Metalloproteinase inhibitors, nonantimicrobial chemically modified tetracyclines, and ilomastat block Bacillus anthracis lethal factor activity in viable cells

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.

FIG. 1.

Dose-dependent inhibition of synthetic peptide cleavage with LF. LF was preincubated with the indicated quantities of inhibitors (▪, 0 μM; •, 1 μM; ▴, 10 μM; ⧫, 100 μM) for 10 min at room temperature in a 500-μl volume of Dulbecco's phosphate-buffered saline (pH 8.2). The peptidolytic reaction was started by adding MAPKKide (o-ABZ/DNP), a synthetic peptide containing a specific cleavage site for B. anthracis lethal factor, into the mixtures of LF and inhibitors, and fluorescence intensity was recorded over time using a λ (ex) value of 320 nm and a λ (em) value of 420 nm in an LS-5 fluorescence spectrophotometer. The reactions were monitored for a 2-h duration to establish the period over which the apparent velocity could be used as an estimate of initial velocity.

FIG. 2.

Dixon plots of reciprocal velocity against inhibitor concentrations in presence of fixed levels of LF and substrate. LF was preincubated with CMT-300 (0, 2, 10, or 50 μM), CMT-308 (0, 5, 10, or 20 μM) and Ilomastat (0, 6.875, 13.75, or 27.5 μM) for 10 min at room temperature in DPBS (pH 8.2). Reactions were started by adding different fixed concentrations of MAPKKide (o-ABZ/DNP) (▪, 15 μM; ▴, 20 μM; •, 25 μM) into 100-μl aliquots of the LF inhibitor mixtures in 96-well microplates, and fluorescence intensity was recorded for 10 min using a λ (ex) value of 320 nm and a λ (em) value of 420 nm in a SpectraMax M2 microplate reader. The reactions were carried out at room temperature. The reciprocal values of the initial velocities of peptide cleavage determined from the fluorescence readings (1/v) were plotted versus the inhibitor concentrations at which the determinations were made, [I]. Experiments were repeated at least three times. The figure shows one set of results for each inhibitor at two of the three substrate concentrations.

FIG. 3.

LF proteolytic activity towards MEK-2 (A) and MEK-6 (B) in vitro. Ten nanograms of LF was incubated with indicated amounts of inhibitors for 15 min at room temperature. Then, LF proteolytic activity was evaluated using MonoMac 6 cell lysate (5 μg total protein) as a source of MEKs. The reactions were stopped at the indicated times, and the products were subsequently separated by SDS-PAGE and transferred to nitrocellulose membranes. Blots were probed with polyclonal antibodies specific for the MEK-2 N terminus (A) and the MEK-6 C terminus (B). Experiments were repeated at least three times.

FIG. 4.

Inhibition of LF proteolytic activity towards MonoMac 6 cell lysates. One nanogram of LF was incubated with 0, 5, 10, or 15 μM CMT-300, CMT-308, or Ilomastat at room temperature for 15 min. Proteolytic activity of LF which had been preincubated with the specified agents was then evaluated against MEK-6 in MonoMac 6 lysate (5 μg protein) for 1 h at 37°C in a total reaction volume of 10 μl as described in Materials and Methods. Reaction products were separated and transferred as described for Fig. 3. Blots were incubated with a polyclonal antibody specific for MEK-6. Experiments were repeated three times.

FIG. 5.

Preexposure prophylactic inhibition of LF proteolytic activity on MEK-2 in intact cells. LF was first incubated with indicated inhibitors (10 μM CMT-300 or CMT-308; 11 μM Ilomastat or GM1489) at room temperature for 10 min. (A) MonoMac 6 cells were incubated with LF plus PA (50 plus 500 ng/ml) in the presence of the inhibitors or with an uninhibited mixture of LF plus PA for the indicated times. Cells were lysed as described in Materials and Methods, and reaction products in 5-μg total protein aliquots were separated and transferred as for Fig. 3. Blots were probed with a polyclonal antibody specific for the MEK-2 N terminus. (B) Human monocytes were incubated with LF plus PA (100 plus 200 ng/ml) in the presence or absence of inhibitors for the indicated times. Reaction products were separated and transferred as described for Fig. 3. Blots were probed with a polyclonal antibody specific for the MEK-2 N terminus. (C) Human dendritic cells were incubated with LF plus PA (100 plus 200 ng/ml) in the presence or absence of inhibitors for the indicated times. Reaction products were separated and transferred as described for Fig. 3. Blots were probed with a polyclonal antibody specific for MEK-2 N terminus. All experiments were repeated three times.

FIG. 6.

Postexposure prophylactic inhibition by CMTs and Ilomastat towards LF proteolytic activity within intact cells. MonoMac 6 cells were incubated with a mixture of 50 ng/ml LF and 200 ng/ml PA for 45 min. Cells were washed once with HBSS (HyClone) containing the indicated amounts of inhibitors (CMT-300 and CMT-308, 10 μM; Ilomastat and GM1489, 11 μM) at room temperature and were centrifuged. The cells were then suspended in Macrophage SFM (Gibco) containing the same concentrations of inhibitors at 37°C, and incubation was continued at 37°C for the indicated times. Cells were lysed as described in Materials and Methods, and reaction products in 5-μg total protein aliquots were separated and transferred as described for Fig. 3. (A) Blots were probed with a polyclonal antibody specific for the MEK-2 N terminus. Experiments were repeated four times. (B) Blots were probed with GAPDH antibody to confirm equivalent loading of cell proteins into all wells of the gel.