HSP60/10 Chaperonin Systems Are Inhibited by a Variety of Approved Drugs, Natural Products, and Known Bioactive Molecules
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HSP60/10 chaperonin systems are inhibited by a variety of approved drugs, natural products, and known bioactive molecules Item Type Article Authors 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 Citation Stevens, M., Abdeen, S., Salim, N., Ray, A. M., Washburn, A., Chitre, S., ... & Johnson, S. M. (2019). HSP60/10 chaperonin systems are inhibited by a variety of approved drugs, natural products, and known bioactive molecules. Bioorganic & medicinal chemistry letters. DOI 10.1016/j.bmcl.2019.02.028 Publisher PERGAMON-ELSEVIER SCIENCE LTD Journal BIOORGANIC & MEDICINAL CHEMISTRY LETTERS Rights © 2019 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license. Download date 05/10/2021 15:14:53 Item License https://creativecommons.org/licenses/by-nc-nd/4.0/ Version Final published version Link to Item http://hdl.handle.net/10150/632031 Bioorganic & Medicinal Chemistry Letters 29 (2019) 1106–1112 Contents lists available at ScienceDirect Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl HSP60/10 chaperonin systems are inhibited by a variety of approved drugs, T natural products, and known bioactive molecules Mckayla Stevensa,e, Sanofar Abdeena,e, Nilshad Salima, Anne-Marie Raya, Alex Washburna, Siddhi Chitrea, Jared Sivinskib, Yangshin Parka,c,d, Quyen Q. Hoanga,c,d, Eli Chapmanb, ⁎ Steven M. Johnsona, a Indiana University School of Medicine, Department of Biochemistry and Molecular Biology, 635 Barnhill Dr., Indianapolis, IN 46202, United States b The University of Arizona, College of Pharmacy, Department of Pharmacology and Toxicology, 1703 E. Mabel St., PO Box 210207, Tucson, AZ 85721, United States c Stark Neurosciences Research Institute, Indiana University School of Medicine. 320 W. 15th Street, Suite 414, Indianapolis, IN 46202, United States d Department of Neurology, Indiana University School of Medicine. 635 Barnhill Drive, Indianapolis, IN 46202, United States ARTICLE INFO ABSTRACT Keywords: All living organisms contain a unique class of molecular chaperones called 60 kDa heat shock proteins (HSP60 – GroEL also known as GroEL in bacteria). While some organisms contain more than one HSP60 or GroEL isoform, at least GroES one isoform has always proven to be essential. Because of this, we have been investigating targeting HSP60 and HSP60 GroEL chaperonin systems as an antibiotic strategy. Our initial studies focused on applying this antibiotic HSP10 strategy for treating African sleeping sickness (caused by Trypanosoma brucei parasites) and drug-resistant Molecular chaperone bacterial infections (in particular Methicillin-resistant Staphylococcus aureus – MRSA). Intriguingly, during our Chaperonin Proteostasis studies we found that three known antibiotics – suramin, closantel, and rafoxanide – were potent inhibitors of Small molecule inhibitors bacterial GroEL and human HSP60 chaperonin systems. These findings prompted us to explore what other Natural products approved drugs, natural products, and known bioactive molecules might also inhibit HSP60 and GroEL cha- peronin 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, allof which were nearly equipotent against human HSP60. These findings illuminate the notion that targeting cha- peronin 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. Molecular chaperones are a class of proteins that cells have devel- of this, we hypothesize that these chaperonin systems are viable anti- oped to help fold polypeptides to their native states, or target them for biotic targets. Since HSP60/10 and GroEL/ES chaperonin systems are degradation.1–7 The 60 kDa heat shock proteins (HSP60 – also known as highly conserved across all organisms, we envision that this could be a GroEL in bacteria) are a unique class of the molecular chaperone family broad-spectrum antibiotic strategy. While human cells also contain an that function by encapsulating unfolded polypeptides in the central HSP60/10 homolog, located in mitochondria, our accumulating evi- cavity of an HSP60 ring, allowing them to fold while sequestered from dence supports that even if compounds can inhibit HSP60/10 in vitro, the cellular milieu.8–14 HSP60 chaperonins accomplish this in an ATP- many of these inhibitors are non-toxic to human cells and in vivo.32–35 dependent fashion with the assistance of co-chaperones called HSP10 No drugs have been developed to specifically inhibit HSP60/10 or (GroES in bacteria), which are “lid” structures that cap off folding-ac- GroEL/ES chaperonins, and thus we believe that targeting these mole- tive HSP60 rings (hence, we typically refer to them as HSP60/10 and cular machines offers significant promise to address the problem of GroEL/ES chaperonin systems). While some organisms contain more antibiotic-resistant infectious organisms. than one HSP60 or GroEL isoform, one has always proven to be es- As a first step in our research, we previously conducted ahigh- sential, at least in the micro-organisms thus far evaluated.15–31 Because throughput screen of ∼700,000 small molecules and identified 235 ⁎ Corresponding author. E-mail address: [email protected] (S.M. Johnson). e Co-first author. https://doi.org/10.1016/j.bmcl.2019.02.028 Received 11 December 2018; Received in revised form 23 February 2019; Accepted 26 February 2019 Available online 28 February 2019 0960-894X/ © 2019 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/). M. Stevens, et al. Bioorganic & Medicinal Chemistry Letters 29 (2019) 1106–1112 Fig. 1. Structures of compounds previously found to inhibit E. coli GroEL/ES and/or human HSP60/10 chaperonin systems. inhibitors of the prototypical GroEL/ES chaperonin system from veterinary medicine, closantel and rafoxanide, were also potent GroEL/ Escherichia coli.36 One lead hit, the bis-sulfonamido-2-ar- ES and HSP60/10 inhibitors.35,38 In addition, other groups have iden- ylbenzimidazole compound A (Fig. 1), was isostructural to another tified a handful of natural products, such as epolactaene and myrtu- molecule identified in the PubChem database (compound B), which was commulone, that were able to target the human HSP60/10 chaperonin reported to inhibit the growth of Leishmania parasites.37 As an exten- system.39–42 Taken together, these accumulating findings prompted us sion, we developed a library of compound B analogs and screened them to consider the possibility that targeting chaperonin systems with small against Trypanosoma brucei, a related parasite that causes African molecule inhibitors may be more common than we previously thought. sleeping sickness.33 Surprisingly, in that study we found that suramin To shed further light on this possibility, we designed the present study (28), the first-line treatment for T. brucei infections in humans, was to identify what other approved drugs, natural products, or known capable of inhibiting Escherichia coli GroEL/ES (which we typically use bioactive molecules might also inhibit HSP60/10 and/or GroEL/ES as a surrogate for compound evaluation) as well as human HSP60/10. chaperonin systems. To further support that this interaction was real, in the present study, In this study, we screened against the Library of Pharmaceutically we analyzed the suramin-GroEL binding properties using Isothermal Active Compounds (LOPAC) and the MicroSource Spectrum libraries, Titration Calorimetry (see the Supporting Information for a detailed which together contain 3680 approved drugs, natural products, and protocol for this experiment). An isotherm for a representative suramin- known bioactive molecules. For the primary high-throughput screen, GroEL binding analysis is presented in Fig. 2, with the thermodynamic we developed a new protocol that combined our traditional GroEL/ES- parameters, binding affinities, and binding stoichiometries averaged dMDH refolding and chaperonin-mediated ATPase assays into one from triplicate analyses presented in Table 1. We found that suramin multiplexed assay. A schematic representation of this multiplexed assay had a Kd of 21 μM for binding to E. coli GroEL, which corresponds is presented in Fig. 3, with a detailed description of the protocol pro- reasonably well with the IC50 values for suramin inhibition in our as- vided in the Supporting Information. Using this assay, we screened the says that monitor GroEL/ES-mediated refolding of dMDH and dRho. 3680 compound library and identified 219 compounds that in- While this analysis shows the suramin-GroEL interaction is indeed real, hibited > 50% of the reporter enzymatic reaction, and thus putatively what remains to be seen is what contribution suramin’s binding to the the refolding of the MDH reporter enzyme by the GroEL/ES chaperonin three HSP60s in T. brucei make to its anti-trypanosomal effects. Future system. We found that none of the hits reduced ATPase activity, which studies will need to explore