Protective antigen having fluorinated histidine residues

#Not applicable.

#1. Field of the Invention

#In general, the invention is directed to compounds derived from protective antigen and methods for the treatment of anthrax and for understanding the mechanisms involved in anthrax infection.

#FIG. 1 shows the ESI-MS analysis of wild-type (“WT”) PA83 (FIG. 1A) and 2-FHisPA83 (FIG. 1B). FIG. 1C shows the amino acid sequence (SEQ ID NO: 1) of the full-length PA83 protein =including the pelB leader peptide, which directs the protein for export to the periplasm and is cleaved off to give the mature polypeptide (MW=83748.4 Da). The ESI spectra were acquired on a Finnigan LTQ-FT mass spectrometer.

#FIG. 2 shows cytotoxicity as determined by protein synthesis inhibition mediated by PA83 or 2-FHis PA83 and LFN-DTA (fusion of LFN and the catalytic domain of diphtheria toxin) into the cytosol of CHO-K1 cells. Plotted are averages of three or more experiments with error bars showing the standard deviation. Lines shown are data fitted to a standard EC50 equation. Error bars represent the standard deviation. The insert shows an autoradiogram for a western blot of an SDS-PAGE of the cell lysate (right panel) of CHO-K1 cells incubated with either 5 μg WT PA83 (lanes 1 and 3) or 2FHis PA83 (lanes 2 and 4) as indicated and probed using an anti-PA antibody.

#FIG. 3A shows urea denaturation of PA83 (●) and 2-FHis PA83 (▪) measured using fluorescence (excitation 280 nm). Spectra represent the normalized emission intensity at 330 nm (ex. 280 nm). The data were fit using non-linear least squares analysis to a three-state NIU model. The inset in FIG. 3A shows the urea gradient gel electrophoresis of PA83 and 2-HisPA83. Urea gradient gels were pre-run for 30 minutes at a constant current of 20 mA. Samples (25 μg in 75 μL) were applied to the top of the gels, and gels run for 16 hours at 20° C., 20 mA. FIG. 3B shows urea denaturation of WT PA83 (●) and 2-FHis PA83 (▪) as measured in FIG. 3A, recording the emission wavelength maxima at each urea concentration. All spectra were recorded at 20° C., in 20 mM Bis-Tris/HEPES/cacodylic acid pH 8.0, and represent the average of 10 scans.

#In the present invention, the effects of the biosynthetic incorporation of 2-FHis in place of histidine on the biochemical properties of the PA83 and the heptameric (PA63)7, the ability to form a pore, and the ability to function in planar lipid bilayers and in CHO-K1 cells was determined. There are a total of ten histidines in the wild-type PA83 and four of the five histidine residues in domain 2 are part of the long β-barrel which comprises the transmembrane channel. The compound 2-FHis (shown below) is an isosteric analog of histidine with a side-chain pKa of about 1. These experiments are based upon a theory that protonation of histidine residues are important for the pre-pore to pore conversion of (PA63)7.

#In the present invention, it was shown that the modified PA proteins blocked translocation of a model cellular effector, LFN-DTA, and also blocked cytotoxicity of the model cellular effector LFn-DTA, which upon entry into the cell cytosol through the pore blocks protein synthesis. However, the modified PA protein retains the ability to form a pore in the absence of the VWA domain of the cellular receptor, indicating that the structural features required for pore formation are retained. In addition, the structural properties of the protein as assessed by circular dichroism spectroscopy and stability to the chemical denaturant urea indicate that there is little perturbation to the structure of the protein as a result of 2-FHis incorporation.

#By an “effective amount” is meant herein an amount that is effective to elicit a desired response. For example, an effective amount of an immunogenic composition is an amount that is effective to elicit a detectable immune response. An effective dose can be determined empirically, according to conventional procedures, taking into account well-known factors such as the age, weight, and/or clinical condition of the subject, the method of and scheduling of administration, and the like.