Biochemistry & Molecular Biology Department Theses and Dissertations

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Browsing Biochemistry & Molecular Biology Department Theses and Dissertations by Author "Absalon, Sabrina"

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    The Mechanisms by Which Small Molecules Modulate the HSP60/10 Chaperonin System to Elicit Antimicrobial Effects
    (2023-06) Stevens, Mckayla Marie; Johnson, Steven; Turchi, John; Hoang, Quyen; Wek, Ronald; Absalon, Sabrina
    Heat Shock Protein 60/10 (HSP60/10, or GroEL/ES in bacteria) chaperonin systems play a critical role in protein homeostasis through facilitating proper folding of misfolded or partially folded polypeptides that are otherwise prone to aggregation. HSP60 chaperonins are highly conserved and essential in nearly all organisms studied thus far, making them a promising target for antibiotic development. Early high-throughput screens in the Johnson lab have identified five main scaffolds that, though hit-to-lead development, have been optimized for chaperonin inhibition and antimicrobial effects. While these initial studies have shown promising evidence to support the viability of a chaperonin-targeting antibiotic strategy, it was unclear whether the conservation of human HSP60 (48% identity to bacterial GroEL) would hinder this therapeutic strategy from advancing due to potential toxicity associated with off-target inhibition of the human homolog. Additionally, while chaperonin inhibition often correlated with cytotoxicity to the various pathogens studied, there was a clear need to investigate inhibitor mechanisms to 1) verify on-target effects, and 2) guide future development of more potent and selective chaperonin-targeting antibiotic candidates. Herein, we conduct a medium-throughput screening of known bioactive molecules, approved drugs, and natural products against both bacterial GroEL and human HSP60, demonstrating that most molecules exhibited low-to-no toxicity to human cells in culture, despite being near equipotent inhibitors of human HSP60 and E. coli GroEL in our refolding assays. Thus, sequence conservation between human HSP60 and bacterial GroELs does not necessarily predict toxicity in vivo. We then investigate inhibitory mechanisms of our most well-established inhibitor series, the phenylbenzoxazole (PBZ) series, identifying three binding sites whereby PBZ molecules modulate GroEL folding and ATPase functions in a site-specific manner, predominately through its ability to interact with its co-chaperone GroES. Finally, we demonstrate that two standard of care drugs for T. brucei infections, suramin and nifurtimox, may elicit their trypanocidal effects through inhibiting HSP60. Due to structural similarities, we then screened our N-acylhydrazone (NAH) and α,β-unsaturated ketone (ABK) series of HSP60 inhibitors against T. brucei, finding that they are highly potent and selective trypanocidal agents. Together, these studies further support HSP60 as a viable drug target for antibiotic development.
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    Small Molecule Inhibitors of GroEL That Disrupt Active Replication of Mycobacterium Tuberculosis and ESKAPE Bacteria
    (2022-07) Tepper, Katelyn; Johnson, Steven M.; Georgiadis, Millie; Motea, Edward; Absalon, Sabrina
    Globally, millions of people die every year due to complications involving infections from antibiotic-resistant bacteria. Of these infections, the most common organisms are Mycobacterium tuberculosis (Mtb) and a group of bacteria known as the ESKAPE pathogens (an acronym that stands for Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa, Enterobacter species). Unfortunately, as the need for antibiotics increases, industrial antibiotic development programs are drying up. However, unique antibiotic candidates targeting new pathways may be better for addressing antibacterial resistance. A target that is currently not the focus of any drug on the market is the bacterial GroEL chaperonin system. GroEL chaperonins are complex, oligomeric proteins that are upregulated in the cell under stressful conditions and prevent the misfolding and aggregation of other proteins. All bacteria have one homolog that performs protein folding functions – such is the case for E. coli and the ESKAPE bacteria – while others, like M. tuberculosis, contain additional GroEL isoforms that appear to perform non-canonical functions that are not well understood. The canonical isoforms are essential for survival under all conditions; thus, these chaperonins represent excellent targets for antibiotic development. This study aimed to identify inhibitors of GroEL in the ESKAPE bacteria and Mtb from a library of compounds with known antibiotic properties that was provided by the Medicines for Malaria Venture. Using two orthogonal assays that assess GroEL activity via its refolding of denatured enzymes Malate Dehydrogenase and Rhodanase, 37 inhibitors of E. coli GroEL were identified. Of these, 33 were examined in dose response testing in in vitro biochemical and cell viability assays. Compound 23 stood out in potency for inhibiting GroEL functions and actively-replicating Mtb bacteria, and thus a small panel of analogs were evaluated to develop structure-activity relationships (SAR) and study their mechanism. Two cysteine residues were identified as covalently modified by compound 23 and one of the lead analogs, giving insight into inhibitory sites on GroEL. Another lead analog bearing a nitrofuran moiety exhibited inhibition of actively-replicating E. coli, S. aureus, and Mtb bacteria. Importantly, this study identified new classes of GroEL inhibitors to explore for optimization as antibacterial candidates.