The sulfur-containing amino acid, L-cysteine, is indispensable for pathogen virulence and survival. Sulfur for biosyn- thesis is largely derived from the assimilation of inorganic sulfate by microorganisms. Genes required for sulfate as- similation are essential to pathogen survival but absent in the human genome and, therefore, represent potential new targets for therapeutic intervention. In the previous funding cycle, we defined key features in the catalytic cycle of sulfonucleotide reductases (SRs), enzymes that catalyze the first committed step in sulfate reduction for de novo synthesis of cysteine and other reduced-sulfur containing biomolecules. Our studies provided fundamental insights into how thioredoxins (Trxs)?central antioxidant enzymes that maintain protein thiols in their reduced state? recognize their cellular targets. We learned that the iron-sulfur cluster in APS reductase (APSR) plays an essential role active-site preorganization and substrate activation, expanding knowledge on the catalytic activities of Fe-S pro- teins and on the divergent evolution of PAPS reductase (PAPR), which lacks this cofactor. These insights led to molecules that target APSR in a new way, via interaction with the iron-sulfur metallocenter. We also discovered first- in-class inhibitors of sulfate reduction that exhibit potent bactericidal activity against drug-resistant clinical isolates of M. tuberculosis. Given the importance of SRs in pathogen oxidative stress resistance and virulence, we propose here to address research areas where the biggest open questions and greatest unmet needs remain: well-validated chemical probes that acutely inhibitor essential steps in microbial reductive sulfate assimilation (Aim 1); defining the non-redundant functions of pathogenic Trxs to facilitate sulfur reduction and cope with redox stress (Aim 2); and ad- vancing knowledge in trafficking and delivery of reactive sulfur species (RSS) for microbial cysteine biosynthesis. Collectively, the experiments in this renewal application will provide fundamental information on one of the most under-studied metabolic chemistries?reductive sulfate assimilation?in the context of one of the most devastat- ing pathogens?M. tuberculosis. We have established outstanding collaborations, with a proven track record of productivity, in order to support these efforts and ensure timely completion. Our efforts will address areas cen- tral to role of sulfur metabolism in pathogen oxidative stress resistance and virulence that have not received suf- ficient attention despite their importance, ultimately leading to a new level of understanding and leverage in the battle against infectious disease.
The sulfur-containing amino acid, L-cysteine, is indispensable for pathogen virulence and survival. Sulfur for biosyn- thesis is largely derived from the assimilation of inorganic sulfate by microorganisms. Genes required for sulfate as- similation are essential to pathogen survival but absent in the human genome and, therefore, represent potential new targets for therapeutic intervention. Our research supports studies to develop selective probes to elucidate the role of cysteine metabolism in pathogenesis and address the central hypothesis that the biosynthetic pathways that lead this building block represent an ?Archilles' heel? that can be exploited to develop agents that can attenuate the virulence of serious human pathogens.
|Paritala, Hanumantharao; Palde, Prakash B; Carroll, Kate S (2016) Functional Site Discovery in a Sulfur Metabolism Enzyme by Using Directed Evolution. Chembiochem 17:1873-1878|
|Palde, Prakash B; Bhaskar, Ashima; Pedró Rosa, Laura E et al. (2016) First-in-Class Inhibitors of Sulfur Metabolism with Bactericidal Activity against Non-Replicating M. tuberculosis. ACS Chem Biol 11:172-84|
|Palde, Prakash B; Carroll, Kate S (2015) A universal entropy-driven mechanism for thioredoxin-target recognition. Proc Natl Acad Sci U S A 112:7960-5|
|Paritala, Hanumantharao; Suzuki, Yuta; Carroll, Kate S (2015) Design, synthesis and evaluation of Fe-S targeted adenosine 5'-phosphosulfate reductase inhibitors. Nucleosides Nucleotides Nucleic Acids 34:199-220|
|Bhaskar, Ashima; Chawla, Manbeena; Mehta, Mansi et al. (2014) Reengineering redox sensitive GFP to measure mycothiol redox potential of Mycobacterium tuberculosis during infection. PLoS Pathog 10:e1003902|
|Paritala, Hanumantharao; Carroll, Kate S (2013) New targets and inhibitors of mycobacterial sulfur metabolism. Infect Disord Drug Targets 13:85-115|
|Paritala, Hanumantharao; Suzuki, Yuta; Carroll, Kate S (2013) Efficient microwave-assisted solid phase coupling of nucleosides, small library generation and mild conditions for release of nucleoside derivatives. Tetrahedron Lett 54:1869-1872|
|Paritala, Hanumantharao; Carroll, Kate S (2013) A continuous spectrophotometric assay for adenosine 5'-phosphosulfate reductase activity with sulfite-selective probes. Anal Biochem 440:32-9|
|Bhave, Devayani P; Hong, Jiyoung A; Keller, Rebecca L et al. (2012) Iron-sulfur cluster engineering provides insight into the evolution of substrate specificity among sulfonucleotide reductases. ACS Chem Biol 7:306-15|
|Holsclaw, Cynthia M; Muse 3rd, Wilson B; Carroll, Kate S et al. (2011) Mass Spectrometric Analysis of Mycothiol levels in Wild-Type and Mycothiol Disulfide Reductase Mutant Mycobacterium smegmatis. Int J Mass Spectrom 305:151-156|
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