Note: Descriptions are shown in the official language in which they were submitted.
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BUFORIN I AS A SPECIFIC INHIBITOR AND THERAPEUTIC AGENT FOR
BOTULINUM TOXIN B AND TETANUS NEUROTOXINS
Acknowledgment of Government Interest
This invention was made by employees of the United States Army. The government
has rights in the invention.
Technical Field
The invention relates to a class of peptide and peptide-like compounds,
"Buforinins"
which inhibit the enzymatic activity of Botulinum toxin B and Tetanus
neurotoxins.
Background of the Invention
The Botulinum toxins (Bttxs) are among the most potent toxins to animals, e.
g. the
LDSO in mice is about 1 ng/kg. Bttxs comprise a family of seven distinct
serotypes (A-G).
Bttxs are composed of two subunits comprising a 100 kdal nerve-cell targeting
heavy chain
and a 50 kdal endoproteolytically active light chain. These toxins are Zn-
metalloproteases
and contain a Zn-protein binding motif HEXXH.
However, Zn-metalloprotease inhibitors, such as angiotensin converting enzyme
inhibitors, captopril and phosphoramidon, are not effective inhibitors of
Bttxs. Although Zn-
chelators inhibit Bttx protease activity in vitro, they merely delay the
protease activity in vivo
and in tissue preparations comprising intact nerve and muscles cells and/or
tissues.
Furthermore, some Zn-chelators are toxic at concentrations necessary to delay
the Bttx
protease activity. Although dithiocarbamates inhibit other Zn-containing
proteins such as
SOD, they are ineffective against the Bttx serotype B (BttxB). Clearly,
inhibitors of the
various Bttx serotypes, such as BttxB, are needed.
BttxB specifically cleaves synaptobrevin (VAMP2) between glutamine 76 and
phenylalanine 77 (QF bond or cleavage site). There is an obligatory
requirement for a
relatively long substrate for the in vivo target VAMP2 as shown by efforts to
produce a
minimum length substrate. It has been shown that 30 amino acids of VAMP2 are
required
and 40 amino acids of VAMP2 are required for optimum cleavage. See Shone, C.
C. et al.
(1993) Eur. J. Biochem. 217:965-971. V2, a peptide derived from VAMP2, is a
sequence of
amino acids located 4 residues upstream from the cleavage site, and was found
to inhibit
Bttx activity. See Pellizzari R. et al. (1996) J. Biol. Chem. 271:20353-20358.
In VAMP2, a
mutation of the C-terminal amino acids had little effect; whereas a helix
disrupting
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substitution of Pro for Ala inhibited BttxB activity by 28%. Further,
replacement of several
negatively charged amino acids led to almost complete inactivity. See
Whitcome, M, et al.
(1996) FEBS Let. 386:133-136).
Computer-aided secondary structure analysis of VAMP2 predicted two stretches
of a-
helical structure flanking the cleavage site QF. See Witcome, M. R. et al.
(1996) FEBS Let.
386: 133-136. Computer-aided tertiary structure analysis indicates that the
two helices could
self associate to form a supersecondary structure of a helix bundle with the
helices separated
by a reverse turn. See Lebeda F. J., et al. (1996) Med. Defense Biosci. Rev.
204.
The above results indicate that more than just the QF bond is required to be
recognized by the toxin for substrate cleavage.
Recently, a new class of compounds have been discovered, which have a
characteristic conformation and the QF bond, that inhibit the Bttx protease
activity. These
compounds and their uses are disclosed herein below.
Summary of the Invention
The invention is directed to a class of peptides and peptide-like compounds,
Buforinins, which have an internal QF bond and the ability to inhibit BttxB
protease
activities. As the tetanus toxin cleavage site is the same as BttxB,
Buforinins may also
competitively inhibit tetanus protease activity.
Thus, in one aspect, the invention is directed to compounds of the formula:
X1 X2B3X4BSX*6X7XSB9X10B 1 1X12B 13X14B I SX16B 17X* 18X* 19B20X21
X22X23Q24F25z*26
X27X28B29X30B31B32X33X34B35B36X37Z38Z39 (1)
and the salts, esters, amides, and acyl forms thereof. Up to 15 amino acids
may be
truncated from the N-terminus and up to 6 amino acids may be truncated from
the C-
terminus. Each position represented by a letter indicates a single amino acid
residue wherein
B is a basic or polar/large amino acid or a modified form thereof; X is a
small or hydrophobic
amino acid or a modified form thereof; X* is a small or polar/large amino acid
or a modified
form thereof; Z is a polar/large or hydrophobic amino acid or a modified form
thereof; Z* is
Proline or a polar/large or hydrophobic amino acid or a modified form thereof.
As described
below, one or more of the peptide linkages between the amino acid residues may
be replaced
by a peptide linkage mimic.
In other aspects, the invention is directed to recombinant materials useful
for the
production of those peptides of the invention that contain gene-encoded amino
acids, as well
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as plants or animals modified to contain expression systems for the production
of these
peptides. The invention also includes methods to prepare and manipulate these
recombinant
materials.
In addition, the invention is directed to pharmaceutical compositions
containing the
compounds of the invention as active ingredients and to compositions which
contain
expression systems for the production of the peptides. The invention is also
directed to
methods to prepare the invention compounds synthetically, to antibodies
specific for these
compounds, and to the use of the compounds as preservatives, therapeutics, and
prophylactics.
The invention is also directed to the use of the compounds of the invention in
assays
for detection of BttxB and Tttx by the use of selective inhibition and for
determining
inhibitors and substrates for a given toxin.
The present invention relates to materials, compositions, kits and methods for
inhibiting the enzymatic activity of Botulinum toxin B and Tetanus
neurotoxins.
The invention further relates to materials, compositions, kits and methods for
preventing or treating toxic poisoning such as Botulinum toxin B and tetanus
poisoning. The
kits can provide single or multiple dosage and can include other conventional
ancillary
materials such as instructions, solutions and compositions needed for
operation. The
compositions and solutions may be placed in containers, test tubes, etc.
Containers could be
similar to those employed in insect/snake bite kits that includes an injector
which provides
the Buforinin, and TCEP in separate chambers. If chaotropes are present, they
are separately
included in one or more containers.
A kit for determining whether a sample contains a Buforinin, the amount of
said
Buforinin or the type of said Buforinin may include antibodies immunospecific
for
Buforinins.
A kit for determining whether a sample contains a Botulinum toxin or the type
of the
Botulinum toxin may include antibodies immunospecific for at least one
Buforinin having an
interaction with a Botulinum toxin. Likewise, a kit for determining whether a
sample
contains a Tetanus toxin would include antibodies immunospecific for at least
one Buforinin
having an interaction with a Tetanus toxin.
Another embodiment includes Buforin I along with one or more known peptide
inhibitors associated with the decontamination of Botulinum B and/or Tetanus
toxins.
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Additionally, the kits may also include a stable peptide mixture or powder
which includes
Buforinin for sprinkling over food or wounds for detoxification.
Description of the Drawings
This invention is further understood by reference to the drawings wherein:
Figure 1 shows that Substance P is not a substrate of BttxB.
Figure 2 shows that Buforinins are not substrates of Bttx B.
Figure 3 shows a double reciprocal plot of inhibition of BttxB endoprotease
activity
by Buforin I.
Figure 4 illustrates the inhibition of BttxB endoprotease activity by various
Buforinins.
Figure 5 illustrates the X-ray crystallographic structure of avian chromosomal
protein
histone octamer H2A residues LyslS-Try39 produced by Brookhaven Protein
Database
# 1 HIO.
In Figure 6, Helix 1 and 2 are the helices predicted for sequence upstream and
downstream of the QF site respectively (see Table 2). Mutants were selected to
increase the
amphipathicity of the helix indicated. B-I Helix 2 is shown as the companion
to which Helix
1 is predicted to associate. A. Amino acid sequences. B. Helical wheel
projections of
Buforin-I. Helix-1 is the predicted upstream helix of the QF site and Helix-2
is the
downstream helix. The helix amino acids are indicated in the wheel center. C.
Helical
wheel projections of mutant Buforinins. The amino acid order is indicated by
the concentric
numbering. For A, B, and C the color code is as follows: dark gray:
hydrophobic; light gray:
hydrophilic; stippled gray: other; with the amino acids indicated within the
circles. Figure
6A is a comparison of the amino acid sequences of Buforin I, and mutant B-I Rl
1L and
mutant B-I R11L, K15L, S18L. Figure 6B shows helical wheel projections for
Buforin I of
Helix I and Helix 2. Figure 6C shows helical wheel projections for Helix 1 of
mutants B-I
R11L and B-I R11L, K15L, S18L.
Figure 7 shows inhibition of Botulinum toxin B endoprotease activity with
peptide
OSP (Sequence: TRSRAKGLQFPGLLVHRLLRKGNY).
Figure 8 shows the rapid uptake of Buforin I in to blood over time.
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Detailed Description of the Invention
In our search for BttxB inhibitors, peptides that contain the QF cleavage site
but are
not identical in primary sequence to VAMP2 surrounding the QF site were
investigated.
Substance P, an 11 amino acid peptide containing the QF bond, is not a
substrate of BttxB.
See Example 1 and 2; and Figure 1. This result supports the preferred helix-
turn-helix and/or
long substrate hypothesis.
Buforin I (B-I) is a peptide isolated from the stomach of the Asian toad Bufo
bufo
gargarizans which has a QF bond. Therefore, the endopeptidase assay, below,
was used to
determine if B-I is a substrate or an inhibitor of BttxB protease activity. B-
I was found not
to be a substrate for BttxB and that B-I dose-dependently and competitively
inhibits BttxB
activity. See Figs. 2 and 3. The extent of inhibition gave an ICSO = 1 x 10-6
M. See Figure 4.
This was a surprising result as B-I is only 18% homologous for conserved amino
acids with
VAMP2 55-94. See Table I.
TABLE I
Sequence
alignment
of VAMP2,
Buforin
I and Buforin
I derivative
peptides
Peptide Sequence
VAMP25s-9a ERDQKLSELDDRADALQAGASQFETSAAKLKRKYWWKNLK
Buforin AGRGKQGGKVRAKAKTRSSRAGLQFPVGRVHRLLRKGNY
Ia
Buforin TRSSRAGLQFPVGRVHRLLRK
IIb
Peptide24' TRSSRAGLQFPVGRVHRLLRKGNY
Peptide36' AGRGKQGGKVRAKAKTRSSRAGLQFPVGRVHRLLRK
aArcher,B.T.
IIL, et
al. (1990)J.
Biol. Chem.
265 (28),
17267-17273.
bPark C.
B.,et al.(1996).
'Garcia,
G. E. et
al.(1998).
Truncated B-I peptides were evaluated with our endopeptidase activity assay.
The
truncated peptides evaluated were Peptide 36 which contains amino acids 1-36
of B-I and
Peptide 24 which contains amino acids 16-39 of B-I. Like B-I, these truncated
peptides were
not substrates of BttxB; however, the truncated peptides are less effective
inhibitors of BttxB
activity as B-I. See Figure 2. Peptide 36 was about 50% as effective as B-I.
Peptide 24 was
about 25% as effective as B-I. Buforin II (B-II), which contains amino acids
16-36 of B-I,
was also evaluated and found to be 25% as effective as B-I.
B-I is derived from histone protein 2A (H2A) of the toad which is nearly
identical to
the sequence of avian H2A. See Table 2 and see Park, C.B., et al. ( 1996)
Biochem. Biophys.
Res. Comm. 218:408-413. X-ray crystallographic analysis of the chicken histone
protein
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particle shows that, for the region K15 to Y39, there are helices upstream and
downstream of
the QF site. See Fig 5 and see Arents, G., et al. (1991) PNAS 88:10148-52 and
Wang, S. W.,
et al. (1985) Nucleic Acids Res. 13:1369-138. Also, NMR analysis of B-II shows
that the
region upstream from the QF site could form a-helix. See Yi, et al. (1996)
FEBS Lett.
398:87-90.
Table II. H2A
comparison
of chicken
to toad for
relevant amino
acid sequences
Database~B
Source Accession Homologya
no.
ufo bufo gagarizansBBU70133 GRGKQGGKVRAKAKTRSSRAGLQFPVGRVHRLLRKGNY
Gallus gallus X02218 GRGKQGGKARAKAKSRSSRAGLQFPVGRVHRLLRKGNY100
i ne sumx -- sigrnnes accession numoers m the ~entsanK oatabase.
aHomology to toad sequence. Similarity; basic: Arg, Lys; acidic: Asp, Glu;
polar: Asn, Gln; hydrophobic: Ala,
Ile, Leu, Met, Val; aromatic: Phe, Tyr, Trp; size: Ala, Ser, Thr.
[1] Kim, H. S., Park, C. B., Kim, M. S., Kim, S. C. (96) Biochem. Biophys.
Res. Comm. 229:381-387.
[2] Wang, S. W., Robins, A. J., d=Andrea, R. Wells, J. R. (85) Nucleic Acids
Res. 13:1369-1387.
These results indicate that there is potential for long Buforinins to form a
similar
supersecondary structure of a reverse turn with helix bundling. See Table 3.
Therefore, a
new class of peptides, "Buforinins" which includes Buforin I (39 amino acids),
Buforin II (21
amino acids), Peptide 36 and Peptide 24, and other analogous peptides having a
QF bond,
that competitively inhibit BttxB protease activity was defined.
TABLE III
Computer-Aided Secondary
Structure Predictions
VAMP255_gq ERDQKLSELDDRADALQAGASQFETSAAKLKRKYWWKNLK
Gibratb HHHHHHHHHHHHHHHHHCCHHHHHHHHHHHHHHTTHHTCT
Nnpredict --------HH-HHHHHHH---HHHHHHHHHHHHHHH----
B-I AGRGKQGGKVRAKAKTRSSRAGLQFPVGRVHRLLRKGNY
Gibrat HTTTTTCCEEEEHHHHHHHHHCTEEEEHHHHEEEETTTC
Nnpredict --------EHE-----------E----HHHHHHH----
_B-I
truncated 5 amino acids QGGKVRAKAKTRSSRAGLQFPVGRVHRLL
on both ends
Gibrat HCCHHEEHHHHHHHHHCCEEEECHEHEEE
Nnpredict --------E----HHHHHH-
aH, helix; E, sheet; C,
coil; T, turn; -, no
prediction. QF cleavage
site is indicated in
bold.
bGarnier J. et al. (1987)
J. Mol. Biol. 120:97-120.
'McCleland, D. G, Rumelhart
D. E. In Explorations
in Parallel Distributed
Processing. 3:318-362.
1988. MIT Press, Cambridge
MA; Kneller D. G., et
al. (1990) J. Mol. Biol.
214:171-182.
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These Buforinins are generally described by the formula:
X1X2B3X4BSX*6X7X8B9X10B11XI2B13X14B15XI6B17X*18X*19B20X21X22X23Q24F25Z*26
X27X28B29X30B31B32X33X34B35B36X37Z38z39 (1)
and the salts, esters, amides, and acyl forms thereof. Up to 15 amino acids
may be
truncated from the N-terminus and up to 6 amino acids may be truncated from
the C-
terminus. Each position represented by a letter indicates a single amino acid
residue wherein
B is a basic or polar/large amino acid or a modified form thereof; X is a
small or hydrophobic
amino acid or a modified form thereof; X* is a small or polar/large amino acid
or a modified
form thereof; Z is a polar/large or hydrophobic amino acid or a modified form
thereof; Z* is
Proline or a polar/large or hydrophobic amino acid or a modified form thereof.
As described
below, one or more of the peptide linkages between the amino acid residues may
be replaced
by a peptide linkage mimic.
The invention compounds include those represented by formula ( 1 ) as well as
analogous peptides. "Analogous" forms are peptides which retain the ability to
form the
supersecondary structure, alpha-helical-turn-alpha-helical configuration and
inhibit BttxB
protease activity in reaction with the toxin (since it apparently has no
secondary structure in
aqueous solution). "Analogous" forms also include peptides having amino acid
sequences
which mimic the conformational structure of either B-I or B-II and interact
with BttxB to
inhibit its protease activity. "Analogous" forms also include peptides which
are isolatable
from the amphibian stomach and inhibit BttxB protease activity.
The amino terminus of the peptide may be in the free amino form or may be
acylated
by a group of the formula RCO-, wherein R represents a hydrocarbyl group of 1-
6C. The
hydrocarbyl group is saturated or unsaturated and is typically, for example,
methyl, ethyl,
i-propyl, t-butyl, n-pentyl, cyclohexyl, cyclohexene-2-yl, hexene-3-yl, hexyne-
4-yl, and the
like.
The C-terminus of the peptides of the invention may be in the form of the
underivatized carboxyl group, either as the free acid or an acceptable salt,
such as the
potassium, sodium, calcium, magnesium, or other salt of an inorganic ion or of
an organic ion
such as caffeine. The carboxyl terminus may also be derivatized by formation
of an ester
with an alcohol of the formula ROH, or may be amidated by an amine of the
formula NH3, or
RNH2, or RZNH, wherein each R is independently hydrocarbyl of 1-6C as defined
above.
Amidated forms of the peptides wherein the C-terminus has the formula CONHZ
are
preferred.
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The peptides of the invention may be supplied in the form of the acid addition
salts.
Typical acid addition salts include those of inorganic ions such as chloride,
bromide, iodide,
fluoride or the like, sulfate, nitrate, or phosphate, or may be salts of
organic anions such as
acetate, formate, benzoate and the like. The acceptability of each of such
salts is dependent
on the intended use, as is commonly understood.
The amino acids in the peptides of the invention may be those encoded by the
gene or
analogs thereof, and may also be the D-isomers thereof. A preferred embodiment
is a
compound of the formula (1) wherein the compound is resistant to protease
activity by having
at least some of its residues in the D-configuration, yet retains the ability
to inhibit BttxB
protease activity.
The amino acid notations used herein are conventional and are as follows:
Amino Acid One-Letter SymbolThree-Letter Symbol
Alanine A Ala
Arginine R Arg
Asparagine N Asn
Aspartic acid D Asp
Cysteine C Cys
Glutamine Q Gln
Glutamic acid E Glu
Glycine G Gly
Histidine H His
Isoleucine I Ile
Leucine L Leu
Lysine K ~,ys
Methionine M Met
Phenylalanine F Phe
Proline P Pro
Serine S Ser
Threonine T Thr
Tryptophan W Trp
Tyrosine Y Tyr
Valine V Val
The compounds of the invention are peptides or peptide-like compounds which
are
partially defined in terms of amino acid residues of designated classes. Amino
acid residues
can be generally subclassified into major subclasses as follows:
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Acidic: The residue has a negative charge due to loss of H ion at
physiological pH
and the residue is attracted by aqueous solution so as to seek the surface
positions in the
conformation of a peptide in which it is contained when the peptide is in
aqueous medium at
physiological pH.
Basic: The residue has a positive charge due to association with H ion at
physiological pH or within one or two pH units thereof (e.g., histidine) and
the residue is
attracted by aqueous solution so as to seek the surface positions in the
conformation of a
peptide in which it is contained when the peptide is in aqueous medium at
physiological pH.
Hydrophobic: The residues are not charged at physiological pH and the residue
is
repelled by aqueous solution so as to seek the inner positions in the
conformation of a peptide
in which it is contained when the peptide is in aqueous medium.
Neutral/polar: The residues are not charged at physiological pH, but the
residue is not
sufficiently repelled by aqueous solutions so that it would seek inner
positions in the
conformation of a peptide in which it is contained when the peptide is in
aqueous medium.
This description also characterizes certain amino acids as "small" since their
side
chains are not sufficiently large, even if polar groups are lacking, to confer
hydrophobicity.
"Small" amino acids are those with four carbons or less when at least one
polar group is on
the side chain and three carbons or less when not.
It is understood, of course, that in a statistical collection of individual
residue
molecules some molecules will be charged, and some not, and there will be an
attraction for
or repulsion from an aqueous medium to a greater or lesser extent. To fit the
definition of
"charged," a significant percentage (at least approximately 25%) of the
individual molecules
are charged at the relevant pH. The degree of attraction or repulsion required
for
classification as polar or nonpolar is arbitrary and, therefore, amino acids
specifically
contemplated by the invention have been classified as one or the other. Most
amino acids not
specifically named can be classified on the basis of known behavior.
Amino acid residues can be further subclassified as cyclic or noncyclic, and
aromatic
or nonaromatic, self explanatory classifications with respect to the side-
chain substituent
groups of the residues, and as small or large. The residue is considered small
if it contains a
total of four carbon atoms or less, inclusive of the carboxyl carbon, provided
an additional
polar substituent is present; three or less if not. Small residues are, of
course, always
nonaromatic.
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For the naturally occurnng protein amino acids, subclassification according to
the
foregoing scheme is as follows.
Acidic Aspartic acid and Glutamic acid
Basic Noncyclic: Arginine, Lysine
Cyclic: Histidine
Small Glycine, Serine, Alanine, Threonine
Polar/largeAsparagine, Glutamine
HydrophobicTyrosine, Valine, Isoleucine, Leucine, Methionine,
Phenylalanine,
Tryptophan
The gene-encoded secondary amino acid proline is a special case due to its
known
effects on the secondary conformation of peptide chains, i.e. helix structure
disruptions.
Therefore, proline may only be allowed in position 26 where it would help to
disrupt the
helix structures found on both sides of the QF cleavage site and force the
helix-turn-helix
structure.
Cysteine residues are also not included in these classifications since their
capacity to
form disulfide bonds to provide secondary structure may overnde the general
polarity/nonpolarity of the residue. However, if a cysteine, which is,
technically speaking, a
small amino acid, is modified so as to prevent its participation in secondary
structure, those
locations indicated "S" in the compound of formula (1) may be inhabited by
such modified
cysteine residues.
There are no cysteine or methionine in any of the sequences (VAMP2 substrate,
B-I,
B-II, Peptide 24, Peptide 36). The side chain of cysteine is somewhat
hydrophobic, but it is
highly reactive. The sulfur moiety has the potential to react with the sulfur
in other cysteine
to from a cystine or disulfide bond. If a single cysteine is introduced then
dimerization may
occur between two buforoxins. If more than one cysteine is introduced then
polymerization
could occur. Clearly, the substitution of cysteine near the cleavage site with
resultant
dimerization or polymerization could interfere with the helix-turn-helix
structure thought to
be necessary for inhibition and produce steric interference for the
interaction of the target
protein and buforinin. Another problem associated with cysteine and methionine
residues is
the potential for oxidation to cysteic acid or methionine sulfoxide or
methionine sulfone
respectively. These conversions would significantly alter the peptide
properties since a
hydrophobic weakly polar or ionizable form would be converted to an acidic or
strongly
polarized form. However, it may be advantageous to incorporate cysteine on
either end of a
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Buforinin for use as a reactive site to label a Buforinin with fluorescent
markers where the
aforementioned problems may be minimized
The "modified" amino acids that may be included in the Buforinins are gene-
encoded
amino acids which have been processed after translation of the gene, e.g., by
the addition of
methyl groups or derivatization through covalent linkage to other substituents
or oxidation or
reduction or other covalent modification. The classification into which the
resulting modified
amino acid falls will be determined by the characteristics of the modified
form. For example,
if lysine were modified by acylating the, -amino group, the modified form
would not be
classed as basic but as polar/large amino acid.
Certain commonly encountered amino acids, which are not encoded by the genetic
code, include, for example, beta-alanine (beta-Ala), or other omega-amino
acids, such as
3-aminopropionic, 2,3-diaminopropionic (2,3-diaP), 4-aminobutyric and so
forth, alpha-
aminisobutyric acid (Aib), sarcosine (Sar), ornithine (Orn), citrulline (Cit),
t-butylalanine
(t-BuA), t-butylglycine (t-BuG), N-methylisoleucine (N-MeIle), phenylglycine
(Phg), and
cyclohexylalanine (Cha), norleucine (Nle), 2-naphthylalanine (2-Nal); 1,2,3,4-
tetrahydroisoquinoline-3-carboxylic acid (Tic); ~-2-thienylalanine (Thi);
methionine
sulfoxide (MSO); and homoarginine (Har). These also fall conveniently into
particular
categories.
Based on the above definitions,
Sar, beta-Ala and Aib are small;
t-BuA, t-BuG, N-MeIle, Nle, Mvl, Cha, Phg, Nal, Thi and Tic are hydrophobic;
2,3-diaP, Orn and Har are basic;
Cit, Acetyl Lys and MSO are neutral/polar/large.
The various omega-amino acids are classified according to size as small (beta-
Ala and
3-aminopropionic) or as large and hydrophobic (all others).
Other amino acid substitutions, which are not gene encoded, are included in
peptide
compounds within the scope of the invention and can be classified within this
general scheme
according to their structure. For example, D-amino acid substitutions would be
desirable to
circumvent potential stability problems due to endogenous protease activity;
especially
important for an oral dosage route.
In all of the Buforinins of the invention, one or more amide linkages (-CO-NH-
) may
optionally be replaced with another linkage which is an isostere such as -
CHZNH-, -CHZS-,
-CHZCHZ, -CH=CH- (cis and trans), -COCHZ-, -CH(OH)CHz- and -CHZSO-. This
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replacement can be made by methods known in the art. The following references
describe
preparation of peptide analogs which include these alternative-linking
moieties: Spatola,
A.F., Vega Data (March 1983), Vol. 1, Issue 3, "Peptide Backbone
Modifications" (general
review); Spatola, A.F., in "Chemistry and Biochemistry of Amino Acids Peptides
and
Proteins," B. Weinstein, eds., Marcel Dekker, New York, p. 267 (1983) (general
review);
Morley, J.S., Trends Pharm Sci (1980) pp. 463-468 (general review); Hudson,
D., et al., Int J
Pept Prot Res (1979) 14:177-185 (-CH2NH-, -CH2CH2-); Spatola, A.F., et al.,
Life Sci (1986)
38:1243-1249 (-CH2-S); Hann, M.M., J Chem Soc Perkin Trans I (1982) 307-314 (-
CH-CH-,
cis and trans); Almquist, R.G., et al., JMed Chem (1980) 23:1392-1398 (-COCH2-
);
Jennings-White, C., et al., Tetrahedron Lett (1982) 23:2533 (-COCH2-); Szelke,
M., et al.,
European Application EP 45665 (1982) CA:97:39405 (1982) (-CH(OH)CH2-);
Holladay,
M.W., et al., Tetrahedron Lett (1983) 24:4401-4404 (-C(OH)CH2-); and Hruby,
V.J., Life Sci
(1982) 31:189-199 (-CH2-S-).
In preferred embodiments of the compounds of the formula (1):
X 1 X2B3X4BSX* 6X7X8B9X 1 OB 11 X 12B 13X14B I SX 16B 17X* 18X* 19B20X21
X22X23 Q24F25Z * 26
X27X28B29X30B31B32X33X34B35B36X37Z38Z39 (1)
X1 is Glycine, Serine, Threonine, Isoleucine, Leucine, Valine, or preferably
Alanine;
X2 is Alanine, Serine, Threonine, Isoleucine, Leucine, Valine, or preferably
Glycine;
B3 is Histidine, Lysine, Asparagine, Glutamine, or preferably Arginine;
X4 is Alanine, Serine, Threonine, Isoleucine, Leucine, Valine, or preferably
Glycine;
BS is Arginine, Histidine, Asparagine, Glutamine, or preferaisly Lysine;
X*6 is Alanine, Glycine, Serine, Threonine, Asparagine, or preferably
Glutamine;
X~ is Alanine, Serine, Threonine, Isoleucine, Leucine, Valine, or preferably
Glycine;
Xg Alanine, Serine, Threonine, Isoleucine, Leucine, Valine, or preferably
Glycine;
B9 is Arginine, Histidine, Asparagine, Glutamine, or preferably Lysine;
Xlo is Alanine, Glycine, Serine, Threonine, Isoleucine, Leucine, or preferably
Valine;
B11 is Histidine, Lysine, Asparagine, Glutamine, or preferably Arginine;
X12 is Glycine, Serine, Threonine, Isoleucine, Leucine, Valine, or preferably
Alanine;
B 13 is Arginine, Histidine, Asparagine, Glutamine, or preferably Lysine;
X14 is Glycine, Serine, Threonine, Isoleucine, Leucine, Valine, or preferably
Alanine;
B15 is Arginine, Histidine, Asparagine, Glutamine, or preferably Lysine;
X16 is Alanine, Glycine, Serine, Isoleucine, Leucine, Valine, or preferably
Threonine;
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B1~ is Histidine, Lysine, Asparagine, Glutamine, or preferably Arginine;
X*18 is Alanine, Asparagine, Glutamine, Glycine, Threonine, or preferable
Serine;
X*19 is Alanine, Asparagine, Glutamine, Glycine, Threonine, or preferable
Serine;
BZO is Histidine, Lysine, Asparagine, Glutamine, or preferably Arginine;
XZ~ is Glycine, Serine, Threonine, Isoleucine, Leucine, Valine, or preferably
Alanine;
XZZ is Alanine, Serine, Threonine, Isoleucine, Leucine, Valine, or preferably
Glycine;
X23 is Asparagine, Glutamine, Alanine, Serine, Threonine, Isoleucine, Glycine,
Valine, or preferably Leucine;
Z*26 is Asparagine, Glutamine, Phenylalanine, Tryptophan, Tyrosine or
preferably
Proline;
XZ~ is Alanine, Serine, Threonine, Isoleucine, Leucine, Glycine , or
preferably Valine;
X28 is Alanine, Serine, Threonine, Isoleucine, Leucine, Valine, or preferably
Glycine;
B29 is Asparagine, Glutamine, Histidine, Lysine, or preferably Arginine;
X3o is Alanine, Glycine, Leucine Serine, Threonine, Isoleucine or preferably;
Valine;
B31 1S Arginine, Lysine, Asparagine, Glutamine, or preferably Histidine;
B32 is Arginine, Histidine, Asparagine, Glutamine, or preferably Lysine
X33 is Alanine, Glycine, Serine, Threonine, Isoleucine, Valine, or preferably
Leucine;
X34 is Alanine, Glycine, Serine, Threonine, Isoleucine, Valine, or preferably
Leucine;
B35 is Lysine , Histidine, Asparagine, Glutamine, or preferably Arginine;
B36 is Arginine, Histidine, Asparagine, Glutamine, or preferably Lysine;
X3~* is Alanine, Glutamine, Serine, Threonine, Asparagine, or preferably
Glycine
Z3g is Glutamine, Phenylalanine, Tryptophan, Tyrosine or preferably
Asparagine; and
Z39 is Asparagine, Glutamine, Phenylalanine, Tryptophan, or preferably
Tyrosine.
Typical compounds within the scope of the Buforinins are:
AGRGKQGGKVRAKAKTRSSRAGLQFPVGRVHRLLRKGNY (SEQ ID NO:1)
TRSSRAGLQFPVGRVHRLLRK (SEQ ID N0:2)
TRSSRAGLQFPVGRVHRLLRKGNY (SEQ ID N0:3)
AGRGKQGGKVRAKAKTRSSRAGLQFPVGRVHRLLRK (SEQ ID N0:4)
TRAARAGLQFPVGRVHRLLRK (SEQ ID NO:S)
TRLLRAGLQFPVGRVHRLLRK (SEQ ID N0:6)
"Active" Buforinins are defined as those peptides that fit the invention
sequence
description and inhibit BttxB and/or Tttx protease activities. The
conformation of the
Buforinins may be determined by circular dichroism and FT-IR. See CBnaves, J.
M., et al.
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(1998) J. Biol. Chem. 273:43214-34221. Proton NMR may also be used. See Yi, G.
et al.
(1996) FEBS Lett. 398:87-90. X-ray crystallography may also be used. See
Sutton, R. B., et
al. (1998) Nature 395, 347-353.
"Derivatives" of Buforinins are defined as those peptides fitting the
invention
description that have amino acid modifications such as Buforinin peptides
containing
'unnatural' amino acids other than the known 21 amino acids (20 common, and
then
selenocysteine, which is an uncommon but naturally occurring non-gene encoded
amino acid)
or additions such as cysteine and lysine on termini to provide a reactive
center for
conjugation to other chemicals, labels or proteins.
"Truncated" Buforinins include compounds of the formula (1) such as B-II.
Amino
acids can be truncated, asymmetrically, upstream and downstream while
maintaining the
helix-turn-helix supersecondary structure. B-II could be optimized by amino
acid
substitutions to promote a helical structure upstream of the QF site. See,
e.g. SEQ ID NO:S
and SEQ N0:6.
Preparation of the Invention Compounds
The invention compounds, often designated herein "Buforinins" are essentially
peptide backbones which may be modified at the N- or C-terminus.
Standard methods can be used to synthesize peptides similar in size and
conformation
to the Buforinins. Most commonly used currently are solid phase synthesis
techniques;
indeed, automated equipment for systematically constructing peptide chains can
be
purchased. Solution phase synthesis can also be used but is considerably less
convenient.
When synthesized using these standard techniques, amino acids not encoded by
the gene and
D-enantiomers can be employed in the synthesis.
In addition to providing the peptide backbone, the N- and/or C-terminus can be
modified with conventional chemical techniques. The compounds of the invention
may
optionally contain an acyl or an acetyl group at the amino terminus. Methods
for acetylating
or, more generally, acylating, the free amino group at the N-terminus are
generally known in
the art.
At the carboxy terminus, the carboxyl group may be present in the form of a
salt; and
in the case of pharmaceutical compositions, the salt will be a
pharmaceutically acceptable
salt. Suitable salts include those formed with inorganic ions such as NH4+,
Na+, K+, Mg++,
Ca++, and the like as well as salts formed with organic canons such as those
of caffeine and
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other highly substituted amines. The carboxy terminus may also be esterified
using alcohols
of the formula ROH wherein R is hydrocarbyl (1-6C) as defined above.
Similarly, the
carboxy terminus may be amidated so as to have the formula -CONHZ, -CONHR, or
-CONR2, wherein each R is independently hydrocarbyl (1-6C) as herein defined.
Techniques
for esterification and amidation as well as neutralizing in the presence of
base to form salts
are all standard organic chemical techniques.
If the peptides of the invention are prepared under physiological conditions,
the side-
chain amino groups of the basic amino acids will be in the form of the
relevant acid addition
salts.
If the peptide backbone is comprised entirely of gene-encoded amino acids, or
if some
portion of it is so composed, the peptide or the relevant portion may also be
synthesized using
recombinant DNA techniques. The DNA encoding the peptides of the invention may
be
synthesized using standard techniques in the art such as solid phase DNA
synthesis with
conventional equipment that includes, for example, an ABI 3948 Nucleic Acid
Synthesis
System (Perkin Elmer Applied Biosystems, Foster City, CA) utilizing
phosphoramidite
synthesis chemistry (Beaucage, S. L. et al. (81) Tetrahedorn Lett. 22:1859-
1862). DNA
oligomers would be synthesized with overlapping matching complimentary
sequences.
Annealing of these sequences would form a double-stranded synthetic gene.
Building on this
process would give larger and larger double-stranded products till the
requisite gene is built.
Alternatively, DNA recombinant means would be employed by cloning Buforinins,
or like-
fragment of H2A protein, and then modifying by site-directed mutagenesis or
DNA-cassette
replacement or other means in the art (Methods Enzymology vol. 152; Eds. S. L.
Berge and
A. R. Kimmel, Academic Press, Inc., Orlando, FL, 1998) to achieve the
modification
desired. Codon choice can be integrated into the synthesis depending on the
nature of the
host.
For recombinant production, the DNA encoding the Buforinins is included in an
expression system which places these coding sequences under the control of a
suitable
promoter and other control sequences which are compatible with an intended
host cell.
Types of host cells available span almost the entire range of the plant and
animal kingdoms.
Thus, the Buforinins of the invention could be produced in bacteria or yeast
(to the extent that
they can be produced in a nontoxic or refractile form or utilize resistant
strains) as well as in
animal cells, insect cells and plant cells.
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The Buforinins can be produced in a form that will result in their secretion
from the
host cell by fusing to the DNA encoding the Buforinin, a DNA encoding a
suitable signal
peptide, or may be produced intracellularly. They may also be produced as
fusion proteins
with additional amino acid sequence which may or may not need to be
subsequently removed
prior to the use of these compounds as an inhibitor of BttxB protease
activity.
Thus, the Buforinins of the invention can be produced in a variety of
modalities
including chemical synthesis and recombinant production or some combination of
these
techniques.
Any members of the Buforinin class which occur naturally are supplied in
purified
and isolated form. By "purified and isolated" is meant free from the
environment in which
the peptide normally occurs (in the case of such naturally occurring peptides)
and in a form
where it can be used practically. Thus, "purified and isolated" form means
that the peptide is
substantially pure, i.e., more than 90% pure, preferably more than 95% pure
and more
preferably more than 99% pure or is in a completely different context such as
that of a
pharmaceutical preparation.
The invention is also directed to the screening assays for the Buforinin
analogues and
assays utilizing the analogues.
The invention is also directed to the use of Buforinins as intracellular
inhibitors of
BttxB. Bttxs specifically target nerve cells because of the receptor-like
recognition of cell
surface gangliosides and synaptogamin by the nerve-cell targeting heavy chain
(HC) subunit
of the toxin. See Kozaki, S., et al. (1998) Microb. Pathog. 25:91-99. Once
bound, the toxin
is internalized by a mechanism not completely understood but appar~e~~tly
requires
acidification of the endosome and cleavage of the disulfide bond linking the
HC and the
endoproteolytically active light chain (LC).
The specificity of this delivery system would be useful for delivery of
Buforinins to
those cell types poisoned or potentially poisoned with BttxB and could be used
as a 'magic
bullet' since the magic bullet approach is becoming a reality. See e.g.
Pastan, L, et al. ( 1994)
Ann. Rev. Biochem. 61:331-354 and Engert, A., et al. (1998) Curr. Top.
Microbial.
Immnunol. 234:13-33 (Introduction of immunotoxins linked to Diptheria toxin or
Ricin A
chain).
Therefore, Buforonins may be linked to BttxB HC with a linkage such as a
disulfide
bond. Alternatively, Buforinins may be linked to BttxB HC with a earner
protein such as
human albumin or another bridge to form a mufti-protein conjugate. This
conjugate should
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then target the susceptible cells in a manner similar to BttxB. Once inside
the cell, the
conjugate may inhibit BttxB or the linkage may be cleaved to free the
Buforinins or carner-
Buforinins to inhibit BttxB.
Antibodies
Antibodies to the Buforinins may be produced using standard immunological
techniques for production of polyclonal antisera and, if desired,
immortalizing the antibody-
producing cells of the immunized host for sources of monoclonal antibody
production.
Techniques for producing antibodies to any substance of interest are well
known. It may be
necessary to enhance the immunogenicity of the substance, particularly as
here, where the
material is only a short peptide, by coupling the hapten to a Garner. Suitable
Garners for this
purpose include substances which do not themselves produce an immune response
in the
mammal to be administered the hapten-Garner conjugate. Common carriers used
include
keyhole limpet hemocyanin (KLH), diphtheria toxoid, serum albumin, and the
viral coat
protein of rotavirus, VP6. Coupling of the hapten to the Garner is effected by
standard
techniques such as contacting the Garner with the peptide in the presence of a
dehydrating
agent such as dicyclohexylcarbodiimide or through the use of linkers such as
those available
through Pierce Chemical Company, Chicago, IL.
The Buforinins in immunogenic form are then injected into a suitable mammalian
host and antibody titers in the serum are monitored.
Polyclonal antisera may be harvested when titers are sufficiently high.
Alternatively,
antibody-producing cells of the host such as spleen cells or peripheral blood
lymphocytes
may be harvested and immortalized. The immortalized cells are then cloned as
individual
colonies and screened for the production of the desired monoclonal antibodies.
The genes
encoding monoclonal antibodies secreted by selected hybridomas or other cells
may be
recovered, manipulated if desired, for example, to provide multiple epitope
specificity or to
encode a single-chain form and may be engineered for expression in alternative
host cells,
such as CHO cells.
Thus, as used herein, "antibodies" also includes any immunologically reactive
fragment of the immunoglobulins such as Fab, Fab' and F(ab')2 fragments as
well as
modified immunoreactive forms such as Fv regions, which are produced by
manipulation of
the relevant genes (isolatable, for example, from the appropriate hybridoma).
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The antibodies of the invention are, of course, useful in immunoassays for
determining the amount or presence of the Buforinins. Such assays are
essential in quality
controlled production of compositions containing the Buforinins of the
invention. In
addition, the antibodies can be used to assess the efficacy of recombinant
production of the
Buforinins, as well as for screening expression libraries for the presence of
Buforinin
encoding genes. They may also be used as affinity ligands for purifying and/or
isolating the
Buforinins. They may also be used for detecting and measuring Buforinins in
sera or plasma
by methods well known in the art such as RIA and ELISA. Therefore, one may
monitor
circulating Buforinin levels to assure sufficient dosage.
Compositions Containing the Buforinins and Methods of Use
The Buforinins are effective in inhibiting the protease activity of BttxB and
tetanus
neurotoxins. Accordingly, they can be used in prevention, prophylaxis and
therapies for
BttxB and Tttx poisoning. For use in such contexts, a Buforinin may be
administered alone,
or a variety of Buforinins may be administered, or the Buforinin or the
variety of Buforinins
may be administered as a mixture with additional protease inhibitors or
adjunct chemicals
such as tris-(2-carboxyethl)phosphine (TCEP).
TCEP is a non-odorous, non-sulfhydryl containing reducing agent that is
relatively
non-toxic in animals (P-CHZCHzCOOH)3HCl; Molecular Probes, Inc. Eugene OR).
TCEP
can reduce the disulfide bond between the HC and LC and allow the dissociation
of the BttxB
or Tttx subunits. TCEP would work on any BOT. This dissociation increases the
availability
of the active QF site to Buforinins. Additionally, the disassociation of the
toxin prevents
nerve cell penetration. Other reducing agents such as dithiothreitol (DTT) may
be used;
however, they may be objectionable due to their distinctive odors and
toxicity. TCEP can be
used in conjunction with chaotropes. Therefore, TCEP is preferred.
The peptides of the invention are also useful as standards in monitoring
assays and in
assays for evaluating the effectiveness of later-generation Buforinins. This
could be done by
utilizing the endopeptidase activity assay for BttxB. In this endopeptidase
assay, one may
evaluate whether potential peptides function as inhibitors or substrates of
BttxB by the ability
to cleave of a synthetic peptide substrate comprising amino acids 55-94 of the
intracellular
target VAMP2. The cleavage products may be separated by a C18 reverse-phase
HPLC
column and detected by absorbance at 205 nm.
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For preventing the initial intoxication or further poisoning caused by BttxB
and Tttx
in animal subjects, the Buforinins can be formulated as pharmaceutical or
veterinary
compositions. Depending on the subject to be treated, the mode of
administration, and the
type of treatment desired -- e.g., prevention, prophylaxis, therapy; the
Buforinins are
formulated in ways consonant with these parameters. A summary of such
techniques is
found in Remington's Pharmaceutical Sciences, latest edition, Mack Publishing
Co., Easton,
PA.
In general, for use in treatment or prophylaxis, the Buforinins may be used
alone or in
combination with other compounds which inhibit protease activity such as
VAMP2. Use of
the enantiomeric forms containing all D-amino acids may confer advantages such
as
resistance to those proteases, such as trypsin and chymotrypsin.
The Buforinins can be administered singly or as mixtures of several Buforinins
or in
combination with other pharmaceutically active components, and in single or
multiple
administrations. The formulations may be prepared in a manner suitable for
systemic
administration. Systemic formulations include those designed for injection,
e.g.
intramuscular, intravenous or subcutaneous injection, or may be prepared for
transdermal,
transmucosal, or oral administration. The formulation will generally include a
diluent as well
as, in some cases, adjuvants, buffers, preservatives and the like. The
Buforinins can be
administered also in liposomal compositions or as microemulsions using
conventional
techniques.
If orally administered, the Buforinins of the invention must be protected from
degradation in the stomach using a suitable enteric coating. This may be
avoided to some
extent by utilizing amino acids in the D-configuration, thus providing
resistance to protease.
However, the peptide is still susceptible to acid hydrolysis; thus, some
degree of enteric
coating may still be required.
The manner of administration and formulation of the compounds useful in the
invention and their related compounds will depend on the nature of the
condition, the severity
of the condition, the particular subject to be treated, and the judgement of
the practitioner;
formulation will depend on mode of administration. As the compounds of the
invention are
small molecules, they are conveniently administered by oral administration by
compounding
them with suitable pharmaceutical excipients so as to provide tablets,
capsules, syrups, and
the like. Suitable formulations for oral administration may also include minor
components
such as buffers, flavoring agents and the like. Typically, the amount of
active ingredient in
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the formulations will be in the range of 5%-95% of the total formulation, but
wide variation
is permitted depending on the Garner. Suitable carriers include sucrose,
pectin, magnesium
stearate, lactose, peanut oil, olive oil, water, and the like.
The compounds useful in the invention may also be administered through
suppositories or other transmucosal vehicles. Typically, such formulations
will include
excipients that facilitate the passage of the compound through the mucosa such
as
pharmaceutically acceptable detergents.
The compounds may also be administered topically, for topical conditions such
as
psoriasis, or in formulation intended to penetrate the skin. These include
lotions, creams,
ointments and the like which can be formulated by known methods.
The compounds may also be administered by injection, including intravenous,
intramuscular, subcutaneous or intraperitoneal injection. Typical formulations
for such use
are liquid formulations in isotonic vehicles such as Hank's solution or
Ringer's solution.
Suitable alternative formulations also include nasal sprays, liposomal
formulations,
slow-release formulations, and the like.
Any suitable formulation may be used. A compendium of art-known formulations
is
found in Remington's Pharmaceutical Sciences, latest edition, Mack Publishing
Company,
Easton, PA. Reference to this manual is routine in the art.
A preferred means to deliver the Buforinins would include the use TCEP. Since
TCEP cleaves the holotoxin which yields a site available to the Buforinin.
TCEP also
disassociates the toxins into individual components which prevents nerve cell
penetration.
Also, the Buforinins could be coupled to a variety of compounds including a
BttxB
heavy chain, which excludes the toxin light chain, to target the Buforinin to
the toxin affected
cells.
The dosages of the compounds of the invention will depend on a number of
factors
which will vary from patient to patient.
The following examples are intended to illustrate but not to limit the
invention.
Example 1
Endopeptidase Activity Assay
The toxin was activated immediately prior to use by incubating at 25°C
for 30
minutes in an activation mixture that contained, in a volume of 7.5 ~1 per
digest: 2.4pg (16
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pmol) of toxin, 30 mM NaHEPES buffer, pH 7.3, and 5 mM DTT or TCEP. A
substrate
peptide mix was prepared that contained 1 nmol of the substrate peptide (VAMP2
55-94), 4%
DMSO, 4% Triton X-100, and 80 mM NaHEPES buffer, pH 7.3, per digest. The final
reaction mix was made by adding 25 ~,1 of the substrate peptide mix, 4.5 gl of
fresh 10 mM
DTT, 13 ~,1 HZO or test peptide, and 7.5 g,l of activation mixture. The
reaction was initiated
by incubation at 37°C. The reaction was stopped by the addition of 1
vol trifluoroacetic acid
(TFA) to 0.25%. The samples were clarified by centrifugation.
In this assay, 16 pmol of BttxB digested 1 nmol of the substrate to completion
in less
than 45 min. at 37°C.
Example 2
Reverse Phase HPLC Analysis of Digestion Products
Digested peptide products were fractionated by RP-HPLC on a Waters :Bondapak
analytical C1g column (3.9 mm x 30 cm) attached to Beckman 126 pumps and a
model 168
Diode Array Detector, controlled by Beckman System Gold Ver 8.1 software. The
solvent
system consisted of buffer A (BA; H20 - 0.1 % TFA) and buffer B (BB; CH3CN -
0.1 %TFA).
The development program consisted of the following: 97% BA, 0-1 min; to 33%
BB, 1-30
min; then wash with 97% BB for 5 min, followed by equilibration in 97% BA for
10 min.
The flow rate was ml miri I except during the wash and equilibrium phase where
it was 1.5 ml
miri ~. 75 ~l injections were made with a Waters Intelligent Sample Processor
(WISP Model
712). The effluent was monitored at dual wavelengths of 205 and 280 nm.
Initially, digestion products are identified by peptide sequencing using
automated
Edman-degradation on an ABI 477A protein sequences attached in-line with a
HPLC (ABI
model 120A) for detection of phenlythiohydratoin derivatized amino acids. The
extent of
digestion was determined by comparison of peak areas of undigested controls
(no added
toxin) and total digests (digests allowed to go to completion, typically 2-3
h). The extent of
inhibition or digestion will be determined from examination of the
chromatograms by peak
area comparison with standards and/or products formed compared with quantified
standards
or digests without added inhibitor that have gone to completion.
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Example 3
Secondary Structure Predictions
Secondary structures were predicted by using the nnpredict, and the Gibrat
(GOR2)
programs. See McCleland, D. G, Rumelhart D. E. In Explorations in Parallel
Distributed
Processing. vol. 3:318-362. 1988. MIT Press, Cambridge MA; Kneller D. G., et
al. (1990) J.
Mol. Biol. 214:171-182; Gamier, J. et al. (1978) J. Mol. Biol 198;425-443;
Gamier J. et al.
(1987) J. Mol. Biol. 120:97-120; Gamier, J., et al. (1996) Methods Enzymol.
266:540-553.
Helical wheel projections were made using the Antheprot program Ver 4. See
Deleage, G.,
Instit de Biologie et Chimi des Proteins, Lyon, France.
The Gibrat program predicts that B-I could form an a-helical-turn-a-helical
configuration similar to that of VAMP2. See Table 3. The result that Buforin I
may form a
secondary structure similar to VAMP2 then suggests that B-I may also form a
similar
supersecondary structure of a reverse turn with helix bundling similar to
VAMP2. See
Lebeda, et. al. (1996). In support of this prediction, it was found that the
diminishing
inhibition of BttxB activity and its helical content as Buforin-I was
truncated, mirrors the
diminishing activity of BttxB for substrate deletions. See data for Buforin-II
in Table l and
Figure 4.
Example 4
Preparation of Buforinins
The Buforinins may be obtained from amphibian stomach by gut lavage using
methods as described by Park, C. B. et al. See Park, C.B., et al. (19~>ii)
Biochem. Biophys.
Res. Comm. 218:408-413.
The Buforinins may be synthesized by solid-phase peptide synthesis (SSPS) as
described by L.A. Carpino, J. Am. Chem. Soc. 79,4427 (1957), C.D. Chang et
al., Int. J. Pept.
Protein Res. 11, 246 (1978), E. Atherton, et al., J. Chem Soc. Chem. Commun.,
537 (1978)
and R.B. Merrifield, J. Am. Chem. Soc. 85, 2149 (1963) and Barlos, K., et al.,
(1989)
Tetrahedron Lett. 30:3947.
The Buforinins may also be produced by DNA recombinant means commonly known
in the art whereby a suitable promoter for expression in heterologous systems,
i.e. bacterial,
fungi, insect, or mammalian cell cultures may be used. The DNA sequence may be
optimized for the particular host and tRNA content.
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Example 5
Inhibition of Protease Activity by Buforinins
The endopeptidase assay and reverse phase HPLC as described in Examples l and
2
may be used to detect the cleavage products and the extent of protease
inhibition. Briefly,
potential inhibitors may be added to the substrate peptide mix immediately
before the
addition of the activation mix containing the toxin as described in Garica, et
al. After
incubation for 45 min at 37°C, the reaction should be stopped and the
digestion products may
be analyzed by using RP HPLC. If a fluorescent-labeled substrate is used then
product
formation will be determined with an in-line fluorescent detector.
The extent of inhibition or digestion will be determined as described in
Example 2 of
undigested substrate remaining and/or products formed compared with quantified
standards
or digests without added inhibitor that have gone to completion.
Alternative means can be used include densitometry wherein the substrates and
products separated by electrophoresis and stained with protein specific dyes,
i. e. Coomassie
brilliant blue, and measured. One may also perform immunoassays to determine
the extent of
inhibition or digestion by utilizing substrate or product specific antibodies.
Alternatives also include in vivo protection or tissue-specific function
assays. For
example, an experimental animal would be dosed with the inhibitor with or with
out adjuncts
and then challenged with the toxin, e.g. i.v. injection of a Buforinin with a
reducing agent
such as TCEP. The onset of symptoms or an alteration of the LDSO would then be
evaluated.
Tissue protection assays would employ an intact nerve-muscle preparation
wherein muscle
twitch response to nerve cell stimulation would be evaluated. The toxin would
be
preincubated with a Buforinin and adjuncts and are then added to the tissue
preparation.
Example 6
Designing Buforinins with an Effect on BttxB Protease Activity
By using standard methods and techniques, the peptides of the invention may be
modified by either making mutations or substitutions which include
substituting ProZb with
glutamine to make the active site more like the substrate, or other amino
acid, that favors turn
formation without the turn constraint imposed by Pro. Such substitutions are
predicted to
result in more effective helix bundling for toxin association to occur. Other
amino acid
substitutions or mutations in the helix region could be made so that either
the helix becomes
more amphipathic to improve helix bundling or improve interaction with the
toxin. Such
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WO 00/69895 PCT/iTS00/12909
changes would include a substitution of R11 with L or another helix favoring
amino acid. See
Figs. 6A and B. Similarly, multiple substitutions R11L, K15L, and S18L or
other amino acids
could be made to favor helix formation and bundling.
Alternatively, B-II which lacks the predicted upstream helix of B-I may be
modified
to enhance and improve its ability to inhibit BttxB protease activity. For
example, a peptide
having substitutions S3A and S4A (SEQ ID NO:S) has a predicted helix upstream
of the QF
site. Another example would be a peptide having substitutions S2L and S4L (SEQ
ID N0:6).
Likewise, this peptide has a predicted helix upstream of the QF site.
Example 7
Buforinin Pretreatment
Buforinins may be used to pretreat food and liquids that might be contaminated
with
BttxB or Tttx. For example, an effective amount of a Buforinin may be mixed
into water
having BttxB to inhibit the protease activity of the BttxB, e.g. 100 ml of
water containing 1
ug of BttxB would be treated with 100ug of Buforinin and 0.1 mmol reducing
agent, i.e.
TCEP in tablet, powder, or liquid form.
These various forms would comprise of a Buforinin, reducing agent such as
TCEP,
and other fillers and stabilizers. A liquid form could be made from a tablet
or powder that is
pre-dissolved prior to use. A Buforinin solution may be applied on the surface
of solid food
having BttxB on the surface. Alternatively, an effective amount of a Buforinin
may be used
to treat solid food which has been ground into small particles in order to
allow the Buforinin
access to amounts of BttxB which is not found on the surface of the food.
Contaminated or suspect non-food surfaces may also be washed with solutions of
Buforinin. Buforinins could be applied as a spray, foam, towelette, or sponge
used to soak or
wipe the surface. The amounts would be typically 200 ug per ml of solution
applied;
however, the concentrations required would depend on the extent of
contamination and the
appropriate Buforinin concentration may be adjusted as needed.
Example 8
Prophylaxis Uses
Buforinins could be used as a prophylactic against BttxB or Tttx poisoning.
Subjects
could be treated with Buforinins prior to entering situations where they are
likely to be in
contact with BttxB or Tttx. The dosage mode and amount could be dependent on
the amount
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WO 00/69895 PCT/US00/12909
of toxin expected to contact and the time in which contact might occur. The
preferred
administration for immediate contact would be i.v. The preferred form
administration for a
slower and more prolonged exposure would be by ingestion. However, other slow
release
forms of delivery such as a patch may be used.
Example 9
Prevention of Aerosol Contamination
Buforinins may be incorporated into a disposable, moist-filter, breathing mask
for
inactivating BttxB in aerosol form. The toxin would be trapped in moist-filter
whereupon it
would inactivated by a Buforinin.
Such a filter design would protect against toxin particles smaller than
bacteria, e.g. 1
micron such as HEPA. The filters could be supplied premoistened and
impregnated with
Buforinins and adjunct chemicals such as TCEP. Alternatively, the filters
could be prepared
by wetting a dry filter pre-impregnated or by soaking the filter in a solution
of Buforinins.
Enclosed areas that have air processing capabilities may also be protected in
this fashion with
appropriate sized filters.
Example 10
Wound Treatment
Open lesions could be treated with topical applications having Buforinins to
inhibit
BttxB or Tttx poisoning before the toxin has a chance to be absorbed into the
body. A
powder mixture containing Buforinins and adjuncts which include a reducing
agent and other
stabilizers or fillers may be applied directly to the wound. This approach
relies on the wound
weeping to dissolve the Buforinins. Alternatively, an ointment, liquid, spray,
foam, or
towelette having Buforinins may be applied to the wound surface. The towelette
could be
supplied or made in a similar manner as the filters of Example 10.
Example 11
Post Exposure
Subjects already suffering from BttxB or Tttx poisoning could be treated with
Buforinins. These of treatments would scavenge accessible toxin not yet
compartmentalized
into susceptible cells. Intoxication of susceptible cells leads to cell
function inhibition but is
not itself lethal to the cells. Given sufficient time the cells can recover
and become
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WO 00/69895 PCT/US00/12909
functional again. This recovery process may last up to several months.
Therefore, treatment
with Buforinins will aid in the recovery of the subject and reduce the need of
alternative life
supporting measures. The treatment may comprise use of Buforinin-BttxB HC or
other like
conjugates. The Bttx-HC portion would specifically direct the conjugate to
susceptible cells
where uptake would occur in a manner similar as the toxin. Inside the cell,
the Buforinins
would access to the toxin and inhibit the protease activity, thereby
protecting the cell against
further toxin damage until the toxin is removed from the cells by endogenous
proteolysis.
Example 12
Identification of a Botulinum Toxin Subclass
Buforinins may be used for the identification of BttxB or Tttx. An unknown
Bttx or
Tttx would be incubated with substrates and a Buforinin that would
specifically inhibit BttxB
and Tttx if present. Detection of uncleaved substrate or reduction of digest
products would
allow the identification of the toxin.
This may be useful as a confirming assay since the inhibition is specific. For
example, a C-terminal fluorescent-labeled substrate, such as VAMP2, would be
attached to
microtiter plates. See Hallis, B., et al. (1996) J. Clin. Microbiol. 34:1934-
1938. The
unknown sample is then added to the well and allowed to incubate. The reaction
would be
stopped and the well rinsed. Reduction of fluorescence would indicate
susceptibility of the
substrate to the toxin. If Buforinins are included in the digest mix then
BttxB or Tttx toxin
would be specifically inhibited and the fluorescence levels would be higher
than those
reactions containing BttxB without inhibitor.
Example 13
Long-lived peptide in vivo
To establish efficacy for the use of buforin I as treatment of botulinum B
toxicity, the
pharmacokinetic parameters of buforin I in the blood were examined. Since
human studies
are not possible, a rat model was used. Buforin I was injected
intraperitoneally at a dose of
100 ng/kg containing radiolabeled ~ZSI-buforin 1 (2,000 Ci/mmol) as a tracer
with the
radioactive dose constant at 11 ~Ci/kg. Blood (100 ~.1) was collected at timed
intervals from
the tail vein and flash frozen on dry-ice. At the time of analysis, the blood
was quickly
thawed and spiked with 1 ~g of cold buforin 1, and the cells were lysed and
solubilized by the
addition of NCS tissue solubilizer. CPM (counts per minute) were determined
for 20 ~1
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aliquots in a Packard Tri-Carb and the results are shown in Figure 7. The
pharmacokinetic
parameters are shown in Table 4.
The results show that there is a rapid uptake of buforin I into the blood
stream over
time. See Figure 8. The time to reach 1/2 the absorbed maximal value is 7.7
minutes. The
maximal- plateau level was reached within 40 min and was maintained for up to
4 h. The
relatively minor differences in absolute responses between the two animals are
probably due
to injection variation or animal variability, nevertheless, both animals
display a long steady
state level of buforin 1.
These results indicate that buforin I would have a long life in vivo and
therefore be an
effective therapeutic agent since it is distributed to the blood in a rapid
manner and the level
of buforin I persists over time at a high steady-state level.
Table
4.
Pharmacokinetic
characteristics
of
,zsl-buforin
I after
IP
injection
Mean SEM n
Bmax 182.6 50.0 CPM 2
Bmax 7.7 0.2 min 2
rPlateau40 min 2
Example 14
Phosphorylation of Peptides
Phosphorylation provides peptides with additional properties that could
improve the
circulatory half life, solubility, resistance to degradation, and the
interaction of the peptide
with the active site of the toxin, making it a more potent inhibitor. Indeed,
the natural
substrate VAMP2 has been found to be a good phosphorylation substrate and
whose function
may be affected by its phosphorylation state (Neilander, HB, et al. (95) J.
Neurochem.
65:1712-20). Another possible mechanism for inhibition of toxin protease
activity by
phosphorylated amino acids i.e., phospho-Ser, -Thr and/or -Tyr, would be that
once the
peptide enters the active site of the toxin, the strong charge associated with
the phosphate
group might form a salt linkage with the zinc found in the active site of the
toxin. This
binding then would block access of the natural proteins to the catalytic site
of the toxin,
neutralizing the toxic effects of the molecule. Peptides containing phospho-
Ser, -Thr and/or -
Tyr(s) can be readily made during solid phase peptide synthesis (White, P and
Beythien, J. in
"Innovations & Perspectives in Solid Phase Synthesis & Combinatorial
Libraries, 4h
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International Symposium", Epton, R. (Ed.), Mayflower Scientific Ltd.,
Birmingham 1996, pp.
557; Wakamiya, T., et al. Chem Lett, 1099 (1994)), or after synthesis by
incubating the
peptide with kinases specific for each of these amino acids (Risinger, C.,
Bennett, MK. (99)
J. Neurochem. 72:614-24).
Example 15
General botulinum toxin/tetanus toxin inhibitor in vivo
Use of TCEP and chaotropes
Disruption of non-covalent interactions between the light and heavy chains of
neurotoxins. A replacement for the foul-smelling and toxic sulfhydryl reducing
agents such
as 2-mercaptoethanol that functions equivalently to activate the neurotoxin in
vitro was
found. Once treated with TCEP, the disulfide bond covalently joining the heavy
and light
chains is broken. However, the neurotoxin chains apparently remain together
due to strong
hydrophobic interactions. In conjunction with TCEP, the use of biocompatible
chaotropes
will aid in completely separating holotoxins into its two chains, then the
light chain would be
effectively diluted in the body and could not target neuronal cells (or other
cell types). Some
biocompatible chaotropes include hydroxyurea or 2-oxo-1 pyrrolidine acetamide,
compounds
that are used for treatment of sickle cell anemia. The combination of TCEP and
biocompatible chaotropes to open the active site of all botulinum serotypes
and similar toxins
to pharmacological intervention before translocation into target cells will
provide more
effective and serotype nonspecific therapeutic peptides.
Incorporation by Reference
To the extent necessary to understand or complete the disclosure of the
present
invention, all publications, patents, and patent applications mentioned herein
are expressly
incorporated by reference therein to the same extent as though each were
individually so
incorporated.
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