Identifying Novel Inhibitors of RpFabG in Typhus-inducing Rickettsia prowazekii

Date
2019
Authors
Panatpur, Aparna
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Abstract

Epidemic typhus is a rickettsial disease that is contracted via ticks and lice found on the flying squirrel. The disease is caused by Rickettsia prowazekii, an intracellular, gram-negative coccobacillus. The biosynthetic pathways of Rickettsia prowazekii and its host are intertwined. Therefore, the ideal antipathogenic drugs would not target a protein that is found within the biochemical pathway of the human host. There is a type II fatty acid synthase pathway that is unique to Rickettsia prowazekii, which can be distinguished from the multienzyme type I fatty- acid synthase pathway used in humans. Fab G 3-ketoacyl-(acyl-carrier-protein) reductase (RpFabG) is a protein that is specific to this type II pathway. Hence, this research is focused on finding a small molecule drug that inhibits RpFabG. The coding DNA sequence for the RpFabG protein was previously cloned into a pNICJBsa4 plasmid, which was transformed into BL21(DE3
Escherichia coli and Dh5α competent cells. The transformed bacteria were cultured in LB media, and the cells were harvested. The expressed protein was purified via Ni-NTA affinity chromatography, made possible by a His6 tag on the vector. Gel electrophoresis was performed to determine the purity of the obtained protein sample; due to indication of slight contamination, gel filtration fast protein liquid chromatography was run on a concentrated protein sample. Genetic Optimization of Ligand Docking (GOLD is a molecular docking software package that was used to rank potential inhibitors of RpFabG according to binding strength; ethyl acetoacetate (EAA and acetoacyl coenzyme A (AAC) were determined to have high binding strengths and thus determined to be strong potential inhibitors. An enzyme assay was run to determine the functionality of the enzyme, with EAA and AAC as substrates. AAC successfully decreased the enzymatic activity of RpFabG, suggesting its potential as a novel drug. DSF assay results were equivocal, indicating that inhibition assays should be run on AAC to further assess its potential as an inhibitor.

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