Mortality in AIDS patients is due to overwhelming opportunistic infections caused by bacteria, fungi, protozoa, and viruses. The long term goal of this project is to develop lead compounds useful for the treatment of bacterial opportunistic infections in AIDS by developing strong inhibitors of alanine racemase, an essential bacterial enzyme involved in cell wall biosynthesis. These compounds will be engineered in a collaborative structure based drug design program based at the University of Houston. Three common opportunistic pathogens will be targeted, Mycobacterium tuberculosis, Mycobacterium avium, and Streptococcus pneumoniae, but several other pathogens will be studied as well. To date, overexpressing clones of alanine racemase from E. coli, Mycobacterium tuberculosis, Pseudomonas aeruginosa, Staphylococcus aureus, and Streptococcus pneumoniae have been used to produce purified protein. These enzymes are being characterized kinetically. Crystallization of all of these racemases will be attempted, and alanine racemase from E. coli, Pseudomonas aeruginosa and Mycobacterium tuberculosis have already been crystallized. The crystals from the E. coli enzyme are of sufficient quality to allow for a three-dimensional structure determination. These structures will serve as target molecules for a comprehensive structure based drug design effort. Promising lead compounds will be synthesized and tested in vitro. Their strength and specificity will be improved through structural determinations of inhibitor-enzyme complexes followed by molecular dynamics simulations. Studies of mutant racemases will be used to help anticipate common pathways for developing resistance to these compounds. While the treatment of opportunistic infections in AIDS patients will benefit most from this research, development of highly effective alanine racemase inhibitors may broadly impact the treatment of other infectious diseases, including those caused by multi-drug resistant bacteria.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI046340-02
Application #
6170670
Study Section
Special Emphasis Panel (ZAI1-VSG-A (S1))
Program Officer
Laughon, Barbara E
Project Start
1999-09-30
Project End
2004-08-31
Budget Start
2000-09-01
Budget End
2001-08-31
Support Year
2
Fiscal Year
2000
Total Cost
$504,272
Indirect Cost
Name
University of Houston
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
800771594
City
Houston
State
TX
Country
United States
Zip Code
77204
Huang, Hung-Chung; Jupiter, Daniel; Qiu, Meikang et al. (2008) Cluster analysis of hydration waters around the active sites of bacterial alanine racemase using a 2-ns MD simulation. Biopolymers 89:210-9
Strych, Ulrich; Davlieva, Milya; Longtin, Joseph P et al. (2007) Purification and preliminary crystallization of alanine racemase from Streptococcus pneumoniae. BMC Microbiol 7:40
Shelburne 3rd, Samuel A; Visnegarwala, Fehmida; Adams, Carlos et al. (2005) Unusual manifestations of disseminated Histoplasmosis in patients responding to antiretroviral therapy. Am J Med 118:1038-41
LeMagueres, Pierre; Im, Hookang; Ebalunode, Jerry et al. (2005) The 1.9 A crystal structure of alanine racemase from Mycobacterium tuberculosis contains a conserved entryway into the active site. Biochemistry 44:1471-81
Mustata, Gabriela; Briggs, James M (2004) Cluster analysis of water molecules in alanine racemase and their putative structural role. Protein Eng Des Sel 17:223-34
LeMagueres, Pierre; Im, Hookang; Dvorak, Anna et al. (2003) Crystal structure at 1.45 A resolution of alanine racemase from a pathogenic bacterium, Pseudomonas aeruginosa, contains both internal and external aldimine forms. Biochemistry 42:14752-61
Kim, Myoung Goo; Strych, Ulrich; Krause, Kurt et al. (2003) N(2)-substituted D,L-cycloserine derivatives: synthesis and evaluation as alanine racemase inhibitors. J Antibiot (Tokyo) 56:160-8
Mustata, Gabriela Iurcu; Soares, Thereza A; Briggs, James M (2003) Molecular dynamics studies of alanine racemase: a structural model for drug design. Biopolymers 70:186-200
Strych, Ulrich; Benedik, Michael J (2002) Mutant analysis shows that alanine racemases from Pseudomonas aeruginosa and Escherichia coli are dimeric. J Bacteriol 184:4321-5
Mustata, Gabriela Iurcu; Briggs, James M (2002) A structure-based design approach for the identification of novel inhibitors: application to an alanine racemase. J Comput Aided Mol Des 16:935-53

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