Browsing by Subject "GroEL"
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Item 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 MedicineGroES/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.Item Analogs of nitrofuran antibiotics are potent GroEL/ES inhibitor pro-drugs(Elsevier, 2020) Stevens, Mckayla; Howe, Chris; Ray, Anne-Marie; Washburn, Alex; Chitre, Siddhi; Sivinski, Jared; Park, Yangshin; Hoang, Quyen Q.; Chapman, Eli; Johnson, Steven M.; Biochemistry and Molecular Biology, School of MedicineIn two previous studies, we identified compound 1 as a moderate GroEL/ES inhibitor with weak to moderate antibacterial activity against Gram-positive and Gram-negative bacteria including Bacillus subtilis, methicillin-resistant Staphylococcus aureus, Klebsiella pneumonia, Acinetobacter baumannii, and SM101 Escherichia coli (which has a compromised lipopolysaccharide biosynthetic pathway making bacteria more permeable to drugs). Extending from those studies, we developed two series of analogs with key substructures resembling those of known antibacterials, nitroxoline (hydroxyquinoline moiety) and nifuroxazide/nitrofurantoin (bis-cyclic-N-acylhydrazone scaffolds). Through biochemical and cell-based assays, we identified potent GroEL/ES inhibitors that selectively blocked E. faecium, S. aureus, and E. coli proliferation with low cytotoxicity to human colon and intestine cells in vitro. Initially, only the hydroxyquinoline-bearing analogs were found to be potent inhibitors in our GroEL/ES-mediated substrate refolding assays; however, subsequent testing in the presence of an E. coli nitroreductase (NfsB) in situ indicated that metabolites of the nitrofuran-bearing analogs were potent GroEL/ES inhibitor pro-drugs. Consequently, this study has identified a new target of nitrofuran-containing drugs, and is the first reported instance of such a unique class of GroEL/ES chaperonin inhibitors. The intriguing results presented herein provide impetus for expanded studies to validate inhibitor mechanisms and optimize this antibacterial class using the respective GroEL/ES chaperonin systems and nitroreductases from E. coli and the ESKAPE bacteria.Item Constructing Kinetically Controlled Denaturation Isotherms of Folded Proteins Using Denaturant-Pulse Chaperonin Binding(Springer Nature, 2018-10-20) O’Neil, Pierce T.; Machen, Alexandra J.; Thompson, Jackie A.; Wang, Wei; Hoang, Quyen Q.; Baldwin, Michael R.; Khar, Karen R.; Karanicolas, John; Fisher, Mark T.; Biochemistry and Molecular Biology, School of MedicineMethods to assess the kinetic stability of proteins, particularly those that are aggregation prone, are very useful in establishing ligand induced stabilizing effects. Because aggregation prone proteins are by nature difficult to work with, most solution based methods are compromised by this inherent instability. Here, we describe a label-free method that examines the denaturation of immobilized proteins where the dynamic unfolded protein populations are captured and detected by chaperonin binding.Item Dual-targeting GroEL/ES chaperonin and protein tyrosine phosphatase B (PtpB) inhibitors: A polypharmacology strategy for treating Mycobacterium tuberculosis infections(Elsevier, 2019-07-01) Washburn, Alex; Abdeen, Sanofar; Ovechkina, Yulia; Ray, Anne-Marie; Stevens, Mckayla; Chitre, Siddhi; Sivinski, Jared; Park, Yangshin; Johnson, James; Hoang; Hoang, Quyen Q.; Chapman, Eli; Parish, Tanya; Johnson, Steven M.; Biochemistry and Molecular Biology, School of MedicineCurrent treatments for Mycobacterium tuberculosis infections require long and complicated regimens that can lead to patient non-compliance, increasing incidences of antibiotic-resistant strains, and lack of efficacy against latent stages of disease. Thus, new therapeutics are needed to improve tuberculosis standard of care. One strategy is to target protein homeostasis pathways by inhibiting molecular chaperones such as GroEL/ES (HSP60/10) chaperonin systems. M. tuberculosis has two GroEL homologs: GroEL1 is not essential but is important for cytokine-dependent granuloma formation, while GroEL2 is essential for survival and likely functions as the canonical housekeeping chaperonin for folding proteins. Another strategy is to target the protein tyrosine phosphatase B (PtpB) virulence factor that M. tuberculosis secretes into host cells to help evade immune responses. In the present study, we have identified a series of GroEL/ES inhibitors that inhibit M. tuberculosis growth in liquid culture and biochemical function of PtpB in vitro. With further optimization, such dual-targeting GroEL/ES and PtpB inhibitors could be effective against all stages of tuberculosis – actively replicating bacteria, bacteria evading host cell immune responses, and granuloma formation in latent disease – which would be a significant advance to augment current therapeutics that primarily target actively replicating bacteria.Item Exploring Dual-Targeting GroEL/ES & PtpB Inhibitors as a New Antibiotic Strategy for Tuberculosis(2019-05) Washburn, J. Alex; Johnson, Steven M.; Georgiadis, Millie M.; Hoang, Quyen Q.Current Mycobacterium tuberculosis (Mtb) treatments suffer from an increase in antibiotic resistance strains and the lack of efficacy against latent state tuberculosis, thus novel approaches targeting different mechanisms of action are needed. One strategy to target Mtb is to target protein homeostasis pathways by inhibiting molecular chaperones, in particular, GroEL/ES (HSP60/10) chaperonin systems. Mtb has two homologs of GroEL, of which GroEL1 is not essential, but is important for cytokine-dependent granuloma formation, and GroEL2 is essential for survival and the likely canonical housekeeping chaperonin. Another strategy to target Mtb is to target the protein tyrosine phosphatase B (PtpB) virulence factor that Mtb secretes into host cells to help evade immune responses. Thus, we envisioned that this analog series might also be capable of inhibiting Mtb PtpB along with GroEL. By developing compound 1 inhibitors that could act on all of GroEL1, GroEL2, and PtpB, we could have an antibiotic candidate that targets all stages of tuberculosis: actively replicating bacteria, bacteria evading host cell immune response, and granuloma formation in latent disease. In the Johnson lab, previous studies explored GroEL/ES inhibitors, with compound 1 being one of the most potent inhibitors, inhibiting both Trypanosoma brucei and Staphylococcus aureus proliferation. In the present study, we have screened previously developed compound 1 analogs, as well as a series of newly synthesized analogs that we term “half-molecules”. In this study, our results indicated two potential avenues to explore for future research. The first is a series of carboxyl-bearing compound 1 inhibitors, compounds 2m-o, 2m-m, and 2m-p, which act solely on Mtb PtpB phosphatase activity without inhibiting GroEL. The second is a series of compound 1 inhibitors (e.g. 20R and 20L) that are able to inhibit both the PtpB phosphatase and GroEL/ES chaperonin system. Thus, this exploratory study showed the possibility of pursuing such a polypharmacological antibiotic strategy against Mtb infections and with further optimization, such dual-targeting GroEL/ES and PtpB inhibitors could be effective against all stages of tuberculosis.Item 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 MedicineWe 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.Item HSP60/10 chaperonin systems are inhibited by a variety of approved drugs, natural products, and known bioactive molecules(Elsevier, 2019-05-01) Stevens, Mckayla; Abdeen, Sanofar; Salim, Nilshad; Ray, Anne-Marie; Washburn, Alex; Chitre, Siddhi; Sivinski, Jared; Park, Yangshin; Hoang, Quyen Q.; Chapman, Eli; Johnson, Steven M.; Biochemistry and Molecular Biology, School of MedicineAll living organisms contain a unique class of molecular chaperones called 60 kDa heat shock proteins (HSP60 - also known as GroEL in bacteria). While some organisms contain more than one HSP60 or GroEL isoform, at least one isoform has always proven to be essential. Because of this, we have been investigating targeting HSP60 and GroEL chaperonin systems as an antibiotic strategy. Our initial studies focused on applying this antibiotic strategy for treating African sleeping sickness (caused by Trypanosoma brucei parasites) and drug-resistant bacterial infections (in particular Methicillin-resistant Staphylococcus aureus - MRSA). Intriguingly, during our studies we found that three known antibiotics - suramin, closantel, and rafoxanide - were potent inhibitors of bacterial GroEL and human HSP60 chaperonin systems. These findings prompted us to explore what other approved drugs, natural products, and known bioactive molecules might also inhibit HSP60 and GroEL chaperonin systems. Initial high-throughput screening of 3680 approved drugs, natural products, and known bioactives identified 161 hit inhibitors of the Escherichia coli GroEL chaperonin system (4.3% hit rate). From a purchased subset of 60 hits, 29 compounds (48%) re-confirmed as selective GroEL inhibitors in our assays, all of which were nearly equipotent against human HSP60. These findings illuminate the notion that targeting chaperonin systems might be a more common occurrence than we previously appreciated. Future studies are needed to determine if the in vivo modes of action of these approved drugs, natural products, and known bioactive molecules are related to GroEL and HSP60 inhibition.Item 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 MedicineWe 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.Item 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, SabrinaGlobally, 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.Item 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 MedicineExtending 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).