Publication Date
2023
Document Type
Dissertation/Thesis
First Advisor
Hagen, Timothy J.
Second Advisor
Horn, James R.
Degree Name
Ph.D. (Doctor of Philosophy)
Legacy Department
Department of Chemistry and Biochemistry
Abstract
There is a crucial need for new antibacterial agents to combat the increase in antibiotic resistance. Methionine aminopeptidase (MetAp) is an enzyme that removes the amino-terminal methionine residue from newly synthesized proteins, an important step in the maturation of proteins. As such, MetAp is a target for the development of antibacterial and anticancer agents. One such bacterial target is Rickettsia prowazekii (Rp), the causative agent of epidemic typhus. To discover new inhibitors of RpMetAp1, the MMV Malaria Box, which is a collection of 400 compounds with drug-like and probe-like compounds, was screened using a high throughput fluorescence-based RpMetAp1 enzyme inhibition assay. A total of 12 hits were identified from the screen that possessed inhibition ≥45% at 10 μM and were analyzed by dose-response curves to obtain IC50 values. After filtering and removing pan-assay interference compounds (PAINS), five drug-like compounds were identified to serve as novel leads for further development. In addition, the MMV Pandemic Response Box, which is a collection of 400 antifungal, antibacterial, and antiviral compounds, was screened using the high throughput fluorescence based RpMetAp1 enzyme inhibition assay. Nineteen compounds possessed inhibition ≥60% at 10 μM. Dose-response screening of the nineteen compounds allowed the determination of IC50 values, which ranged from 300 nM to 10 μM. Compounds identified through screening were subjected to molecular docking studies (performed by Dr. Drashti Daraji) to better understand the potential binding mechanism.Attractive targets for new antibacterial agents include enzymes from the MEP (methylerythritol phosphate) pathway, which produces isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP). The IPP and DMAPP products are important building blocks for a wide range of isoprenoids, such as natural rubber and flavors. The MEP pathway is essential for most bacteria and is not present in humans. The enzyme 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase, also called IspF, catalyzes the fifth step in the MEP pathway from the species Burkholderia pseudomallei (Bp) and is the target for this study. Burkholderia pseudomallei is the causative agent of a resistant infection called melioidosis. To identify new lead compounds targeting BpIspF, it is important to have a high-throughput screening method in place. However, the available inhibition assays are time-consuming and labor-intensive. A thermal shift assay provides an alternate strategy to screen compounds that bind a target protein, as long as one can experimentally observe the protein unfolding event with and without the presence of a ligand. The high thermal stability of BpIspF (Tm ≥ 95℃), rules out this technique, as positive shifts in Tm cannot be accurately measured. Previously, several single-point mutations were introduced into the BpIspF trimer to test whether the enzyme could be destabilized to enable the use of thermal shift assays to screen potential ligands. Two mutations, Q151E and N130D, which are located within the hydrophobic monomer-monomer interfaces of the IspF trimer, destabilized the enzyme and lowered the Tm values to 74℃. Here, a double mutant BpIspF Q151E-N130D was developed to test whether enzyme destabilization could be enhanced through the tandem introduction of the two charged residues. As compared to the single mutant BpIspF Q151E, the double mutant resulted in an additional decrease in Tm of ~20℃. As expected, the double mutant exhibited stability that was pH dependent. The melting temperature of the BpIspF Q151E-N130D was determined in the presence of known BpIspF ligands (Ethoxzolamide, L-tryptophan hydroxamate, and CDP) with all the three compounds binding to the active site and had known Kd for BpIspF. Shifts in the melting temperature of BpIspF Q151E-N130D were observed to be as expected because of the known affinities of the compounds to BpIspF. The largest upward shift was observed with Ethoxzolamide, followed by L-tryptophan hydroxamate, and the last CDP. These compounds, L-tryptophan hydroxamate, and CDP were tested using Isothermal Titration Calorimetry (ITC) to determine their binding affinities for comparison to the data generated with the BpIspF wild type. Investigation of Kd demonstrated the same affinities as the BpIspF wild type with L-tryptophan hydroxamate and CDP, where they bound in the same rank ordering as expected due to their known Kd. With more than 620 million cases and 6 million deaths worldwide due to SARS-CoV-2, there is an urgent need to discover therapeutic agents that are effective against SARS-CoV-2. Several targets are essential for the SARS-CoV-2 life cycle: 3CL Proteases. Thermal shift assay is a straightforward and high throughput assay technique that can be utilized to screen hundreds or thousands of compounds. Here, DSF assay conditions were optimized for 3CL Proteases. A library of compounds from the Hagen group was screened that was previously tested in an inhibition assay at Purdue University and found to have low micromolar activity against 3CL Protease. Downward shifts were observed with increasing concentrations of the compounds because they were covalent inhibitors and formed covalent bonds with the target. Solubility issues were also encountered with the compounds and further screening is required, to have a better solubility of the compounds. The assay conditions developed in this study can be used to test other compounds in a high throughput manner.
Recommended Citation
Sharma, Ishpriya, "Identification of Rickettsia Prowazekii Methionine aminopeptidase Inhibitors and Development of Thermal Shift assays for Burkholderia Pseudomallei Ispf and Sars-Cov-2 3CL Protease" (2023). Graduate Research Theses & Dissertations. 7354.
https://huskiecommons.lib.niu.edu/allgraduate-thesesdissertations/7354
Extent
158 pages
Language
eng
Publisher
Northern Illinois University
Rights Statement
In Copyright
Rights Statement 2
NIU theses are protected by copyright. They may be viewed from Huskie Commons for any purpose, but reproduction or distribution in any format is prohibited without the written permission of the authors.
Media Type
Text
