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Selective Inhibitor of Endosomal Trafficking Pathways Exploited By Selective inhibitor of endosomal trafficking pathways exploited by multiple toxins and viruses Eugene J. Gillespiea, Chi-Lee C. Hoa, Kavitha Balajib, Daniel L. Clemensc, Gang Dengd, Yao E. Wanga, Heidi J. Elsaessera, Batcha Tamilselvame,AmandeepGargie, Shandee D. Dixona,BryanFrancea,f, Brian T. Chamberlaind,f,StevenR.Blankee, Genhong Chenga, Juan Carlos de la Torreg, David G. Brooksa, Michael E. Jungd,f, John Colicellib, Robert Damoiseauxf, and Kenneth A. Bradleya,f,1 aDepartment of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, CA 90095; bDepartment of Biological Chemistry, David Geffen School of Medicine, University of California, Los Angeles, CA 90095; cDepartment of Medicine, Division of Infectious Diseases, David Geffen School of Medicine, University of California, Los Angeles, CA 90095; dDepartment of Chemistry and Biochemistry, University of California, Los Angeles, CA 90095; eDepartment of Microbiology, Institute for Genomic Biology, University of Illinois at Urbana–Champaign, Champaign, IL 61801; fCalifornia NanoSystems Institute, University of California, Los Angeles, CA 90095; and gDepartment of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, CA 92037 Edited by R. John Collier, Harvard Medical School, Boston, MA, and approved October 7, 2013 (received for review February 5, 2013) Pathogenic microorganisms and toxins have evolved a variety of by host proteases into 63- and 20-kDa fragments, allowing olig- mechanisms to gain access to the host-cell cytosol and thereby omerization of the toxin into a prepore (13). The PA oligomer exert virulent effects upon the host. One common mechanism of can then bind up to four monomers of LF, forming a holotoxin cellular entry requires trafficking to an acidified endosome, which complex (14, 15). Two cellular toxin receptors, TEM8 and CMG2, promotes translocation across the host membrane. To identify small- mediate toxin binding and endocytic uptake (16, 17). Acidification molecule inhibitors that block this process, a library of 30,000 small of the lumen of the late endosome drives a conformational change molecules was screened for inhibitors of anthrax lethal toxin. Here in the prepore, resulting in insertion into the endosomal membrane N we report that 4-bromobenzaldehyde -(2,6-dimethylphenyl)semi- and translocation of LF into the cytosol (18–20). Alternatively, LF fi carbazone, the most active compound identi ed in the screen, inhib- may be translocated to the interior of intraluminal vesicles and its intoxication by lethal toxin and blocks the entry of multiple other transported to the late endosome via multivesicular bodies in acid-dependent bacterial toxins and viruses into mammalian cells. a process dependent on COPI and ALIX (21). The vesicular This compound, which we named EGA, also delays lysosomal target- membranes then fuse with the limiting endosomal membrane ing and degradation of the EGF receptor, indicating that it targets fi and thereby deliver LF to the cytosol (21). host-membrane traf cking. In contrast, EGA does not block endo- fi somal recycling of transferrin, retrograde trafficking of ricin, Despite substantial effort to de ne binding and entry mecha- phagolysosomal trafficking, or phagosome permeabilization by nisms used by lethal toxin (LT), much is still unknown about how Franciscella tularensis. Furthermore, EGA does not neutralize acidic it, and indeed many other toxins and viruses, gain access to the organelles, demonstrating that its mechanism of action is distinct host cytosol. To address this, we performed a high-throughput from pH-raising agents such as ammonium chloride and bafilomy- screen to identify small molecules that block cellular entry of cin A1. EGA is a powerful tool for the study of membrane trafficking LT. Here, we report the identification and characterization of and represents a class of host-targeted compounds for therapeutic a compound that blocks trafficking of various toxins and development to treat infectious disease. viruses to acidified endosomes. Results he success of a broad array of microbial pathogens depends fi Ton their ability to gain entry into and/or transport proteins High-Throughput Screen Identi es Potent Anthrax Toxin Inhibitor. To into the cytosol of host cells. Intracellular-acting bacterial toxins identify inhibitors of toxin entry, we used a cellular intoxication have evolved to take advantage of numerous host-mediated entry mechanisms (1), making these toxins ideal tools for studying Significance endocytosis and vesicular trafficking. Indeed, the use of bacterial toxins has contributed to many key discoveries, including mem- Bacterial and viral infections are a significant public health brane recycling, clathrin-independent endocytosis, and retro- burden. To corrupt normal host cellular functions, many bac- grade transport (2). Compounds that inhibit entry of ricin, Shiga terial toxins and all viruses must gain entry to host cells, a toxin, and Pseudomonas aeruginosa exotoxin A (ExoA) into host process that exploits the host’s own cellular machinery. In this cells have been identified (3–5). These small molecules exhibit study, we use high-throughput technologies to screen for varied mechanisms of action, including blockade of retrograde chemical inhibitors of bacterial toxin and viral entry. We report toxin trafficking at the early endosome–trans Golgi network (TGN) the discovery of a small molecule that inhibits several viruses junction, morphological disruption of the Golgi apparatus, and and bacterial toxins. In addition to the therapeutic potential, inhibition of the toxin active site. Small molecules that disrupt toxin this compound represents a powerful probe for dissecting the binding, entry, trafficking, and host response can serve not only as mechanisms of mammalian membrane trafficking processes. probes to dissect such eukaryotic cellular pathways, but also are potential therapeutics for infectious and genetic diseases. Author contributions: E.J.G., C.-L.C.H., K.B., D.L.C., G.D., Y.E.W., H.J.E., B.T., S.D.D., S.R.B., Bacillus anthracis, the causative agent of the disease anthrax, G.C., D.G.B., M.E.J., J.C., R.D., and K.A.B. designed research; E.J.G., C.-L.C.H., K.B., D.L.C., G.D., Y.E.W., H.J.E., B.T., A.G., S.D.D., B.F., B.T.C., and R.D. performed research; Y.E.W., B.T.C., secretes binary toxins that enter host cells and disrupt physio- G.C., J.C.d.l.T., and D.G.B. contributed new reagents/analytic tools; E.J.G., C.-L.C.H., K.B., 2+ logical processes. Lethal factor (LF) is a Zn -dependent met- D.L.C., G.D., Y.E.W., H.J.E., B.T., S.D.D., S.R.B., M.E.J., J.C., R.D., and K.A.B. analyzed data; alloprotease that cleaves mitogen-activated protein kinase and E.J.G. and K.A.B. wrote the paper. kinases (MAPKKs) 1–4, 6, and 7 (6, 7) and Nlrp1b (8–10) and The authors declare no conflict of interest. reproduces many pathologies of anthrax when injected into This article is a PNAS Direct Submission. laboratory animals (11, 12). The cellular entry of LF is de- 1To whom correspondence should be addressed. E-mail: [email protected]. pendent on a cell-binding and translocation subunit known as This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. protective antigen (PA). PA is an 83-kDa protein that is cleaved 1073/pnas.1302334110/-/DCSupplemental. E4904–E4912 | PNAS | Published online November 4, 2013 www.pnas.org/cgi/doi/10.1073/pnas.1302334110 Downloaded by guest on September 28, 2021 assay using an immortalized macrophage cell line that undergoes To determine if the effect of EGA extended beyond established PNAS PLUS a rapid cell death known as pyroptosis in response to anthrax LT. cell lines, primary bone marrow-derived macrophages (BMDMs) Pyroptosis is a proinflammatory, caspase-1–dependent cell death from mice with an LT-sensitive Nlrp1b transgene were intoxi- that occurs in response to LT in macrophages and dendritic cells cated in the presence or absence of EGA. Whereas BMDMs encoding certain alleles of the inflammasome gene Nlrp1b (22). treated with DMSO vehicle were killed efficientlybyLT,BMDMs This rapid cytolytic response occurs within 2–3 h of toxin addi- treated with EGA were protected (Fig. 1D). tion and provides a convenient assay for toxin entry. A total of NMR and liquid chromatography–mass spectrometry (LC-MS) 30,000 small molecules from a commercially available compound analysis confirmed purity and structure of commercial EGA (Fig. library were screened for their ability to inhibit LT-mediated S2 A and B), reducing the possibility that the activity of EGA is cytotoxicity. Hits were defined based on percentage of survival due to a contaminant from its synthesis and/or from a breakdown relative to untreated controls. All compounds that yielded sur- product. Resynthesis of EGA yielded similar activity as that ob- vival greater than 7% (∼0.1% hit rate) were selected for initial tained from the commercial source (Fig. S2C), further validating revalidation. Thirty-seven initial hits were picked from the the identity of the compound. As an initial investigation of the source library, assembled onto a single master plate, and retested structure–activity relationship, a series of related compounds from for protection in the LT macrophage cytotoxicity assay. Com- Chembridge was assayed. Interestingly, none of the commercially pounds that increased survival at least three SDs above controls available EGA analogs were active (Fig. 2 and Fig. S3). These treated with LT and vehicle were considered verified. Thirty-two compounds revealed critical roles for four-position substitution compounds exhibited activity in validation assays, whereas five on the benzaldehyde, as well as 2,6 substitutions on the aniline failed to reconfirm. Of the 32 confirmed hits, fresh powder stocks ring. Furthermore, the semicarbazone linker appeared to con- were ordered for 8 compounds, including the 5 that displayed the tribute to compound activity.
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