Browsing by Author "Johnson, Steven M."

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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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    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 Medicine
    In 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.
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    Antibiotic Discovery Targeting Bacterial GroEL/GroES Chaperonin Systems
    (2018-07-29) Kunkle, Trent A.; Johnson, Steven M.; Georgiadis, Millie M.; Hoang, Quyen Q.
    The Centers for Disease Control (CDC) and World Health Organizations (WHO) have highlighted six species of highly drug-resistant bacteria, commonly termed the ESKAPE pathogens, that new antibacterials are urgently needed to treat). The ESKAPE pathogens account for over two-million infections and have healthcare costs upwards of $20 billion dollars annually. Over the past several decades, pharmaceutical companies have drastically reduced their research programs for developing new antibacterial agents. As well, bacteria are predisposed to rapidly generate resistance against these “me too” drugs, making this strategy a temporary stop-gap in our ability to fight these pathogens. This has left the burden to identify new antibiotics that function through fundamentally unique mechanisms of action to academia. Towards this goal, we are developing a unique antibacterial strategy that functions through targeting the bacterial GroEL chaperonin systems. GroEL is a molecular chaperone that helps fold proteins into their functional states. Being an essential protein, inhibiting GroEL activity leads to global aggregation and bacterial cell death. We previously reported a high-throughput screening effort that identified 235 GroEL inhibitors. A subsequent study with a subset of these inhibitors identified several that kill bacteria. To follow-up, we have synthesized 43 analogs of a hit-to-lead molecule, compound 1, containing systematic deletions of substituents and substructures to determine the essential parts of the scaffold for inhibiting GroEL and killing bacteria. Along with inhibiting GroEL, several compound 1 analogs exhibit >50-fold therapeutic windows between antibacterial efficacy and cytotoxicity to human liver and kidney cells in cell culture. Evaluation of two lead candidates (1 and 11) in a gain-of-resistance assay indicated that MRSA bacteria were not able to easily generate resistance to this compound class. Compound 1 also exhibited the ability to permeate through already established S. aureus biofilms and maintain its bactericidal effects, whereas vancomycin could not. Having established initial structure-activity relationships for the compound 1 substituents and substructures in this study, future efforts will focus on optimizing the antibacterial effects of lead candidates and reducing their off-target toxicity to human cells.
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    Chemically induced partial unfolding of the multifunctional Apurinic/apyrimidinic endonuclease 1
    (bioRxiv, 2023-06-29) Rai, Ratan; Dawodu, Olabode I.; Johnson, Steven M.; Vilseck, Jonah Z.; Kelley, Mark R.; Ziarek, Joshua J.; Georgiadis, Millie M.; Biochemistry and Molecular Biology, School of Medicine
    Targeting of the multifunctional enzyme apurinic/apyrimidinic endonuclease I/redox factor 1 (APE1) has produced small molecule inhibitors of both its endonuclease and redox activities. While one of the small molecules, the redox inhibitor APX3330, completed a Phase I clinical trial for solid tumors and a Phase II clinical trial for Diabetic Retinopathy/Diabetic Macular Edema, the mechanism of action for this drug has yet to be fully understood. Here, we demonstrate through HSQC NMR studies that APX3330 induces chemical shift perturbations (CSPs) of both surface and internal residues in a concentration-dependent manner, with a cluster of surface residues defining a small pocket on the opposite face from the endonuclease active site of APE1. Furthermore, APX3330 induces partial unfolding of APE1 as evidenced by a time-dependent loss of chemical shifts for approximately 35% of the residues within APE1 in the HSQC NMR spectrum. Notably, regions that are partially unfolded include adjacent strands within one of two beta sheets that comprise the core of APE1. One of the strands comprises residues near the N-terminal region and a second strand is contributed by the C-terminal region of APE1, which serves as a mitochondrial targeting sequence. These terminal regions converge within the pocket defined by the CSPs. In the presence of a duplex DNA substrate mimic, removal of excess APX3330 resulted in refolding of APE1. Our results are consistent with a reversible mechanism of partial unfolding of APE1 induced by the small molecule inhibitor, APX3330, defining a novel mechanism of inhibition.
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    Discovery, Characterization, and Development of Small Molecule Inhibitors of Glycogen Synthase
    (2020-06) Tang, Buyun; Hurley, Thomas D.; Roach, Peter J.; Georgiadis, Millie M.; Johnson, Steven M.; Elmendorf, Jeffrey S.
    The over-accumulation of glycogen appears as a hallmark in various glycogen storage diseases (GSDs), including Pompe, Cori, Andersen, and Lafora disease. Glycogen synthase (GS) is the rate-limiting enzyme for glycogen synthesis. Recent evidence suggests that suppression of glycogen accumulation represents a potential therapeutic approach for treating these diseases. Herein, we describe the discovery, characterization, and development of small molecule inhibitors of GS through a multicomponent study including biochemical, biophysical, and cellular assays. Adopting an affinity-based fluorescence polarization assay, we identified a substituted imidazole molecule (H23), as a first-in-class inhibitor of yeast glycogen synthase 2 (yGsy2) from the 50,000 ChemBridge DIVERSet library. Structural data derived from X-ray crystallography at 2.85 Å, and enzyme kinetic data, revealed that H23 bound within the uridine diphosphate glucose binding pocket of yGsy2. Medicinal chemistry efforts examining over 500 H23 analogs produced structure-activity relationship (SAR) profiles that led to the identification of potent pyrazole and isoflavone compounds with low micromolar potency against human glycogen synthase 1 (hGYS1). Notably, several of the isoflavones demonstrated cellular efficacy toward suppressing glycogen accumulation. In an alternative effort to screen inhibitors directly against human GS, an activity-based assay was designed using a two-step colorimetric approach. This assay led to the identification of compounds with submicromolar potency to hGYS1 from a chemical library comprised of 10,000 compounds. One of the hit molecules, hexachlorophene, was crystallized bound to the active site of yGsy2. The structure was determined to 3.15 Å. Additional kinetic, mutagenic, and SAR studies validated the binding of hexachlorophene in the catalytic pocket and its non-competitive mode of inhibition. In summary, these two novel assays provided feasible biochemical platforms for large-scale screening of small molecule modulators of GS. The newly-developed, potent analogs possess diverse promising scaffolds for drug development efforts targeting GS activity in GSDs associated with excess glycogen accumulation.
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    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 Medicine
    Current 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.
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    Examining the Potential of Targeting the HSP60 Chaperonin System as a Broadly Applicable Chemotherapeutic Strategy
    (2023-12) Liechty, Hope Lauren; Johnson, Steven M.; Motea, Edward A.; Turchi, John J.; Vilseck, Jonah Z.
    This study methodically examined our diversity set of GroEL and HSP60 inhibitors to identify lead candidates that exhibited the most potent and selective cytotoxicity to colon cancer cells over non-cancer cells in vitro. While several structurally distinct candidates were identified, we found that our nitrofuran and hydroxyquinoline-containing N-acylhydrazone series (NF-NAH and HQ-NAH, respectively) were among the most potent and selective. Subsequent screenings across an NCI panel of cancer cell lines of different origins revealed the superior efficacy of the NF-NAH and HQ-NAH series as chemotherapeutic candidates, in contrast to the ABK-based inhibitors we previously reported, which showed poor efficacy across the panel. Given the emerging evidence of the role of mis-localized HSP60 in cancer cell survival, this study also compared the structure and function of naive cHSP60 (the aberrant form presumed to be in the cytosol) with that of mHSP60 (the processed and mature form that is in mitochondria). Analytical size exclusion chromatography revealed cHSP60 is a more stable oligomer consisting of both single and double-ring complexes. Intriguingly, cryoEM analyses revealed that cHSP60 formed a unique face-to-face double-ring complex, as opposed to the structures of other double-ring GroEL and HSP60 chaperonins where their rings stack back-to-back with one another. Subsequent assays demonstrated similar ATPase activities for both mHSP60 and cHSP60, with stimulatory effects observed in the presence of HSP10 for both. Despite the apparent engagement of HSP10, cHSP60 was unable to refold the denatured MDH client protein efficiently, suggesting potential functional divergence in vivo. These enticing results offer novel insights into the physiological importance of the cHSP60 complex and its possible role in cancer progression. As our previous studies examined inhibitors that were developed as GroEL-targeting antibacterial candidates, and given the unique structural/functional differences of cHSP60 compared to GroEL and other chaperonins, including mHSP60, the findings from this study underscore the need for future to identify and optimize inhibitors specifically for targeting cHSP60 to enhance chemotherapeutic effectiveness.
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    Exploiting the HSP60/10 Chaperonin System as a Chemotherapeutic Target for Colorectal Cancer
    (Elsevier, 2021) Ray, Anne-Marie; Salim, Nilshad; Stevens, Mckayla; Chitre, Siddhi; Abdeen, Sanofar; Washburn, Alex; Sivinski, Jared; O’Hagan, Heather M.; Chapman, Eli; Johnson, Steven M.; Biochemistry and Molecular Biology, School of Medicine
    Over the past few decades, an increasing variety of molecular chaperones have been investigated for their role in tumorigenesis and as potential chemotherapeutic targets; however, the 60 kDa Heat Shock Protein (HSP60), along with its HSP10 co-chaperone, have received little attention in this regard. In the present study, we investigated two series of our previously developed inhibitors of the bacterial homolog of HSP60/10, called GroEL/ES, for their selective cytotoxicity to cancerous over non-cancerous colorectal cells. We further developed a third "hybrid" series of analogs to identify new candidates with superior properties than the two parent scaffolds. Using a series of well-established HSP60/10 biochemical screens and cell-viability assays, we identified 24 inhibitors (14%) that exhibited > 3-fold selectivity for targeting colorectal cancer over non-cancerous cells. Notably, cell viability EC50 results correlated with the relative expression of HSP60 in the mitochondria, suggesting a potential for this HSP60-targeting chemotherapeutic strategy as emerging evidence indicates that HSP60 is up-regulated in colorectal cancer tumors. Further examination of five lead candidates indicated their ability to inhibit the clonogenicity and migration of colorectal cancer cells. These promising results are the most thorough analysis and first reported instance of HSP60/10 inhibitors being able to selectively target colorectal cancer cells and highlight the potential of the HSP60/10 chaperonin system as a viable chemotherapeutic target.
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    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.