A peptide-based fluorescence resonance energy transfer assay for Bacillus anthracis lethal factor protease

Abstract

A fluorescence resonance energy transfer assay has been developed for monitoring Bacillus anthracis lethal factor (LF) protease activity. A fluorogenic 16-mer peptide based on the known LF protease substrate MEK1 was synthesized and found to be cleaved by the enzyme at the anticipated site. Extension of this work to a fluorogenic 19-mer peptide, derived, in part, from a consensus sequence of known LF protease targets, produced a much better substrate, cleaving approximately 100 times more efficiently. This peptide sequence was modified further on resin to incorporate donor͞quencher pairs to generate substrates for use in fluorescence resonance energy transfer-based appearance assays. All peptides cleaved at similar rates with signal͞background ranging from 9 -16 at 100% turnover. One of these substrates, denoted (Cou)Consensus(K(QSY-35)GG)-NH2, was selected for additional assay optimization. A plate-based assay requiring only low nanomolar levels of enzyme was developed for screening and inhibitor characterization.F atalities resulting from the recent bioterrorist attacks in the U.S. have highlighted the inadequacy of available medical treatments for anthrax infection. Although Bacillus anthracis, the causative agent of anthrax, can be cleared from the host with antibiotics, the ongoing action of the secreted anthrax toxin limits the clinical efficacy of these drugs (1). A pharmacological agent that directly targets the toxin might thus prove to have valuable synergy with more traditional antibacterial treatments.The anthrax toxin consists of three proteins, a receptorbinding component designated protective antigen, and two enzymatic components termed edema factor and lethal factor (LF) (2). Edema factor is a calmodulin-dependent adenylate cyclase, whereas LF is a zinc-dependent metalloprotease that has been shown to cleave near the N termini of several MAP kinase kinases (MKKs). Although the complete mechanism of pathogenesis is unclear, the disruption of key signaling pathways mediated by MKKs seems to lead first to the lysis of macrophages and later to the death of the host. The pivotal role of LF in the virulence of the toxin suggests that inhibitors of the enzyme may provide protection against cytotoxicity.The only known inhibitors of LF are weak hydroxamates (IC 50 s Ͼ300 M) and chelating agents such as EDTA and ortho-phenanthroline (3). Existing assays for LF activity, such as SDS͞PAGE (4, 5) or HPLC (3), are impractical for highthroughput screening of compound collections. Identification of potent and selective LF inhibitors requires an assay that is less labor-intensive, has faster turnaround, and utilizes low levels of purified enzyme. In this article we describe the development of a peptide-based fluorescence resonance energy transfer (FRET) assay for LF protease that meets these criteria. Materials and MethodsSubstrate Synthesis. Peptide synthesis was carried out on an ABI Model 433A peptide synthesizer using FastMoc chemistry with increased acylation times on a 250-mol scale. All reagents, including...

Anthrax protective antigen is a calcium-dependent serine protease

“…Additionally, PA 83 is able to hydrolyze a protease substrate of which the sequence is based on MAPKK [20], suggesting that PA 83 might possess LF-like activity. Thus it is hypothesized that by inhibiting the proteolytic activity of PA 83 it may be possible to decrease the pathogenic potential of B. anthracis .…”

“…20 µg/mL PA 83 was incubated with 16 µM FRET-peptide PEK-054 (FITC-NleKKKKVLPIQLNAATDK-KDbc) [20] in the presence of varying concentrations of either protease inhibitors or lethal toxin formation inhibitors including 4-bromobenzaldehyde N -(2,6-dimethylphenyl) semicarbazone (EGA), calpain-inhibitor MDL281700, furin inhibitor II (hexa- d -arginine (D6R)), EDTA, ZnCl 2 , N-ethylmaleimide (NEM) and aprotinin in HEPES buffer (20 mM HEPES, 0.05% Tween-20, pH 8.2). To study the effect of metal ions on PA 83 proteolytic activity, 20 µg/mL PA 83 was incubated with 16 µM FRET-peptide PEK-054 in HEPES buffer (20 mM HEPES, 0.05% Tween-20, pH 8.2) supplemented with 1 mM EDTA.…”

Progress in rapid screening of Bacillus anthracis lethal factor activity

“…Such drugs could be used in association with antibiotics to treat anthrax. The need for specific therapeutics to treat anthrax was shown clearly during the recent bioterrorist attacks in the United States as underlined by Cummings et al (12) in this issue of PNAS.…”

“…Nevertheless, the rate of substrate cleavage and sensitivity of the assay are adequate for inhibitor screening, although the assay can be disturbed by the fluorescence of tested inhibitors. As a result, Cummings et al are looking for other fluorophore͞quencher pairs (12). As yet, no efficient inhibitor of LF has been described, which contrasts with tetanus and botulinum toxins for which molecules with a thiol group acting as a zincchelating reagent were reported recently to exhibit micromolar and nanomolar affinities, respectively (15,16).…”

“…Selective cleavage by LF at a single site was verified by HPLC, and the sequences of the two metabolites formed were established by mass spectrometry and not by the more traditional method of comparing the retention times in HPLC of synthetic metabolites. The efficacies of cleavage of these substrates were determined, and one with a rate of cleavage Ϸ100-fold higher than the native MEK-1 substrate was selected (12).…”