This application seeks to merge chemical biology, structural biology and pathogen biology (including metabolomics in vitro and infections in mice) for insight into the function of two enzymes in Mtb's intermediary metabolism, lipoamide dehydrogenase (Lpd) and 2-hydroxy-3-oxoadipate (HOA) synthase (HOAS). Lpd participates in 3 enzyme complexes in intermediary metabolism and anti-oxidant defense. Its knockout severely attenuates Mtb in the mouse. We have identified and co-crystallized a species-selective Lpd inhibitor and will identify (and subsequently co-crystallize) others by applying orthogonal high throughput screens: one that tests 2 million compounds against the recombinant enzyme and the other using the hits from the first screen against replicating and non-replicating Mtb. HOAS, which we have crystallized, carries out a newly identified function of the reportedly essential gene Rv1248c, originally mis-annotated as E1 of 1-ketoglutarate (1-KG ) dehydrogenase. This is the first identification of a specific gene product that catalyzes carboligation of 1-KG and glyoxylate, producing HOA and its spontaneous decarboxylation product, hydroxylevulinate. We will replace Rv1248c with a Tet-regulated allele have developed a novel strategy to synthesize HOAS inhibitors from an analog of the cofactor thiamine diphosphate. This work will provide insights about the metabolic constraints faced by Mtb in the mouse, furnish tool compounds for the study of the roles of Lpd and HOAS together with other enzymes, establish if Lpd and HOAS warrant consideration as drug targets, and reveal new aspects of Mtb's intermediary metabolism. PHS 398/2590 (Rev. 06/09) Page Continuation Format Page

Public Health Relevance

Most antibiotics target biosynthetic processes and kill replicating bacteria. A major global health need is to eradicate substantially non-replicating subpopulations of Mtb with new chemophores active against new targets. Compounds developed here will inhibit two enzymes that are required by non-replicating Mtb (Lpd) or seem likely to be so (HOAS) and along with metabolomic studies will shed fundamental new light on Mtb's intermediary metabolism.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI064768-08
Application #
8402564
Study Section
Special Emphasis Panel (ZRG1-BACP-B (09))
Program Officer
Boyce, Jim P
Project Start
2005-06-01
Project End
2015-11-30
Budget Start
2012-12-01
Budget End
2013-11-30
Support Year
8
Fiscal Year
2013
Total Cost
$865,364
Indirect Cost
$254,401
Name
Weill Medical College of Cornell University
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
060217502
City
New York
State
NY
Country
United States
Zip Code
10065
Balakrishnan, Anand; Jordan, Frank; Nathan, Carl F (2013) Influence of allosteric regulators on individual steps in the reaction catalyzed by Mycobacterium tuberculosis 2-hydroxy-3-oxoadipate synthase. J Biol Chem 288:21688-702
Mazloum, Nayef; Stegman, Melanie A; Croteau, Deborah L et al. (2011) Identification of a chemical that inhibits the mycobacterial UvrABC complex in nucleotide excision repair. Biochemistry 50:1329-35
de Carvalho, Luiz Pedro S; Ling, Yan; Shen, Chun et al. (2011) On the chemical mechanism of succinic semialdehyde dehydrogenase (GabD1) from Mycobacterium tuberculosis. Arch Biochem Biophys 509:90-9
Rhee, Kyu Y; de Carvalho, Luiz Pedro Sorio; Bryk, Ruslana et al. (2011) Central carbon metabolism in Mycobacterium tuberculosis: an unexpected frontier. Trends Microbiol 19:307-14
Venugopal, Aditya; Bryk, Ruslana; Shi, Shuangping et al. (2011) Virulence of Mycobacterium tuberculosis depends on lipoamide dehydrogenase, a member of three multienzyme complexes. Cell Host Microbe 9:21-31
de Carvalho, Luiz Pedro S; Zhao, Hong; Dickinson, Caitlyn E et al. (2010) Activity-based metabolomic profiling of enzymatic function: identification of Rv1248c as a mycobacterial 2-hydroxy-3-oxoadipate synthase. Chem Biol 17:323-32
Bryk, Ruslana; Arango, Nancy; Venugopal, Aditya et al. (2010) Triazaspirodimethoxybenzoyls as selective inhibitors of mycobacterial lipoamide dehydrogenase . Biochemistry 49:1616-27
Zhao, Hong; de Carvalho, Luiz Pedro S; Nathan, Carl et al. (2010) A protecting group-free synthesis of deazathiamine: a step toward inhibitor design. Bioorg Med Chem Lett 20:6472-4
Wang, Tao; Li, Hua; Lin, Gang et al. (2009) Structural insights on the Mycobacterium tuberculosis proteasomal ATPase Mpa. Structure 17:1377-85