Metalloproteinase Inhibitors, Nonantimicrobial Chemically Modified Tetracyclines, and Ilomastat Block Bacillus anthracis Lethal Factor Activ
http://www.100md.com
感染与免疫杂志 2005年第11期
Program in Cellular and Molecular Biology
Department of Oral Biology & Pathology
Department of Biochemistry
Department of Pathology
Institute for Cell and Developmental Biology, State University of New York at Stony Brook, Stony Brook, New York 11794
CollaGenex Pharmaceuticals, Inc., Newtown, Pennsylvania 18940
ABSTRACT
Lethal toxin, produced by the bacterium Bacillus anthracis, is a major contributor to morbidity and mortality in animals and humans who have contracted anthrax. One component of this toxin, lethal factor (LF), proteolytically inactivates members of the mitogen-activated protein kinase kinase (MAPKK or MEK) family. In this study we show that CMT-300, CMT-308, and Ilomastat, agents initially characterized as matrix metalloproteinase inhibitors which are in early stages of development as pharmaceuticals, effectively inhibit the zinc metalloproteinase activity of LF. All three inhibitors, CMT-300, CMT-308, and Ilomastat, inhibit LF-mediated cleavage of a synthetic peptide substrate based on the N-terminal domain of MEKs. Inhibition of LF-mediated MEK proteolysis by all three agents was also achieved using lysates of the human monocytoid line MonoMac 6 as sources of MAPKKs and visualization of the extent of cleavage after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by detection by Western blotting. Finally, we have demonstrated inhibition of intracellular MEKs in viable human monocytes and MonoMac 6 cells by these agents after incubation of the cells with a reconstituted preparation of recombinant lethal toxin. All three agents are effective inhibitors when incubated with LF prior to exposure to cells, while the CMTs, but not Ilomastat, are also effective when added after LF has already entered the viable cell targets. These results offer promise for strategies to combat effects of the lethal toxin of B. anthracis.
INTRODUCTION
Anthrax is a disease of animals and humans caused by Bacillus anthracis (28). The bacterium secretes three proteins: protective antigen (PA), edema factor (EF), and lethal factor (LF), which are harmless individually but which combine to form two toxins, lethal toxin and edema toxin (4). PA binds independently to EF and LF to facilitate their entry into the cytosol of targeted cells (7, 40). PA binds initially to cell surface molecules which function as receptors (e.g., anthrax toxin receptor or human capillary morphogenesis protein 2) and is then selectively cleaved and activated by furin or furin-like proteases (2, 3, 24, 47). The activated form of PA (PA63) can assemble into a heptameric pore-forming complex to which LF and EF bind. The entire complex is delivered to an acidic compartment within the cell by endocytosis, where the low pH causes a conformational change, resulting in membrane insertion and pore formation to deliver LF and EF to the cytosol (19). EF is a calmodulin- and Ca2+-dependent adenylate cyclase which elevates the cAMP level in the cytosol (4, 7, 28). LF has a HExxH zinc binding motif characteristic of metalloproteinases (23, 32). The purified protein has been shown to cleave within the N-terminal domain of mitogen-activated protein kinase kinase (MAPKK) protein family members, thereby disrupting their interactions with mitogen-activated protein kinases, which in turn results in inhibition of the signaling pathway (11, 21, 34, 45, 46). LF-deficient strains of B. anthracis fail to trigger fatal complications of infection, and mutations in the zinc binding motif of LF diminish its toxicity in animal models, substantiating the hypothesis that the proteolytic activity of LF is critical for the mortality and morbidity associated with B. anthracis infection (7, 23, 28).
Although the antimicrobial activity of the tetracycline family of antibiotics is well established, the observation that the tetracyclines are also inhibitors of matrix metalloproteinases is more recent (14, 16, 26, 37). A pivotal clarification of the distinction between these two modes of action of the tetracyclines was achieved when a series of nonantimicrobial chemically modified tetracyclines (CMTs) which retained inhibitory activity towards matrix metalloproteinases (MMPs) was reported (5, 15, 18, 27, 30, 41). Two of the most effective antiproteolytic CMTs are CMT-300 [6-dimethyl-6-deoxy-4-de(dimethylamino) tetracycline; CMT-3, COL-3] and CMT-308 [9-amino-6-demethyl-6-deoxy-4-de(dimethylamino) tetracycline; COL-308]. Orally administered CMT-300 is currently in a number of Phase I and II clinical trials with human patients for treatment of solid tumors and Kaposi's sarcoma and for management of rosacea and periodontitis. The only significant toxicity of CMT-300 in humans which has been observed at the maximum tolerated doses in the phase I trials is associated with the well-known cutaneous photosensitivity typical of many tetracyclines. CMT-308 fails to display photosensitivity in animal models and in the 3T3 in vitro model of phototoxicity but has not been evaluated for human use at this time (48).
Ilomastat [HONHCOCH2CH(i-Bu)CO-L-Trp-NHMe; GM6001, Galardin] is a potent MMP inhibitor of the hydroxamate family which binds to the critical active-site zinc atom present in all members of this class of proteinases (12, 17). The isobutyl group and tryptophan side chain are believed to bind to the subsites on the target enzymes which normally bind extracellular matrix proteins (12). In addition to its inhibition of MMPs, Ilomastat inhibits bacterial metalloproteinases, such as thermolysin and Pseudomonas aeruginosa elastase (1, 8, 17, 20). A nonhydroxamic acid analogue of Ilomastat, GM 1489, can still inhibit MMPs but fails to inhibit bacterial metalloproteinases. Ilomastat has been shown to inhibit angiogenesis in a chick chorioallantoic membrane model, to diminish neovascularization of the rat cornea stimulated by an implanted pellet containing a tumor extract, and to reduce the inflammation and proliferation resulting from application of phorbol esters to the skin of rats (12, 13). Human clinical trials for ophthalmic applications of Ilomastat have been conducted without reported toxicities (12).
MATERIALS AND METHODS
LF and PA. Recombinant anthrax LF and PA were purchased from List Biological Laboratories, Inc. (Campbell, CA). The purity of LF and PA were 90% and 100%, respectively, as reported by the manufacturer. The specific activity of LF was evaluated by the manufacturer, using its own oligopeptide substrate MAPKKide in a fluorescence resonance energy transfer (FRET)-based assay of peptidolytic activity: 5 μM substrate was reported to be cleaved by 5 μM LF at a rate of 1.0 to 1.5 relative fluorescence units per second in 20 mM HEPES, pH 8.2, at 37°C. Other known biological and enzymatic activities of LF and PA were verified qualitatively by the manufacturer.
Inhibitors. Ilomastat (GM 6001) of 95% purity and GM 1489 of 95% purity were purchased from Calbiochem (La Jolla, CA). CMT-300 and CMT-308 of >98% purity were supplied by Collagenex Pharmaceuticals, Inc. (Newtown, PA). 1,10-Phenanthroline (o-phenanthroline) of 95% purity was purchased from Aldrich (St. Louis, MO). All inhibitors were dissolved in dimethyl sulfoxide (DMSO). All inhibitors were tested for cellular toxicity in a tetrazolium salt reduction assay as described below and were employed at concentrations which did not diminish viability of the cells employed in these studies.
FRET assay. Assays of LF peptidolytic activity based on cleavage of the FRET-quenched substrate MAPKKide were carried out according to a modification of the method of Cummings et al. (9). MAPKKide (o-aminobenzoyl [o-ABZ]/2,4-dinitrophenyl [DNP]), a synthetic peptide containing the o-ABZ donor and DNP acceptor groups separated by a cleavage site specific for anthrax LF, was purchased from List Biological Labs. Digestion of MAPKKide by LF was carried out in Dulbecco's phosphate-buffered saline (DPBS) (HyClone, Logan Utah), pH 8.2, as recommended by the manufacturer and was followed in a SpectraMax M2 microplate reader (Molecular Devices, Sunnyvale, CA) or in a, LS-5 fluorescence spectrophotometer (Perkin-Elmer, Wellesley, MA) using a excitation (ex) value of 320 nm and a emission (em) value of 420 nm. LF was preincubated with indicated concentrations of putative inhibitors for 10 min at room temperature, and the reaction was initiated by addition of indicated concentrations of the substrate to a 100-μl or 500-μl reaction mixture.
Cells. MonoMac 6 cells (DSMZ, Braunschweig, Germany) were maintained in RPMI 1640 (HyClone) medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 1% nonessential amino acids, 1 mM oxaloacetate, 0.45 mM pyruvate, 0.2 U/ml insulin (OPI media supplement; Sigma, St. Louis, MO), and 1% (vol/vol) penicillin-streptomycin solution (final concentrations, 100 IU penicillin G and 100 μg streptomycin per ml; Sigma) (49). Cells were washed with Hanks ' balanced salt solution (HBSS) (HyClone) at 37°C once prior to commencing experiments, which were all carried out in serum-free media without antibiotics.
Human dendritic cells (Ready-to-use Dendritic Cells DC-100-RT) were purchased from MatTek Corporation (Ashland, MA) and prepared for culture according to the manufacturer's protocol. Upon arrival, cells were pelleted (200 to 300 g) at room temperature for 10 min, resuspended in fresh warm medium supplied by the manufacturer, and held at 37°C for 15 min before repelleting. This washing procedure was repeated three times before resuspension of the cells a final time for counting and plating on 24-well plates at a density of 1 x 105 cells/ml. Cells were incubated for 1 h at 37°C in a humidified atmosphere containing 5% CO2 before experiments were initiated.
Monocyte isolation. Human monocytes were isolated from leukocyte concentrates according to a modification of the method of Levy and Edgington (25). The buffy coat was mixed with an equal volume of DPBS-3 mM EDTA at room temperature and layered onto 1.5 volumes of Lymphoprep (Accurate Chemical & Scientific Corp., Westbury, NY) in 50-ml centrifuge tubes. The tubes were then centrifuged at 650 x g for 30 min at 25°C. The mononuclear cell layer was diluted into 50 ml DPBS and recentrifuged at 250 x g for 10 min at 25°C. The pellet was subjected to hypotonic NaCl (0.2% [wt/vol] for no more than 1 min at 4°C) to lyse contaminating erythrocytes, and the medium was promptly restored to isotonicity with an equal volume of 1.6% NaCl. The mononuclear cells were pelleted by centrifugation at 250 x g for 10 min at 25°C and were resuspended in serum-free medium (Macrophage SFM, Gibco, Grand Island, NY). The cells were plated at a density of 5 x 106/well in 24-well microplates which had been precoated with human serum for 1 h at 37°C. Cells were incubated in serum-coated plates at 37°C for 4 h in a humidified atmosphere containing 5% CO2 and the nonadherent cells removed with several washes of Macrophage SFM. The adherent monocytes were used directly for further experiments.
Preparation of cell lysates. MonoMac 6 cells were washed once with DPBS at room temperature and were lysed by three successive freeze-thaw cycles in DPBS. LF was preincubated for 15 min with CMT-300 (5 to 1,000 μM in DMSO), CMT-308 (5 to 1,000 μM in DMSO), Ilomastat (5 to 257 μM in DMSO), o-phenanthroline (10 mM in DMSO), or with DMSO alone at room temperature, and the reaction mixtures were then incubated with MonoMac 6 cell lysates containing a total protein concentration of 0.5 μg/μl for 1 h at 37°C in a final reaction volume of 10 μl. Vitale et al. (45) incubated LF with inhibitor for 30 min at room temperature; however, we found that a 10- to 15-min incubation is sufficient to reach steady-state inhibition of LF by the active inhibitors we have employed. The reactions were stopped by adding DPBS containing 10 mM o-phenanthroline followed by heating at 70°C for 10 min.
LF proteolytic activity in viable cells. MonoMac 6 cells were washed with warm DPBS and were resuspended in Macrophage SFM for plating at a density of 1 x 106/ml/well in 24-well microplates. After addition of recombinant PA and LF, the cells were incubated for different periods of time, and at each designated time point a 200-μl aliquot of cell suspension was removed. Cells were pelleted by centrifugation and were then lysed in a buffer containing 0.1% Nonidet P-40 (NP-40), 150 mM NaCl, 40 mM Tris (pH 7.2), 10% glycerol, 5 mM NaF, 1 mM Na pyrophosphate, 1 mM Na o-vanadate, 10 mM o-phenanthroline, and 100 ng/ml phenylmethylsulfonyl fluoride at 70°C for 10 min.
Determination of inhibitory efficacy of CMTs and Ilomastat in viable cells. CMTs and Ilomastat were evaluated for their capacity to inhibit the proteolytic activity of LF prior to exposure to cells by incubating the inhibitors with LF for 10 min, followed by addition of PA and then by addition of the mixture to target cells. To evaluate inhibitory activity towards lethal factor which had already entered cells, MonoMac 6 cells were first incubated with a mixture of 50 ng/ml LF and 200 ng/ml PA for 45 min in the absence of inhibitors. Cells were washed once with warm HBSS (HyClone, Logan, Utah) containing the indicated amounts of the inhibitors to be tested. The cells were then centrifuged and suspended in Macrophage SFM (Gibco, Grand Island, NY) containing the same indicated amounts of inhibitors at 37°C. After further incubation, the cells were lysed as described above, and the lysates were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Blots were probed with a polyclonal antibody specific for the MEK-2 N terminus.
Western blotting and antibodies. All cell lysates were analyzed by SDS-PAGE, using 10% polyacrylamide gels (Novex, Carlsbad, CA), followed by transfer to nitrocellulose membranes in an electrophoretic transfer apparatus (Novex) at 30 V for 1 h. Aliquots of 7.5 to 10 μg protein per well were applied to minimize loading artifacts. The membranes were blocked with 5% low-fat milk in Tris-buffered saline buffer (pH 7.6) containing 20 mM Tris, 137 mM NaCl, and 0.1% Tween 20 for 1 h at room temperature and then probed with anti-MEK-2 rabbit polyclonal antibody with specificity for the N terminus (Santa Cruz; Santa Cruz, CA) at a dilution of 1:2,000 or with anti-MEK-6 rabbit polyclonal antibody with specificity for the C terminus (Stressgen Biotechnologies Corp., Victoria, BC Canada) at a dilution of 1 μg/ml. To serve as an internal loading control, levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the samples were visualized with a rabbit polyclonal anti-GAPDH antibody (FL-335; Santa Cruz, CA) which was used at a dilution of 1:1,000. Goat antirabbit immunoglublin G (KPL, Gaithersburg, MD) was used at a 1:2,000 dilution as the secondary antibody. Blots were washed and processed by enhanced chemiluminescence detection (Amersham Pharmacia, Piscataway, NJ) in accordance with the protocol suggested by the manufacturer.
Cell viability assay. The CellTiter 96 AQueous One Solution cell proliferation assay (Promega, Madison, WI), a colorimetric method for determining numbers of viable cells in proliferation, cytotoxicity, or chemosensitivity assays based on the capacity of viable cells to convert a tetrazolium salt [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy-methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium chloride (MTS)] to its reduced formazan product, was employed to evaluate cytotoxicity of the agents employed in this study after 24 h of exposure to target cells. This assay generates a soluble formazan and can be performed directly on cells in multiwell microplates. The yield of formazan was quantitated by measuring the absorbance at 490 nm.
In our studies, 500 μl fresh serum-free medium was added to 1 x 106 adherent monocytes, followed by addition of 100 μl MTS solution, or in the case of MonoMac 6 cells, 100 μl of a suspension of 1 x 106 cells per ml was mixed with 20 μl MTS solution. Cells were incubated at 37°C for 1 to 4 h, and absorbance was recorded at 490 nm at hourly intervals for up to 4 h.
RESULTS
Fluorometric measurements of inhibition of anthrax LF peptidolytic activity by CMTs and Ilomastat. The inhibitory potencies of two CMTs, CMT-300 and its less-photosensitizing derivative, CMT-308, were evaluated along with an MMP inhibitor of the hydroxamate class, Ilomastat (GM-6001), and its nonhydroxamate analog, G-1489, using recombinant LF as a source of zinc metalloproteinase activity and the synthetic FRET-quenched oligopeptide MAPKKide as a substrate. Assays were performed using fixed concentrations of LF (0.1 μM) and MAPKKide (10 μM) in the presence of different concentrations of the inhibitors, and peptidolytic activity was monitored by following the rate of increase in fluorescence intensity at 420 nm with 320-nm excitation as the substrate was hydrolyzed. CMT-300, CMT-308, and Ilomastat (GM-6001) all produced dose-dependent inhibition of LF peptidolytic activity, whereas at doses up to 100 μM, GM-1489 showed less than 6% inhibitory activity (Fig. 1). These observations are consistent with the reported lack of inhibitory activity of GM-1489 towards other bacterial metalloproteinases (17). The mode of inhibition was determined, and the magnitude of Ki for the different inhibitors was estimated by analysis of Dixon plots (39), using the enzyme kinetics package Enzfitter (Biosoft, Cambridge, United Kingdom). Inspection of Dixon plots of CMTs and Ilomastat determined at different substrate concentrations (15, 20, and 25 μM) showed lines that consistently intersected above the x axis, consistent with a competitive mode of inhibition for all three agents (Fig. 2). The linearity of the Dixon plots is also consistent with a mechanism in which the inhibitors and the substrate rapidly reach a steady state in their interaction with the proteinase. The apparent Ki values for CMT-300, CMT-308, and Ilomastat were computed from the intersection of the Dixon plots at different substrate concentrations to be 6.86 ± 0.18, 5.23 ± 1.14, and 2.74 ± 0.86 μM, respectively.
Inhibition of LF-mediated degradation of MEK-2 and MEK-6 in MonoMac 6 cell lysates by CMTs and Ilomastat. To provide a qualitative test of the capacity of the agents used in this study to inhibit LF proteolytic activity against natural substrates in a cell-free system, the recombinant protease was preincubated with the agents and the reaction mixtures were then incubated with MonoMac 6 cell lysates. LF-mediated cleavage of MAPKK family members was then evaluated by SDS-PAGE, transfer to nitrocellulose membranes, and immunodetection with antibodies directed to the N terminus of MEK-2 or the C terminus of MEK-6 as described in Materials and Methods.
As shown in Fig. 3A (blot 1), after incubation of LF with MonoMac 6 lysate, a progressively increasing fraction of the protein reacting with anti-MEK-2 antibody underwent a shift to increased electrophoretic mobility and a decrease in immunoreactivity, consistent with cleavage of the MEK-2 protein at its N and C termini. No alterations in electrophoretic mobility or immunoreactivity of MEK-2 were seen after incubation of MonoMac 6 lysates with LF that had been preincubated with the metal chelator and metalloproteinase inhibitor o-phenanthroline (Fig. 3A, blot 2) or the putative metalloproteinase inhibitors CMT-300 (Fig. 3A, blot 3), CMT-308 (Fig. 3A, blot 4), and Ilomastat (Fig. 3A, blot 5). In contrast, the immunoblot of MonoMac 6 lysate incubated with LF which had been preincubated with GM-1489 indicated only slightly diminished cleavage of MEK-2; we conclude that GM 1489 is an ineffective inhibitor of LF proteolytic activity.
Immunoblots of MonoMac 6 lysates which had been incubated with LF revealed that a band reacting with anti-MEK-6 antibody underwent increased electrophoretic mobility but did not diminish in intensity (Fig. 3B, blot 1), indicating that MEK-6 protein was cleaved near its N terminus without modification or removal of the C-terminal epitope to which the antibody had been raised. No alterations in electrophoretic mobility of the anti-MEK-6 reactive band from that of untreated control lysate were detected in blots of lysates which had been incubated with LF that had been pretreated with o-phenanthroline (Fig. 3B, blot 2), CMT-300 (Fig. 3B, blot 3), CMT-308 (Fig. 3B, blot 4), or Ilomastat (Fig. 3B, blot 5). The presence of a double band shows evidence of a slightly diminished extent of apparent cleavage of MEK-6 in MonoMac 6 lysates after incubation with LF that had been pretreated with GM-1489, consistent with the interpretation that this nonhydroxamate analog of Ilomastat had markedly reduced inhibitory activity towards LF (Fig. 3B, blot 6).
These results indicate that LF cleaves MEK-2 and MEK-6 in MonoMac 6 lysates at specific sites and that the proteolytic activity of LF which is responsible for these cleavages can be inhibited by the CMTs and by Ilomastat.
Dose-dependent inhibition of LF proteolytic activity. To determine the dose dependence of inhibition of LF proteolytic activity by the agents which we found to have significant inhibitory activity in the qualitative studies described above, we assayed changes in the electrophoretic mobility of MEK-6 after incubation of MonoMac 6 lysates with recombinant LF that had been preincubated with different concentrations of CMT-300, CMT-308, or Ilomastat. After separation by SDS-PAGE followed by transfer to nitrocellulose, the reaction products were probed with antibody directed against the C terminus of MEK-6. A 5 μM dose of CMT-300, CMT-308, or Ilomastat was sufficient to inhibit the cleavage of MEK-6 completely within the limits of sensitivity of the immunoblots in MonoMac 6 lysates produced by 1 ng LF in vitro (Fig. 4). These inhibitor doses are within the range of concentrations of CMT-300 which have been shown to be reached in the plasma of human subjects (6, 33, 38, 43; personal communication from Bruce J. Dezube, Harvard Medical School).
CMTs and Ilomastat can inhibit LF activity in viable MonoMac 6 cells, human peripheral blood monocytes, and human dendritic cells. To test the inhibitory affects of the agents in this study against the proteolytic activity of LF in intact viable cells, LF was preincubated with the putative metalloproteinase inhibitors for 15 min at room temperature before being combined with recombinant protective antigen (PA). The mixtures of LF and PA were then incubated with MonoMac 6 cells, adherent monocytes isolated from peripheral blood, and human dendritic cells for durations ranging from 1 h to 4 h. The cells were then lysed, and the lysates were assayed for changes in the electrophoretic mobility of MEK-2.
As shown in Fig. 5A (blot 1), after treatment of viable cells with LF and PA in the absence of inhibitors, we detected a decrease in the intensity of bands recognized by MEK-2 antibody, consistent with LF-mediated cleavage at the N terminus of this target protein. Although we had detected bands resulting from apparent additional C-terminal cleavages in MonoMac 6 lysates after incubation with LF, only the proteolytic event resulting in loss of the N terminus of MEK-2 could be visualized after cleavage by LF within intact viable cells. No diminution in intensity of the immunoreactive MEK-2 band could be detected in MonoMac 6 cells after preincubation of LF with the metalloproteinase inhibitors CMT-300 (Fig. 5A, blot 5), CMT-308 (Fig. 5A, blot 4), or Ilomastat (Fig. 5A, blot 6). Moreover, incubation of viable cells with LF alone or with PA alone failed to result in diminished immunoreactivity of MEK-2. We observed that, as in the case of the in vitro studies with cell lysates described above, preincubation of LF with GM 1489 cannot totally inhibit its proteolytic activity in viable MonoMac 6 cells (Fig. 5A, blot 7). We observed results similar to those obtained with MonoMac 6 cells when we incubated human peripheral blood monocytes (Fig. 5B) or human dendritic cells (Fig. 5C) with PA and LF that had been untreated or had been preincubated with CMTs or Ilomastat. In a series of cytotoxicity studies employing reduction of the tetrazolium salt MTS as a measure of viability, a less than 5% diminution of MonoMac 6 cell viability was observed even after 24 h of incubation with LF and PA, indicating that cytotoxicity is not initiated by entry of LF into the cytosol of this cell line (data not shown).
CMTs have greater inhibitory efficacy than Ilomastat when employed in a postexposure prophylactic mode with viable human cells. Our experiments show that a significant time interval elapses (about 1 h depending on concentrations of LF and PA) between the first exposure of intact cells to mixtures of LF and PA and the earliest visual evidence of LF-mediated MEK cleavage (blot 1 and blot 7 of Fig. 5A; blot 1 and blot 7 of Fig. 5B; blot 1, blot 6, and blot 7 of Fig. 6A). This time period presumably reflects the sequence of steps in trafficking of the complex of LF and PA into an acidic intracellular compartment (endosome), subsequent pore formation, and ultimate passage of LF into the cytosol, where it can bind to and cleave the MEKs. Thus, at some point within the first 60 min of incubation of viable cells with mixtures of LF plus PA, LF might have effectively passed from the extracellular environment but might not yet have reached its cytosolic substrates. To investigate whether this interval represented a period of time during which the intracellular LF could be inhibited selectively by agents which could enter target cells, MonoMac 6 cells were incubated with the mixtures of LF and PA in the absence of inhibitors. The cells were washed at different time points (30 min, 45 min, etc.) and were then incubated for an additional 0 to 5 h in fresh medium containing no LF or PA. At the end of each designated time point, a 200-μl aliquot of cell suspension was removed, the cells were then lysed, and the lysates were assayed for changes in the electrophoretic mobility of MEK-2. We did not detect any subsequent decrease in the intensity of bands recognized by MEK-2 antibody after an incubation of viable cells with LF and PA in the absence of inhibitors which was limited to 30 min (data not shown). We did detect subsequent time-dependent decreases in the intensity of bands recognized by MEK-2 antibody after treatment of viable cells with LF and PA for 45 min followed by washing to remove extracellular toxin components, indicating that this duration of exposure of the cells is sufficient for LF to leave the extracellular environment and subsequently to reach its cytosolic targets.
To test the capacity of the agents in this study to block LF-mediated MEK cleavage in a postexposure prophylactic capacity, MonoMac 6 cells were incubated with mixtures of LF and PA. After 45 min, cells were washed with warm HBSS containing the agent to be tested and were then resuspended in fresh medium containing the same concentration of putative inhibitor. After further incubation, the cells were then lysed and the lysates were assayed for alterations in the electrophoretic mobility or immunoreactivity of MEK-2.
As shown in Fig. 6A (blot 1), after treatment of viable cells with LF and PA in the absence of inhibitors for 45 min, we detected a subsequent time-dependent decrease in the intensity of bands recognized by MEK-2 antibody. No apparent subsequent diminution in intensity of the immunoreactive MEK-2 band could be detected in MonoMac 6 cells after removal of extracellular LF plus PA and subsequent addition of CMT-300 or CMT-308 to the cells (Fig. 6A, blots 4 and 5). In contrast, we observed that neither Ilomastat nor GM 1489 can totally inhibit the proteolytic activity of LF in viable MonoMac 6 cells when these agents are added in a postexposure prophylactic mode (Fig. 6A, blot 6 and blot 7). These results show that in 45 min, sufficient amounts of LF can enter the cell to result in subsequent MEK cleavage and that CMT-300 and CMT-308 can inhibit the LF which has already entered the cells before subsequent MEK cleavage can occur. In contrast, Ilomastat, which is as potent an inhibitor of LF as CMT-300 and CMT-308 in a cell-free environment, cannot inhibit LF which has already entered cells as efficiently as the CMTs.
DISCUSSION
Recent cases of anthrax in the United States have redefined the probability that the causative agent, Bacillus anthracis, may be used for hostile purposes. The experience of attempting to manage the 2001 outbreak of anthrax has drawn attention to the exigency of developing strategies for prophylaxis and therapeutic interventions in settings of known or suspected exposure of large populations to B. anthracis. While management plans which employ antibiotics or lysins specifically toxic to B. anthracis may permit effective clearance of the bacterium from the host, the complications resulting from toxins which have already been secreted by microbes which have disseminated in an infected individual are currently addressed only with supportive therapy. The mechanisms responsible for the symptoms of anthrax are still not completely understood, but it is generally agreed that the zinc metalloproteinase activity of LF produced by the vegetative form of Bacillus anthracis is responsible either directly or indirectly for the morbidity and mortality associated with untreated infections in humans (7, 28). Thus, a pharmacological agent which directly inhibits the activity of this most harmful anthrax toxin might offer a promising approach to combatting the pathogen.
Here we propose a strategy to combat the effects of the lethal toxin of Bacillus anthracis based on the actions of CMTs and Ilomastat (GM6001). The CMTs are derivatives of tetracycline with no antimicrobial activity. Ilomastat is also devoid of classical antibiotic activity. We have specifically avoided the incorporation of antimicrobial activity into the agents we have investigated because weaponized strains of B. anthracis may have already been engineered to be resistant to known antibiotics, and administration of antimicrobial agents to large populations which have not actually been infected but are at some risk for exposure to B. anthracis spores could present a potential risk for emergence of antibiotic-resistant strains of pathogens. In contrast to recent reports on identification of small-molecule inhibitors of anthrax LF (10, 31, 44), which are far from being approved for human use, this study demonstrates that existing metalloproteinase inhibitors that have already been used in various human clinical trials offer a promising approach to combatting anthrax.
In this paper we show that CMT-300, CMT-308, and Ilomastat all have the capacity to inhibit B. anthracis LF activity in cell-free systems and in viable cells when the inhibitors are employed prior to addition of LF plus PA to the cells in a protocol consistent with the U.S. Food and Drug Administration's definition of "preexposure prophylaxis." Furthermore, the inhibitory capacity of the CMTs, but not Ilomastat, is retained when the agents are added after LF has entered viable cells and no extracellular toxin components are left. This protocol is consistent with the Food and Drug Administration's definition of "postexposure prophylaxis" (administration after the cells have been challenged with toxin but prior to the appearance of pathophysiological manifestations of toxin action). Our initial characterization of inhibition employed a fluorescence energy transfer assay with an oligopeptide substrate (MAPKKide) specific for the metalloproteinase activity of LF. Using this assay, we have demonstrated that CMT-300, CMT-308, and Ilomastat (GM-6001) inhibit the peptidolytic activity of recombinant LF in a dose-dependent manner. The use of soluble components in an in vitro assay has facilitated kinetic analysis of the mode of inhibition and the apparent dissociation constants of these agents. The two CMTs and Ilomastat display "classical" competitive inhibition in which enzyme, substrate, and inhibitor all rapidly achieve a steady state (39), with apparent Ki values for the agents of less than 7 μM. The nonhydroxamate analog of Ilomastat, GM-1489, shows only negligible inhibitory activity in the peptidolytic assay. These results are consistent with previous findings which indicate that Ilomastat, but not GM-1489, can inhibit other bacterial metalloproteinases, including thermolysin and Pseudomonas aeruginosa elastase (17).
The inhibitory capacity of the CMTs and Ilomastat extends to the proteolytic activity of LF towards its only known natural substrates, the MEK family of MAPKKs. The three agents inhibit LF-mediated cleavage of MEK-2 and MEK-6 in lysates of mononuclear cells as well as in viable cells, as long as entry of LF into the cytosol is facilitated by its association with PA. The dose dependence of inhibition of proteolysis of the MEKs is comparable to that of inhibition of peptidolysis of the synthetic oligopeptide substrate. It is noteworthy that the concentrations of the CMTs that are sufficient to inhibit MEK cleavage completely are well within the concentrations of these agents which have been measured in the circulation of patients with a variety of solid tumors and Kaposi's sarcoma who have received an oral formulation of CMT-300 in phase I and phase II clinical trials, and in normal human subjects who have received oral doses of CMT-300 in phase I trials (6, 33, 38, 43; personal communication with Bruce J. Dezube from Harvard Medical School). The pharmacokinetics of orally administered CMT-300 in humans permit maintenance of blood levels with a simple daily dosing regimen; some individuals in the Kaposi's sarcoma trials have been following this schedule for more than 1 year (6, 33). These properties make CMT-300 an especially attractive candidate for distribution in capsule form without the need for trained medical personnel and for administration to large populations which may have been exposed to B. anthracis spores. The only notable adverse events reported for CMT-300 at the maximum tolerated dose in phase I trials with human subjects were related to photosensitization, which can result in sunburn-like reactions in subjects who have not employed sunscreen products for protection. CMT-308 appears to be devoid of phototoxicity in an NIH 3T3 cell-based in vitro assay and in a rat model but has not yet been evaluated with human subjects (48). This CMT represents a potential first choice for future development, especially in situations where unprotected sun exposure may be unavoidable, such as battlefield settings.
Ilomastat is a slightly better inhibitor than the two CMTs of LF-mediated cleavage of the synthetic oligopeptide substrate MAPKKide and appears to be comparable to the CMTs in its inhibitory efficacy towards LF-mediated MEK cleavage in cell lysates. Ilomastat is also effective as an inhibitor of LF activity in viable cells, but only when it is added to extracellular LF in a preexposure prophylactic mode. In contrast, we found that Ilomastat cannot inhibit LF which has already entered cells as well as the CMTs. It is highly possible that Ilomastat may not penetrate cell membranes as easily as the CMTs. Our result is consistent with previous findings, which indicate that much higher concentrations of Ilomastat are required to inhibit effects of LF in viable cells or animal models than would have been expected on the basis of the Ki of this inhibitor as determined in cell-free assays with synthetic substrates (29). The capacity of the CMTs to inhibit LF which has already entered viable cells is especially noteworthy in view of the capacity of B. anthracis to spread systemically and establish a toxemia rapidly within the infected host, thereby limiting the window of efficacy of an agent which can function only in a preexposure prophylactic mode.
It has been reported that mixtures of LF and PA show toxicity to a subset of cell types in selected animal models (22, 42). In studies to be reported in greater detail subsequently, we have found that mixtures of LF and PA show some dose-dependent cytotoxicity to human monocytes isolated from peripheral blood with a loss of viability of less than 50% after 24 h as judged by MTS reduction but exhibit no significant dose-dependent cytotoxicity to the transformed MonoMac 6 cell line. However, the two CMTs and Ilomastat administered in preexposure prophylactic mode appear to block the LF-induced cytotoxicity which can be detected after 24 h in monocytes, while the CMTs can also diminish cytotoxicity when administered in postexposure prophylactic mode; none of the agents induces cytotoxicity in MonoMac 6 cells at doses which completely inhibit LF-mediated MEK cleavage (data not shown). The mechanisms of cytotoxicity induced by lethal factor remain uncertain and may not be applicable across species (murine mononuclear cells appear to be far more sensitive than human cells [36]). More pertinent to the underlying mechanism of the morbidity and mortality associated with anthrax, a consensus is emerging that the exceptionally rapid dissemination of B. anthracis in infected animals and humans reflects compromise of both the innate and adaptive components of the immune response, only part of which may be attributed to effects on macrophage functions (35). It is noteworthy that the three agents we have evaluated are all effective inhibitors of LF-mediated MEK cleavage in human dendritic cells, which have an essential role in adaptive immunity. It has been speculated that some aspects of the systemic complications of the late stages of anthrax pathology in humans may arise from attack by bacterial components on target cells other than mononuclear phagocytes, resulting in proteolysis of host proteins other than the MEKs (28). Given the uncertainties over the details of the pathway by which infection with B. anthracis triggers such morbidity and mortality in humans, the results reported here are insufficient for us to conclude that the CMTs or Ilomastat will achieve total therapeutic benefit in vivo comparable to their efficacy in the in vitro studies we have undertaken so far. However, the absence of significant toxicity and minimal risk of facilitating antibiotic resistance, combined with high oral availability and potency at circulating levels against the proteolytic activity of the most pathogenic toxin produced by B. anthracis, suggests that the CMTs and, for more restricted applications, Ilomastat offer promise for management of populations which might have been exposed to spores of the anthrax bacillus or are simply at high risk for such exposure.
ACKNOWLEDGMENTS
We are grateful to our colleagues in S. R. Simon's laboratory who have provided assistance in this project. We thank Richard Kew for use of the SpectraMax M2 microplate reader and members of Ute Moll's lab for providing GAPDH antibody. We are also indebted to Collagenex Pharmaceuticals, Inc., for their generous supply of CMT-300 and CMT-308.
This work was supported by NIH (NIAID) R21-AI53524 and was submitted by S.S.K. in partial fulfillment of the requirements for the Ph.D. Program in Molecular and Cellular Biology (State University of New York at Stony Brook).
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Department of Oral Biology & Pathology
Department of Biochemistry
Department of Pathology
Institute for Cell and Developmental Biology, State University of New York at Stony Brook, Stony Brook, New York 11794
CollaGenex Pharmaceuticals, Inc., Newtown, Pennsylvania 18940
ABSTRACT
Lethal toxin, produced by the bacterium Bacillus anthracis, is a major contributor to morbidity and mortality in animals and humans who have contracted anthrax. One component of this toxin, lethal factor (LF), proteolytically inactivates members of the mitogen-activated protein kinase kinase (MAPKK or MEK) family. In this study we show that CMT-300, CMT-308, and Ilomastat, agents initially characterized as matrix metalloproteinase inhibitors which are in early stages of development as pharmaceuticals, effectively inhibit the zinc metalloproteinase activity of LF. All three inhibitors, CMT-300, CMT-308, and Ilomastat, inhibit LF-mediated cleavage of a synthetic peptide substrate based on the N-terminal domain of MEKs. Inhibition of LF-mediated MEK proteolysis by all three agents was also achieved using lysates of the human monocytoid line MonoMac 6 as sources of MAPKKs and visualization of the extent of cleavage after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by detection by Western blotting. Finally, we have demonstrated inhibition of intracellular MEKs in viable human monocytes and MonoMac 6 cells by these agents after incubation of the cells with a reconstituted preparation of recombinant lethal toxin. All three agents are effective inhibitors when incubated with LF prior to exposure to cells, while the CMTs, but not Ilomastat, are also effective when added after LF has already entered the viable cell targets. These results offer promise for strategies to combat effects of the lethal toxin of B. anthracis.
INTRODUCTION
Anthrax is a disease of animals and humans caused by Bacillus anthracis (28). The bacterium secretes three proteins: protective antigen (PA), edema factor (EF), and lethal factor (LF), which are harmless individually but which combine to form two toxins, lethal toxin and edema toxin (4). PA binds independently to EF and LF to facilitate their entry into the cytosol of targeted cells (7, 40). PA binds initially to cell surface molecules which function as receptors (e.g., anthrax toxin receptor or human capillary morphogenesis protein 2) and is then selectively cleaved and activated by furin or furin-like proteases (2, 3, 24, 47). The activated form of PA (PA63) can assemble into a heptameric pore-forming complex to which LF and EF bind. The entire complex is delivered to an acidic compartment within the cell by endocytosis, where the low pH causes a conformational change, resulting in membrane insertion and pore formation to deliver LF and EF to the cytosol (19). EF is a calmodulin- and Ca2+-dependent adenylate cyclase which elevates the cAMP level in the cytosol (4, 7, 28). LF has a HExxH zinc binding motif characteristic of metalloproteinases (23, 32). The purified protein has been shown to cleave within the N-terminal domain of mitogen-activated protein kinase kinase (MAPKK) protein family members, thereby disrupting their interactions with mitogen-activated protein kinases, which in turn results in inhibition of the signaling pathway (11, 21, 34, 45, 46). LF-deficient strains of B. anthracis fail to trigger fatal complications of infection, and mutations in the zinc binding motif of LF diminish its toxicity in animal models, substantiating the hypothesis that the proteolytic activity of LF is critical for the mortality and morbidity associated with B. anthracis infection (7, 23, 28).
Although the antimicrobial activity of the tetracycline family of antibiotics is well established, the observation that the tetracyclines are also inhibitors of matrix metalloproteinases is more recent (14, 16, 26, 37). A pivotal clarification of the distinction between these two modes of action of the tetracyclines was achieved when a series of nonantimicrobial chemically modified tetracyclines (CMTs) which retained inhibitory activity towards matrix metalloproteinases (MMPs) was reported (5, 15, 18, 27, 30, 41). Two of the most effective antiproteolytic CMTs are CMT-300 [6-dimethyl-6-deoxy-4-de(dimethylamino) tetracycline; CMT-3, COL-3] and CMT-308 [9-amino-6-demethyl-6-deoxy-4-de(dimethylamino) tetracycline; COL-308]. Orally administered CMT-300 is currently in a number of Phase I and II clinical trials with human patients for treatment of solid tumors and Kaposi's sarcoma and for management of rosacea and periodontitis. The only significant toxicity of CMT-300 in humans which has been observed at the maximum tolerated doses in the phase I trials is associated with the well-known cutaneous photosensitivity typical of many tetracyclines. CMT-308 fails to display photosensitivity in animal models and in the 3T3 in vitro model of phototoxicity but has not been evaluated for human use at this time (48).
Ilomastat [HONHCOCH2CH(i-Bu)CO-L-Trp-NHMe; GM6001, Galardin] is a potent MMP inhibitor of the hydroxamate family which binds to the critical active-site zinc atom present in all members of this class of proteinases (12, 17). The isobutyl group and tryptophan side chain are believed to bind to the subsites on the target enzymes which normally bind extracellular matrix proteins (12). In addition to its inhibition of MMPs, Ilomastat inhibits bacterial metalloproteinases, such as thermolysin and Pseudomonas aeruginosa elastase (1, 8, 17, 20). A nonhydroxamic acid analogue of Ilomastat, GM 1489, can still inhibit MMPs but fails to inhibit bacterial metalloproteinases. Ilomastat has been shown to inhibit angiogenesis in a chick chorioallantoic membrane model, to diminish neovascularization of the rat cornea stimulated by an implanted pellet containing a tumor extract, and to reduce the inflammation and proliferation resulting from application of phorbol esters to the skin of rats (12, 13). Human clinical trials for ophthalmic applications of Ilomastat have been conducted without reported toxicities (12).
MATERIALS AND METHODS
LF and PA. Recombinant anthrax LF and PA were purchased from List Biological Laboratories, Inc. (Campbell, CA). The purity of LF and PA were 90% and 100%, respectively, as reported by the manufacturer. The specific activity of LF was evaluated by the manufacturer, using its own oligopeptide substrate MAPKKide in a fluorescence resonance energy transfer (FRET)-based assay of peptidolytic activity: 5 μM substrate was reported to be cleaved by 5 μM LF at a rate of 1.0 to 1.5 relative fluorescence units per second in 20 mM HEPES, pH 8.2, at 37°C. Other known biological and enzymatic activities of LF and PA were verified qualitatively by the manufacturer.
Inhibitors. Ilomastat (GM 6001) of 95% purity and GM 1489 of 95% purity were purchased from Calbiochem (La Jolla, CA). CMT-300 and CMT-308 of >98% purity were supplied by Collagenex Pharmaceuticals, Inc. (Newtown, PA). 1,10-Phenanthroline (o-phenanthroline) of 95% purity was purchased from Aldrich (St. Louis, MO). All inhibitors were dissolved in dimethyl sulfoxide (DMSO). All inhibitors were tested for cellular toxicity in a tetrazolium salt reduction assay as described below and were employed at concentrations which did not diminish viability of the cells employed in these studies.
FRET assay. Assays of LF peptidolytic activity based on cleavage of the FRET-quenched substrate MAPKKide were carried out according to a modification of the method of Cummings et al. (9). MAPKKide (o-aminobenzoyl [o-ABZ]/2,4-dinitrophenyl [DNP]), a synthetic peptide containing the o-ABZ donor and DNP acceptor groups separated by a cleavage site specific for anthrax LF, was purchased from List Biological Labs. Digestion of MAPKKide by LF was carried out in Dulbecco's phosphate-buffered saline (DPBS) (HyClone, Logan Utah), pH 8.2, as recommended by the manufacturer and was followed in a SpectraMax M2 microplate reader (Molecular Devices, Sunnyvale, CA) or in a, LS-5 fluorescence spectrophotometer (Perkin-Elmer, Wellesley, MA) using a excitation (ex) value of 320 nm and a emission (em) value of 420 nm. LF was preincubated with indicated concentrations of putative inhibitors for 10 min at room temperature, and the reaction was initiated by addition of indicated concentrations of the substrate to a 100-μl or 500-μl reaction mixture.
Cells. MonoMac 6 cells (DSMZ, Braunschweig, Germany) were maintained in RPMI 1640 (HyClone) medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 1% nonessential amino acids, 1 mM oxaloacetate, 0.45 mM pyruvate, 0.2 U/ml insulin (OPI media supplement; Sigma, St. Louis, MO), and 1% (vol/vol) penicillin-streptomycin solution (final concentrations, 100 IU penicillin G and 100 μg streptomycin per ml; Sigma) (49). Cells were washed with Hanks ' balanced salt solution (HBSS) (HyClone) at 37°C once prior to commencing experiments, which were all carried out in serum-free media without antibiotics.
Human dendritic cells (Ready-to-use Dendritic Cells DC-100-RT) were purchased from MatTek Corporation (Ashland, MA) and prepared for culture according to the manufacturer's protocol. Upon arrival, cells were pelleted (200 to 300 g) at room temperature for 10 min, resuspended in fresh warm medium supplied by the manufacturer, and held at 37°C for 15 min before repelleting. This washing procedure was repeated three times before resuspension of the cells a final time for counting and plating on 24-well plates at a density of 1 x 105 cells/ml. Cells were incubated for 1 h at 37°C in a humidified atmosphere containing 5% CO2 before experiments were initiated.
Monocyte isolation. Human monocytes were isolated from leukocyte concentrates according to a modification of the method of Levy and Edgington (25). The buffy coat was mixed with an equal volume of DPBS-3 mM EDTA at room temperature and layered onto 1.5 volumes of Lymphoprep (Accurate Chemical & Scientific Corp., Westbury, NY) in 50-ml centrifuge tubes. The tubes were then centrifuged at 650 x g for 30 min at 25°C. The mononuclear cell layer was diluted into 50 ml DPBS and recentrifuged at 250 x g for 10 min at 25°C. The pellet was subjected to hypotonic NaCl (0.2% [wt/vol] for no more than 1 min at 4°C) to lyse contaminating erythrocytes, and the medium was promptly restored to isotonicity with an equal volume of 1.6% NaCl. The mononuclear cells were pelleted by centrifugation at 250 x g for 10 min at 25°C and were resuspended in serum-free medium (Macrophage SFM, Gibco, Grand Island, NY). The cells were plated at a density of 5 x 106/well in 24-well microplates which had been precoated with human serum for 1 h at 37°C. Cells were incubated in serum-coated plates at 37°C for 4 h in a humidified atmosphere containing 5% CO2 and the nonadherent cells removed with several washes of Macrophage SFM. The adherent monocytes were used directly for further experiments.
Preparation of cell lysates. MonoMac 6 cells were washed once with DPBS at room temperature and were lysed by three successive freeze-thaw cycles in DPBS. LF was preincubated for 15 min with CMT-300 (5 to 1,000 μM in DMSO), CMT-308 (5 to 1,000 μM in DMSO), Ilomastat (5 to 257 μM in DMSO), o-phenanthroline (10 mM in DMSO), or with DMSO alone at room temperature, and the reaction mixtures were then incubated with MonoMac 6 cell lysates containing a total protein concentration of 0.5 μg/μl for 1 h at 37°C in a final reaction volume of 10 μl. Vitale et al. (45) incubated LF with inhibitor for 30 min at room temperature; however, we found that a 10- to 15-min incubation is sufficient to reach steady-state inhibition of LF by the active inhibitors we have employed. The reactions were stopped by adding DPBS containing 10 mM o-phenanthroline followed by heating at 70°C for 10 min.
LF proteolytic activity in viable cells. MonoMac 6 cells were washed with warm DPBS and were resuspended in Macrophage SFM for plating at a density of 1 x 106/ml/well in 24-well microplates. After addition of recombinant PA and LF, the cells were incubated for different periods of time, and at each designated time point a 200-μl aliquot of cell suspension was removed. Cells were pelleted by centrifugation and were then lysed in a buffer containing 0.1% Nonidet P-40 (NP-40), 150 mM NaCl, 40 mM Tris (pH 7.2), 10% glycerol, 5 mM NaF, 1 mM Na pyrophosphate, 1 mM Na o-vanadate, 10 mM o-phenanthroline, and 100 ng/ml phenylmethylsulfonyl fluoride at 70°C for 10 min.
Determination of inhibitory efficacy of CMTs and Ilomastat in viable cells. CMTs and Ilomastat were evaluated for their capacity to inhibit the proteolytic activity of LF prior to exposure to cells by incubating the inhibitors with LF for 10 min, followed by addition of PA and then by addition of the mixture to target cells. To evaluate inhibitory activity towards lethal factor which had already entered cells, MonoMac 6 cells were first incubated with a mixture of 50 ng/ml LF and 200 ng/ml PA for 45 min in the absence of inhibitors. Cells were washed once with warm HBSS (HyClone, Logan, Utah) containing the indicated amounts of the inhibitors to be tested. The cells were then centrifuged and suspended in Macrophage SFM (Gibco, Grand Island, NY) containing the same indicated amounts of inhibitors at 37°C. After further incubation, the cells were lysed as described above, and the lysates were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Blots were probed with a polyclonal antibody specific for the MEK-2 N terminus.
Western blotting and antibodies. All cell lysates were analyzed by SDS-PAGE, using 10% polyacrylamide gels (Novex, Carlsbad, CA), followed by transfer to nitrocellulose membranes in an electrophoretic transfer apparatus (Novex) at 30 V for 1 h. Aliquots of 7.5 to 10 μg protein per well were applied to minimize loading artifacts. The membranes were blocked with 5% low-fat milk in Tris-buffered saline buffer (pH 7.6) containing 20 mM Tris, 137 mM NaCl, and 0.1% Tween 20 for 1 h at room temperature and then probed with anti-MEK-2 rabbit polyclonal antibody with specificity for the N terminus (Santa Cruz; Santa Cruz, CA) at a dilution of 1:2,000 or with anti-MEK-6 rabbit polyclonal antibody with specificity for the C terminus (Stressgen Biotechnologies Corp., Victoria, BC Canada) at a dilution of 1 μg/ml. To serve as an internal loading control, levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the samples were visualized with a rabbit polyclonal anti-GAPDH antibody (FL-335; Santa Cruz, CA) which was used at a dilution of 1:1,000. Goat antirabbit immunoglublin G (KPL, Gaithersburg, MD) was used at a 1:2,000 dilution as the secondary antibody. Blots were washed and processed by enhanced chemiluminescence detection (Amersham Pharmacia, Piscataway, NJ) in accordance with the protocol suggested by the manufacturer.
Cell viability assay. The CellTiter 96 AQueous One Solution cell proliferation assay (Promega, Madison, WI), a colorimetric method for determining numbers of viable cells in proliferation, cytotoxicity, or chemosensitivity assays based on the capacity of viable cells to convert a tetrazolium salt [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy-methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium chloride (MTS)] to its reduced formazan product, was employed to evaluate cytotoxicity of the agents employed in this study after 24 h of exposure to target cells. This assay generates a soluble formazan and can be performed directly on cells in multiwell microplates. The yield of formazan was quantitated by measuring the absorbance at 490 nm.
In our studies, 500 μl fresh serum-free medium was added to 1 x 106 adherent monocytes, followed by addition of 100 μl MTS solution, or in the case of MonoMac 6 cells, 100 μl of a suspension of 1 x 106 cells per ml was mixed with 20 μl MTS solution. Cells were incubated at 37°C for 1 to 4 h, and absorbance was recorded at 490 nm at hourly intervals for up to 4 h.
RESULTS
Fluorometric measurements of inhibition of anthrax LF peptidolytic activity by CMTs and Ilomastat. The inhibitory potencies of two CMTs, CMT-300 and its less-photosensitizing derivative, CMT-308, were evaluated along with an MMP inhibitor of the hydroxamate class, Ilomastat (GM-6001), and its nonhydroxamate analog, G-1489, using recombinant LF as a source of zinc metalloproteinase activity and the synthetic FRET-quenched oligopeptide MAPKKide as a substrate. Assays were performed using fixed concentrations of LF (0.1 μM) and MAPKKide (10 μM) in the presence of different concentrations of the inhibitors, and peptidolytic activity was monitored by following the rate of increase in fluorescence intensity at 420 nm with 320-nm excitation as the substrate was hydrolyzed. CMT-300, CMT-308, and Ilomastat (GM-6001) all produced dose-dependent inhibition of LF peptidolytic activity, whereas at doses up to 100 μM, GM-1489 showed less than 6% inhibitory activity (Fig. 1). These observations are consistent with the reported lack of inhibitory activity of GM-1489 towards other bacterial metalloproteinases (17). The mode of inhibition was determined, and the magnitude of Ki for the different inhibitors was estimated by analysis of Dixon plots (39), using the enzyme kinetics package Enzfitter (Biosoft, Cambridge, United Kingdom). Inspection of Dixon plots of CMTs and Ilomastat determined at different substrate concentrations (15, 20, and 25 μM) showed lines that consistently intersected above the x axis, consistent with a competitive mode of inhibition for all three agents (Fig. 2). The linearity of the Dixon plots is also consistent with a mechanism in which the inhibitors and the substrate rapidly reach a steady state in their interaction with the proteinase. The apparent Ki values for CMT-300, CMT-308, and Ilomastat were computed from the intersection of the Dixon plots at different substrate concentrations to be 6.86 ± 0.18, 5.23 ± 1.14, and 2.74 ± 0.86 μM, respectively.
Inhibition of LF-mediated degradation of MEK-2 and MEK-6 in MonoMac 6 cell lysates by CMTs and Ilomastat. To provide a qualitative test of the capacity of the agents used in this study to inhibit LF proteolytic activity against natural substrates in a cell-free system, the recombinant protease was preincubated with the agents and the reaction mixtures were then incubated with MonoMac 6 cell lysates. LF-mediated cleavage of MAPKK family members was then evaluated by SDS-PAGE, transfer to nitrocellulose membranes, and immunodetection with antibodies directed to the N terminus of MEK-2 or the C terminus of MEK-6 as described in Materials and Methods.
As shown in Fig. 3A (blot 1), after incubation of LF with MonoMac 6 lysate, a progressively increasing fraction of the protein reacting with anti-MEK-2 antibody underwent a shift to increased electrophoretic mobility and a decrease in immunoreactivity, consistent with cleavage of the MEK-2 protein at its N and C termini. No alterations in electrophoretic mobility or immunoreactivity of MEK-2 were seen after incubation of MonoMac 6 lysates with LF that had been preincubated with the metal chelator and metalloproteinase inhibitor o-phenanthroline (Fig. 3A, blot 2) or the putative metalloproteinase inhibitors CMT-300 (Fig. 3A, blot 3), CMT-308 (Fig. 3A, blot 4), and Ilomastat (Fig. 3A, blot 5). In contrast, the immunoblot of MonoMac 6 lysate incubated with LF which had been preincubated with GM-1489 indicated only slightly diminished cleavage of MEK-2; we conclude that GM 1489 is an ineffective inhibitor of LF proteolytic activity.
Immunoblots of MonoMac 6 lysates which had been incubated with LF revealed that a band reacting with anti-MEK-6 antibody underwent increased electrophoretic mobility but did not diminish in intensity (Fig. 3B, blot 1), indicating that MEK-6 protein was cleaved near its N terminus without modification or removal of the C-terminal epitope to which the antibody had been raised. No alterations in electrophoretic mobility of the anti-MEK-6 reactive band from that of untreated control lysate were detected in blots of lysates which had been incubated with LF that had been pretreated with o-phenanthroline (Fig. 3B, blot 2), CMT-300 (Fig. 3B, blot 3), CMT-308 (Fig. 3B, blot 4), or Ilomastat (Fig. 3B, blot 5). The presence of a double band shows evidence of a slightly diminished extent of apparent cleavage of MEK-6 in MonoMac 6 lysates after incubation with LF that had been pretreated with GM-1489, consistent with the interpretation that this nonhydroxamate analog of Ilomastat had markedly reduced inhibitory activity towards LF (Fig. 3B, blot 6).
These results indicate that LF cleaves MEK-2 and MEK-6 in MonoMac 6 lysates at specific sites and that the proteolytic activity of LF which is responsible for these cleavages can be inhibited by the CMTs and by Ilomastat.
Dose-dependent inhibition of LF proteolytic activity. To determine the dose dependence of inhibition of LF proteolytic activity by the agents which we found to have significant inhibitory activity in the qualitative studies described above, we assayed changes in the electrophoretic mobility of MEK-6 after incubation of MonoMac 6 lysates with recombinant LF that had been preincubated with different concentrations of CMT-300, CMT-308, or Ilomastat. After separation by SDS-PAGE followed by transfer to nitrocellulose, the reaction products were probed with antibody directed against the C terminus of MEK-6. A 5 μM dose of CMT-300, CMT-308, or Ilomastat was sufficient to inhibit the cleavage of MEK-6 completely within the limits of sensitivity of the immunoblots in MonoMac 6 lysates produced by 1 ng LF in vitro (Fig. 4). These inhibitor doses are within the range of concentrations of CMT-300 which have been shown to be reached in the plasma of human subjects (6, 33, 38, 43; personal communication from Bruce J. Dezube, Harvard Medical School).
CMTs and Ilomastat can inhibit LF activity in viable MonoMac 6 cells, human peripheral blood monocytes, and human dendritic cells. To test the inhibitory affects of the agents in this study against the proteolytic activity of LF in intact viable cells, LF was preincubated with the putative metalloproteinase inhibitors for 15 min at room temperature before being combined with recombinant protective antigen (PA). The mixtures of LF and PA were then incubated with MonoMac 6 cells, adherent monocytes isolated from peripheral blood, and human dendritic cells for durations ranging from 1 h to 4 h. The cells were then lysed, and the lysates were assayed for changes in the electrophoretic mobility of MEK-2.
As shown in Fig. 5A (blot 1), after treatment of viable cells with LF and PA in the absence of inhibitors, we detected a decrease in the intensity of bands recognized by MEK-2 antibody, consistent with LF-mediated cleavage at the N terminus of this target protein. Although we had detected bands resulting from apparent additional C-terminal cleavages in MonoMac 6 lysates after incubation with LF, only the proteolytic event resulting in loss of the N terminus of MEK-2 could be visualized after cleavage by LF within intact viable cells. No diminution in intensity of the immunoreactive MEK-2 band could be detected in MonoMac 6 cells after preincubation of LF with the metalloproteinase inhibitors CMT-300 (Fig. 5A, blot 5), CMT-308 (Fig. 5A, blot 4), or Ilomastat (Fig. 5A, blot 6). Moreover, incubation of viable cells with LF alone or with PA alone failed to result in diminished immunoreactivity of MEK-2. We observed that, as in the case of the in vitro studies with cell lysates described above, preincubation of LF with GM 1489 cannot totally inhibit its proteolytic activity in viable MonoMac 6 cells (Fig. 5A, blot 7). We observed results similar to those obtained with MonoMac 6 cells when we incubated human peripheral blood monocytes (Fig. 5B) or human dendritic cells (Fig. 5C) with PA and LF that had been untreated or had been preincubated with CMTs or Ilomastat. In a series of cytotoxicity studies employing reduction of the tetrazolium salt MTS as a measure of viability, a less than 5% diminution of MonoMac 6 cell viability was observed even after 24 h of incubation with LF and PA, indicating that cytotoxicity is not initiated by entry of LF into the cytosol of this cell line (data not shown).
CMTs have greater inhibitory efficacy than Ilomastat when employed in a postexposure prophylactic mode with viable human cells. Our experiments show that a significant time interval elapses (about 1 h depending on concentrations of LF and PA) between the first exposure of intact cells to mixtures of LF and PA and the earliest visual evidence of LF-mediated MEK cleavage (blot 1 and blot 7 of Fig. 5A; blot 1 and blot 7 of Fig. 5B; blot 1, blot 6, and blot 7 of Fig. 6A). This time period presumably reflects the sequence of steps in trafficking of the complex of LF and PA into an acidic intracellular compartment (endosome), subsequent pore formation, and ultimate passage of LF into the cytosol, where it can bind to and cleave the MEKs. Thus, at some point within the first 60 min of incubation of viable cells with mixtures of LF plus PA, LF might have effectively passed from the extracellular environment but might not yet have reached its cytosolic substrates. To investigate whether this interval represented a period of time during which the intracellular LF could be inhibited selectively by agents which could enter target cells, MonoMac 6 cells were incubated with the mixtures of LF and PA in the absence of inhibitors. The cells were washed at different time points (30 min, 45 min, etc.) and were then incubated for an additional 0 to 5 h in fresh medium containing no LF or PA. At the end of each designated time point, a 200-μl aliquot of cell suspension was removed, the cells were then lysed, and the lysates were assayed for changes in the electrophoretic mobility of MEK-2. We did not detect any subsequent decrease in the intensity of bands recognized by MEK-2 antibody after an incubation of viable cells with LF and PA in the absence of inhibitors which was limited to 30 min (data not shown). We did detect subsequent time-dependent decreases in the intensity of bands recognized by MEK-2 antibody after treatment of viable cells with LF and PA for 45 min followed by washing to remove extracellular toxin components, indicating that this duration of exposure of the cells is sufficient for LF to leave the extracellular environment and subsequently to reach its cytosolic targets.
To test the capacity of the agents in this study to block LF-mediated MEK cleavage in a postexposure prophylactic capacity, MonoMac 6 cells were incubated with mixtures of LF and PA. After 45 min, cells were washed with warm HBSS containing the agent to be tested and were then resuspended in fresh medium containing the same concentration of putative inhibitor. After further incubation, the cells were then lysed and the lysates were assayed for alterations in the electrophoretic mobility or immunoreactivity of MEK-2.
As shown in Fig. 6A (blot 1), after treatment of viable cells with LF and PA in the absence of inhibitors for 45 min, we detected a subsequent time-dependent decrease in the intensity of bands recognized by MEK-2 antibody. No apparent subsequent diminution in intensity of the immunoreactive MEK-2 band could be detected in MonoMac 6 cells after removal of extracellular LF plus PA and subsequent addition of CMT-300 or CMT-308 to the cells (Fig. 6A, blots 4 and 5). In contrast, we observed that neither Ilomastat nor GM 1489 can totally inhibit the proteolytic activity of LF in viable MonoMac 6 cells when these agents are added in a postexposure prophylactic mode (Fig. 6A, blot 6 and blot 7). These results show that in 45 min, sufficient amounts of LF can enter the cell to result in subsequent MEK cleavage and that CMT-300 and CMT-308 can inhibit the LF which has already entered the cells before subsequent MEK cleavage can occur. In contrast, Ilomastat, which is as potent an inhibitor of LF as CMT-300 and CMT-308 in a cell-free environment, cannot inhibit LF which has already entered cells as efficiently as the CMTs.
DISCUSSION
Recent cases of anthrax in the United States have redefined the probability that the causative agent, Bacillus anthracis, may be used for hostile purposes. The experience of attempting to manage the 2001 outbreak of anthrax has drawn attention to the exigency of developing strategies for prophylaxis and therapeutic interventions in settings of known or suspected exposure of large populations to B. anthracis. While management plans which employ antibiotics or lysins specifically toxic to B. anthracis may permit effective clearance of the bacterium from the host, the complications resulting from toxins which have already been secreted by microbes which have disseminated in an infected individual are currently addressed only with supportive therapy. The mechanisms responsible for the symptoms of anthrax are still not completely understood, but it is generally agreed that the zinc metalloproteinase activity of LF produced by the vegetative form of Bacillus anthracis is responsible either directly or indirectly for the morbidity and mortality associated with untreated infections in humans (7, 28). Thus, a pharmacological agent which directly inhibits the activity of this most harmful anthrax toxin might offer a promising approach to combatting the pathogen.
Here we propose a strategy to combat the effects of the lethal toxin of Bacillus anthracis based on the actions of CMTs and Ilomastat (GM6001). The CMTs are derivatives of tetracycline with no antimicrobial activity. Ilomastat is also devoid of classical antibiotic activity. We have specifically avoided the incorporation of antimicrobial activity into the agents we have investigated because weaponized strains of B. anthracis may have already been engineered to be resistant to known antibiotics, and administration of antimicrobial agents to large populations which have not actually been infected but are at some risk for exposure to B. anthracis spores could present a potential risk for emergence of antibiotic-resistant strains of pathogens. In contrast to recent reports on identification of small-molecule inhibitors of anthrax LF (10, 31, 44), which are far from being approved for human use, this study demonstrates that existing metalloproteinase inhibitors that have already been used in various human clinical trials offer a promising approach to combatting anthrax.
In this paper we show that CMT-300, CMT-308, and Ilomastat all have the capacity to inhibit B. anthracis LF activity in cell-free systems and in viable cells when the inhibitors are employed prior to addition of LF plus PA to the cells in a protocol consistent with the U.S. Food and Drug Administration's definition of "preexposure prophylaxis." Furthermore, the inhibitory capacity of the CMTs, but not Ilomastat, is retained when the agents are added after LF has entered viable cells and no extracellular toxin components are left. This protocol is consistent with the Food and Drug Administration's definition of "postexposure prophylaxis" (administration after the cells have been challenged with toxin but prior to the appearance of pathophysiological manifestations of toxin action). Our initial characterization of inhibition employed a fluorescence energy transfer assay with an oligopeptide substrate (MAPKKide) specific for the metalloproteinase activity of LF. Using this assay, we have demonstrated that CMT-300, CMT-308, and Ilomastat (GM-6001) inhibit the peptidolytic activity of recombinant LF in a dose-dependent manner. The use of soluble components in an in vitro assay has facilitated kinetic analysis of the mode of inhibition and the apparent dissociation constants of these agents. The two CMTs and Ilomastat display "classical" competitive inhibition in which enzyme, substrate, and inhibitor all rapidly achieve a steady state (39), with apparent Ki values for the agents of less than 7 μM. The nonhydroxamate analog of Ilomastat, GM-1489, shows only negligible inhibitory activity in the peptidolytic assay. These results are consistent with previous findings which indicate that Ilomastat, but not GM-1489, can inhibit other bacterial metalloproteinases, including thermolysin and Pseudomonas aeruginosa elastase (17).
The inhibitory capacity of the CMTs and Ilomastat extends to the proteolytic activity of LF towards its only known natural substrates, the MEK family of MAPKKs. The three agents inhibit LF-mediated cleavage of MEK-2 and MEK-6 in lysates of mononuclear cells as well as in viable cells, as long as entry of LF into the cytosol is facilitated by its association with PA. The dose dependence of inhibition of proteolysis of the MEKs is comparable to that of inhibition of peptidolysis of the synthetic oligopeptide substrate. It is noteworthy that the concentrations of the CMTs that are sufficient to inhibit MEK cleavage completely are well within the concentrations of these agents which have been measured in the circulation of patients with a variety of solid tumors and Kaposi's sarcoma who have received an oral formulation of CMT-300 in phase I and phase II clinical trials, and in normal human subjects who have received oral doses of CMT-300 in phase I trials (6, 33, 38, 43; personal communication with Bruce J. Dezube from Harvard Medical School). The pharmacokinetics of orally administered CMT-300 in humans permit maintenance of blood levels with a simple daily dosing regimen; some individuals in the Kaposi's sarcoma trials have been following this schedule for more than 1 year (6, 33). These properties make CMT-300 an especially attractive candidate for distribution in capsule form without the need for trained medical personnel and for administration to large populations which may have been exposed to B. anthracis spores. The only notable adverse events reported for CMT-300 at the maximum tolerated dose in phase I trials with human subjects were related to photosensitization, which can result in sunburn-like reactions in subjects who have not employed sunscreen products for protection. CMT-308 appears to be devoid of phototoxicity in an NIH 3T3 cell-based in vitro assay and in a rat model but has not yet been evaluated with human subjects (48). This CMT represents a potential first choice for future development, especially in situations where unprotected sun exposure may be unavoidable, such as battlefield settings.
Ilomastat is a slightly better inhibitor than the two CMTs of LF-mediated cleavage of the synthetic oligopeptide substrate MAPKKide and appears to be comparable to the CMTs in its inhibitory efficacy towards LF-mediated MEK cleavage in cell lysates. Ilomastat is also effective as an inhibitor of LF activity in viable cells, but only when it is added to extracellular LF in a preexposure prophylactic mode. In contrast, we found that Ilomastat cannot inhibit LF which has already entered cells as well as the CMTs. It is highly possible that Ilomastat may not penetrate cell membranes as easily as the CMTs. Our result is consistent with previous findings, which indicate that much higher concentrations of Ilomastat are required to inhibit effects of LF in viable cells or animal models than would have been expected on the basis of the Ki of this inhibitor as determined in cell-free assays with synthetic substrates (29). The capacity of the CMTs to inhibit LF which has already entered viable cells is especially noteworthy in view of the capacity of B. anthracis to spread systemically and establish a toxemia rapidly within the infected host, thereby limiting the window of efficacy of an agent which can function only in a preexposure prophylactic mode.
It has been reported that mixtures of LF and PA show toxicity to a subset of cell types in selected animal models (22, 42). In studies to be reported in greater detail subsequently, we have found that mixtures of LF and PA show some dose-dependent cytotoxicity to human monocytes isolated from peripheral blood with a loss of viability of less than 50% after 24 h as judged by MTS reduction but exhibit no significant dose-dependent cytotoxicity to the transformed MonoMac 6 cell line. However, the two CMTs and Ilomastat administered in preexposure prophylactic mode appear to block the LF-induced cytotoxicity which can be detected after 24 h in monocytes, while the CMTs can also diminish cytotoxicity when administered in postexposure prophylactic mode; none of the agents induces cytotoxicity in MonoMac 6 cells at doses which completely inhibit LF-mediated MEK cleavage (data not shown). The mechanisms of cytotoxicity induced by lethal factor remain uncertain and may not be applicable across species (murine mononuclear cells appear to be far more sensitive than human cells [36]). More pertinent to the underlying mechanism of the morbidity and mortality associated with anthrax, a consensus is emerging that the exceptionally rapid dissemination of B. anthracis in infected animals and humans reflects compromise of both the innate and adaptive components of the immune response, only part of which may be attributed to effects on macrophage functions (35). It is noteworthy that the three agents we have evaluated are all effective inhibitors of LF-mediated MEK cleavage in human dendritic cells, which have an essential role in adaptive immunity. It has been speculated that some aspects of the systemic complications of the late stages of anthrax pathology in humans may arise from attack by bacterial components on target cells other than mononuclear phagocytes, resulting in proteolysis of host proteins other than the MEKs (28). Given the uncertainties over the details of the pathway by which infection with B. anthracis triggers such morbidity and mortality in humans, the results reported here are insufficient for us to conclude that the CMTs or Ilomastat will achieve total therapeutic benefit in vivo comparable to their efficacy in the in vitro studies we have undertaken so far. However, the absence of significant toxicity and minimal risk of facilitating antibiotic resistance, combined with high oral availability and potency at circulating levels against the proteolytic activity of the most pathogenic toxin produced by B. anthracis, suggests that the CMTs and, for more restricted applications, Ilomastat offer promise for management of populations which might have been exposed to spores of the anthrax bacillus or are simply at high risk for such exposure.
ACKNOWLEDGMENTS
We are grateful to our colleagues in S. R. Simon's laboratory who have provided assistance in this project. We thank Richard Kew for use of the SpectraMax M2 microplate reader and members of Ute Moll's lab for providing GAPDH antibody. We are also indebted to Collagenex Pharmaceuticals, Inc., for their generous supply of CMT-300 and CMT-308.
This work was supported by NIH (NIAID) R21-AI53524 and was submitted by S.S.K. in partial fulfillment of the requirements for the Ph.D. Program in Molecular and Cellular Biology (State University of New York at Stony Brook).
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