John H. Thurston, Ph.D. » The College of Idaho
||John H. Thurston, Ph.D.
||The College of Idaho
Infectious disease accounts for approximately one-third of the annual worldwide mortality. In the past two decades, increasing rates of drug resistant, and multiple-drug resistant microbial infections underscore the urgent need to develop new antibiotics that are selective and safe. One potential target for novel antibiotics is the microbe specific 5' Methylthioadenosine /S-adenosylhomocysteine nucleosidase (MTN), an enzyme with a central role in nutrient salvage and synthesis of bacterial quorum sensing signals that govern processes such as drug resistance, biofilm formation, and the expression of virulence. In this study, we propose the development of MTN-specific inhibitors and to examine their mechanisms of action on enterohemorragic E. coli O157:H7. In the first aim, MTN inhibitors will be synthesized using known chemistries based on the transition-state core structure and predicted modifications that should confer increased selectivity and activity through improved active site electrostatic interactions. In the second aim, the selectivity and potency of drugs will be tested using cellular and biochemical assays to examine the effect on enzyme activity, microbial viability, virulence and quorum sensing dependent processes. In the third aim, the global antimicrobial mechanism of action of identified lead compounds will be explored through LC/MS based proteomics and metabolomics.
Project 1 – organic chemistry and lab; Project 2 – microbiology and cell and molecular biology are desirable.
Project 1: To synthesize and characterize novel MTN inhibitors. As part of this project, we will:
1) synthesize a library of non-hydrolysable nucleosides with discriminatory 2’ and 5’ substitutions;
2) confirm the chemical identity of inhibitors using crystallographic and spectroscopic techniques;
3) analyze inhibitors for activity against E. coli MTN and human MTAP enzymes
Project 2: To characterize the mechanism of MTN enzyme interruption on specific metabolic, autoinducer signaling, and virulence events in E. coli O157:H7. As part of this project, we will:
1) analyze the antibiotic effects of MTN inhibitors on bacterial growth and biofilm formation;
2) evaluate the effects of MTN inhibitors on MTAP containing mammalian cell lines;
3) quantify the effect of MTN inhibitors on autoinducer-2 production, metabolic constituents (polyamines, vitamins, etc); virulence factors, and in vitro virulence
Project 1 will place a heavy emphasis on synthetic organic chemistry and on methods for the spectroscopic characterization of small molecules, including n.m.r. spectroscopy, mass spectrometry and single crystal x-ray diffractometry. Project 2 will rely on assays from microbiology and molecular biology to assess the efficacy of the drugs developed as part of this study.