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Anthrax Lethal Factor Inhibition Anthrax lethal factor inhibition W. L. Shoop*†, Y. Xiong*, J. Wiltsie*, A. Woods*, J. Guo*, J. V. Pivnichny*, T. Felcetto*, B. F. Michael*, A. Bansal*, R. T. Cummings*, B. R. Cunningham*, A. M. Friedlander‡, C. M. Douglas*, S. B. Patel*, D. Wisniewski*, G. Scapin*, S. P. Salowe*, D. M. Zaller*, K. T. Chapman*, E. M. Scolnick§, D. M. Schmatz*, K. Bartizal*, M. MacCoss*, and J. D. Hermes* *Merck Research Laboratories, Rahway, NJ 07065; ‡United States Army Medical Research Institute of Infectious Diseases, Frederick, MD 21702; and §Department of Biology, Massachusetts institute of Technology, Cambridge, MA 02139 Communicated by William C. Campbell, Drew University, Madison, NJ, April 12, 2005 (received for review November 5, 2004) The primary virulence factor of Bacillus anthracis is a secreted phylactically if intentional release of anthrax were suspected) or, zinc-dependent metalloprotease toxin known as lethal factor (LF) more probably, an LFI would be used to block late stage effects that is lethal to the host through disruption of signaling pathways, of LF during an active infection and increase the probability of cell destruction, and circulatory shock. Inhibition of this proteolyt- host survival. This latter aspect would unquestionably be used in ic-based LF toxemia could be expected to provide therapeutic value adjunct therapy with an antibiotic. Herein, we reveal the crystal in combination with an antibiotic during and immediately after an structure of a hydroxamate LFI and its intimate interaction with active anthrax infection. Herein is shown the crystal structure of an LF and present a sequence of in vitro and in vivo studies, intimate complex between a hydroxamate, (2R)-2-[(4-fluoro-3- including those with active B. anthracis Ames strain infections, methylphenyl)sulfonylamino]-N-hydroxy-2-(tetrahydro-2H-pyran- that indicate this interaction has dramatic protective benefits. 4-yl)acetamide, and LF at the LF-active site. Most importantly, this molecular interaction between the hydroxamate and the LF active Methods site resulted in (i) inhibited LF protease activity in an enzyme assay LFI and Recombinant Toxins. The hydroxamate LFI, (2R)-2-[(4- and protected macrophages against recombinant LF and protective fluoro-3-methylphenyl)sulfonylamino]-N-hydroxy-2-(tetrahy- antigen in a cell-based assay, (ii) 100% protection in a lethal mouse dro-2H-pyran-4-yl)acetamide, was used in all studies herein and toxemia model against recombinant LF and protective antigen, (iii) was synthesized at Merck Research Laboratories (Rahway, NJ). Ϸ50% survival advantage to mice given a lethal challenge of B. Recombinant LF was purified from Escherichia coli (R. J. anthracis Sterne vegetative cells and to rabbits given a lethal Collier, Harvard Medical School, Cambridge, MA) and com- challenge of B. anthracis Ames spores and doubled the mean time pared with LF isolated from B. anthracis (S. Leppla, National to death in those that died in both species, and (iv) 100% protection Institutes of Health, Bethesda, MD). LFI showed identical against B. anthracis spore challenge when used in combination inhibition versus LF isolated from either source. Recombinant therapy with ciprofloxacin in a rabbit ‘‘point of no return’’ model protective antigen (PA) was purified from E. coli (R. J. Collier). for which ciprofloxacin alone provided 50% protection. These results indicate that a small molecule, hydroxamate LF inhibitor, as N-Terminally Truncated LF. Forward and reverse PCR primers revealed herein, can ameliorate the toxemia characteristic of an (5ЈGGATCCAGGCATGCTGTCAAGATATGAAAAAT- active B. anthracis infection and could be a vital adjunct to our GGGAAAAG-3Ј and 5Ј-GGATCCTTGCTGCCGCGGG- ability to combat anthrax. GCACCAGTGAGTTAATAATGAACTTAATCTGA-3Ј, re- spectively) were designed to remove a stop codon, add a 3Ј Bacillus anthracis ͉ hydroxamate thrombin site, and add BamHI restriction sites to the DNA sequence encoding amino acids 264–776 of LF. The PCR product was amplified from pET15b-LF (10) and cloned into acillus anthracis, the etiological agent of anthrax, has been pET23ϩ (Novagen). To add a GST tag to the LF C-terminal developed as a bioweapon by countries and terrorists largely B coding region, a double-stranded adapter formed by annealing because of a combination of the spore’s durability and the lethal two oligonucleotides (5Ј-GATCTAAGGATCCGC-3Ј and 5Ј- toxemia of the vegetative stage. This Gram-positive bacterium GGCCGCGGATCCTTA-3Ј) was inserted between the BamHI forms spores resistant to adverse environmental conditions and and NotI sites of vector pGEX-4T-3 (Amersham Pharmacia), can survive for decades in pastures (1). If ingested or inhaled, and the resulting vector was linearized with BamHI before the even in small numbers, the spores germinate to establish explo- LF BamHI fragment from the pET23ϩ construct was inserted. sive vegetative growth and a resulting toxemia that is usually fatal This plasmid encodes a GST–LF (264–776) fusion protein with to the host (2–4). The primary virulence factor is a secreted thrombin cleavage sites at the GST–LF junction and the LF C zinc-dependent metalloprotease toxin known as lethal factor terminus. (LF), which is lethal to the host through disruption of signaling pathways, cell destruction, and circulatory shock. The only X-Ray Crystallography. Crystals of the truncated LF:LFI complex existing therapeutic intervention for naturally acquired or wea- were obtained by the vapor diffusion method in hanging drops ponized anthrax is antibiotic treatment that must be given early with 20–22% polyethylene glycol 8000͞100 mM Mg(OAc)2͞100 after infection and at a time when victims may experience only mM sodium cacodylate, pH 6.8, as precipitant. Crystals were mild flu-like symptoms (5–9). Delay of treatment, even by hours, orthorhombic, with unit cell parameters a ϭ 57.3 Å, b ϭ 75.96 substantially reduces survival of infected patients (1, 5). To date, Å, and c ϭ 139.0 Å. Data were collected on an ADSC Q210 physicians have antibiotic options to eliminate an anthrax infec- tion, but they have no therapeutic options to combat the LF-mediated toxemia and tissue destruction during an ongoing Freely available online through the PNAS open access option. infection or the residual toxemia that persists even after the Abbreviations: LF, lethal factor; LFI, LF inhibitor; PA, protective antigen; DB, Dutch-belted; bacteria have been eliminated by antibiotics. t.i.d., three times a day; b.i.d., two times a day; MTD, mean time to death. It is envisaged that, depending on how and when administered, Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, either an LF inhibitor (LFI) could block the proteolytic protec- www.pdb.org (PDB ID code 1YQY). tion provided by LF in the macrophage and allow that cell to †To whom correspondence should be addressed. E-mail: wesley࿝[email protected]. eliminate spores early in infection (which could be used pro- © 2005 by The National Academy of Sciences of the USA 7958–7963 ͉ PNAS ͉ May 31, 2005 ͉ vol. 102 ͉ no. 22 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0502159102 Downloaded by guest on September 26, 2021 charge-coupled device detector at beamline 17-ID in the facil- saline at 250 ␮g⅐100 ␮lϪ1⅐hϪ1. The mice were challenged i.p. with ities of the Industrial Macromolecular Crystallography Associ- 108 colony-forming units of B. anthracis Sterne strain 24 h after ation-Collaborative Access Team at the Advanced Photon infusion began, and continuous infusion of LFI or saline was Source (Argonne National Laboratory, Argonne, IL) from a maintained for 9 days. crystal that was flash frozen in a liquid nitrogen stream. The cryoprotectant was 25% ethylene glycol in mother liquor. Data Monotherapeutic LFI Protection of Rabbits Infected with B. anthracis were processed with HKL-2000 (11). The resulting data set was Ames Spores. Dutch-belted (DB) rabbits (weight, 2 kg; age, 16 wk) 98.9% complete and 7-fold redundant to 2.3 Å, with an average were purchased from Covance (Princeton, NJ) and B. anthracis I͞␴I of 10.8. The structure was solved by molecular replacement Ames strain spores were obtained from R. Lyons (University of with MOLREP (12) and the coordinates 1J7N.pdb (Protein Data New Mexico, Albuquerque). The initial efficacy test consisted of Bank ID code 1J7N). The refinement was conducted by alter- six DB rabbits dosed s.c. with LFI at 100 mg͞kg t.i.d. in saline for nating computer-based refinement (13) and manual rebuilding 7 days and six rabbits dosed s.c. with saline at the same times. of the model in O (14). The final model had a crystallographic Two hours after the first dose, all rabbits were challenged s.c. ϭ R factor of 19.1% (Rfree 26.9%) and good geometry (rms with 104 Ames spores and observed for 21 days. deviations for bond length and bond angles were 0.011 Å and A second study was conducted to confirm and extend the 1.24°, respectively). The coordinates were deposited in the previous monotherapy finding in the DB rabbit. As in the first Protein Data Bank under ID code 1YQY. trial, a s.c. injection of 104 spores of B. anthracis Ames was used to challenge 12 DB rabbits. LFI monotherapy was delivered at LF Protease Enzyme and Macrophage Cytotoxicity Assays. LF pro- 100 mg͞kg s.c. t.i.d. in saline for 7 days starting at the time of tease activity was determined with a fluorogenic peptide slightly spore challenge to the first group (n ϭ 4) and for 6 days starting modified from earlier work (ref. 10; see also Supporting Materials 24 h after challenge to a second group (n ϭ 4). A third group and Methods, which is published as supporting information on served as a saline-treated control (n ϭ 4). the PNAS web site). Murine J774A.1 macrophage cells (Amer- ican Type Culture Collection) were used in the cytotoxicity Ciprofloxacin and LFI Combination Therapy of Rabbits Challenged assays.
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