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 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; 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 and LFI Combination Therapy of Rabbits Challenged assays. J774A.1 cells were cultured in Dulbecco’s modified with B. anthracis Ames Spores. Ciprofloxacin (Cipro) is a first-line Eagle’s medium (Invitrogen) supplemented with 10% FCS and drug in anthrax therapy, and a ‘‘point of no return’’ model was incubated with LFIs for1hat37°C. LF and PA were then added ͞ ͞ developed in DB rabbits to evaluate whether combination of to make a final concentration of 15 ng ml and 250 ng ml, Cipro with LFI would provide additional useful therapy beyond respectively. After4hofincubation at 37°C, 3-(4,5- what Cipro would do alone. Probe studies with Cipro mono- dimethylthiazol-2-yl)-5–3-carboxymethoxyphenyl)-2-(4-sulfo- therapy at twice daily (b.i.d.) 5 mg͞kg s.c. administration to phenyl)-2H-tetrazolium (Promega) was added, and, after an rabbit groups (n ϭ 4) challenged with 104 spores of B. anthracis additional 2-h incubation at 37°C, the OD was measured. 490 Ames were made stepwise starting at 24 h after challenge and continued through 36, 48, 54, 60, and 66 h after challenge until Mouse Toxemia Model with Recombinant PA and LF. BALB͞c mice the point at which 50% of the Cipro-treated rabbits died was (weight, 22 g; age, 8 wk) were purchased from Taconic Farms. determined. Once that determination was made, a test was done For the definitive toxemia test, 32 mice were allocated at random in 11 DB rabbits to evaluate whether the LFI and Cipro MEDICAL SCIENCES to one of four groups and each of the four groups was then combination would provide additional protection beyond Cipro allocated at random to treatment with LFI at 0, 1, 10, or 30 4 mg͞kg three times a day (t.i.d.) (Ϫ0.25, ϩ1, and ϩ 3 h relative alone. All rabbits were challenged s.c. with 10 Ames spores at to administration of LF and PA), with saline as the vehicle. At time 0. At the 50% point of no return, the surviving rabbits were time 0, all mice were coinjected i.v. with 100 ␮g of recombinant divided into two equal experimental groups of four rabbits each, LF and 100 ␮g of recombinant PA in a 150-␮l saline mixture into and the remainder was allocated to a third group given saline the mouse tail vein. only. Of the two experimental groups, the first received Cipro monotherapy at 5 mg͞kg s.c. b.i.d. for 2 days starting at the 50% Monotherapeutic LFI Protection of Mice Infected with B. anthracis point of no return, and the second group received combination Sterne Strain Vegetative Cells. Jugular vein-cannulated BALB͞c therapy with Cipro by following the same protocol as group 1 on ͞ mice (weight, 22 g; age, 8 wk) were purchased from Taconic one side of the body and LFI at 100 mg kg s.c. four times a day Farms and used in protection trials versus B. anthracis Sterne for 1 day also starting at the 50% point of no return on the other vegetative cells. A Harvard Apparatus pump-controlled syringe side of the body. continuously infused a precise rate of LFI or saline through 25-gauge polyethylene tubing to each mouse by means of a Animal Welfare and Husbandry. All animal experiments and hous- counterbalanced lever arm and swivel (Instech Solomon, Ply- ing were approved and conducted according to guidelines from mouth Meeting, PA). Merck’s Institutional Animal Care and Use Committee. The acapsular B. anthracis Sterne strain was obtained from Hank Heine (United States Army Medical Research Institute of Results Infectious Diseases, Fort Detrick, MD) and maintained at LF Protease Enzyme and the Macrophage Toxicity Assays. The LF Ϫ70°C. The frozen material was thawed and a loop of material protease enzyme and the macrophage toxicity assays were used was streaked on a sheep red-blood agar plate and incubated for in tandem to identify weak hydroxamate leads from the Merck 18 h at 35°C. Immediately before challenge, the 18-h colonies sample collection and then to guide a medicinal chemistry effort were suspended in saline, vortex mixed vigorously, and diluted to that yielded a potent hydroxamate LFI (Fig. 6A Inset, which is achieve an inoculum previously shown to give 108 colony- published as supporting information on the PNAS web site). LFI forming units per 0.2 ml. To verify the inoculum, 100 ␮l from is a time-dependent, reversible inhibitor of LF protease activity each of the dilution tubes was cultured on sheep red-blood agar with an IC50 value of 60 nM. The kinetic mechanism of inhibition plates, and the colonies were counted 24 h later. for LFI was determined to be competitive with substrate, with a Two mouse tests evaluating the monotherapy of LFI against B. Ki value of 24 nM (Fig. 7, which is published as supporting anthracis Sterne strain vegetative cells were conducted. Each information on the PNAS web site). LFI also blocked LF- consisted of one group of 10 BALB͞c mice infused with saline induced cytotoxicity in mouse macrophage J774A.1 cells with an at 100 ␮l͞h and a second group of 10 mice infused with LFI in IC50 of 160 nM (Fig. 6B).

Shoop et al. PNAS ͉ May 31, 2005 ͉ vol. 102 ͉ no. 22 ͉ 7959 Downloaded by guest on September 26, 2021 Table 1. LFI protection of mice from recombinant LF- and PA-mediated death Dose, Survivors Survival Compound mg͞kg t.i.d. n after 48 h rate, %

LFI 30 8 8 100 LFI 10 8 7 87.5 LFI 1 8 1 12.5 None 0 8 0 0

A mixture of 100 ␮g of recombinant LF and 100 ␮g of recombinant PA was injected into the tail vein of BALB͞c mice. LFI in saline was administered at various doses i.p. 15 min before and 1 and 3 h after intoxication. Neither LF nor PA was toxic alone.

hydrophobic pocket adjacent to the catalytic center (Fig. 1B). From the synthesis and testing of Ͼ500 inhibitor analogs, the 4-fluoro-3-methylphenyl group appeared to optimally fill the hydrophobic pocket identified on LF. The tetrahydropyran moiety of LFI was positioned in the large cavity between domains III and IV, but made limited interactions with the protein. Thus, this modification would have little consequence on LF affinity.

Mouse Toxemia Model Using Recombinant PA and LF. LFI was tested for its ability to protect BALB͞c mice from a lethal mixture of recombinant PA and LF. Probe studies showed that i.v. coin- ␮ ␮ ␣ jection of 100 g of recombinant LF and 100 g of recombinant Fig. 1. Crystal structure of LFI bound to LF. (A) Overlay of a C trace of ͞ full-length apo LF (Protein Data Bank ID code 1J7N, blue) and the truncated PA in saline into the tail vein of BALB c mice led to 100% (264–776) version (green) used in this study. The domains are numbered in red. mortality within 48 h and was chosen as the standard toxin LFI and the zinc ion are represented as ball-and-stick models (yellow, carbon; challenge. To identify a dosage regime that would maintain green, sulfur; red, oxygen; blue, nitrogen; cyan, fluorine; and gray, zinc; the trough micromolar blood levels for several hours, a range-finding same coloring scheme is used in all three models). Despite the absence of pharmacokinetic analysis was undertaken. LFI was administered domain I in our construct, the overall structure of domains II–IV is conserved. in saline to mice i.p. at 1, 10, or 30 mg͞kg t.i.d. (0, ϩ1.25, and (B) Molecular surface of LF around the inhibitor binding site. The surface is ϩ 3.25 h), and sufficient ranges of trough levels of 0.2–0.3, colored according to potential (red, negative; blue, positive). The S1Ј site is 1.1–2.4, or 1.9–3.1 ␮M, respectively, and cmax levels of 1.6–2.2, labeled. (C) Stereoview of LFI bound to LF. Residues within6Åofthebound 12.3–13.4, or 20.0–29.9 ␮M, respectively, were obtained. ligand are displayed. Water molecules are omitted for clarity. The electron Ϫ Therefore, LFI was examined in the toxemia model by i.p. density surrounding the inhibitor is from a 2Fo Fc difference map contoured ͞ Ϫ ϩ ϩ at 1.5 s. The orientation is similar to that of A. administration at 0, 1, 10, or 30 mg kg t.i.d. ( 0.25, 1, and 3 h relative to toxin administration) in saline. After a 48-h evaluation, none of the eight mice survived in the group receiv- Crystal Structure of LF in Complex with LFI. In parallel with the ing saline vehicle, whereas one of eight (12.5%), seven of eight enzymatic and cell-based assays, the 3D structures of LF com- (87.5%), and eight of eight (100%) mice survived in the 1, 10, or plexed with various hydroxamate-based compounds were deter- 30 mg͞kg t.i.d. LFI-treated groups, respectively (Table 1). mined by x-ray crystallography and used to design inhibitors with Although seven of eight mice survived in the 10 mg͞kg t.i.d. improved potency and solubility. The N-terminally truncated group, clinical signs typical of anthrax infection, such as ruffled ͞ form of LF, composed of residues 264–776, had identical hair and huddling, were evident, whereas mice in the 30 mg kg protease activity compared with full-length LF, contained the t.i.d. group appeared normal throughout the trial. entire protease active site with all inhibitor contact points, and yielded much better diffracting crystals than full-length LF. Monotherapeutic LFI Protection of Mice Infected with B. anthracis Although the truncated form of LF lacked PA-binding domain Sterne Strain Vegetative Cells. The follow-up to the toxemia model I, the overall architecture of domains II–IV was maintained (Fig. was to examine the ability of LFI to protect mice from an active B. anthracis infection. Probe studies showed that an injection of 1A). The major differences between the unliganded and inhib- 108 B. anthracis Sterne vegetative cells into the peritoneal cavity ited structures were the overall position of domain III and the resulted in Ͼ90% mortality of BALB͞c mice within 2–3 days. conformation of the loops spanning residues 427–437, 645–651, The time course for LF production in mouse blood was similar and 673–680. Domain III moved in a rigid body motion and in infections with either Sterne or Ames strains in our laboratory. appeared to be coupled to the movement of the 673–680 loop, For both anthrax strains in mice, maximum plasma levels of Ϸ by 30° upon inhibitor binding. The movements of loops 427– 10–15 nM LF as determined by LF protease activity were 437 and 645–651 were probably related to different crystal reached Ϸ12 h before death of the host. packing of the two forms of the enzyme rather than a direct Individual injections of LFI that would maintain micromolar consequence of inhibitor binding. trough levels for multiple days against bacterially generated LFI bound in the groove at the interface of LF domains III and toxins were eliminated from consideration because of a terminal IV (catalytic domain; Fig. 1A). The oxygen atoms (ϪCO- 0.4-h half-life of the drug in mice. Probe pharmacokinetic ϩ NHOH) of the hydroxamate chelated the Zn2 ion in a biden- studies, however, showed that mice continuously infused with tate, planar conformation; the other ligands to Zn2ϩ were LFI by means of a jugular vein catheter at 250 ␮g⅐100 ␮lϪ1⅐hϪ1 His-686, His-680, and Asp-735. The substituted phenyl ring of in saline maintained LFI plasma levels of 8–10 ␮M. LFI (corresponding to the P1Ј position) was bound in a deep In the first evaluation of LFI in this model, 10 jugular-vein-

7960 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0502159102 Shoop et al. Downloaded by guest on September 26, 2021 Fig. 2. LFI protection of BALB͞c mice infected with B. anthracis Sterne strain Fig. 3. LFI protection of rabbits infected with B. anthracis Ames spores. DB vegetative cells. Intravenous continuous infusion of LFI at a rate of 250 mg⅐100 rabbits were dosed s.c. with LFI at 100 mg͞kg t.i.d. (Œ)(n ϭ 6) or saline vehicle ␮lϪ1⅐hϪ1 (circles) or saline at 100 ␮l͞h (squares) was started 1 day before (E)(n ϭ 6) starting 2 h before (arrow) infection with 104 B. anthracis Ames (arrow) infection of groups of 10 mice with 108 cells of B. anthracis Sterne spores (day 0). Dosing was discontinued on day 7. Surviving rabbits were strain (day 0). Open and filled symbols represent two independent studies. monitored to day 21. One LFI-treated rabbit died on day 7 while its blood was Inhibitor dosing was discontinued on day 9, and the animals were killed. The drawn. The two survival curves are statistically different (P ϭ 0.013). proportion of mice surviving when dosed with LFI is significantly greater than those on vehicle alone (P ϭ 0.0019). died while obtaining a blood sample at the end of the dosing period, but the three remaining rabbits survived and appeared cannulated BALB͞c mice were continuously infused with saline completely normal at the end of the experiment, day 19, when at 100 ␮l͞h, and 10 were similarly infused with LFI at 250 ␮g⅐100 they were killed. Ϫ Ϫ ␮l 1⅐h 1. All 20 mice were challenged with 108 Sterne vegetative To confirm and extend the previous monotherapy finding in cells 24 h after the start of the infusions. Three days after the rabbit, a second study was conducted. As in the first trial, a challenge, all 10 mice infused with saline developed clinical signs s.c. injection of 104 spores of B. anthracis Ames was used to of anthrax and died. A mean time to death (MTD) of 65 h was challenge 12 DB rabbits. LFI monotherapy was delivered at 100 calculated for the 10 saline-infused mice, compared with 91 h for mg͞kg s.c. t.i.d. for 7 days starting at the time of spore challenge the five mice that died in the LFI-infused group. Importantly, to the first group (n ϭ 4) and for 6 days starting 24 h after five LFI-treated mice survived to the termination of dosing on challenge to a second group (n ϭ 4). A third group served as day 9 (Fig. 2, solid symbols). These five surviving mice under- saline-treated control (n ϭ 4). All saline-treated control rabbits went an anthrax crisis, albeit delayed and reduced relative to the died by 80 h after challenge, with an MTD of 72 h (Fig. 4). The saline-infused mice, from days 5–7 after challenge, but these first rabbit in the LFI monotherapy group dosed at time of MEDICAL SCIENCES mice appeared to resolve the crisis and were normal and healthy challenge did not die until 112 h after challenge, and the second at day 9. A repeat study was conducted by using the same died at 246 h, providing an MTD of 179 h and more than conditions with nearly identical results (Fig. 2, open symbols). doubling the survival time relative to the control group. The two other rabbits in this group that received LFI treatment starting Monotherapeutic LFI Protection of Rabbits Infected with B. anthracis at time of challenge survived for 21 days and were healthy and Ames Spores. Lethality in rabbit models of anthrax using B. normal. anthracis Ames spores is dominated by effects of toxin; therefore, In the second arm of this monotherapy trial, the LFI group the rabbit is accepted as the best nonprimate model for human with 24-h delayed treatment also showed an increase in MTD anthrax disease (15, 16). To determine the challenge inoculum, relative to control (Fig. 4). The first rabbit from this delayed 4 probe studies showed that a s.c. injection of 10 B. anthracis Ames therapy group died at 128 h, and two additional rabbits died at strain spores was Ͼ90% lethal to DB rabbits by 72 h, and that 152 h, giving an MTD of 144 h. Thus, even when LFI treatment inoculum was used in all rabbit studies. To determine dose, pharmacokinetic studies indicated that LFI had a terminal half-life of 2.0 h in the rabbit that necessitated 100 mg͞kg s.c. t.i.d. injections to provide trough blood levels of 3–6 ␮M and cmax levels of 8–12 ␮M (the latter approximated that of the mouse continuous-infusion experiment described above). The initial rabbit efficacy test consisted of 12 DB rabbits: six were dosed s.c. with LFI at 100 mg͞kg t.i.d. for 7 days and six were dosed s.c. with saline at the same times. Two hours after the first dose, all rabbits were challenged s.c. with 104 Ames spores. From 48 to 72 h, five of six saline-treated rabbits underwent an anthrax crisis and died (Fig. 3). Time to death and the number of deaths in the LFI-treated rabbit group were delayed and reduced: one died at 96 h and one other died at 120 h. The MTD for rabbits dying in the saline group was 65 h, whereas for the two LFI-treated rabbits, it was nearly double (111 h). Surviving rabbits in the LFI-treated group underwent a diminished anthrax Fig. 4. LFI protection of rabbits infected with B. anthracis Ames spores. Twelve DB rabbits were challenged with 104 Ames spores s.c. at time 0. Four crisis from day 4 to day 6, but all rabbits appeared normal by day rabbits were given saline s.c. t.i.d. starting at time of challenge for 7 days (E). 7. When dosing ended at day 7, five of the six (83%) saline- Four rabbits were given LFI at 100 mg͞kg s.c. t.i.d. starting at time of challenge treated rabbits were dead compared with only two of the six for 7 days (Œ). Four rabbits were given LFI at 100 mg͞kg s.c. t.i.d. starting 24 (33%) LFI-treated rabbits. One additional LFI-treated rabbit h after time of challenge for 6 days (ᮀ).

Shoop et al. PNAS ͉ May 31, 2005 ͉ vol. 102 ͉ no. 22 ͉ 7961 Downloaded by guest on September 26, 2021 antibiotic. Initial LFI monotherapy experiments in mice against lethal cocktails of recombinant LF and PA showed a distinct dose titration whereby all mice receiving saline died and all mice receiving LFI at 30 mg͞kg i.p. t.i.d. survived. This finding was proof of concept that inhibition of LF by the hydroxamate LFI had potential to lead to useful therapy against anthrax toxemia; however, to be a practical success, an LFI must deal not with a single pulse of LF but with continuous secretion. To test LFI against continuous secretion of LF, a murine model was developed by using the B. anthracis Sterne strain. Probe studies showed that an i.p. inoculum of 108 vegetative colony-forming units were consistently lethal to mice in 72 h and produced a bacteremia and circulating LF level equivalent to a 103 Ames vegetative inoculum that killed in the same time Fig 5. LFI or LFI͞Cipro protection in rabbits infected with B. anthracis Ames period. The initial murine study against the Sterne strain dem- spores when dosing began at 66 h after challenge. Eleven DB rabbits were onstrated that continuous infusion of LFI by itself delayed MTD challenged with 104 Ames spores s.c. at time 0. Three rabbits were given saline in all Sterne-infected mice, suggesting that LFI prophylaxis could only starting at 66 h after challenge (E). Four rabbits were given Cipro at 5 provide valuable additional survival time for sterilizing antibiotic mg͞kg b.i.d. for 48 h starting at 66 h after challenge (■). Four rabbits were ͞ therapy to be used. The second unanticipated benefit of LFI given Cipro as above in combination with LFI at 100 mg kg s.c. four times a day monotherapy was that half of the LFI-infused mice survived. It for 24 h starting 66 h after challenge (Œ). is noteworthy that even the surviving mice underwent a delayed but obvious anthrax crisis from day 5 to day 7 but then resolved was delayed for 24 h, there was still a doubling of MTD relative the crisis and appeared normal at the end of the study. These to vehicle-treated control rabbits. In addition, a single rabbit data were repeated with almost identical results. from this group survived for 21 days and appeared normal at trial Success against B. anthracis Sterne vegetative cells created an end. urgency to evaluate LFI directly against B. anthracis Ames spores, and a rabbit model was chosen for such an evaluation. 4 Cipro and LFI Combination Therapy of Rabbits Challenged with B. Probe efforts showed that 10 B. anthracis Ames spores delivered anthracis Ames Spores. A Cipro point of no return model was s.c. were consistently lethal to rabbits in 72 h. Dose regimens of developed in DB rabbits to evaluate whether combination of LFI that produced blood concentration levels similar to those Cipro with LFI would provide additional useful therapy beyond observed in the Sterne model in mice were targeted. The first of what Cipro would do alone. Probe studies with Cipro mono- these studies was done prophylactically with LFI dosing begin- therapy at 5 mg͞kg s.c. b.i.d. for 2 days showed it to be completely ning 2 h before the s.c. Ames spore challenge. LFI treatment protective to rabbit groups (n ϭ 4) challenged with 104 spores of produced a doubling of MTD, and half the group survived. These B. anthracis Ames when dosing started at 24, 36, 48, or 54 h after surviving rabbits underwent an anthrax crisis through days 5–7 but resolved the crisis by day 9 with apparent, full recovery. challenge. However, one of four rabbits died when Cipro therapy These surviving animals were observed for 21 days before being was delayed to 60 h, and two of four died when therapy was killed. A follow-up study with LFI dosing beginning at the same delayed to 66 h, and this latter time was defined as the 50% point time of the Ames spore challenge confirmed these data in of no return for Cipro. rabbits. Moreover, a separate group in the follow-up rabbit study In the definitive point of no return test, 11 DB rabbits were extended the MTD and survival data to include rabbits in which challenged s.c. with 104 Ames spores at time 0. At 66 h after LFI treatment had been delayed by a critical 24 h beyond spore challenge, the surviving rabbits were divided into two equal challenge. experimental groups of four rabbits each, and the remainder was The final rabbit salvage study in which antibiotic therapy with given saline only. The first experimental groups received Cipro ͞ Cipro was combined with LFI demonstrated the complementary monotherapy at 5 mg kg s.c. b.i.d. for 2 days starting at 66 h, and benefit these two different mechanisms of action offer against the second experimental group received combination therapy anthrax infection. Much probe work with Cipro was done in with Cipro by following the same protocol as the first group on rabbits to identify the 66-h postchallenge time to be the 50% ͞ one side of the body and LFI at 100 mg kg s.c. four times a day point of no return. Once that time point was identified and for one day on the other side. The three rabbits that received dosing began, the Cipro and LFI combination saved all rabbits nothing or saline died by 96 h (Fig. 5), and, when their peritoneal at a point at which Cipro alone saved only half. Not only did the cavities were swabbed and cultured, each was positive for B. Cipro͞LFI combination-treated rabbits survive, but few signs of anthracis Ames. Cipro protected half of the Cipro monotherapy anthrax were observed. It was particularly striking that the two group and extended the MTD of those dying to 144 h. It is Cipro-treated rabbits that died were found to have sterile blood noteworthy that the two rabbits that died in the Cipro mono- and peritoneal cultures. These observations illustrated the pri- therapy group were negative for B. anthracis Ames, indicating mary rationale for a toxin inhibitor, because, although Cipro that the four Cipro treatments given to each rabbit, although successfully eliminated anthrax infection in the rabbits, the sterilizing, did not prevent death. Most importantly, there was rabbits still died. not only complete protection in the combination group receiving Strategically, LF may represent both a prophylactic and ther- Cipro and LFI in survival terms, but few clinical signs of anthrax apeutic target because it has been reported to play a vital role were observed in these rabbits. The six surviving DB rabbits from at two different points in anthrax infection. Intracellularly, LF the two experimental groups were killed on day 11; peritoneal has a protective role for the spore after ingestion by macrophages cultures were made, and all were negative for B. anthracis. (17). Mutant B. anthracis lacking LF are destroyed in macro- phages, indicating that efficient neutralization of LF early in Discussion infection could allow host cells to eliminate the initial insult at The in vivo experiments revealed herein have demonstrated the locus of infection. Extracellularly, where LF is believed to be efficacy of the hydroxamate LFI when given as prophylactic the primary virulence factor, it interferes with signal transduc- monotherapy or in late-stage combination therapy with an tion in host defense cells and destroys the integrity of endothelial

7962 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0502159102 Shoop et al. Downloaded by guest on September 26, 2021 vessels, which leads to circulatory shock and death of the host. sition of anthrax drug resistance by this mechanism, through There is little doubt that the LFI in the studies reported here is either deliberate engineering or bacterial evolution, would ap- expressing an effect in the extracellular phase, which is indicated pear unlikely. in the mouse studies using vegetative cells, because there was no Systemic anthrax, although rare as a natural disease in hu- intracellular phase possible and in the rabbit studies using spores mans, has recently gained substantial notoriety as an agent of in which treatment was delayed 24 h with monotherapy and 66 h and terrorism. Mortality from inhalational with combination therapy. The role of LFI in the intracellular anthrax is very high, even with aggressive antimicrobial therapy. phase remains to be elucidated, but the fact that surviving mice In the attacks after September 11th, 2001, mortality of those with and rabbits prophylactically dosed with LFI went through an- inhalational anthrax was 45% (5͞11) (19). Specific antimicrobial thrax crises and resolved them may hint at an intact immune agents (e.g., Cipro and doxycycline) are very effective against B. response and a block of the early influence that LF has in turning anthracis, but, if given too late, they cannot address the primary off normal immune responses. virulence of anthrax, its systemic toxemia. It is envisaged that The structure-activity relationships observed in the medicinal complementary combination of an antimicrobial mechanism and chemistry program concur with x-ray crystallographic results a LF-inhibiting mechanism as reported here has the potential to showing that the binding of LFI to LF is driven by the contacts provide additional means of therapeutic intervention and sub- between the hydroxamic acid moiety and the 4-fluoro-3- stantially reduce mortality. methylphenyl substituent of the compound with the active site Zn2ϩ and S1Ј pocket of enzyme, respectively. An examination of We thank R. J. Collier and C. T. Walsh (Harvard Medical School, the substrate cleavage sites in naturally occurring mitogen- Cambridge, MA) for hospitality during the early phase of this project and activated protein kinase kinases and synthetic peptides from a R. J. Collier, S. Leppla, H. Heine, and R. Lyons for materials and strains. Ј Use of the Industrial Macromolecular Crystallography Association- large combinatorial library (18) indicates that the P1 position is Collaborative Access Team beamline 17-ID at the Advanced Photon Source important for selectivity. The common protein binding sites for was supported by the Industrial Macromolecular Crystallography Associ- inhibitor and substrate suggest that it may be difficult to find LF ation through a contract with the Illinois Institute of Technology. Use of the mutations that weaken inhibitor binding without a concomitant Advanced Photon Source was supported by the United States Department compromise of natural substrate recognition. Thus, the acqui- of Energy, Office of Science, under Contract W-31-109-Eng-38.

1. Friedlander, A. M. (2000) Curr. Clin Top. Infect. Dis. 20, 335–349. 11. Otwinowski, Z. & Minor, W. (1997) Methods Enzymol. 276, 307–326. 2. Dixon, T. C., Fadl, A. A., Koehler, T. M., Swanson, J. A. & Hanna, P. C. (2000) 12. Vagin, A. & Teplyakov, A. (1997) J. Appl. Cryst. 30, 1022–1025. Cell Microbiol. 2, 453–463. 13. Vonrhein, C. B. (2004) AUTOBUSTER, An Automated System for Macromo- 3. Guidi-Rontani, C. (2002) Trends Microbiol. 10, 405–409. lecular Refinement Using BUSTER-TNT (0.0.7) (Cambridge University, Cam- 4. Hanna, P. C. & Ireland, J. A. (1999) Trends Microbiol. 7, 180–182. bridge, U.K.). 5. Bartlett, J. G., Inglesby, T. V., Jr., & Borio, L. (2002) Clin. Infect. Dis. 35, 14. Jones, T. A. & Kjeldgaard, M. (1997) Methods Enzymol. 277, 173–208. 851–858. 15. Zaucha, G. M., Pitt, L. M., Estep, J., Ivins, B. E. & Friedlander, A. M. (1998) 6. Inglesby, T. V., O’Toole, T., Henderson, D. A., Bartlett, J. G., Ascher, M. S., Arch. Pathol. Lab. Med. 122, 982–992. Eitzen, E., Friedlander, A. M., Gerberding, J., Hauer, J., Hughes, J., et al. 16. Fellows, P. F., Linscott, M. K., Ivins, B. E., Pitt, M. L., Rossi, C. A., Gibbs, P. H. (2002) J. Am. Med. Assoc. 287, 2236–2252. & Friedlander, A. M. (2001) 19, 3241–3247.

7. Meyerhoff, A. & Murphy, D. (2002) J. Am. Med. Assoc. 288, 1848–1849. MEDICAL SCIENCES 17. Guidi-Rontani, C., Weber-Levy, M., Labruyere, E. & Mock, M. (1999) Mol. 8. Wein, L. M., Craft, D. L. & Kaplan, E. H. (2003) Proc. Natl. Acad. Sci. USA 100, 4346–4351. Microbiol. 31, 9–17. 9. Brookmeyer, R. & Blades, N. (2002) Science 295, 1861. 18. Turk, B. E., Wong, T. Y., Schwarzenbacher, R., Jarrell, E. T., Leppla, S. H., 10. Cummings, R. T., Salowe, S. P., Cunninghan, B. R., Wiltsie, J., Park, Y. W., Collier, R. J., Liddington, R. C. & Cantley, L. C. (2004) Nat. Struct. Mol. Biol. Sonatore, L. M., Wisniewski, D., Douglas, C. M., Hermes, J. D. & Scolnick, 11, 60–66. E. M. (2002) Proc. Natl. Acad. Sci. USA 99, 6603–6606. 19. Atlas, R. M. (2002) Annu. Rev. Microbiol. 56, 167–185.

Shoop et al. PNAS ͉ May 31, 2005 ͉ vol. 102 ͉ no. 22 ͉ 7963 Downloaded by guest on September 26, 2021