We propose a pharmacologic strategy to combat the effects of LeTx of Bacillus anthracis, the cause of anthrax, based on the pleiotropic actions of non-antimicrobial CMTs. The CMTs are inhibitors of zinc matrix metalloproteases (MMPs), but they also can inhibit zinc metalloproteases of bacterial origin. Lethal Factor (LeF), the proteolytic subunit of LeTx, is a zinc metalloprotease with a catalytic domain similar to thermolysin and is a potential target of the CMTs as well. The human cell most sensitive to LeTx is the macrophage, which is activated through proteolysis of multiple MAPK kinases (MAPKKs) to trigger a systemic inflammatory response that may develop potentially fatal pulmonary complications of Acute Respiratory Distress Syndrome (ARDS). We have shown that, in addition to their inhibition of MMPs, the CMTs downregulate responses of activated macrophages, including release of MMPs and generation of nitric oxide (NO). With collaborators at SUNY Syracuse, we have shown that in multiple porcine models of ARDS, administration of CMT-3 prevents the otherwise fatal progression of lung damage while leukocyte respiratory burst activity, leukocyte infiltration of the lung alveolar spaces, and levels of leukocyte-derived proteases are all diminished. In vitro treatment with CMT-3 of the blood from human patients whose leukocytes were activated in vivo also results in a diminished leukocyte respiratory burst. We therefore propose to evaluate the inhibitory potency of CMT-3 and CMT-308 (less cytotoxic and photosensitizing than CMT-3) against the proteolytic activity of purified LeF, initially using a novel fluorogenic synthetic oligopeptide substrate specific for this metalloprotease. We will then evaluate the capacity of the CMTs to inhibit LeF-mediated cleavage of the natural macrophage substrates Mek-1 and Mek-2, using human macrophage Iysates as sources of the MAPKKs and detection by Western blotting. Finally, we will evaluate the capacity of the CMTs to suppress the inflammatory response of LeF-exposed human monocytes, monocyte-derived macrophagess, and a human monocytoid cell line, Mono Mac 6, by introducing LeF alone directly into the macrophage cytosol with a protein transfection agent (Chariot(tm), Active Motif Corp.). We will measure release of reactive oxygen species by DGFH oxidation, NO release with ozone chemiluminescence, secretion of MMPs by zymography with subsequent quantitation by ELISAs, and release of cytokines, also by ELISAs, as indicators of extent of LeF-triggered macrophage activation in the absence and presence of CMTs. Because GMT-3 has already been shown to be safe in Phase I trials with normal volunteers, if efficacy can be shown, it may potentially be rapidly deployed for protection in cases of possible anthrax exposure.
| Kocer, S S; Matic, M; Ingrassia, M et al. (2008) Effects of anthrax lethal toxin on human primary keratinocytes. J Appl Microbiol 105:1756-67 |
| Kocer, Salih S; Walker, Stephen G; Zerler, Brad et al. (2005) Metalloproteinase inhibitors, nonantimicrobial chemically modified tetracyclines, and ilomastat block Bacillus anthracis lethal factor activity in viable cells. Infect Immun 73:7548-57 |
