Virtual drug screening of potential and novel inhibitors of Escherichia coli dihydrofolate reductase

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Virtual drug screening of potential and novel inhibitors of Escherichia coli dihydrofolate reductase

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Title: Virtual drug screening of potential and novel inhibitors of Escherichia coli dihydrofolate reductase
Author: Nguyen, Adam Van
Abstract: Dihydrofolate Reductase (DHFR) is a major drug target because it is an enzyme that acts in a reaction mechanism to help produce tetrahydrofolic acid, which is then used by all cells to synthesize DNA precursors. The activity of this enzyme has also been implicated in the functions of cancerous cells, as they tend to over-replicate and thus require the enzyme more. Virtual Drug Screening is a relatively cheap and efficient method of screening through large libraries of compounds to find ones that have the highest binding affinities to the target protein/enzyme of interest. Specifically in this project, the virtual screening of ligands that can bind and possibly inhibit ecDHFR (Esherichia coli dihydrofolate reductase) was performed. E. Coli provides a target for developing antibiotic drugs since it can be responsible for certain illnesses caused in humans. At the same time, it is particularly difficult to develop antibacterial to E. Coli due to the sheer number of strains present. The actual ecDHFR enzyme was cloned into a pNIC-Bsa4 expression vector, expressed in BL21(DE3) cells, purified using Ni-NTA affinity chromatography, and characterized on a PAGE-gel. Concurrent with the wet lab, virtual screening of over 400,000 ligands also took place. A non-inhibitor enzyme assay was performed to validate the activity of the grown-up ecDHFR enzyme. Further testing in the wet-lab of some of the top-scoring ligands from the virtual screening (NRB00358 from Maybridge/Ryan Scientific; 5282931, 7722615, and 6407567 from cb-306_3d) via spectrophotometric enzyme inhibition assays showed no inhibition of ecDHFR activity. This shows that virtual screening is an imperfect measure of the ligand’s binding affinity, and may not necessarily predict its inhibitory qualities on the target protein. Even as such, it is hoped that further analysis of other top ligands from the virtual screening results will yield potential hits against this enzyme.
Department: Biological Sciences, School of
Subject: virtual drug screening ligands inhibitors novel Escherichia coli E. coli dihydrofolate reductase College of Natural Sciences
URI: http://hdl.handle.net/2152/15617
Date: 2012

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