Browsing by Author "Ambrose, Andrew J."

Now showing 1 - 6 of 6
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    Allosteric differences dictate GroEL complementation of E. coli
    (Wiley, 2022) Sivinski, Jared; Ngo, Duc; Zerio, Christopher J.; Ambrose, Andrew J.; Watson, Edmond R.; Kaneko, Lynn K.; Kostelic, Marius M.; Stevens, Mckayla; Ray, Anne-Marie; Park, Yangshin; Wu, Chunxiang; Marty, Michael T.; Hoang, Quyen Q.; Zhang, Donna D.; Lander, Gabriel C.; Johnson, Steven M.; Chapman, Eli; Biochemistry and Molecular Biology, School of Medicine
    GroES/GroEL is the only bacterial chaperone essential under all conditions, making it a potential antibiotic target. Rationally targeting ESKAPE GroES/GroEL as an antibiotic strategy necessitates studying their structure and function. Herein, we outline the structural similarities between Escherichia coli and ESKAPE GroES/GroEL and identify significant differences in intra- and inter-ring cooperativity, required in the refolding cycle of client polypeptides. Previously, we observed that one-half of ESKAPE GroES/GroEL family members could not support cell viability when each was individually expressed in GroES/GroEL-deficient E. coli cells. Cell viability was found to be dependent on the allosteric compatibility between ESKAPE and E. coli subunits within mixed (E. coli and ESKAPE) tetradecameric GroEL complexes. Interestingly, differences in allostery did not necessarily result in differences in refolding rate for a given homotetradecameric chaperonin. Characterization of ESKAPE GroEL allostery, ATPase, and refolding rates in this study will serve to inform future studies focused on inhibitor design and mechanism of action studies.
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    Functional Differences between E. coli and ESKAPE Pathogen GroES/GroEL
    (American Society for Microbiology, 2021-01-12) Sivinski, Jared; Ambrose, Andrew J.; Panfilenko, Iliya; Zerio, Christopher J.; Machulis, Jason M.; Mollasalehi, Niloufar; Kaneko, Lynn K.; Stevens, Mckayla; Ray, Anne-Marie; Park, Yangshin; Wu, Chunxiang; Hoang, Quyen Q.; Johnson, Steven M.; Chapmana, Eli; Biochemistry and Molecular Biology, School of Medicine
    As the GroES/GroEL chaperonin system is the only bacterial chaperone that is essential under all conditions, we have been interested in the development of GroES/GroEL inhibitors as potential antibiotics. Using Escherichia coli GroES/GroEL as a surrogate, we have discovered several classes of GroES/GroEL inhibitors that show potent antibacterial activity against both Gram-positive and Gram-negative bacteria. However, it remains unknown if E. coli GroES/GroEL is functionally identical to other GroES/GroEL chaperonins and hence if our inhibitors will function against other chaperonins. Herein we report our initial efforts to characterize the GroES/GroEL chaperonins from clinically significant ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species). We used complementation experiments in GroES/GroEL-deficient and -null E. coli strains to report on exogenous ESKAPE chaperone function. In GroES/GroEL-deficient (but not knocked-out) E. coli, we found that only a subset of the ESKAPE GroES/GroEL chaperone systems could complement to produce a viable organism. Surprisingly, GroES/GroEL chaperone systems from two of the ESKAPE pathogens were found to complement in E. coli, but only in the strict absence of either E. coli GroEL (P. aeruginosa) or both E. coli GroES and GroEL (E. faecium). In addition, GroES/GroEL from S. aureus was unable to complement E. coli GroES/GroEL under all conditions. The resulting viable strains, in which E. coli groESL was replaced with ESKAPE groESL, demonstrated similar growth kinetics to wild-type E. coli, but displayed an elongated phenotype (potentially indicating compromised GroEL function) at some temperatures. These results suggest functional differences between GroES/GroEL chaperonins despite high conservation of amino acid identity.
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    GroEL/ES inhibitors as potential antibiotics
    (Elsevier, 2016-07) Abdeen, Sanofar; Salim, Nilshad; Mammadova, Najiba; Summers, Corey; Frankson, Rochelle; Ambrose, Andrew J.; Anderson, Gregory G.; Schultz, Peter G.; Horwich, Arthur L.; Chapman, Eli; Johnson, Steven M.; Department of Biochemistry & Molecular Biology, IU School of Medicine
    We recently reported results from a high-throughput screening effort that identified 235 inhibitors of the Escherichia coli GroEL/ES chaperonin system [Bioorg. Med. Chem. Lett.2014, 24, 786]. As the GroEL/ES chaperonin system is essential for growth under all conditions, we reasoned that targeting GroEL/ES with small molecule inhibitors could be a viable antibacterial strategy. Extending from our initial screen, we report here the antibacterial activities of 22 GroEL/ES inhibitors against a panel of Gram-positive and Gram-negative bacteria, including E. coli, Bacillus subtilis, Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter cloacae. GroEL/ES inhibitors were more effective at blocking the proliferation of Gram-positive bacteria, in particular S. aureus, where lead compounds exhibited antibiotic effects from the low-μM to mid-nM range. While several compounds inhibited the human HSP60/10 refolding cycle, some were able to selectively target the bacterial GroEL/ES system. Despite inhibiting HSP60/10, many compounds exhibited low to no cytotoxicity against human liver and kidney cell lines. Two lead candidates emerged from the panel, compounds 8 and 18, that exhibit >50-fold selectivity for inhibiting S. aureus growth compared to liver or kidney cell cytotoxicity. Compounds 8 and 18 inhibited drug-sensitive and methicillin-resistant S. aureus strains with potencies comparable to vancomycin, daptomycin, and streptomycin, and are promising candidates to explore for validating the GroEL/ES chaperonin system as a viable antibiotic target.
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    A High Throughput Substrate Binding Assay Reveals Hexachlorophene as an Inhibitor of the ER-resident HSP70 Chaperone GRP78
    (Elsevier, 2019-07) Ambrose, Andrew J.; Zerio, Christopher J.; Sivinski, Jared; Schmidlin, Cody J.; Shi, Taoda; Ross, Alison B.; Widrick, Kimberly J.; Johnson, Steven M.; Zhang, Donna D.; Chapman, Eli; Biochemistry and Molecular Biology, School of Medicine
    Glucose-regulated protein 78 (GRP78) is the ER resident 70 kDa heat shock protein 70 (HSP70) and has been hypothesized to be a therapeutic target for various forms of cancer due to its role in mitigating proteotoxic stress in the ER, its elevated expression in some cancers, and the correlation between high levels for GRP78 and a poor prognosis. Herein we report the development and use of a high throughput fluorescence polarization-based peptide binding assay as an initial step toward the discovery and development of GRP78 inhibitors. This assay was used in a pilot screen to discover the anti-infective agent, hexachlorophene, as an inhibitor of GRP78. Through biochemical characterization we show that hexachlorophene is a competitive inhibitor of the GRP78-peptide interaction. Biological investigations showed that this molecule induces the unfolded protein response, induces autophagy, and leads to apoptosis in a colon carcinoma cell model, which is known to be sensitive to GRP78 inhibition.
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    Hydroxybiphenylamide GroEL/ES Inhibitors Are Potent Antibacterials against Planktonic and Biofilm Forms of Staphylococcus aureus
    (ACS, 2018-11) Kunkle, Trent; Abdeen, Sanofar; Salim, Nilshad; Ray, Anne-Marie; Stevens, McKayla; Ambrose, Andrew J.; Victorino, José; Park, Yangshin; Hoang, Quyen Q.; Chapman, Eli; Johnson, Steven M.; Biochemistry and Molecular Biology, School of Medicine
    We recently reported the identification of a GroEL/ES inhibitor (1, N-(4-(benzo[d]thiazol-2-ylthio)-3-chlorophenyl)-3,5-dibromo-2-hydroxybenzamide) that exhibited in vitro antibacterial effects against Staphylococcus aureus comparable to vancomycin, an antibiotic of last resort. To follow up, we have synthesized 43 compound 1 analogs to determine the most effective functional groups of the scaffold for inhibiting GroEL/ES and killing bacteria. Our results identified that the benzothiazole and hydroxyl groups are important for inhibiting GroEL/ES-mediated folding functions, with the hydroxyl essential for antibacterial effects. Several analogs exhibited >50-fold selectivity indices between antibacterial efficacy and cytotoxicity to human liver and kidney cells in cell culture. We found that MRSA was not able to easily generate acute resistance to lead inhibitors in a gain-of-resistance assay and that lead inhibitors were able to permeate through established S. aureus biofilms and maintain their bactericidal effects.
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    Sulfonamido 2 arylbenzoxazole GroEL/ES inhibitors are potent antibacterials against methicillin resistant Staphylococcus aureus (MRSA)
    (ACS, 2018) Abdeen, Sanofar; Kunkle, Trent; Salim, Nilshad; Ray, Anne-Marie; Mammadova, Najiba; Summers, Corey; Stevens, Mckayla; Ambrose, Andrew J.; Park, Yangshin; Schultz, Peter G.; Horwich, Arthur L.; Hoang, Quyen; Chapman, Eli; Johnson, Steven M.; Biochemistry and Molecular Biology, School of Medicine
    Extending from a study we recently published examining the antitrypanosomal effects of a series of GroEL/ES inhibitors based on a pseudosymmetrical bis-sulfonamido-2-phenylbenzoxazole scaffold, here, we report the antibiotic effects of asymmetric analogs of this scaffold against a panel of bacteria known as the ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species). While GroEL/ES inhibitors were largely ineffective against K. pneumoniae, A. baumannii, P. aeruginosa, and E. cloacae (Gram-negative bacteria), many analogs were potent inhibitors of E. faecium and S. aureus proliferation (Gram-positive bacteria, EC50 values of the most potent analogs were in the 1–2 μM range). Furthermore, even though some compounds inhibit human HSP60/10 biochemical functions in vitro (IC50 values in the 1–10 μM range), many of these exhibited moderate to low cytotoxicity to human liver and kidney cells (CC50 values > 20 μM).