Dissertation
Rational design and experimental characterization of small molecule inhibitors of the bacterial stringent response activating enzyme RelA
Doctor of Philosophy (Ph.D.), Drexel University
Sep 2020
DOI:
https://doi.org/10.17918/00000308
Abstract
Medical biofilms can be defined as an infectious organized community of bacteria that are living as a multicellular entity. Bacteria have evolved multiple strategies to ensure their survival in these communities. One of these strategies is called the "stringent response" and is activated almost universally among most bacterial species in response to various nutrient limitations. The keystone enzyme in triggering the bacterial stringent response which produces a state of relative metabolic quiescence is RelA. RelA is a highly conserved pyrophosphotransferase that is encoded by the relA gene. This enzyme is responsible for sensing amino acid starvation at the ribosome and is self-regulating within a bacterial biofilm. RelA catalyzes the reaction between GTP/GDP and ATP to form the "alarmones" pppGpp (guanosine pentaphosphate) and ppGpp (guanosine tetraphosphate). These molecules trigger a transcriptional change within the cell causing an upregulation in oxidative damage combating enzymes as well as a reduction in replication machinery. Cells which have gone through these metamorphic changes are called persister cells. Persister cells, while not genetically resistant to antibiotics, can withstand up to 1000x the antibiotic concentrations of their planktonic counterparts. This work outlines the task of developing a RelA inhibitor which would both prophylactically stop persister cells formation and would convert already formed persister cells to their antibiotic-susceptible state allowing for the synergetic treatment of medical biofilms using a combination therapy. An in silico pipeline approach to identify hit compounds for the inhibition of RelA was developed. Using this approach, combined with biochemical, molecular biological and microbiological methods we have analyzed these in silico hit compounds and identified a set of lead compounds that inhibit RelA and reduce biofilm persistence when the biofilm is simultaneously treated with antibiotics and the RelA inhibitors. We have developed and synthesized several analogs of these lead compounds and have tested them in SAR studies to show how these compounds interact in the inhibition of the RelA enzyme. Through this approach, we have gained both a better understanding of the stringent response and the ability to combat bacteria in medical biofilm infections.
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Details
- Title
- Rational design and experimental characterization of small molecule inhibitors of the bacterial stringent response activating enzyme RelA
- Creators
- Donald C. Hall Jr.
- Contributors
- Haifeng Ji (Advisor)
- Awarding Institution
- Drexel University
- Degree Awarded
- Doctor of Philosophy (Ph.D.)
- Publisher
- Drexel University; Philadelphia, Pennsylvania
- Number of pages
- xix, 193 pages
- Resource Type
- Dissertation
- Language
- English
- Academic Unit
- College of Arts and Sciences; Chemistry; Drexel University
- Other Identifier
- 991014695547304721