorganism, it is crucial that antibiotic therapy begins prophylactically, therefore effective drugs need to be inexpensive and tolerated in a large population. Inhibitors of dihydrofolate reductase (DHFR) have been used for decades to treat bacterial infections, however, trimethoprim, a clinically used DHFR inhibitor, is ineffective against B. anthracis due to poor interactions with the Bacillus enzyme. However, alternative DHFR inhibitors that exhibit high potency against the Bacillus enzyme should be excellent therapeutics to treat anthrax infections. We have designed a number of new DHFR inhibitors and found that several inhibit DHFR from B. anthracis and promote B. anthracis cell death while maintaining low mammalian cell toxicity. Novel methods for the syntheses of these compounds have resulted in an inhibitor with a submicromolar IC50 value and a MIC99 value of 20 5g/mL with no mammalian cell toxicity. Recent efforts have delivered more potent compounds with MIC99 values as low as 3.5 5g/mL. The goal of the work in this application is to develop a lead and a backup compound that are potent and selective inhibitors of B. anthracis growth. To do this, we have four specific aims: one, synthesize a focused library of inhibitors intended to increase potency and determine resistance generation;two, build on the potent inhibitors to synthesize a library of selective inhibitors;three, determine the crystal structure of Bacillus anthracis DHFR bound to a lead compound in order to evaluate protein:ligand interactions;and four, develop and synthesize advanced leads based on results from protein binding and P450 enzyme activity assays as well as additional crystal structures of resistant proteins.

Public Health Relevance

There is a critical need for new antibiotics effective against anthrax infections. Small molecule inhibitors of a key metabolic enzyme have been synthesized and proven to be potent without being toxic to mammalian cells. The goal of this proposal is to use the structure of the enzyme to further develop those compounds to effectively inhibit the growth of Bacillus anthracis Sterne at low concentrations.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI073375-02
Application #
7623525
Study Section
Special Emphasis Panel (ZRG1-IDM-H (02))
Program Officer
Xu, Zuoyu
Project Start
2008-06-01
Project End
2013-05-31
Budget Start
2009-06-01
Budget End
2010-05-31
Support Year
2
Fiscal Year
2009
Total Cost
$334,616
Indirect Cost
Name
University of Connecticut
Department
Pharmacology
Type
Schools of Pharmacy
DUNS #
614209054
City
Storrs-Mansfield
State
CT
Country
United States
Zip Code
06269
Lamb, Kristen M; G-Dayanandan, Narendran; Wright, Dennis L et al. (2013) Elucidating features that drive the design of selective antifolates using crystal structures of human dihydrofolate reductase. Biochemistry 52:7318-26
Anderson, Amy C (2012) Winning the arms race by improving drug discovery against mutating targets. ACS Chem Biol 7:278-88
Zhou, Wangda; Viswanathan, Kishore; Hill, Dennis et al. (2012) Acetylenic linkers in lead compounds: a study of the stability of the propargyl-linked antifolates. Drug Metab Dispos 40:2002-8
Wright, Dennis L; Anderson, Amy C (2011) Antifolate agents: a patent review (2006 - 2010). Expert Opin Ther Pat 21:1293-308
Algul, Oztekin; Paulsen, Janet L; Anderson, Amy C (2011) 2,4-Diamino-5-(2'-arylpropargyl)pyrimidine derivatives as new nonclassical antifolates for human dihydrofolate reductase inhibition. J Mol Graph Model 29:608-13
Anderson, Amy C; Pollastri, Michael P; Schiffer, Celia A et al. (2011) The challenge of developing robust drugs to overcome resistance. Drug Discov Today 16:755-61
Beierlein, J M; Anderson, A C (2011) New developments in vaccines, inhibitors of anthrax toxins, and antibiotic therapeutics for Bacillus anthracis. Curr Med Chem 18:5083-94
Frey, Kathleen M; Lombardo, Michael N; Wright, Dennis L et al. (2010) Towards the understanding of resistance mechanisms in clinically isolated trimethoprim-resistant, methicillin-resistant Staphylococcus aureus dihydrofolate reductase. J Struct Biol 170:93-7
Beierlein, Jennifer M; Karri, Nanda G; Anderson, Amy C (2010) Targeted mutations of Bacillus anthracis dihydrofolate reductase condense complex structureýýýactivity relationships. J Med Chem 53:7327-36
Frey, Kathleen M; Liu, Jieying; Lombardo, Michael N et al. (2009) Crystal structures of wild-type and mutant methicillin-resistant Staphylococcus aureus dihydrofolate reductase reveal an alternate conformation of NADPH that may be linked to trimethoprim resistance. J Mol Biol 387:1298-308

Showing the most recent 10 out of 12 publications