WO1999042473A1 - Inhibition de la translocation d'une toxine - Google Patents

Inhibition de la translocation d'une toxine Download PDF

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WO1999042473A1
WO1999042473A1 PCT/US1999/003457 US9903457W WO9942473A1 WO 1999042473 A1 WO1999042473 A1 WO 1999042473A1 US 9903457 W US9903457 W US 9903457W WO 9942473 A1 WO9942473 A1 WO 9942473A1
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toxin
pore
forming
mutant
mts
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PCT/US1999/003457
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WO1999042473A9 (fr
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R. John Collier
Erika L. Benson
Alan Finkelstein
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President And Fellows Of Harvard College
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention relates generally to the prevention and treatment of toxicity due to pore- forming toxins, particularly A-B toxins.
  • Intracellularly acting bacterial toxins enzymatically modify specific intracellular constituents of eukaryotic target cells. Intracellularly acting bacterial toxins must cross a membrane barrier of host cells to reach their cytosolic targets. Most intracellularly acting toxins may be classified as A-B toxins, where the B moiety binds to the surface of the host cell and translocates the enzymatic A moiety into the cytosol (Gill, D. M., in Bacterial Toxins and Cell Membranes, Jeljaszewicz, J., & Wadstrom, T., Eds., pp 291-322, Academic Press, New York, 1978).
  • membrane translocation of the A moiety involves insertion of the B moiety into the host membrane, resulting in the formation of an ion-conducting pore.
  • X-ray crystal structures have been solved for the water soluble forms of two pore-forming A-B toxins and also of other toxins that function simply by forming pores at the cell surface (Parker, M. . et al., Nature 337:93-96, 1989; Li, J. D. et al, Nature 353:815-21, 1991; Choe, S. et al., Nature 357:216-22, 1992; Parker, M. W. et al., Nature 367, 292-95, 1994; Petosa, C.
  • bacterial toxins such as diphtheria toxin
  • a and B moieties of binary toxins are contained in separate proteins which self-assemble at the mammalian cell surface.
  • An example of a binary toxin is anthrax.
  • the B moiety Protective Antigen (PA)
  • PA Protective Antigen
  • EF Edema Factor
  • LF Lethal Factor
  • Insertion of the PA 63 heptamer into the endosomal membrane is believed to mediate translocation of EF and LF. Elucidation of the insertion mechanism of pore - forming toxins would allow prevention of the translocation process, which, in turn, could provide an effective means for reducing toxicity associate with these viruses.
  • the invention features a purified protein that includes a mutant pore-forming toxin, wherein the toxin comprises a mutation of an amino acid that forms the transmembrane pore of the toxin.
  • the mutation is a missense mutation or a deletion, is genetically engineered, and reduces the toxicity of a pore- forming toxin.
  • the mutation inhibits the translocation of the wild-type pore-forming toxin across the transmembrane pore, and decreases conductance measured at the transmembrane pore.
  • Additional embodiments of this aspect include a mutant pore- forming toxin that is a mutant B-moiety of an A-B toxin, a mutant B-moiety of a binary toxin, preferably, the B-moiety is an anthrax toxin protective antigen (PA), or its C-terminal 63 kDa tryptic fragment (PA 63 ).
  • PA anthrax toxin protective antigen
  • PA 63 C-terminal 63 kDa tryptic fragment
  • the mutation is located in the amino acid sequence that includes the transmembrane pore of the pore-forming toxin, preferably, at the hydrophilic face of a transmembrane pore, preferably, in the D2L2 loop, more preferably, in the D2L2 loop of PA or PA 63 , and, most preferably, in amino acid residue E302, H304, N306, E308, H310, S312, F313, F314, D315, G317, S319, S321, G323, or S325 of the D2L2 loop.
  • mutant pore-forming toxin is streptolysin O, -toxin, A-B toxin (staph- ⁇ or thiol-activated), or binary toxin
  • the mutant pore-forming toxin is anthrax.
  • the invention features a substantially pure nucleic acid sequence encoding the amino acid sequence of the mutant pore- forming toxin protein, wherein the toxin has a mutation in an amino acid that forms the transmembrane pore of said toxin.
  • the mutation in the pore-forming protein is a missense mutation or a deletion, is genetically engineered, and reduces the toxic response to a pore- forming toxin.
  • the mutation in the pore- forming toxin inhibits the translocation of the wild-type pore-forming toxin across the transmembrane pore, and decreases conductance measured at the transmembrane pore.
  • Additional embodiments of this aspect include a nucleic acid encoding a mutant pore-forming toxin that is a mutant B-moiety of an A-B toxin, a mutant B- moiety of a binary toxin, preferably, the B-moiety is an anthrax toxin protective antigen (PA), or its C-terminal 63 kDa tryptic fragment (PA 63 ).
  • PA anthrax toxin protective antigen
  • PA 63 C-terminal 63 kDa tryptic fragment
  • the mutation is located in the amino acid sequence that includes the transmembrane pore of the pore- forming toxin, preferably, at the hydrophilic face of a transmembrane pore, preferably, in the D2L2 loop, more preferably, in the D2L2 loop of PA or PA 63 , and, most preferably, in amino acid residue E302, H304, N306, E308, H310, S312, F313, F314, D315, G317, S319, S321, G323, or S325 of the D2L2 loop.
  • nucleic acid encodes a mutant pore- forming toxin that is streptolysin O, ⁇ -toxin, A-B toxin (staph- ⁇ or thiol-activated), or binary toxin (diptheria or exotoxin A), preferably, the mutant pore-forming toxin is anthrax.
  • the invention features a method of decreasing the toxic response to a pore-forming toxin, that includes administering a protein that is a mutant pore- forming toxin, wherein the toxin includes a mutation of an amino acid that forms the transmembrane pore of the toxin, in a dose sufficient to inhibit translocation of a pore-forming toxin.
  • the mutant protein functions as a dominant mutant to inhibit translocation of a wild- type pore-forming toxin, the protein is administered prophylactically, and the protein generates antibody production directed against a wild-type sequence pore-forming toxin.
  • the toxin's mutation is a missense mutation or a deletion, is genetically engineered, and reduces the toxic response to a pore-forming toxin.
  • the mutation inhibits the translocation of the wild- type pore-forming toxin across the transmembrane pore, and decreases conductance measured at the transmembrane pore.
  • Additional embodiments of this aspect include administering a mutant pore- forming toxin that is a mutant B-moiety of an A-B toxin, a mutant B-moiety of a binary toxin, preferably, the B-moiety is an anthrax toxin protective antigen (PA), or its C-terminal 63 kDa tryptic fragment (PA 63 ).
  • PA anthrax toxin protective antigen
  • PA 63 C-terminal 63 kDa tryptic fragment
  • the mutation is located in the amino acid sequence that includes the transmembrane pore of the pore-forming toxin, preferably, at the hydrophilic face of a transmembrane pore, preferably, in the D2L2 loop, more preferably, in the D2L2 loop of PA or PA 63 , and, most preferably, in amino acid residue E302, H304, N306, E308, H310, S312, F313, F314, D315, G317, S319, S321, G323, or S325 of the D2L2 loop.
  • Additional embodiments provide for administering mutant pore-forming toxin that is streptolysin O, ⁇ -toxin, A-B toxin (staph- ⁇ or thiol-activated), or binary toxin (diptheria or exotoxin A), preferably, the mutant pore-forming toxin is anthrax.
  • “Mutation” means an alteration in the nucleic acid sequence such that the amino acid sequence encoded by the nucleic acid sequence has at least one amino acid alteration from the wild-type sequence.
  • the mutation may, without limitation, be an insertion, deletion, frameshift mutation, or a missense mutation, or other modification which alters protein function as described herein.
  • Dominant mutant toxin means a mutant toxin which effectively inhibits the translocation of wild type toxin by forming transmembrane pore hybrid heptamers with the wild-type pore-forming toxin proteins and interfering with normal pore formation or translocation of toxin.
  • Pore-forming toxin means a toxin which traverses the membrane barrier through its transmembrane pore and enzymatically modifies specific intracellular substrates of a host cell.
  • Pore-forming A-B toxin means a pore-forming toxin with two functional moieties; one moiety (B) which forms a pore in a host cell barrier membrane, and the other (A) traverses the membrane barrier through the transmembrane pore and enzymatically modifies specific intracellular substrates of a host cell.
  • Binary toxin means a pore-forming A-B toxin in which the A and B moieties of the pore- forming toxin inhabit separate proteins, and interact during the intoxication of host cells.
  • An example of a binary toxin is anthrax toxin.
  • B moiety means the component of a pore- forming A-B toxin which binds a specific host cell-surface receptor, forms a transmembrane pore in the host cell membrane, binds the A moiety toxin, and functions to translocate the A moiety into the host cell cytoplasm.
  • PA Protective antigen
  • PA 63 means the carboxy- terminal portion that results from proteolytic cleavage of a 20 kDa N-terminal segment from the PA polypeptide. PA 63 forms a heptameric prepore and binds the two alternative A moieties, edema factor (EF) and lethal factor (LF). The entire complex is trafficked to the endosome, where PA 63 inserts into the membrane, forms a transmembrane pore, and translocates EF and LF into the host cell cytoplasm.
  • EF edema factor
  • LF lethal factor
  • Transmembrane pore means the ⁇ -barrel channel formed by alternating hydrophilic and hydrophobic residues of the pore- forming toxin protein such that the hydrophobic residues form an exterior membrane-contiguous surface of the barrel, and the hydrophilic residues face an aqueous lumen of a pore that spans across the host cell membrane.
  • D2L2 loop means the amphipathic loop which connects strands 2 ⁇ 2 and 2 ⁇ 3 of PA polypeptide and PA 63 polypeptide as described herein.
  • "Decreased conductance measured at the transmembrane pore” means a reduction in conductance at the transmembrane pore of the toxin in response to derivatization of an amino acid residue on the hydrophilic face by a positively charged reagent, as measured by macroscopic or single channel analysis.
  • the positively charged reagent is methanethiosulfonate ethyl sulfate (MTS-ET).
  • the decrease in conductance is preferably at least 20% relative to the conductance across the transmembrane pore of a wild-type pore- forming toxin in the presence of the positively charged reagent. More preferably, the decrease is at least 40%.
  • “Inhibit translocation” means a reduction in the process by which the toxin moiety of pore- forming toxin is transferred through the transmembrane pore into the host cell cytoplasm. This decrease in toxin translocation is positively correlated with, and could be predicted by, a decrease in conductance measured at the transmembrane pore in response to derivatization by a positively charged mutant (as previously described at page 9, line 14 to page 10, line 1).
  • the decrease in translocation results in decreased toxin transfer by at least 20% relative to toxin transfer by wild-type PA 63 . More preferably, the decrease is at least 40%.
  • Fig. 1A shows the amphipathic sequences of the D2L2 loop of PA and the Gly-rich loop of ⁇ -hemolysin (aHL).
  • Residues that form the hydrophobic face of the ⁇ barrel in a-hemolysin, or have been proposed to form the hydrophobic face of the PA 63 pore are underscored with a solid line.
  • Residues that form the hydrophilic face in ⁇ -hemolysin, or have been proposed to form the hydrophilic face of the PA 63 pore are underscored with a dotted line.
  • Fig. IB shows the proposed model for pore formation by PA 63 .
  • the D2L2 loops move to the base of the heptamer and combine to form a 14-stranded transmembrane ⁇ barrel.
  • Fig. 2 shows the effect of MTS-ET on N306C-induced macroscopic conductance.
  • the current record (with the voltage held at +20 mV) begins 7 minutes after addition of trypsin-nicked PA N306C to a final concentration of 1.1 nM.
  • MTS-ET was added trans to a concentration of 38 rnM. After the initial artifactual increase in conductance due to the addition, the current is seen to decrease 6-fold within seconds.
  • Fig. 3 shows the reduction of conductance produced by trans MTS-ET as a function of location of Cys mutants within the D2L2 loop. Percent reduction was calculated as [1-(IPA+MTS/IPA)] x 100, where IPA was the current immediately before MTS addition, and IPA+MTS was the lowest current observed within three minutes following addition of MTS-ET. Values are reported as the mean +/- standard error of 2-4 experiments. For A307C and 1316C, MTS-ET-induced reduction in current was only seen in some experiments. Note the alternating pattern of reduction in conductance, except for the consecutive MTS-ET-responsive residues 312-315.
  • Fig. 4A shows the effect of MTS-ET on conductance of a single N306C channel.
  • the trace begins after a single channel has opened with a conductance of 90 pS (at a holding potential of +50 mV).
  • MTS-ET was then added to a concentration of 8 mM trans, and the current record was briefly obscured during stirring.
  • One or more reactions occurred during stirring, since after the stirring was stopped the conductance of the channel was about half that before MTS-ET addition.
  • the arrows indicate stepwise decreases in single channel conductance consistent with the reaction of MTS-ET with cysteines within the channel.
  • Fig. 4B shows that DTT reverses the MTS-ET effect.
  • the trace begins five minutes after the final MTS-ET reaction was observed in Fig. 4A.
  • DTT was added trans to a concentration of 1.2 mM, and the single channel conductance immediately increased during stirring.
  • the arrows indicate further stepwise increases in single channel conductance, consistent with reduction of the mixed disulfides formed upon reaction of the cysteines with MTS-ET.
  • the transition at the second arrow appears to be composed of 2-4 stepwise increases in conductance. (The last break in the record is 90 seconds.)
  • Fig. 5 A shows that MTS-ET does not restrict the accessibility of cis Bu4N+ to its binding site.
  • Current records (with voltage held at +20 mV) begin 3-5 minutes after addition of trypsin-nicked PA S325C.
  • the arrows mark addition of Bu4N+.
  • the addition of Bu4N+ cis to a concentration of 15 mM produced a 3.5-fold reduction in conductance.
  • Fig. 5B shows that MTS-ET does not restrict the accessibility of trans Bu4N+ to its binding site. Addition of Bu4N+ trans to a concentration of 500 mM produced a 2-fold reduction in conductance.
  • Fig. 5C shows that addition of Bu4N+ cis to a concentration of 15 mM to S325C channels previously modified with MTS-ET produced the same 3.5-fold fall in conductance as in Fig. 5A, despite the conductance having previously been substantially reduced due to modification of the channels' cysteines with MTS-ET.
  • Fig. 5D shows that addition of 500 mM Bu4N+ trans to S325C channels previously reacted with MTS-ET had essentially no effect on conductance; subsequent addition of 15 mM Bu4N+ cis produced its usual 3.5-fold fall in conductance.
  • cysteines at positions 325 modified with MTS-ET Bu4N+ added trans is prevented from reaching its binding site, whereas Bu4N+ added cis is not.
  • Fig. 6 shows a model for the orientation of D2L2 within the membrane.
  • the filled boxes indicate residues that are responsive to MTS-ET, based upon reduction of channel conductance.
  • the open boxes indicate residues that show little or no effect upon MTS-ET addition.
  • the pattern is consistent with each D2L2 contributing two antiparallel ⁇ strands to make a 14-stranded ⁇ barrel.
  • the invention provides an antidote for use in treating exposure to pore forming toxins.
  • the proteins of the invention also have immunogenic properties which may enhance host response to pore forming toxin exposure.
  • PA 63 forms cation-selective channels in artificial membranes (Blaustein, R. O. et al, Proc Natl Acad Sci U S A 86:2209-13, 1989; Koehler, T. M, & Collier, R. J, Mol. Microbiol. 5: 1501-506, 1991) and in cell membranes (Milne, J. C. & Collier, R. J, Molec. Micro. 10:647-53, 1993).
  • Purified PA 63 was originally shown by electron microscopy to form ring-shaped heptamers (Milne, J. C. et al, J. Biol. Chem. 269: 20607-12, 1994), and recently the X-ray crystal structure of a heptameric, water soluble form of PA 63 was determined (Petosa, C. et al. Nature 385;833-38,1997). Since this structure shows no regions of hydrophobicity that might mediate membrane insertion, this water soluble form structure may represent an intermediate, or prepore, in the insertion process.
  • the crystal structures of native PA and the PA 63 prepore reveal the presence of a disordered, amphipathic loop (D2L2), which has alternating hydrophilic and hydrophobic residues reminiscent of the Gly-rich loop of the ⁇ -hemolysin (Fig. 1).
  • This loop which connects strands 2 ⁇ 2 and 2 ⁇ 3 within domain 2, projects outward from the side of domain 2 of each monomer within the water soluble heptamer (Petosa, C. et al. Nature 385;833-38,1997).
  • a significant conformational rearrangement would be needed for this loop to participate in barrel formation, but a plausible mechanism for such a rearrangement has been proposed (Fig. 2) (Petosa, C. et al. Nature 385;833-38,1997).
  • each hairpin is in turn derived from a Gly-rich amphipathic loop in the monomeric water-soluble protein.
  • the loop contains alternating hydrophilic and hydrophobic residues.
  • MTS-ET is only 6 A, only residues lining the channel have accessibility to the reagent (Akabas, M. H. et al. Science 258:307-310, 1992).
  • PA is ideally suited to this method, in that the native protein is devoid of cysteines. Should the cysteine of interest line the ion-conducting pathway, derivatization with the positively-charged reagent methanethiosulfonate ethyltrimethylammonium (MTS-ET) would introduce a positive charge within the cation-selective channel and likely result in reduction of channel conductance.
  • MTS-ET positively-charged reagent methanethiosulfonate ethyltrimethylammonium
  • MTS-ET bromide and sodium MTS-ES are available from Toronto Research Chemicals (North York, Ontario, Canada); Bu4N bromide, puriss grade, was obtained from Fluka (Buchs, Switzerland).
  • Wild type PA containing a conservatively introduced Sal I site at 792 bp, was cloned into the Bam Hl-Xho I sites of the Escherichia coli expression vector pET22b+ from Novagen (Madison, WI), which directs for periplasmic expression.
  • Site directed mutants were created from this template via two-step recombinant PCR using appropriate primers, and a 195 base pair Sal I-Eco RI fragment was subcloned back into the wild type vector.
  • the ligation products were transformed into E. coli XL 1 -Blue from Stratagene (La Jolla, CA).
  • the plasmid DNA was amplified, purified, and sequenced to confirm the presence of the mutation.
  • Confirmed mutant plasmids were then transformed into the E. coli expression host BL21(DE3). Cultures were grown in Luria broth containing ampicillin at 37°C to an OD 600 of 0.6-1.0, and protein expression was induced by addition of isopropyl ⁇ -D-thiogalactopyranoside (1 mM) for 3 hours at 30 °C. Periplasmic proteins were extracted by first resuspending pelleted cells in 4 ml 20% sucrose, 5 mM EDTA, 150 mg/ml lysozyme, 20 mM Tris-HCl, pH 8.0, per gram of cells. After incubation on ice for 40 minutes, 80 ml of
  • Mutant proteins were diluted to 0.5 mg/ml in buffer A, and trypsin was added to a final concentration of 1 mg/ml. After incubation for 5 minutes at 37 °C, soybean trypsin inhibitor was added to a final concentration of 10 mg/ml. Nicked proteins were stored on ice or at -80 °C until use. Although the mutant proteins were stored in
  • MTS-ET was added either to the cis chamber to a final concentration of 4 mM or to the trans chamber to a final concentration of 4-160 mM; these concentrations of MTS-ET had no effect on the current induced by nicked wild type PA.
  • the effects of MTS-ET on mutant PA-induced conductance generally occurred over a period of tens of seconds.
  • the percent decline of conductance produced by MTS-ET was calculated as [ 1 -(IP A+MTS/IPA)] 100, where IP A was the current immediately before MTS-ET addition, and IPA+MTS was the lowest current observed within three minutes following addition of MTS-ET. Values are reported as the mean standard error of 2-4 experiments.
  • this reagent was added to a final concentration of 15 mM cis or 500 mM trans. All experiments were done under voltage clamp conditions (with the cis chamber held at +20 mV with relative to the trans chamber), using a single pair of Ag/AgCl electrodes that made electrical contact with the solutions in the chambers through 3 M KC1 agar bridges. The current responses were filtered at 10 Hz and displayed on a Narco (Houston, TX) physiograph chart recorder. Single channel experiments.
  • Planar bilayers were formed at room temperature from a 1% solution of diphytanoylphosphatidylcholine in hexane using a modification of the folded film method (Montal, M, Methods in Enzymology 32:545-554, 1974) across a 90-100 mm hole in a polystyrene cup (Wonderlin, W. F. et al, Biophys. J. 58:289-297,1990) as previously described (Silverman, J. A. et al, J. Membr. Biol. 137:17-28, 1994).
  • Fig. 2 shows a typical trace for a MTS-ET-responsive mutant.
  • Addition of 38 mM MTS-ET trans to channels formed by the N306C mutant reduced macroscopic current nearly 6-fold within seconds.
  • Subsequent trans addition of 4 mM DTT caused a reversal of the effect.
  • the current induced by most other MTS-ET-responsive mutants was also maximally reduced within seconds following MTS-ET addition. With some mutants, the decrease was more gradual, but in all cases was essentially complete within three minutes. The reduced current persisted with most mutants for the duration of the experiment.
  • the decline in G317C- and S319C-induced current produced by MTS-ET added trans was followed by a linear rise in current to levels exceeding that which was observed before MTS-ET addition.
  • Fig. 3 shows the maximal reduction of macroscopic current attained by each mutant within the first three minutes following trans-addition of MTS-ET.
  • D2L2 E302C-A311C and 1316C-S325C
  • alternating positions displayed a reduction of current following the addition of MTS-ET.
  • All hydrophilic positions within these stretches were responsive to MTS-ET, whereas all hydrophobic positions displayed little to no MTS-ET effect. (The only exceptions were positions A307C and I316C, where a slow effect was seen in some experiments.)
  • These stretches were bridged by a region of consecutive MTS-ET-responsive residues, from S312C-D315C.
  • the pattern of conductance inhibition by MTS-ET was similar in all single- channel experiments: a decrease in conductance occurred during or immediately following addition of MTS-ET, and additional jumps to lower conductance states occurred afterwards. Up to five transitions to lower conductance states were resolved within some single channels. The state of lowest conductance ranged from 28 pS to 12 pS (72%-88% reduction), depending upon the experiment, and the magnitudes of individual transitions varied from experiment to experiment. In the putative heptameric channel, 19 spatial combinations would be allowed for derivatization of 1-7 cysteines (Braga, O. et al, Chem Biol 4:497-505, 1997).
  • the second MTS-ET could modify cysteines at any of three different locations relative to the first — an adjacent residue, or one or two monomers removed.
  • Each configuration may result in a different reduction of channel conductance through a combination of steric and electrostatic effects.
  • reaction with MTS-ET and reversal by DTT could apparently occur from the trans compartment while the channel was temporarily closed.
  • a channel often transiently closed for a few seconds.
  • the channel closed in the presence of trans MTS-ET it re-opened to a lower conductance state.
  • DTT a closed channel sometimes reopened to a higher conductance state.
  • D2L2 forms a channel which is gated at a site cis relative to position 306.
  • A-B toxins such as diphtheria and anthrax toxins
  • the B moiety inserts into membranes under translocation conditions to form a pore which may serve as a conduit for A-chain translocation.
  • MTS-ET The probe used for assessing cysteine assessibility, MTS-ET, has been shown to be bilayer-impermeant by liposome leakage and excised patch experiments
  • the ⁇ turn region within D2L2 of protective antigen is presumably different than that adopted by the Staphylococcus aureus ⁇ -hemolysin: in PA 63 , the i and i+3 residues of the turn need to lie on the same face of the hairpin, while in the a-hemolysin, i and i+3 apparently lie on opposite sides of the barrel. Only two minor anomalies in the overall pattern of inhibition were observed: in some experiments, a weak effect of MTS-ET was detected at A307C and 1316C. This may reflect greater flexibility of the ⁇ structure at these locations.
  • the variation in stepwise changes in conductance from experiment to experiment may reflect the large number of possible configurations of reacted cysteines within the channel.
  • Up to 19 configurations of 1-7 reacted cysteines within a heptamer are theoretically possible (Braga, O. et al, Chem Biol 4:497-505, 1997), and each configuration may have a unique effect on conductance resulting from steric and electrostatic effects.
  • Multiple MTS reactions within a single channel have also been observed in analysis of the ryanodine receptor channel (Quinn, K. E, & Ehrlich, B.
  • the D2L2 loops move to the base of the heptameric prepore, where they form a membrane-inserted ⁇ barrel (Petosa, C. et al. Nature 385:833-8, 1997).
  • the globular domains of the heptameric ring would then extend the pore above the cis leaflet of the membrane, as seen with the Staphylococcus aureus a-hemolysin pore.
  • the channel blocker Bu4N+ is prevented from reaching its binding site when added to the trans (but not the cis) compartment of S325C channels pre-derivatized with MTS-ET.
  • the lumen of the prepore is predominately negatively charged (Petosa, C. et al. Nature 385:833-8, 1997), and assuming there is little change in that lumen during the prepore-to-pore conversion, it represents a likely locus for binding of the Bu4N+ ion.
  • the ability of trans MTS-ET and DTT to react within the channel in its closed state suggests that the gate lies cis to the membrane-inserted region defined here, and this gate may also reside within the globular cap of the pore.
  • the D2L2 loops are located midway up the globular domains and are flanked by the 2 ⁇ 2 and 2 ⁇ 3 strands of domain 2.
  • ⁇ and 2b3 tear away from the body of domain 2 under the influence of low pH (Petosa, C. et al. Nature 385:833-8, 1997).
  • Residues of the 2 ⁇ 2 and 2 ⁇ 3 strands may also contribute to the channel by forming an extension of the ⁇ barrel formed by D2L2, and this extension may serve as a link between the membrane-inserted and globular regions. This extension represents an alternative locus for Bu4N+ and/or channel gating sites.
  • Pore-forming bacterial toxins undergo a variety of conformational changes in the transition from a secreted hydrophilic protein to one capable of penetrating a hydrophobic membrane. Such transitions may involve the creation of entirely new surfaces, such as the ⁇ barrel for anthrax protective antigen, as presently described, and for the membrane-associated ⁇ -hemolysin (Song, L. et al. Science 274: 1859-66, 1996).
  • a pore-forming toxin protein had a deletion of the amino acid sequence that forms the transmembrane pore, such a protein could to neither effectively form a transmembrane pore nor translocate toxin into a host cell.
  • Such a mutant could also function as a dominant mutant, effectively inhibiting the translocation of wild type toxin by forming hybrid heptamers with the wild-type toxin proteins and interfering with normal pore formation.

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Abstract

L'invention concerne en général une toxine porogène mutante qui comprend une mutation dans un acide aminé formant le pore transmembranaire de ladite toxine. L'invention concerne également un acide nucléique sensiblement pur qui code ladite toxine porogène mutante, ainsi que des procédés qui permettent de diminuer la toxicité d'une toxine porogène par administration d'une toxine porogène mutante à une dose suffisante pour inhiber la translocation d'une toxine porogène.
PCT/US1999/003457 1998-02-18 1999-02-18 Inhibition de la translocation d'une toxine WO1999042473A1 (fr)

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AU27710/99A AU2771099A (en) 1998-02-18 1999-02-18 Inhibition of toxin translocation

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US7528698P 1998-02-18 1998-02-18
US60/075,286 1998-02-18

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WO2005004791A2 (fr) * 2002-11-08 2005-01-20 President And Fellows Of Harvard College Composes et procedes de traitement et de prevention d'infections bacteriennes
US7037503B2 (en) 2000-05-04 2006-05-02 President And Fellows Of Harvard College Compounds and methods for the treatment and prevention of bacterial infection

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7037503B2 (en) 2000-05-04 2006-05-02 President And Fellows Of Harvard College Compounds and methods for the treatment and prevention of bacterial infection
WO2005004791A2 (fr) * 2002-11-08 2005-01-20 President And Fellows Of Harvard College Composes et procedes de traitement et de prevention d'infections bacteriennes
WO2005004791A3 (fr) * 2002-11-08 2009-07-09 Harvard College Composes et procedes de traitement et de prevention d'infections bacteriennes

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