Bimodal Modulation of the Botulinum Neurotoxin Protein-Conducting Channel
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Bimodal modulation of the botulinum neurotoxin protein-conducting channel Audrey Fischera,1, Yuya Nakaib,1, Lisa M. Eubanksb, Colin M. Clancyc, William H. Teppc, Sabine Pellettc, Tobin J. Dickersonb, Eric A. Johnsonc, Kim D. Jandab,2, and Mauricio Montala,2 aSection of Neurobiology, Division of Biological Sciences, University of California San Diego, La Jolla, CA 92093; bDepartments of Chemistry and Immunology, The Skaggs Institute for Chemical Biology, and Worm Institute for Research and Medicine, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037; and cFood Research Institute, University of Wisconsin, 1925 Willow Drive, Madison, WI 53706 Communicated by Sydney Brenner, The Salk Institute for Biological Studies, La Jolla, CA, December 16, 2008 (received for review November 17, 2008) Clostridium botulinum neurotoxin (BoNT) is the causative agent of internalized toxin across intracellular membranes to reach its botulism, a neuroparalytic disease. We describe here a semisyn- cytosolic targets (7, 8), and that the structure of toosendanin thetic strategy to identify inhibitors based on toosendanin, a would likely preclude it from inhibiting the BoNT/A LC metal- traditional Chinese medicine reported to protect from BoNT intox- loprotease (13) nor BoNT binding to cells (11) suggests that ication. Using a single molecule assay of BoNT serotypes A and E toosendanin could operate by hindering LC translocation out of light chain (LC) translocation through the heavy chain (HC) channel the endosome. Herein, we report that the mechanism of action in neurons, we discovered that toosendanin and its tetrahydrofu- of toosendanin stems from its ability to alter LC translocation ran analog selectively arrest the LC translocation step of intoxica- through the HC channel/chaperone; addition of toosendanin at tion with subnanomolar potency, and increase the unoccluded HC the onset of LC translocation arrests this process, whereas channel propensity to open with micromolar efficacy. The inhibi- exposure to toosendanin after completion of cargo translocation tory profile on LC translocation is accurately recapitulated in 2 modulates HC channel activity, increasing the time the channel different BoNT intoxication assays, namely the mouse protection resides in the open state. These findings disclose an unprece- and the primary rat spinal cord cell assays. Toosendanin has an dented bimodal action of a small molecule on a transmembrane unprecedented dual mode of action on the protein-conducting chaperone: cargo-dependent inhibitor of translocation and car- channel acting as a cargo-dependent inhibitor of translocation and go-free channel activator. as cargo-free channel activator. These results imply that the bi- modal modulation by toosendanin depends on the dynamic inter- Results actions between channel and cargo, highlighting their tight inter- Semisynthetic Analysis and Analog Preparation. Based on first play during the progression of LC transit across endosomes. principles, it was not clear how to dissect the complex molecular architecture of toosendanin and elucidate the mechanistic na- natural product ͉ protein translocation ͉ small molecule modulator ture of its antibotulinal properties. Hence, we turned to semi- synthesis to generate a set of rationally designed toosendanin he BoNTs comprise a family of 7 immunologically distinct analogs. Examination of the molecular architecture of toosen- Tproteins synthesized by strains of anaerobic bacteria. These danin reveals 3 key functionalities that could possibly contribute toxins (BoNT/A-G) are the most lethal poisons known, with to the observed biological activity: (i) the hemiacetal bridge BoNT serotype A (BoNT/A) having a LD50 for a 70-kg human spanning C10 and C4 of the core ABCD fused ring system; (ii) of a mere 0.7–0.9 g by inhalation (1). All BoNT serotypes are the heterocyclic furan attached to C17; and (iii) the epoxide at disulfide-linked di-chain proteins consisting of a 50-kDa light C14 and C15 (Fig. 1). Each of these moieties is rare in charac- chain (LC) Zn2ϩ-metalloprotease and a 100-kDa heavy chain terized steroidal structures, with the latter two being common (HC). The HC encompasses the translocation domain (TD) and only to the limonoid family of triterpenoids. Trapping of the the receptor-binding domain (RBD), conventionally denoted as hemiacetal was accomplished by treatment of the parent com- HN and HC (2, 3). The conspicuously specific activity of BoNT pound with N-methylmorpholine N-oxide (NMO) and catalytic to selectively disable synaptic vesicle exocytosis has transformed amount of tetra-n-propylammonium perruthenate (TPAP) (14) this protein into the first bacterial toxin approved by the FDA for to oxidize all alcohols, thus disallowing the open hemiacetal and treatment of a number of diseases characterized by abnormal generating lactone analog 2 (Fig. 1). Heterocyclic furan func- muscle contraction, a blockbuster cosmeceutical, and a highly tionalities are generally considered toxic to mammalian species feared bioweapon (1, 4, 5). Functionally, these clostridial toxins (15). To specifically remove the furan, toosendanin was treated inhibit the release of acetylcholine at neuromuscular junctions with palladium on alumina and hydrogenated using standard through a multistep mechanism that ultimately culminates in the conditions to give tetrahydrofuran analog 3 (THF-toosendanin) cleavage of Soluble N-ethylmaleimide-sensitive fusion protein (Fig. 1). Epoxide moieties are uncommon in natural products in attachment protein receptor proteins, resulting in progressive light of the ensuing high reactivity, and in the case of toosen- flaccid paralysis (6–8). danin, this reactivity should be significantly enhanced due the The major limonoid constituent of the bark of the tree M. toosendan, the triterpenoid toosendanin 1 (Fig. 1), has been reported to possess activities ranging from ascarifuge to anti- Author contributions: A.F., Y.N., L.M.E., W.H.T., S.P., E.A.J., K.D.J., and M.M. designed research; A.F., Y.N., L.M.E., C.M.C., W.H.T., S.P., K.D.J., and M.M. performed research; A.F., botulinum (9–12). Specifically, studies conducted over twenty K.D.J., and M.M. contributed new reagents/analytic tools; A.F., Y.N., L.M.E., W.H.T., S.P., years ago purported that toosendanin could protect monkeys T.J.D., E.A.J., K.D.J., and M.M. analyzed data; and A.F., L.M.E., S.P., T.J.D., E.A.J., K.D.J., and from BoNT/A, BoNT/B, and BoNT/E-induced death in a dose- M.M. wrote the paper. dependent fashion when coadministered with, or several hours The authors declare no conflict of interest. after, neurotoxin administration (9–12). These reports, although 1A.F. and Y.N. contributed equally to this work. mechanistically unclear, were still intriguing in that toosendanin 2To whom correspondence may be addressed. E-mail: [email protected] or mmontal@ucsd. might provide the basis of an unmet challenge, a small molecule This article contains supporting information online at www.pnas.org/cgi/content/full/ therapeutic for the treatment of botulinum intoxication. Given 0812839106/DCSupplemental. that a primary event for intoxication is the translocation of © 2009 by The National Academy of Sciences of the USA 1330–1335 ͉ PNAS ͉ February 3, 2009 ͉ vol. 106 ͉ no. 5 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0812839106 Downloaded by guest on September 30, 2021 Fig. 1. Semisynthetic preparation of toosendanin analogs. Synthetic mod- ifications were chosen to selectively remove the desired functional group without alteration of the remainder of the molecule. unfavorable ring strain imparted by the [6,5] CD fused ring system. However, this ring strain can also be exploited to drive a [1,2]-rearrangement and the subsequent formation of a ther- modynamically more stable ketone 4 (Fig. 1). In addition to probing these rare structural features, toosendanin also pos- Fig. 2. In vivo and in vitro tests of toosendanin activity. (A) Toosendanin and sesses other functional groups anticipated to have low in vivo THF-toosendanin extend the time to death of mice challenged with lethal doses of BoNT/A. In all groups, animals (n ϭ 10) were administered the desired stability. In particular, the acetate functionalities at C3 and C12 toosendanin analog (2.5 mM, 0.1 mL, i.v.) immediately followed by BoNT would be labile and could be rapidly hydrolyzed by serum Ͻ challenge (5LD50, i.p.). *, P 0.001 compared with toxin-only control. (B) esterases, including butyrylcholinesterase and liver carboxyles- Western blot analysis of SNAP-25 cleavage by BoNT/A in primary rat spinal cord terases (16). To mimic this reaction, the two acetates were cells treated with the indicated concentrations of toosendanin and 250 pg (5.6 excised without impinging upon the other functionalities under pM) of BoNT/A. (C) Quantitative depiction of inhibition of BoNT/A activity by BIOPHYSICS hydrolysis conditions optimized to selectively hydrolyze the toosendanin in the primary rat spinal cord cells assay. Bands corresponding to Ͻ desired esters without altering other base-labile sites to yield uncleaved and cleaved SNAP-25 were quantified by densitometry. *, P 0.05 deacylated toosendanin analog 5 (Fig. 1). compared with toxin-only control. In Vivo Testing of Toosendanin Analogs. The previously reported toosendanin (TSDN) results in gradual preservation of intact, antibotulinal activity of toosendanin was confirmed in a mouse uncleaved SNAP-25 (synaptosomal-associated protein with lethality bioassay for BoNT/A intoxication. This model has been Mr ϭ 25 kDa), the intracellular BoNT/A and BoNT/E substrate, CHEMISTRY well-established as the FDA standard for assessing BoNT po- becoming practically complete above 200 nM (Fig. 2 B and C); tency and is widely used in the study of BoNT antagonists (17). the Effective Dose to achieve 50% of the response (ED ) for Ϸ 50 Toosendanin extended the time to death (TTD) 4-fold from a BoNT/A was 55 Ϯ 2.7 nM. Similar studies conducted with single injection at the highest toosendanin dose (7.1 h in control BoNT/E demonstrated nearly complete SNAP-25 protection at Ͼ animals versus 28 h TTD, using 2.5 mM toosendanin– 1 M(Fig.