Inhibition of the Proteolytic Activity of Anthrax Lethal Factor by Aminoglycosides

Abstract

The anthrax lethal factor (LF), a Zn-dependent endopeptidase, is considered the dominant virulence factor of anthrax. Because pharmacological inhibition of the catalytic activity of LF is considered a plausible mechanism for preventing the lethality of anthrax, a high-throughput screening experiment based on LF-catalyzed cleavage of a fluorescent substrate was performed to identify novel inhibitors of LF. The RNA-targeting antibiotics, neomycin B and some synthetic dimeric aminoglycosides, were found to be nanomolar active-site inhibitors of LF.

High-Affinity, Human Antibody-Like Antibody Fragment (Single-Chain Variable Fragment) Neutralizing the Lethal Factor (LF) of Bacillus anthracis by Inhibiting Protective Antigen-LF Complex Formation

“…A cooperative effect between an anti-LF and an anti-PA antibody has been described previously (7), so we looked for such cooperation between 2LF and our anti-PA Fab, 35PA 83 (26). The assays were performed in vitro, because not enough Fab was available for in vivo tests.…”

“…For these reasons, and because a cooperative effect between an anti-LF and an anti-PA antibody has already been described in vivo (6), we have developed an anti-LF scFv which is apparently the first recombinant antibody fragment of this specificity ever obtained. This contrasts with both (i) the large number of anti-PA antibodies that have been developed (17,36,38,50,56), sometimes by exploiting the availability of lymphocyte donors immunized with anthrax vaccines composed mainly of PA, and (ii) the many synthetic molecules that have been developed to inhibit the enzymatic (proteolytic) activity of LF (15,24,26,32,37,42,52,55) but whose bioavailability and tolerance are not guaranteed.…”

“…However, no recombinant antibody directed against LF has been reported, as far as we are aware. Furthermore, although many synthetic molecules have been designed to inhibit the enzymatic activity of LF (15,24,26,32,37,42,52,55), neither their bioavailability nor their tolerance is guaranteed. We report here the first recombinant neutralizing antibody directed against LF, obtained after immunization of a nonhuman primate (NHP) and showing human-like framework regions (FRs), as expected (10,31).…”

Microsphere‐based protease assays and screening application for lethal factor and factor Xa

“…FRET studies using small peptides demonstrated 100-fold faster cleavage of a consensus cleavage sequence (derived from MAPKK cleavage sites) when compared with natural sites (8). Using FRET peptides, small molecule inhibitors with K i 's ranging from 0.5 lM to 7 nM have been isolated (13,14). Absorbance methods using p-nitroanilide coupled peptides in HTS screens were used to select the peptide inhibitor IN-2-LF (9).…”

Development of a cell-based fluorescence resonance energy transfer reporter for Bacillus anthracis lethal factor protease

“…Interestingly, several natural products, including defensin peptides [33; 34] and aminoglycosides [35], have been recently shown to inactivate LF factor.…”