Note: Descriptions are shown in the official language in which they were submitted.
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W
CONOTOXINS
This invention relates to relatively short
peptides, and more particularly to peptides between about
16 and about 46 residues in length, which are naturally
available in minute amounts in the venom of the cone
snails and which may include one or more cyclizing
disulfide linkages.
background of the 'Invention
Mollusks of the genus Conus produce a highly
toxic venom which enables them to carry out their unique
predatory lifestyle. Prey are immobilized by the venom
which is injected by means of a highly specialized venom
apparatus, a disposable hollow tooth which functions both
in the manner of a harpoon and a hypodermic needle.
Few interactions between organisms are more
striking than those between a venomous animal and its
envenomated victim. Venom may be used as a primary
weapon to capture prey or as a defense mechanism. These
venoms disrupt essential organ systems in the envenomated
animal, and many of these venoms contain molecules
directed to receptors and ion channels of neuromuscular
systems.
The predatory cone snails (Conus) have
developed a unique biological strategy. Their venom
contains relatively small peptides that are targeted to
various neuromuscular receptors and may be equivalent in
their pharmacological diversity to the alkaloids of
plants or secondary metabolites of microorganisms. Many
of these peptides are among the smallest nucleic
acid-encoded translation products having defined
conformations, and as such they are somewhat unusual
because peptides in this size range normally equilibrate
CA 02420184 2003-03-12
among many conformations for proteins having a fixed
conformation are generally much larger.
The cone snails that produce these toxic
peptides, which are generally referred to as conotoxins
or conotoxin peptides, are a large genus of venomous
gastropods comprising approximately 500 species. All
cone snail species are predators that inject venom to
capture prey, and the spectrum of animals that the genus
as a whole can envenomate is broad. A wide variety of
hunting strategies are used; however, every Conus species
uses fundamentally the same basic pattern of
envenomation.
The major paralytic peptides in these fish-
hunting cone venoms were the first to be identified and
characterized. In C. qeographus venom, three classes of
disulfide-rich peptides were found: the a-conotoxins
(which target and block the nicotinic acetylcholine
receptors); the ~-conotoxins (which target and block the
skeletal muscle Na' channels); and the ~-conotoxins (which
target and block the presynaptic neuronal Ca2' channels).
However, there are multiple homologs in each toxin class;
for example, at least five different ~-conotoxins are
present in C. qeographus venom alone. Considerable
variation in sequence is evident, and when different ~-
conotoxin sequences were first compared, only the
cysteine residues that are involved in disulfide bonding
and one glycine residue were found to be invariant.
Another class of conotoxins found in C. Qeoaraphus venom
is that referred to as the conantokins which cause sleep
in young mice and hyperactivity in older mice and are
targeted to the NMDA receptor. Each cone venom appears
to have its own distinctive group or signature of
different conotoxin sequences.
Many of these peptides have now become fairly
standard research tools in neuroscience. The
conotoxins, because of their ability to preferentially
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block muscle but not axonal Na° channels, are convenient
tools for immobilizing skeletal muscle without affecting
axonal or synaptic events. The ~a-conotoxins have become
standard pharmacological reagents for investigating
voltage-sensitive Ca2° channels and are used to block
presynaptic termini and neurotransmitter release. The Za-
conotoxin GVIA from C. aeoaraphus venom, which binds to
neuronal voltage-sensitive Ca2° channels, is an example of
such. The affinity (Kd) of i~-conotoxin GVIA for its high-
l0 affinity targets is sub-picomolar; it takes more than 7
hours for 50% of the peptide to dissociate. Thus the
peptide can be used to block synaptic transmission
virtually irreversibly because it inhibits presynaptic
Caz° channels. However, i~-conotoxin is highly tissue-
specific. In contrast to the standard Ca2° channel-
blocking drugs (e.g. the dihydropyridines, such as
nifedipene and nitrendipene, which are widely used for
angina and cardiac problems), which can bind Caz° channels
in smooth, skeleteal, and cardiac muscle as well as
neuronal tissue, ~-conotoxins generally bind only to a
subset of neuronal CaZ° channels, primarily of the N
subtype. The discrimination ratio for ~-conotoxin
binding to voltage-sensitive Ca2' channels in neuronal
versus nonneuronal tissue ge.g. skeletal or. cardiac
muscle) is greater than io$ in many cases.
Additional conotoxin peptides having these
general properties continue to be sought.
Summary of the Invention
The present invention provides a group of
bioactive conotoxin peptides which are extremely potent
inhibitors of synaptic transmission at the neuromuscular
junction and/or which are targeted to specific ion
channels. They are useful as pesticides, and many of
them or closely related analogs thereof are targeted to
specific insects or other pests. Therefore, the DNA
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°4-
encoding such conotoxin peptides can be advantageously
incorporated into plants as a plant-defense gene to
render plants resistant to specified pests.
These conotoxin peptides have the formulae set
forth hereinafter. Moreover, examination of the formulae
shows an indication of two new classes of conotoxin
peptides in addition to those classes hereinbefore
described. Class A includes peptides SEQ ID NO:1 to
N0:6; each has 6 Cys residues which are interconnected by
3 disulfide linkages, with the 2 Cys residues nearest the
N-terminus being part of a sequence -Cys-Cys-Gly-. All 6
members have at least one 4Hyp residue and a C-terminus
which appears to be amidated. There are 2 amino acid(AA)
residues separating the 3rd and 4th ~Cys as numbered (from
the N°terminus) and a single AA residue spacing the 4th
Cys from the 5th Cys. Moreover, the 2nd Cys is usually
separated from the 3rd Cys by either 6 or 7 AA residues
in this class, whereas there can be from about 3 to about
6 AA residues separating the 5th and 6th Cys residues.
Class E is exemplified by SEQ ID N0:7, wherein there is a
central sequence of 5 AA residues having 2 pairs of Cys
residues flanking a center residue which is preferably
Asn and wherein there are 2 additional. pairs of spaced-
apart Cys residues located, respecti'rely, N-terminally
~5 and C-terminally of this central sequence. SEQ ID N0:8
appears to be a member of the known class of a-
conotoxins. SEQ ID N0:9 appears to be a member of the
known class of ~-conotoxins. SEQ ID NO:10 and N0:11 may
be members of the class of ~°conotoxins. SEQ ID NO:12
30 appears to be a member of the class of conantokins
characterized by the N-terminal sequence Gly-Glu-Gla-Gla,
and SEQ ID N0:13 may be a member of a heretofore
uncharacterized class which causes sluggish behavior.
The individual formulae of these conotoxins are as
35 follows:
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°5-
Gly-Cys-Cys-Gly-Ser-Tyr-Pro-Asn-Ala-Ala-Cys-His-Pro-Cys-
Ser-Cys-Lys-Asp-Arg-Xaa-Ser-Tyr-Cys-Gly-Gln (SEQ ID NO:1)
(J-020), wherein Xaa is 4Hyp (4-hydro~yproline) and the
C-terminus is amidated;
Glu-Lys-Ser-Leu-Val-Pro-Ser-Val-Ile-Thr-Thr-Cys-Cys-Gly-
Tyr-Asp-Xaa-Gly-Thr-Met-Cys-Xaa-Xaa-Cys-Arg-Cys-Thr-Asn-
Ser-Cys (SEQ ID No:2) (J-005) wherein Glu in the 1-
position is pGlu, Xaa is 4Hyp and the C-terminus is
amidated; Ser in the 7-position may be glycosylated;
Cys-Cys-Gly-Val-Xaa-Asn-Ala-Ala-Cys-Pro-Xaa-Cys-Val-Cys-
Asn-Lys-Thr-Cys-Gly (SEQ ID N0:3) (OB-34) wherein Xaa is
4Hyp and the C-terminus is amidated;
Gly-Cys-Cys-Gly-Ser-Tyr-Xaa-Asn-Ala-Ala-Cys-His-Xaa-Cys-
Ser-Cys-Lys-Asp-Arg-Xaa-Ser-Tyr-Cys-Gly-Gln (SEQ ID N0:4)
(J-019) wherein Xaa is 4Hyp and the C-terminus is
amidated;
Gly-Cys-Cys-Gly-Ser-Tyr-Xaa-Asn-Ala-Ala-Cys-His-Pro-Cys-
Ser-Cys-Lys-Asp-Arg-Xaa-Ser-Tyr-Cys-Gly-Gln (SEQ ID
N0:5) (J-026) wherein Xaa is 4Hyp and the C-terminus is
amidated;
Cys-Cys-Gly-Val-Xaa-Asn-Ala-Ala-Cys-His-Xaa-Cys-Val-Cys-
Lys-Asn-Thr-Cys (SEQ ID N0:6) (OB-26) wherein Xaa is 4Hyp
and the C-terminus is amidated;
Gly-Xaa-Ser-Phe-Cys-Lys-Ala-Asp-Glu-Lys-Xaa-Cys-Glu-Tyr-
His-Ala-Asp-Cys-Cys-Asn-Cys-Cys-Leu-Ser-Gly-Ile-Cys-Ala-
Xaa-Ser-Thr-Asn-Trp-Ile-Leu-Pro-Gly-Cys-Ser-Thr-Ser-Ser-
Phe-Phe-Lys-Ile (sEQ ID No:7) (J-o29) wherein xaa is
4Hyp; the C-terminus may optionally be amidated;
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Gly-Cys-Cys-Ser-His-Pro-Ala-Cys-Ser-GIy-Lys-Tyr-Gln-Xaa-
Tyr-Cys-Arg-Xaa-Ser (SEQ ID N0:8) (OH-20) wherein Xaa is
Gla and the C-terminus is amidated;
His-Xaa-Xaa-Cys-Cys-Leu-Tyr-Gly-Lys-Cys-Arg-Arg-Tyr-Xaa-
Gly-Cys-Ser-Ser-Ala-Ser-Cys-Cys-Gln (sEQ zD No:9) (J-o21)
wherein Xaa is 4Hyp;
Cys-Lys-Thr-Tyr-Ser-Lys-Tyr-Cys-Xaa-Ala-Asp-Ser-Xaa-Cys-
Cys-Thr-Xaa-Gln-Cys-Val-Arg-Ser-Tyr-Cys-Thr-Leu-Phe (SEQ
ID NO:10) (J-010) wherein Xaa is Gla and the C-terminus
is amidated;
Ser-Thr-Ser-Cys-Met-Glu-Ala-Gly-Ser-I'yr-Cys-Gly-Ser-Thr-
Thr-Arg-Ile-Cys-Cys-Gly-Tyr-Cys-Ala-Tyr-Phe-Gly-Lys-Lys-
Cys-Ile-Asp-Tyr-Pro-Ser-Asn (SEQ ID NO:11) (J-008);
Gly-Glu-Xaa-Xaa-Val-Ala-Lys-Met-Ala-Ala-Xaa-Leu-Ala-Arg-
Xaa-Asn-Ile-Ala-Lys-Gly-Cys-Lys-Val-Asn-Cys-Tyr-Pra (SEQ
ID N0:12) (J-017) wherein Xaa is Gla (y-carboxyglutmate);
and
Glu-Ser-Glu-Glu-Gly-Gly-Ser-Asn-Ala-Thr-Lys-Lys-Pra-Tyr-
Ile-Leu (SEQ ID No:l3) (J-004), wherein Glu in the 1-
position is pGlu (pyroglutamic) and the C-terminus may be
amidated; Thr may be glycosylated.
Accordingly in one aspect, the invention
provides conotoxin peptides having the general formula:
Xaa'-Cys-Cys-Gly-Xaaz-Cys-Xaa3-Xaa4-Cys-Xaa~-Cys-Xaag-Cys-
Xaa7-NH2 (SEQ ID N0:14) wherein Xaa1 is des-Xaa~ or Gly or
pGlu-Lys-Ser-Leu-Val-Pro-Ser-Val-Ile-Thr-Thr; XaaZ is
Ser-Tyr-Pro-Asn-Ala-Ala or Tyr-Asp-4Hyp-Gly-Thr-Met or
Val-4Hyp-Asn-Ala-Ala or Ser-Tyr-4Hyp-Asn-Ala-Ala; Xaa3 is
His, 4Hyp or Pro; Xaa4 iS Pro or 4Hyp,° Xaa~ is Ser, Arg or
VaI; Xaab is Lys-Asp-Arg-4Hyp-Ser-Tyr or Thr-Asn-Ser or
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Asn-Lys-Thr or Lys-Asn-Thr~ and Xaal is des-Xaa~ or Gly or
Gly-Gln.
In another aspect, the invention provides
conotoxin peptides having 6 Cys residues interconnected
by 3 disulfide bonds, with the 2 Cys residues nearest the
N-terminus being part of the sequence Cys-Cys-Gly and
with the 3rd, 4th and 5th residue being spaced apart by 2
residues and 1 residue, respectively, said two residues
being selected from His, Pro and 4Hyp, said single
residue being Ser, Arg or Val and with the C-terminus
being amidated, said conotoxin binding to the
acetylcholine receptor.
In yet another aspect, the invention provides
conotoxin peptides having 8 Cys residues interconnected
by 4 disulfide bonds with the central 4 Cys residues
being part of the sequence Cys-Cys-Asn-Cys-Cys (SEQ ID
N0:15), said conotoxin causing immediate paralysis when
administered intercranially to laboratory mice.
These peptides, which are generally termed
conotoxins, are sufficiently small to be chemically
synthesized. General chemical syntheses for preparing
the foregoing conotoxins are described hereinafter along
with specific chemical syntheses of several conotoxins
and indications of biological activities of these
synthetic products. Various of these conotoxins can also
be obtained by isolation and purification from specific
conus species using the technique described in U.S.
Patent NO: 4,447,356 (May 8, 1.984)'
Many of these conotoxin peptides are extremely
potent inhibitors of synaptic transmission at the
neuromuscular junction, while at the same time lacking
demonstrable inhibition of either nerve or muscle action
potential propagation. They are considered useful to
relax certain muscles during surgery.
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The activity of each of these conotoxin
peptides is freely reversible upon dilution or removal of
the toxin from the affected muscle. Moreover, toxicity
of the cyclic peptides is generally destroyed by agents
which disrupt disulfide bonds in the cyclic conotoxins,
suggesting that correct disulfide bonding appears
essential for biological activity~ however, correct
folding and~or rearrangement of a conotoxin may occur in
vivo so that in some cases the linear peptide may be
administered for certain purposes. In general, however,
the synthetic linear peptides fold spontaneously when
exposed to air-oxidation at cold room temperatures to
create the correct disulfide bonds to confer biological
activity, and such processing is accordingly preferred.
The conotoxins exhibit activity on a 'wide range of
vertebrate animals, including humans, and on insects, and
many are useful to reversibly immobilize a muscle or
group of muscles in humans or other vertebrate species.
Many of these conotoxins and derivatives thereof are
further useful for detection and measurement of
acetylcholine receptors and other specific receptors
which are enumerated hereinafter with respect to various
particular peptides.
Many of these conotoxin peptides are also
useful in medical diagnosis. For example, an
immunoprecipitation assay with radiolabeled ~-conotoxin
can be used to diagnose the Lambent-Eaton myasthenic
syndrome, which is a disease in which autoimmune
antibodies targeted to endogenous Ca24 channels are
inappropriately elicited, thereby causing muscle weakness
and autonomic dysfunction.
Various of these conotoxin peptides are further
useful for the treatment of neuromuscular disorders and
for rapid reversible immobilization of muscles in
vertebrate species, including humans, thereby
facilitating the setting of fractures and dislocations.
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These conotoxins generally inhibit synaptic transmission
at the neuromuscular junction and bond strongly to the
acetylcholine receptor of the muscle end plate, and many
are therefore especially suitable for detection and assay
of acetylcholine receptors. Such measurements are of
particular significance in clinical diagnosis of
myasthenia gravis, and various of these conotoxins, when
synthesized with a radioactive label or as a fluorescent
derivative, provide improved quantitation and sensitivity
to in acetylcholine receptor assays.
Detailed Desori~ti~n of the Preferred Embodiments
Although the conotoxins can be obtained by
purification from the enumerated cone snails, because the
amounts of conotoxins obtainable from individual snails
are very small, the desired substantially pure conotoxins
are best practically obtained in commercially valuable
amounts by chemical synthesis. For example, the yield
from a single cone snail may be about l~ micrograms or
less of conotoxin. By substantially pure is meant that
the peptide is present in the substantial absence of
other biological molecules of the same type; it is
preferably present in an amount at least about 85~ by
weight and more preferably at least about 95% of such
biological molecules of the same type which are present,
i.e. water, buffers and innocuous small molecules may be
present. Chemical synthesis of biologically active
conotoxin peptide depends of course upon correct
determination of the amino acid sequence, and these
sequences have now been determined and are set forth in
the preceding summary.
Many of the conotoxins have approximately the
same level of activity, and comparison of them suggests a
reasonable tolerance for substitution near the carboxy
terminus of these peptides. Accordingly, equivalent
molecules can be created by the substitution of
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equivalent residues in this region, and such
substitutions are useful to create particular
invertebrate-specific conotoxins.
Cysteine residues are present in a majority of
these conotoxins, and several of the conotoxins disclosed
herein exhibit similar disulfide cross-linking patterns
to that of erabutoxin, a known protein toxin of sea snake
venom. The fact that biological activity of these
particular compounds is destroyed by agents which break
to disulfide bonds, such as sodium borohydride or ~-
mercaptoethanol, indicates that a specific folded
configuration induced by disulfide cross-links, is
essential for bioactivity of these particular conotoxins.
It has been found that air-oxidation of the linear
peptides for prolonged periods under cold room
temperatures results in the creation of a substantial
amount of the bioactive, disulfide-linked molecules.
Therefore, the preferable procedure for making these
peptides is to oxidize the linear peptide and then
2~ fractionate the resulting product, uGing reverse-phase
high performance liquid chromatography (HPLC) or the
like, to separate peptides having different linked
configurations. Thereafter, either by comparing these
fractions with the elution of the native material or by
using a simple assay, the particular fraction having the
correct linkage for maximum biological potency is easily
determined. It is also found that the linear peptide, or
the oxidized product having more than one fraction, can
sometimes be used for in vivo administration, because the
3o cross-linking and/or rearrangement which occurs in vivo
has been found to create the biologically potent
conotoxin molecules however, because of the dilution
resulting from the presence '~f other fractions of less
biopotency, a somewhat higher dosage may be required.
These conotoxins disclosed herein generally
inhibit synaptic transmission at the neuromuscular
CA 02420184 2003-03-12
°11-
junction by binding the acetylcholine receptor at a
muscle end plate. A particularly useful characteristic
of a number of these conotoxins is their high affinity
for particular macromolecular receptors, accompanied by a
narrow receptor-target specificity. A major problem in
medicine results from side effects which drugs very often
exhibit, some of which are caused by the drug binding not
only to the particular receptor subtype that renders
therapeutic value, but also to closely related,
therapeutically irrelevant receptor subtypes which can
often cause undesirable physiological effects. In
contrast to most drugs, these conotoxins generally
discriminate among closely related receptor subtypes.
The peptides are synthesized by a suitable
method, such as by exclusively solid-phase techniques, by
partial solid-phase techniques, by fragment condensation
or by classical solution couplings. The employment of
recently developed recombinant DNA techniques may be used
to prepare these peptides, particularly the longer ones
containing only natural amino acid residues which do not
require post-translational processing steps.
In conventional solution phase peptide
synthesis, the peptide chain can be prepared by a series
of coupling reactions in which the constituent amino
acids are added to the growing peptide chain in the
desired sequence. The use of various N-protecting
groups, various coupling reagents, e.g.,
dicyclohexylcarbodiimide or carbonyldimidazole, various
active esters, e.g., esters of N-hydroxypthalimide or N-
hydroxy-succinimide, and the various cleavage reagents,
to carry out reaction in solution, with subsequent
isolation and purification of intermediates, is well
known classical peptide methodology. Classical solution
synthesis is described in detail in the treatise
'°Methoden der Organischen Chemie (Houben-Weyl): Synthese
von Peptiden~°, E. Wunsch (editor) (1974) Georg Thieme
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Verlag, Stuttgart, W. Ger. Techniques of exclusively
solid-phase synthesis are set forth in the textbook
''Solid-Phase Peptide Synthesis", Stewart & Young, Freeman
& Co., San Francisco, 1969, and are exemplified by the
disclosure of U.S. Patent No. 4,105,603, issued August 8,
1978 to Vale et al. The fragment condensation method of
synthesis is exemplified in U.S. Patent No. 3,972,859
(August 3, 1976). Other available syntheses are-
exemplified by U.S. Patent No. 3,842,067 (October 15,
1974) and U.S. Patent No. 3,862,925 (~anuary 28, 1975).
Common to such chemical syntheses is the
protection of the labile side chain groups of the various
amino acid moieties with suitable protecting groups which
will prevent a chemical reaction from occurring at that
site until the group is ultimately removed. Usually also
common is the protection of an alpha-amino group on an
amino acid or a fragment while that entity reacts at the
carboxyl group, followed by the selective removal of the
alpha-amino protecting group to allow subsequent reaction
to take place at that location. Accordingly, it is
common that, as a step in such a synthesis, an
intermediate compound is produced which includes each of
the amino acid residues located in its desired sequence
in the peptide chain with appropriate side-chain
protecting groups linked to various of the residues
having labile side chains.
As far as the selection of a side chain-amino
protecting group is concerned, generally one is chosen
which is not removed during deprotection of the a-amino
groups during the synthesis. However, for some amino
acids, e.g. His, protection is not generally necessary.
In selecting a particular side chain protecting group to
be used in the synthesis of the peptides, the following
general rules are followed: (aj the protecting group
preferably retains its protecting properties and is not
split off under coupling conditions, (b) the protecting
CA 02420184 2003-03-12
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group should be stable under the reaction conditions
selected for removing the a-amino protecting group at
each step of the synthesis, and (c) the side chain
protecting group must be removable, upon the completion
of the synthesis containing the desired amino acid
sequence, under reaction conditions that will not
undesirably alter the peptide chain.
It should be possible to prepare many, or even
all, of these peptides using recombinant DNA technology~
however, when peptides are not so prepared, they are
preferably prepared using the Merrifield solid phase
synthesis, although other equivalent chemical syntheses
known in the art can also he used as previously
mentioned. Solid-phase synthesis is commenced from the
C-terminus of the peptide by coupling a protected a-amino
acid to a suitable resin. Such a starting material. can
be prepared by attaching an a-amino-protected amino acid
by an ester linkage to a chloromethylated resin or a
hydroxymethyl resin, or by an amide bond to a
benzhydrylamine (BHA} resin or paramethylbenzhydrylamine
(MBHA) resin. The preparation of the hydroxymethyl resin
is described by Bodansky et al., Chem. Ind. (London} 38,
1597-98 (1966). Chloromethylated resins are commercially
available from Bio Rad Laboratories, Richmond, California
and from Lab. Systems, Inc. The preparation of such a
resin is described by Stewart et al., "Solid Phase
Peptide Synthesis", supra. BHA and MBHA resin supports
are commercially available and are generally used when
the desired polypeptide being synthesized has an
unsubstituted amide at the C-terminus. Thus, solid resin
supports may be any of those known in the art, such as
one having the formulae -o-CH2-resin support, -NH BHA
resin support or -NH-MBHA resin suppart. When the
unsubstituted amide is desired, use of a BHA or MBHA
resin is preferred, because cleavage directly gives the
amide. In case the N-methyl amide is desired, it can be
CA 02420184 2003-03-12
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generated from an N-methyl BHA resin. Should other
substituted amides be desired, the teaching of U.S.
Patent No. 4,569,967 can be used, or should still other
groups than the free acid be desired at the C-terminus,
it may be preferable to synthesize the peptide using
classical methods as set forth in the Houben-Weyl text.
The C-terminal amino acid, protected by Boc and
by a side-chain protecting group, if appropriate, can_be
first coupled to a chloromethylated resin according to
the procedure set forth in Chemistry Letters, K. Horiki
et al. 165-168 (1978), using KF in DNTF at about 60°C. for
24 hours with stirring, when a peptide having free aced
at the C-terminus is to be synthesized. Following the
coupling of the BOC-pratected amino acid to the resin
support, the a-amino protecting group is removed, as by
using trifluoroacetic acid(TFA) in methylene chloride or
TFA alone. The deprotection is carried out at a
temperature between about 0°C and room temperature. Other
standard cleaving reagents, such as HCl in dioxane, and
conditions for removal of specific a-amino protecting
groups may be used as described in Schroder & Lubke, "The
Peptides", 1 pp 72-75, Academic Press (1965).
After remotral of the a-amino protecting group,
the remaining a-amino- and side chain-protected amino
acids are coupled step-wise in the desired order to
obtain the intermediate compound defined hereinbefore, or
as an alternative to adding each amino acid separately in
the synthesis, some of them may be coupled to one another
prior to addition to the solid phase reactor. The
selection of an appropriate coupling reagent is within
the skill of the art. Particularly suitable as a coupling
reagent is N,N'-dicyclohexyl carbodiimide (DCC).
The activating reagents used in the solid phase
synthesis of the peptides are well known in the peptide
art. Examples of suitable activating reagents are
carbodiimides, such as N,N'-diisopropylcarbodiimide and
CA 02420184 2003-03-12
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N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide. Other
activating reagents and their use in peptide coupling are
described by Schroder & Lubke supra, in Chapter III and
by Kapoor, J. Phar. Sci., 59, pp 1-27 (1970).
Each protected amino acid or amino acid
sequence is introduced into the solid phase reactor in
about a twofold or more excess, and the coupling may be
carried out in a medium of dimethylformamide(DMF): CH2C12
(1:1) or in DMF or CHZClZ alone. In cases where
incomplete coupling occurs, the coupling procedure is
repeated before removal of the a-amino protecting group
prior to the coupling of the next amino acid. The
success of the coupling reaction at each stage of the
synthesis, if performed manually, is preferably monitored
by the ninhydrin reaction, as described by E. Kaiser et
al., Anal. Biochern. 34, 595 (1970). The coupling
reactions can be performed automatically, as on a Beckman
990 automatic synthesizer, using a program such as that
reported in Rivier et al. Biopolymers, 1978, 17, pp
1927-1938.
After the desired amino acid sequence has been
completed, the intermediate peptide can be removed from
the resin support by treatment with a reagent, such as
liquid hydrogen fluoride, which not only cleaves the
peptide from the resin but also cleaves all remaining
side chain protecting groups and also the a-amino
protecting group at the N-terminus if it was not
previously removed to obtain the peptide in the form of
the free acid. If Met is present in the sequence, the
Boc protecting group is preferably first removed using
trifluoroacetic acid(TFA)/ethanedithiol prior to cleaving
the peptide from the resin with HF to eliminate potential
S-alkylation. when using hydrogen fluoride for cleaving,
one or more scavengers, such as anisole, cresol, dimethyl
sulfide, and methylethyl sulfide are included in the
reaction vessel.
CA 02420184 2003-03-12
~1~-
Cyclization of the linear peptide is preferably
effected, as opposed to cyclizing the peptide while a part
of the peptidoresin, to create bonds between Cys residues.
To effect such a disulfide cyclizing linkage, the fully
protected peptide can be cleaved from a hydroxymethylated
resin or a chloromethylated resin support by ammonolysis, as
is well known in the art, to yield the fully protected amide
intermediate, which is thereafter suitably cyclized and
deprotected; alternatively, deprotection as well as cleavage
of the peptide from the above resins or a benzhydrylamine
(BHA) resin or a methyl-benzhydrylamine (MBHA), can take
place at 0°C with hydrofluoric acid (HF), followed by air-
oxidation under high dilution conditions.
Thus, in one aspect, the invention also provides a
35 method for manufacturing a synthetic conotoxin peptide of
interest by carrying out the following steps: (a) forming a
peptide intermediate having the desired amino acid residue
sequence and at least one protective group attached to a
labile side chain of a residue such as Ser, Thr, Tyr, Asp,
GIu, His, Cys, Arg or lays and optionally having its C-
terminus linked by an anchoring bond to resin support; (b)
splitting off the protective group or groups and any
anchoring bond from the peptide intermediate to form a
linear peptide; (c) creating a cyclizing bond between Cys
residues present in the linear peptide to create a cyclic
peptide; and (d) if desired, converting the resulting
cyclic peptide into a nontoxic salt thereof. Particular
side chain protecting groups and resin supports are well
known in the art and are disclosed in the earlier-referenced
patents..
In order to illustrate specific preferred
embodiments of the invention in greater detail, the
following exemplary work is provided.
CA 02420184 2003-03-12
-17-
EXAMPLE 1
Conotoxin SEQ ID NO:1 (also referred to as J-
020), having the chemical formula:
H-Gly-Cys-Cys-Gly-Ser-Tyr-Pro-Asn-Ala-Ala-Cys-His-Pro-
Cys-Ser-Cys-Lys-Asp-Arg-4Hyp-Ser-Tyr-Cys-Gly-Gln-NHZ is
synthesized by stepwise elongation from the carboxyl
terminus, using the solid phase Merrifield peptide
synthesis procedure. Operational details of this general
procedure, which are not set forth hereinafter, can be
found in Stewart, J.M. and Young, J., Solid Phase Peptide
Synthesis, 2nd Ed., Pierce Chemical Co., Rockford, I11.,
(1984), and in Rivier et al., U.S. Patent No. 5,064,939
(Nov. 12, 1991) ,
A methylbenzyhydrylamine resin is used as the
solid phase support and facilitates production of the
amidated peptide. Amino acid residues,, in the form of
their Boc (tart-butyloxycarbonyl) derivatives, are
coupled successively to the resin using
dicyclohexylcarbodiimide (DCC) as the coupling or
condensing agent. At each cycle of stepwise amino acid
addition, the Boc group is removed by acidolysis with 50
percent (v/v) trifluoroacetic acid (TFA) in methylene
chloride, using an appropriate scavenger, such as 1,2
ethanedithiol, thereby exposing a new cx-amino group for
the subsequent coupling step. More specifically, when an
automated machine and about 5 grams of resin are used,
following the coupling of each amino acid residue,
washing, deblocking and coupling of the next residue are
preferably carried out according to the following
schedule:
CA 02420184 2003-03-12
-18-
STEP REAGENTS AND OPERATIONS MIX TIMES
- :III N -
1 CH2C12 wash-4 0 ml . ( 2 times ) 3
2 Methanol(MeOH) wash-30 ml. (2 times) 3
3 CHZC12 wash-80 ml. (3 times) 3
4 50 percent TFA plus 5 percent 1,2-ethane-
dithiol in CHZC12-70 ml. (2 times) 12
5 Isopropanol wash-40 ml. (2 times) 3
6 TEA 12.5 percent in CHZC12-7o ml.
(2 times) 5
7 MeOH wash-40 ml. (2 times) 2
8 CHZClZ wash-80 ml. (3 times) 3
9 Boc-amino acid (10 mmoles) in 30 ml. of either
DMF or CHZC12, depending upon the solubility
of the particular protected amino acid, (1 time)
plus DCC (10 mmoles) in CHZCIz 30-300
Side chain protecting groups are generally
chosen from among the standard set of moderately acid-
stable derivatives. Such protecting groups are
preferably ones that are not removed during deblocking
by trifluoroacetic acid in methylene chloride; however,
all are cleaved efficiently by anhydrous hydrofluoric
acid (HF) to release the functional side chains.
Cysteine residues in positions 2, 3, 11, 14, 16 and 23
of the peptide are protected by p-methoxy-benzyl (Mob)
groups so as to expose sulfhydryls upon deprotection.
The phenolic hydroxyl group of Tyr is protected by 2-
bromo-benzyloxycarbonyl (Brzj. The side chain of 9-
hydroxyproline (~Fiyp) is protected by benzyl ether
(OBzlj, and it is commercially available in this
protected form. The side chain of Arg is protected with
Tos (p-toluenesulfonyl). The side chain of Asp is
protected as the cyclohexyl ester (OChxj, and the
CA 02420184 2003-03-12
-19-
primary amino side chain of Lys is protected with 2-
chlorobenzyloxycarbonyl (Clz). The imidazole nitrogen
of His is protected by Tos. Serine is protected by
benzyl ether (OBzl). Asn is coupled without side chain
protection in the presence of hydroxybenzotriazole
(Host).
At the end of the synthesis, the following
peptide intermediate is obtained: Boc-Gly-Cys(Mob)-
Cys(Mob)-Gly-Ser(OBzl)-Tyr(Brz)-Fro-Asn-Ala-Ala-
Cys(Mob)-His(Tos)-Pro-Cys(Mob)-Ser(OBzl)-Cys(Mob)-
Lys(Clz)-Asp(OChx)-Arg(Tos)-4Hyp(Bzl)-Ser(OBzl)-
Tyr(Brz)-Cys(Mob)-Gly-Gln-MBHA resin support. All the
side-chain blocking graups are HF-cleavable.
After removing the N-terminal Boc group with
TFA, the linear peptide is cleaved from the resin and
deprotected with HF, using 15o milliliters of HF, 16 ml
of anisole and about 4 ml dimethyl sulfide for about 1.5
hours at 0°C., which removes all the remaining
protecting groups. Any volatiles are removed by the
application of a vacuum, and the peptide is washed with
ethylether and then dissolved in 5 percent acetic acid.
The solution is then diluted to about 15 liters and pH
is adjusted to about 8.0 with diisopropyl ethylamine.
It is exposed to air-oxidation in a cold room at about
4°C. for 4 days to form the disulfide cross links or
bridges. One drop of mixture is recovered about emery
12 hours and added to one drop of a solution containing
dithio-bis(2-nitrobenzoic) acid in a molar buffer of
K2HP04(pH 8) in order to follow.the progress of the
oxidation reaction (Bllman test). During the whole
reaction, the pH was maintained at 8 by addition of
diisopropylethylamine. After 50 hours, the absence of
yellow coloration is observed in the test with dithio-
bis(2-nitrobenzoic) acid.
CA 02420184 2003-03-12
-2~-
After formation of the disulfide bridges, the
*
cyclized pool of peptides is applied to a Bio-Rex-7o
column (5 x 15 em), washed in distilled water (100 ml),
and eluted with 50% acetic acid. The cyclized peptide
fractions are collected and lyophilized.
The lyophilized peptide fractions are then
purified by preparative or semi-preparative HPLC as
described in Rivier, et al., ~. Chromatography, 288,
303-328 (1984); and Hoeger, et al., BioChromatoqraphy,
2, 3, 134-142 (1987). The chromatographic fractions are
carefully monitored by HPLC, and only the fractions
showing substantial purity are pooled.
The peptide is judged to be homogeneous by
reversed-phase high performance liquid chromatography
*
using a Waters HPLC system with a 0.46 x 25 cm. column
packed with 5~m C~8 silica, 300 pore size. The
determination is run at room temperature using gradient
conditions with 2 buffers. Buffer A is an aqueous
trifluoroacetic acid (TFA) solution consisting of 1.0
ml. of TFA per 1000 ml, of solution. Buffer B is 1 ml
TFA diluted to 400 ml with Hz0 which is added to.600 ml.
of acetonitrile. The analytical HPLC was run under
gradient conditions which vary uniformly from 20 volume
percent (v/o) Buffer B to 35 v/o Buffer B over 10
minutes, at a constant flow rate of 2 ml. per minute;
the retention time for the biologically active cyclic
conotoxin is 10.6 minutes.
The product is also characterized by amino acid
analysis and by toxicity tests. one microgram of the
synthetic toxin injected intracerebrally (IC) in a mouse
is lethal in less than 10 minutes showing that the
synthetic product is highly toxic, and thus synthesis by
the described method, if followed by air-oxidation,
achieves the correct disulfide pairing arrangement to
assure biological activity. The synthetic peptide is
shown to be substantially identical with the native
*Trade-mark
CA 02420184 2003-03-12
-21-
conotoxin as a result of coelution on HPLC, amino acid
analysis and biological activity. This peptide binds to
and inhibits the function of the acetylcholine receptor,
thereby causing paralysis and thereafter death. It can
be used in assays for the acetylcholine receptor.
EXAMPLE 2
Conotoxin SEQ ID No:2 (also referred to as
J-005), having the chemical formula:
H-pGlu-Lys-Ser-Leu-Val-Pro-Ser-Val-Ile-Thr-Thr-Cys-Cys-
Gly-Tyr-Asp-4Hyp-Gly-Thr-Met-Cys-4Hyp-4Hyp-Cys-Arg-Cys-
Thr-Asn-Ser-Cys-NHZ is synthesized by stepwise elongation
from the carboxyl terminus, using the solid phase
synthesis procedure as set forth in Example 1 and the
same methyl benzyhydrylamine resin.
The side chains of hydroxyproline, threonine
and serine are protected by benzyl ether (Bzl).
At the end of the synthesis, the following
peptide intermediate is obtained: Boc-pGlu-Lys(Clz)_
Ser(Bzl)-Leu-Val-Pro-Ser(Bzl)-Val-Ile-Thr(Bzl)-Thr(Bzl)--
Cys(Mob)-Cys(Mob)-Gly-Tyr(Brz)-Asp(OChx)-4Hyp(Bzl)-Gly-
Thr(Bzl)-Met-Cys(Mob)-4Hyp(Bzl)-4Hyp(Bzl)-Cys(Mob)-
Arg(Tos)-Cys(Mob)-Thr(Bzl)-Asn-Ser(Bzl)-Cys(Mob)-MBHA
resin support. All the side-chain blocking groups are
HF-cleavable.
After removing the N-terminal Boc group with
TFA, the linear peptide is cleaved from 3 grams of the
resin and deprotected, using l00 milliliters of HF, 1 ml
of anisole and about 4 ml dimethyl sulfide for about 1.5
hours at 0°C., which removes all the remaining
protecting groups. Any volatiles are removed by the
application of a vacuum, and the peptide i.s washed with
ethylether and then extracted with 10 percent acetic
acid containing 10% cyanomethane. The solution is then
diluted to about 4 liters and a pH of about 6.95. The
solution is exposed to air-oxidation in a cold room at
CA 02420184 2003-03-12
-
about 4°C. for a time sufficient to completely oxidize
by forming disulfide crosslinks or bridges, i.e., a
period of about 1 to 2 weeks.
After formation of the disulfide bridges, the
cyclized pool of peptides is applied to a Bio-Rex-70
column (5 x 15 cm) and eluted with 50o acetic acid. The
cyclized peptide fractions are collected and
lyophilized. The synthetic peptide is shown to be
substantially identical with the native conotoxin as a
result of coelution on HPLC, amino acid analysis and
biological activity, which comparison is made with the
native conotoxin following deglycosylation to remove the
carbohydrate linked to Ser in the 7-position which
increases bioactivity.
When injected IC into mice, the peptide causes
mice to become spastic and to suffer paralysis. It is
thus known to have high affinity and specificity for a
particular receptor and can be used to target this
receptor and in assays for this receptor.
EXAMPLE 3
The peptide ~B-34 (SEQ ID N0:3) is produced by
using the synthesis as generally set forth in Example 1.
The peptide in question has the following formula:
H-Cys-Cys-Gly-Val-4Hyp-Asn-Ala-Ala-Cys-Pro-4Hyp-Cys-Val-
Cys-Asn-Lys-Thr-Cys-Gly-NHz
The synthesis is carried out on an MBHA resin,
and Boc is used to protect the a-amino groups. The same
side chain protecting units are used as described
hereinbefore.
About 4-1/2 grams of the peptide-resin is
treated with 5 milliliters of anisole, 1 milliliter of
methylethyl sulfide, and 6o milliliters of Hf for 1./2
hour at -20°C and 1 hour at 0°C. The peptide is then
extracted and dissolved in 4.5 liters of ammonium
acetate buffer, a solution containing about 10 grams of
CA 02420184 2003-03-12
-23-
ammonium acetate at a pH of about 4.3. pH is adjusted
to about 7.75 with ammonium hydroxide, and the solution
is maintained in a cold room at about 4°C. for a
sufficient length of time to allow complete air-
s oxidation to occur. Purification is then carried out as
previously described with respect to Example 2, and the
purified peptide is subjected to analytical HPLC. It is
found to exhibit a single peak with both a gradient flow
and with isocratic flow of appropriate buffers. The
purity of the compound was estimated to be greater than
about 99 percent. The synthetic peptide coelutes with
the native peptide on HPLC.
Injection of 1 microgram of the synthetic
peptide OB-34 intracerebrally into a mouse shows that
the mouse exhibits a reproducible physical effect
indicative of binding to a specific receptor and
confirms that the air-oxidation produces appropriate
cross-linking so that the synthetic conotoxin exhibits
biological potency. It is thus known to have high
affinity and specificity for a particular receptor and
can be used to target this receptor and in assays for
this receptor.
EXAMPLE 4
The peptide J-019 (SEQ ID N0:4) is synthesized
using the procedure as described with respect to Example
1. The synthetic peptide has the following formula:
H-Gly-Cys-Cys-Gly-Ser-Tyr-4Hyp-Asn-Ala-Ala-Cys-His-4Hyp-
Cys-Ser-Cys-Lys-Asp-Arg-4Hyp-Ser-Tyr-Cys-Gly-Gln-NHZ
An MBHA resin is used, and Boc is used to
protect the a-amino groups of each of the amino acids
employed in the synthesis. Side chain protecting groups
as set forth with respect to Example 1 are similarly
employed.
Cleavage from the resin and air-oxidation to
CA 02420184 2003-03-12
-24-
carry out cyclicization are performed as set forth in
Example 1.
The cyclic peptide is purified using the
procedure set forth in Example 1 and checked for purity
via analytical HPLC, which shows that a substantially
pure synthetic material is obtained. The synthetic
peptide is shown to be substantially identical with the
native conotoxin as a result of coelution on HPLC, amino
acid analysis and biological activity. Injection of the
peptide intracerebrally into a mouse shows an initial
attack of violent scratching followed by paralysis and
ultimate death, confirming that air-oxidation can
produce appropriate cross-linking so that the synthetic
conotoxin exhibits biological potency. It is thus known
to have high affinity and specificity for a particular
receptor and can be used to target this receptor and in
assays for this receptor.
EXAMPLE 5
The procedure of Example 4 is repeated with a
single change of the amino acid in tyre 13-position to
substitute proline for 4-hydroxyproline and thereby
synthesize the peptide J-026 (SEQ ID N0:5~. The
synthetic peptide has the following formula:
H-Gly-Cys-Cys-Gly-Ser-Tyr-4Hyp-Asn-Ala-Ala-Cys-His-Pro-
Cys-Ser-Cys-Lys-Asp-Arg-4Hyp-Ser-Tyr-Cys-Gly-Gln-NH2
Cleavage from.the resin and air-oxidation to
carry out cyclicization are performed as set forth in
Example 1.
Analytical HPLC shows the substantially pure
compound is obtained. The synthetic peptide is shown to
be substantially identical with the native conotoxin as
a result of coelution on HPLC, amino acid analysis and
biological activity. Testing by IC injection into a
mouse results in violent movements followed by paralysis
and death. It is thus known to have high affinity and
CA 02420184 2003-03-12
-2 5-
specificity for a particular receptor and can be used to
target this receptor and in assays far this receptar.
EXAMPLE 6
The synthesis of peptide OB-26 (SEQ ID N0:6) is
carried out using a procedure generally the same as that
described with respect to Examples 1 and :~. The
synthetic peptide has the following formula:
H-Cys-Cys-Gly-Val-4Hyp-Asn-Ala-Ala-Cys-His-4Hyp-Cys-Val-
Cys-Lys-Asn-Thr-Cys-NHz
Cleavage from the MBHA resin and air-oxidation
are carried out as set forth in Example 1. HPLC
purification of the cross-linked peptide is carried out
in a similar manner. The resultant synthetic peptide is
checked by analytical HPLC and shown to constitute a
substantially pure compound. The synthetic peptide is
shown to be substantially identical with the native
conotoxin as a result of coelution on HPLC, amino acid
analysis and biological activity. Injection of 1
microgram of the synthetic peptide IC into a mouse
results in a reproducible physical effect, which
verifies that the appropriate disulfide linkages are
achieved during the air-oxidation step. It is believed
that the peptide has high affinity and specificity for a
particular receptor and that it can be used to target
this receptor and to assay for this receptor.
EXAMPLE 7
Synthesis of the peptide J-029 (SEQ TD N0:7) is
carried.out on a chloromethylated resin in the same
general manner as set forth in Example 6. The synthetic
peptide has the following formula:
H-Gly-4Hyp-Ser-Phe-Cys-Lys-AIa-Asp-GIu-Lys-4Hyp-Cys-Glu-
Tyr-His-Ala-Asp-Cys-Cys-Asn-Cys-Cys-Leu-Ser-Gly-Ile-Cys-
Ala-Hyp-Ser-Thr-Asn-Trp-Ile-Leu-Pro-Gly-Cys-Ser-Thr-Ser-
Ser-Phe-Phe-Lys-Ile-OH
CA 02420184 2003-03-12
-26-
The peptide is cleaved from the resin with
anisole, methylethyl sulfide and HF, and air-oxidation
is then carried out under the conditions as generally
set forth in Example 1 in order to obtain the cyclic
compound. Thereafter, purification is carried out using
HPLC as set forth hereinbefore. Ultimate subjection of
the purified peptide to analytical HPLC shows that a
substantially pure compound is obtained. The synthetic
peptide is shown to be substantially identical with the
native conotoxin as a result of coelution on HPLC, amino
acid analysis and biological activity.
Injection of a dose of about 1 microgram of the
synthetic conotoxin IC into a mouse shows substantially
immediate paralysis occurring. It is thus known to have
high affinity and specificity for a particular receptor
and can be used~to target this receptor and in assays
for this receptor.
EXAMPLE 8
The synthesis of peptide OB-20 (SEQ ID N0:8)
having the formula:
H-Gly-Cys-Cys-Ser-His-Pro-Ala-Cys-Ser-Gly-Lys-Tyr-Gln-
Gla-Tyr-Cys-Arg-Gla-Ser-NHz is carried out generally as
set forth in Example 3 using an Fmoc strategy on a
2,4dimethoxy-alkoxyberizyl amine resin.
The peptide is cleaved from the resin using a
mixture of TFA, thioanisole, water and DCM in the
following volume ratios: 40:10:1:44. Cleavage is
carried out for about 8 hours at 37°C. Following
cleavage., air-oxidation is carried out to cyclize the
peptide as set forth in Example 1.
Purification of the cyclized peptide is carried
out as set forth hereinbefore. Subjection of the
purified peptide to HPLC and amino acid analysis shows
that a peptide having a purity of greater than 95
percent is obtained, which has the expected ratio of
CA 02420184 2003-03-12
-27-
residues when subjectbd to amino acid analysis. The
synthetic peptide coelutes with the native peptide on
HPLC.
Injection of 1 microgram of the synthetic
peptide OB-20 intracerebrally into a mouse shows that
the mouse exhibits a reproducible physical effect and
confirms that air-oxidation produces appropriate cross-
linking so that the synthetic conotoxin e~c:hibits
biological potency. It is thus known to have high
affinity and specificity for a particular receptor and
can be used to target this receptor and in assays for
this receptor.
EXAMPLE 9
A synthesis, as generally set forth in Example
1, is carried out using about 25 grams of a
chloromethylated polystyrene resin of the type generally
commercially available to produce peptide
J-021 (SEQ ID NOs9) which has the following formula:
H-His-4Hyp-4Hyp-Cys-Cys-Leu-Tyr-Gly-Lys-Cys-Arg-Arg-Tyr-
4Hyp-Gly-Cys-Ser-Ser-Ala-Ser-Cys-Cys-Gln-OH.
Similar side chain protecting groups are
provided as described in Example 1, and the hydroxyl
side chain of 4-hydroxyproline is protected as the
benzyl ether. Coupling of the N-terminal His residue is
carried out using Boc-His(Tos) dissolved in DMF and
using about 3 millimoles of benzotriazol-1-yl-oxy-
tris(dimethylamino)phosphonium hexafluorophosphate (BOP)
as a coupling agent.
After the final His residue is coupled to the
peptide-resin, the Boc group is removed using 45 percent
TFA in methylene chloride. The peptide-rea m is then
treated with anisole and methylethyl sulfide and HF.
Five grams of resin are treated with 10 milliliters of
anisole, one ml of methylethyl sulfide and 125 ml of HF
for 1/2 hour at -20°C and 3 hour at 0°C. The cleaved
CA 02420184 2003-03-12
-28-
peptide is then extracted using 200 milliliters of 50
percent acetic acid at a temperature below 0°C.
Thereafter, the extracted peptide is dissolved in 8
liters of 1 percent ammonium acetate at a pH of about
4.35. The pH is raised to about 7.74 with ammonium
hydroxide, and air-oxidation is effected as described in
Example 1.
Purification is carried out as described in
Example 1, and then purity is checked using analytical
HPLC. The peptide is applied to a reversed phase C'$
column, and then eluted by subjecting the column to a
gradient of buffers A and B at a flog rate of shout 0.21
milliliters per minute, which gradient changes uniformly
from 0 percent buffer B to 20 percent buffer B over a
time period of 20 minutes. Buffer A is a 1 percent aqueous
solution of TFA, and buffer B is 0.1~ TFA and 70%
acetonitrile. This HPLC shows that the peptide elutes
at about 18.6 minutes and has a purity of greater than
99 percent. The synthetic peptide coelutes with the
native peptide on HPLC. Amino acid analysis of the
pure peptide shows that the expected residues are obtained.
It is believed that testing will show this peptide
to have high affinity and specificity for a particular
receptor so that it can be used to target this receptor or
to assay for this receptor.
EXAMPLE 10
The peptide J-010 (SEQ ID NO:10) is synthesized
using the procedure as generally set forth with respect
to Example 8 using an Fmoc protection strategy. The
synthetic peptide has the following formula:
H-Cys-Lys-Thr-Tyr-Ser-Lys-Tyr-Cys-Gla-Ala-Asp-Ser-Gla-
Cys-cys-Thr-Gla-Gln-Cys-Val-Arg-Ser-Tyr-Cys-Thr-Leu-Phe-
NH2 .
The peptide is cleaved from the resin using a
mixture of TFA, thioanisole, water and DCM in the
CA 02420184 2003-03-12
-29-
following volume ratios: 40:10:1:44. Cleavage is
carried out for about 8 hours at 37°C. lFollowing
cleavage, air-oxidation is carried out to cyclize the
peptide as previously described.
Purification of the cyclized peptide is carried
out as set forth hereinbefore, and subjection of the
purified peptide to HPLC shows that a substantially pure
peptide is obtained. The synthetic peptide is shown to
be substantially identical with the native conotoxin as
a result of coelution on HPLC, amino acid analysis and .
biological activity. Injection of about 1 microgram of
the synthetic peptide intracerebrally into a mouse shows
that the mouse begins rapid running and stretching,
ultimately resulting in death. It is thus known to have
high affinity and specificity for a particular receptor
and can be used to target this receptor and in assays
for this receptor.
EXAMPLE 11
A synthesis as generally performed in Example l
is carried out to produce peptide J-008 ~SEQ ID N0:11)
having the formula:
H-Ser-Thr-Ser-Cys-Met-Glu-Ala-Gly-Ser-Tyr-Cys-Gly-Ser-
Thr-Thr-Arg-Ile-Cys-Cys-Gly-Tyr-Cys-Ala-Tyr-Phe-Gly-Lys-
Lys-Cys-Ile-Asp-Tyr-Pro-Ser-Asn-OH.
The C-terminal residue in the peptide is Asn in
its free acid form. An MBHA resin was used along with
the incorporation of Boc-protected Asp through its ~-
carboxylic group.
Cleavage from the resin and cyclization is
carried out as in Example 1. The final product is
similarly purified to homogeneity by HPLC, and amino
acid analysis of the purified peptide gives the expected
results. The synthetic peptide coelutes with the native
peptide on HPLC.
CA 02420184 2003-03-12
-30-
The synthetic toxin is injected IC into a
mouse, and it proves lethal in less than 10 minutes,
confirming that the synthetic product is highly toxic
and that the stated synthesis produces a compound having
biological activity. It is thus known to have high
affinity and specificity for a particular receptor and
can be used to target this receptor and in assays for
this receptor.
EXAMPLE 12
Synthesis of conotoxin SEQ ID NO:12 (also
referred to as J-017), having the formula: H-Gly-Glu-
Gla-Gla-Val-Ala-Lys-Met-Ala-Ala-Gla-Leu-Ala-Arg-Gla-Asn-
Ile-Ala-Lys-Gly-Cys-Lys-Val-Asn-Cys-Tyr-Pro-OH is
carried out generally similarly to that of Example 1 but
using the modifications described hereinafter.
A commercially available p-alkoxybenzyl alcohol
resin is used for the synthesis, which is a standard
resin used in solid phase syntheses employing the Fmoc-
amino acid strategy. Fluorenylmethyloxycarbonyl (Fmoc)
is used to protect the a-amino groups of each of the
amino acids, and Boc protection is used for the side-
chain amino groups of Lys. The Tyr side chain is
protected by 0-tBu, and the Cys side chain .is protected
by diphenylmethyl (trityl). The carboxyl side chain of
Glu and the side chains of Gla are protected by O-t-Bu
as described hereinafter. Arg is protected by 4-
methoxy-2,3,6-trimethylbenzenesulfonyl (Mtr).
Fmoc-L-Gla(O-t-Bu)Z-OH is prepared as set forth
hereinafter. Condensation of Z-L-Ser(Tos)-OCH3 with
di-tert-butyl malonate, to give Z-DL-Gla(O-t-Bu)2-OCH3,
is carried out by a modification of the procedure of
Rivier et al. ~iochemistrv 26, 8508-8512 (1987,. Sodium
hydride is rinsed twice with pentane, suspended in
absolute benzene, and then added to the benzene solution
of di-tert-butyl malonate. The reaction is allowed to
CA 02420184 2003-03-12
-31-
proceed to completion with 10 minutes of reflex. The
resulting suspension is cooled in an ice bath, and the
Z-L-Ser(Tos)-OCH~ dissolved in benzene/tetrahydrofuran is
added under an argon atmosphere with vigorous stirring
and continued cooling at 0°C. for 2 hours. Stirring is
maintained for additional 48 hours at room temperature.
At this time, the suspension is cooled and washed
successively with ice water, 1 N HCI, and water. After
rotary evaporation at room temperature, the oil is
l0 dissolved in benzene, and pentane is added to initiate
crystallization. The yield is 40-60a for a preparation
of 0.5 mole. The methyl ester is hydrolyzed by
dissolving in alcohol and adding 1.2 equiv of KOH
dissolved in water/ethanol. The solution is allowed to
remain at room temperature for several days; the
reaction is monitored by HPLC using a C'8 S-um column,
with O.lo TFA-acetonitrile as the solvent. When the
reaction is complete, the solution is evaporated at room
temperature, and the product extracted with ethyl
acetate after the addition of NaHS04. The ethyl acetate
extract is dried over Na2S04 and evaporated under reduced
pressure; the yield is 80-90%.
The D- and L-isomers are resolved by
crystallization of the quinine salt of the D-isomer.
Z-DL-di-t-Bu-Gla-OH in ethyl acetate is reacted with an
equivalent amount of quinine. The crystals are
separated from the mother liquid, and the
Z-D~di-t-Bu-Gla-OH is recrystallized from ethyl acetate.
The quinine salt is suspended in ether, and quinine is
removed by the addition of a 20~ citric acid solution at
0°C. The same process is used to remove quinine from
the liquid phase. The L-isomer is precipitated in the
form of its ephedrine salt from ethyl acetate-pentane
and recrystallized (Marki et al., Helv. Chim. Acta. 60,
798-800, 1977). Elimination of ephedrine by acid
extraction, hydrogenation of the Z group, and
CA 02420184 2003-03-12
-32-
introduction of the Fmoc are all standard laboratory
procedures. Optical purity of the L° and D-isomers of
Fmoc-Gla(O-t-Bu)z-off is assessed after hydrolysis to Glu
(6 N HCl, 110°C, 20 hours), and each is approximately
99~ pure.
The coupling of the Fmoc-protected amino acids
to the resin is accomplished using a schedule generally
similar to that set forth in Example 1 but removing the
Fmoc group via the use of a 20 percent solution (v/vj of
freshly distilled piperidine in dimethylformamide (DMF)
for l0 minutes. Thorough resin washing is accomplished
by repeated application of DMF, methanol, or
dichloromethane (DCM). Couplings are mediated by DCC in
either DCM, DMF, or mixtures thereof, depending upon the
solubility of the particular amino acid derivative.
Fmoc-Asn is incorporated into the peptide with an
unprotected side chain, in the presence of 2 equiv of
HOBT, and is coupled in DMSO/DMF or DMSO/DCM.
The peptide is released from 4 grams of the
peptide resin as the C-terminal free acid by treatment
with a freshly prepared mixture of TFA, thioanisole, H20,
EDT and DCM (40/18/1/2/49) (~0 ml) at about 37°C for 6-8
hours. Trial cleavages on small amounts demonstrate
that the peptide is freed and that all side-chain
protection, including the difficult Mtr group, are
removed while Gla remains intact.
The peptide is precipitated from the cleavage
solution after extraction with methyl tart-butyl. ether.
The peptide is then dissolved in distilled water, the pH
of the resulting solution is adjusted to approximately
7-8 with dilute ammonium hydroxide, after separating the
resin by filtration. Formation of the disulfide cross-
link is carried out on the crude peptide product using a
liquid phase, air-oxidation step in a cold room as
described with respect to Example 1. The crude peptide
is purified by preparative HPLC using a preparative
CA 02420184 2003-03-12
- 33 -
0
cartridge (15-20 ,um, 300 A Vydac~'°' C1$) and a TEAP buffer, pH
2.25, and also with a O.lo TFA buffer using appropriate
gradients of acetonitrile. Highly purified fractions are
pooled and lyophilized, yielding peptide as its TFA salt.
Optical rotation in 1% acetic acid measures [a]D = -64° c = 1)
at 20°C. Amino acid analysis gives the expected values. FAB
mass spectrometry is performed on the peptide, and the
spectrum shows a protonated molecular ion (MH~) at m/z = 3097.4
corresponding to the calculated monoisotopic peptide of
3097.36. A chromatogram of the crude preparation after TFA
cleavage and deprotection illustrates that the major product
is particularly pure and that only a relatively small amount
of hydrophobic impurities are present. Sequence analysis
gives the expected residue at each cycle, except for blanks
with Gla residues, confirming that the pure target peptide is
obtained. The synthetic peptide coelutes with the native
peptide on HPLC.
When injected IC into young mice, it causes sleeping;
however, when injected into older mice, it causes
hyperactivity. It is thus known to have high affinity and
specificity for a particular receptor and can be used to
target this receptor and in assays for this receptor,
tentatively identified as the NMDA receptor. It can be used
to provide neuroprotection.
EXAMPLE 13
A synthesis of the linear peptide J-004 (SEQ ID N0:13) is
carried out on an MBHA resin using the procedure as generally
set forth in Example 1. The linear peptide J-004 has the
following formula:
H-Glu-Ser-Glu-Glu-Gly-Gly-Ser-Asn-Ala-Thr-Lys-Lys-Pro-Tyr-Ile-
Leu-NH2. In a preferred embodiment, the Thr residue may be
glycosylated.
The ultimate linear peptide is purified and subjected to
amino acid analysis; it shows that the
CA 02420184 2003-03-12
_3~_
expected residues are obtained in the peptide sequence.
The synthetic peptide coelutes with the native peptide
on HPLC, after the native conotoxin has been
deglycosylated to remove the carbohydrate which is
linked to Thr in the 10-position which appears to
increase bioactivity. Testing of the synthetic peptide
by injection IC into a mouse shows that the mouse
quickly becomes sluggish and unable to stand or function
normally, which demonstrates that the synthetic peptide
has the expected biological potency. It is thus known
to have high affinity and specificity for a particular
receptor and can be used to target this receptor and in
assays for this receptor.
These synthetic peptides, for administration to
humans, should have a purity of at least about 95
percent (herein referred to a substantially pure), and
preferably have a purity of at least about 98 percent.
Purity for purposes of this application refers to the
weight of the intended peptide as compared to the weight
of all peptide fragments present. These synthetic
peptides, either in the free form or in the form of a
nontoxic salt, are commonly combined with a
pharmaceutically or veterinarily acceptable carrier to
create a composition for administration to animals,
including humans, or for use in in vitro assays. ~n
vivo administration should be carried out by a physician
and the required dosage will vary with the particular
objective being pursued. In this respect, guidelines
have been developed for the use of other conotoxins such
as conotoxin GI and such are well known in this art are
employed for the particular purpose of use.
As indicated hereinbefore, DNA encoding the
amino acid structure of any o.f these conotoxins can be
used to produce the proteins recombinantly as well as to
afford different varieties of plants with pesticidal
properties.
CA 02420184 2003-03-12
-35-
To synthesize a protein having the desired
conotoxin amino acid residue sequence by recombinant
DNA, a double-stranded DNA chain which encodes the
sequence might be synthetically constructed. Although
it is nowadays felt that PCR techniques would be method
of choice to produce DNA chains, a DNA chain encoding
the desired sequence could be designed using certain
particular colons that are more efficient for
polypeptide expression in a certain type of organism,
i.e. selection might employ those colons which are most
efficient for expression in the type of organism which
is to serve as the host for the recombinant vector.
However, any correct set of colons will encode a desired
product, although perhaps slightly less efficiently.
Colon selection may also depend upon vector construction
considerations; for example, it may be necessary to
avoid placing a particular restriction site in the DNA
chain if, subsequent to inserting the synthetic DNA
chain, the vector is to be manipulated using the
2~ restriction enzyme that cleaves at such a site. Also,
one should of course avoid placing restriction sites in
the DNA chain if the host organism, which is to be
transformed with the recombinant vector containing the
DNA chain, is known to produce a restriction enzyme that
would cleave at such a site within the DNA chain.
To assemble such a synthetic, nonchromosomal,
conotoxin-encoding DNA chain, oligonucleotides are
constructed by conventional procedures such as those
described in J. Sambrook et al.; Molecular Cloning,
3o ~aborator~ Manual, Cold Spring Harbor Laboratory Press,
New York (1989) (hereinafter, Sambrook et al.). Sense
and antisense oligonucleotide chains, up to about 70
nucleotide residues long, are synthesized, preferably on
automated synthesizers, such as the Applied Biosystem
*
Inc. Model 380A DNA synthesizer. The oligonucleotide
chains are constructed so that portions of the sense and
*Trade-mark
CA 02420184 2003-03-12
antisense oligonucleotides overlap, associating with
each other through hydrogen bonding between
complementary base pairs and thereby forming double
stranded chains, in most cases with gaps in the strands.
Subsequently, the gaps in the strands are filled in, and
oligonucleotides of each strand are joined end to end
with nucleotide triphosphates in the presence of
appropriate DNA polymerases and/or with ligases.
As an alternative to such stepwise construction
of a synthetic DNA chain, the cDNA corresponding to the
desired conotoxin may be cloned. As is well known, a
cDNA library or an expression library is produced in a
conventional manner by reverse transcription from
messenger RNA (mRNA) from suitable tissue from the cone
snail of interest. To select clones containing desired
sequences, a hybridization probe or a mixed set of
probes which accommodate the degeneracy of the genetic
code and correspond to a selected portion of the protein
of interest are produced and used to identify clones
containing such sequences. Screening of such an
expression library with antibodies made against the
protein may also be used, either alone or in conjunction
with hybrid~i2ation probing, to identify or confirm the
presence of DNA sequences in cDNA library clones which
are expressing the protein of interest. Such techniques
are taught, for example in Sambrook et al., supra.
In addition to the protein-encoding sequences,
a DNA chain should contain additional sequences
depending upon vectar construction considerations.
Typically, a synthesi2ed DNA chain has linkers at its
ends to facilitate insertion into restriction sites
within a cloning vector. A DNA chain may be constructed
so as to encode the protein amino acid sequences as a
portion of a fusion polypeptide; and if so, it will
generally contain terminal sequences that encode amino
acid residue sequences that serve as proteolytic
CA 02420184 2003-03-12
-37-
processing sites, whereby the desired polypeptide may be
proteolytically cleaved from the remainder of the fusion
polypeptide. The terminal portions of the synthetic DNA
chain may also contain appropriate start and stop
signals.
Accordingly, a double-stranded
conotoxin-encoding DNA chain is constructed or modified
with appropriate linkers for its insertion into a
particular appropriate cloning vector. The cloning
vector that is to be recombined to incorporate the DNA
chain is selected appropriate to its viability and
expression in a host organism or cell line, and the
manner of insertion of the DNA chain depends upon
factors particular to the host. For example, if the DNA
chain is to be inserted into a vector for insertion into
a prokaryotic cell, such as E. coli, the DNA chain will
be inserted 3' of a promoter sequence, a Shine-Delgarno
sequence (or ribosome binding' site) that is within a 5'
non-translated portion and an ATG start colon. The ATG
start colon is appropriately spaced from the
Shine-Delgarno sequence, and the encoding sequence is
placed in correct reading frame with the ATG start
colon. The cloning vector also provides a 3'
non-translated region and a translation termination
site. For insertion into a eukaryotic cell, such as a
yeast cell or a cell line obtained from a higher. animal,
the conotoxin-encoding oligonucleotide sequence is
appropriately spaced from a capping site and in correct
reading frame with an ATG start signal. The cloning
vector also provides a 3' non-translated region and a
translation termination site.
Prokaryotic transformation vectors, such as
p8R322, pMB9, Col ~1, pCRl, RP4 and lambda-phage, are
available for inserting a DNA chain of the length which
encodes conotoxin with substantial assurance of at least
some expression of the encoded polypeptide. Typically,
CA 02420184 2003-03-12
-38-
such vectors are constructed or modified to have one or
more unique restriction sites appropriately positioned
relative to a promoter, such as the lac promoter. The
DNA chain may be inserted with appropriate linkers into
such a restriction site, with substantial assurance of
production of desired protein in a prokaryotic cell line
transformed with the recombinant vector. To assure
proper reading frame, linkers of various lengths may be
provided at the ends of the protein-encoding sequences.
Alternatively, cassettes, which include sequences, such
as the 5' region of the lac Z gene (including the
operator, promoter, transcription start site, Shine-
Delgarno sequence and translation initiation signal),
the regulatory region from the tryptophane gene (trp
operator, promoter, ribosome binding site and
translation initiator), and a fusion gene containing
these two promoters called the trp-lac or commonly
called the Tac promoter are available into which the
synthetic DNA chain may be conveniently inserted and
then the cassette inserted into a cloning vector of
choice.
Similarly, eukary~tic transformation vectors,
such as, the cloned bovine papilloma 'virus genome, the
cloned genomes of the marine retroviruses, and
eukaryotic cassettes, such as the pSV-2 gpt system
(described by Mulligan and Berg, Nature 277, 108-114,
1979), the Okayama-Berg cloning system (Mol. Cell Biol.
2_, 161-170, 1982), and the expression cloning vector
recently described by Genetics Institute (Science 228,
810-815,.1985), are available which provide substantial
assurance of at least some expression of conotoxin in
the transformed eukaryotic cell line.
As previously mentioned, a convenient way to
ensure production of a protein of the length of the
conotoxins of interest is to produce the protein
initially as a segment of a gene-encoded fusion protein.
CA 02420184 2003-03-12
-39-
In such case, the DNA chain is constructed so that the
expressed protein has enzymatic processing sites
flanking the conotoxin amino acid residue sequences. A
conotoxin-encoding DNA chain may be inserted, for
example, into the beta-galactosidase gene for insertion
into E_. coli, in which case, the expressed fusion
protein is subsequently cleaved with proteolytic enzymes
to release the conotoxin from beta-galactosidase peptide
sequences.
An advantage of inserting the protein-encoding
sequence so that the desired sequence is expressed as a
cleavable segment of a fusion protein, e.g. as the
conotoxin sequence fused within the beta-galactosidase
peptide sequence, is that the endogenous protein into
which the desired conotoxin sequence is inserted is
generally rendered non-functional, thereby facilitating
selection for vectors encoding the fusion protein.
The conotoxin proteins may also be reproduced
in yeast using known recombinant DNA techniques. For
example, a suitable plasmid, amplified in an _E. coli
clone, is isolated and cleaved with co RI and Sal I.
This digested plasmid is electrophoresed oh an agarose
gel allowing for the separation and recovery of the
amplified insert of interest. The insert is inserted
into the plasmic pYEp, a shuttle vector which can be
used to transform both ,. coli and Saccharamyces
cerevisiae yeast. Insertion of the synthetic DNA chain
at this point assures that the DNA sequence is under the
control of a promoter, in proper reading frame from an
ATG signal and properly spaced relative to a cap site.
The shuttle vector is used to transform URA3, a strain
of S. cerevisiae yeast from which the oratate
monophosphate decarboxylase gene is deleted.
The transformed yeast is grown in medium to
attain log growth. The yeast is separated from its
culture medium, and cell lysates are prepared. Pooled
CA 02420184 2003-03-12
cell lysates are determined by RIA to be reactive with
antibody raised against the conotoxin, demonstrating
that a protein containing protein segment is expressed
within the yeast cells.
The production of conotoxins can be carried out
in both prokaryotic and eukaryotic cell lines to provide
protein for biological and therapeutic use. While
conotoxin synthesis is easily demonstrated using either
to bacteria or yeast cell lines, the synthetic genes should
be insertable for expression in cells of higher animals,
such as mammalian tumor cells, and in plants. Such
mammalian cells may be grown, for example, as peritoneal
tumors in host animals, and certain conotoxins may be
harvested from the peritoneal fluid. The cloned DNA is
insertable into plant varieties of interest where the
plant utilizes it as a plant defense gene, i.e. it
produces sufficient amounts of the pesticide of interest
to ward off insects or the like that are natural
2o predators to such plant species.
Although the above examples demonstrate that
conotoxins can be synthesi2ed through recombinant nNA
techniques, the examples do not purport to have
maximized conotoxin production. It is expected that
~5 subsequent selection of more efficient cloning vectors
and host cell lines will increase the yield, and known
gene amplification techniques for both eukaryotic and
prokaryotic cells may be used to increase production.
Secretion of the gene-encoded protein from the host cell
30 line into the culture medium is also considered to be an
important factor in obtaining certain of the synthetic
proteins in large quantities.
Although the invention has been described with
35 regard to certain preferred embodiments, it should be
understood that various changes and modifications as
CA 02420184 2003-03-12
-4 Z-
would be obvious to one having the ordinary skill in the
art may be made without departing from the scope of the
invention which is set forth in appended claims. For
example, substitution of various of the amino acid
residues depicted in the amino acid sequences by
residues known to be equivalent with those residues can
be effected to produce equivalent peptides having
similar biological activities. Moreover, it is known
that additional substitutions in the amino acid sequence
generally throughout the C-terminal portion of the
peptide, i.e. within about 1/~ of the length of the
conotoxin nearest its C-terminus, can be effected in
order to produce conotoxins having phylogenetic
specificity; thus, such substitutions in this region can
be carried out to produce valuable equivalent
structures. The C-terminus of many of the illustrated
peptides is amidated, and the inclusion of a substituted
amide at the C-terminus of such peptides, as described
hereinbefore, is considered to create an equivalent
conotoxin.
Particular features of the invention are
emphasized in the claims which follow"
CA 02420184 2003-03-12
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: OLIVERA. Baldomero M.
(B) STREET: 1370 Bryan Ave.
(C) CITY: Salt Lake City
(D) STATE: Utah
(E) COUNTRY: United States
(F) POSTAL CODE (ZIP): 84105
(A) NAME: RIVIER, Jean E.F.
(B) STREET: 9674 Blackgold Rd.
(C) CITY: La Jolla
(D) STATE: California
(E) COUNTRY: United States
(F) POSTAL CODE (ZIP): 92037
(A) NAME: CRUZ, Lourdes J.
(B) STREET: 31 M Street, ~4D3
(C) CITY: Salt Lake City
(D) STATE: Utah
(E) COUNTRY: United States
(F) POSTAL CODE (ZIP):' 84103
(A) NAME: ABOGADIE, Fe
(B) STREET: 2225 Ridge Ave., ~'2N
(C) CITY: Evanston
(D) STATE: Illinois
(E) COUNTRY: United States
(F) POSTAL CODE (ZIP): 60201
(A) NAME: HQPKINS. Chris E.
(B) STREET: 1254 E. 500 So.
(C) CITY: Salt Lake City
(D) STATE: Utah
(E) COUNTRY: United States
(F) POSTAL CODE (ZIP): 84102
(A) NAME: DYKERT, John
(B) STREET: 704 $arsby Street
(C) CITY: Vista
(D) STATE: California
(E) COUNTRY: United States
(F) POSTAL CODE (ZIP): 92084
(A) NAME: TORRES, Josep L.
(B) STREET: Pssg. Maragall 296, 2-1
(C) CITY: 08031 Barcelona
(E) COUNTRY: Spain
(F) POSTAL CODE (ZIP): none
(ii) TITLE OF INVENTION: CONOTOXINS I
(iii) NUMBER OF SEQUENCES: 13
(iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentln Release ~1.'D, Version #1.25 (EPO)
(v) PRIOR APPLICATION DATA:
CA 02420184 2003-03-12
-43-
(A) APPLICATION NUMBER: US 08/0$4,848
(B} FILING DATE: June 29, 1993
(2) INFORMATION FOR SEQ ID NO:1:
(ij SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 amino acids
(B) TYPE: amino acid
(D} TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
Gly Cys Cys Gly Ser Tyr Pro Asn Ala Ala Cys His Pro Cys Ser Cys
1 5 10 15
Lys Asp Arg Xaa Ser Tyr Cys Gly Gln
20 25
(2) INFORMATION FOR SEQ ID No:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 amino acids
(B} TYPE: amino acid
(Dj TOPOLOGY: unknown
(ii) MOLECULE TYPE: pept3.de
(xij SEQUENCE DESCRIPTION: SEQ ID N0:2:
Glu Lys Ser Leu Val Pro Ser Val Ile Thr T'hr Cys Cys Gly Tyr Asp
1 5 10 15
Xaa Gly Thr Met Cys Xaa Xaa Cys Arg Cys Thr Asn Ser Cys
20 25 30
(2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 amino acids
(B} TYPE: amino acid
(Dj TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
CA 02420184 2003-03-12
--44-
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:
Cys Cys Gly Val Xaa Asn Ala Ala Cys Pro Xaa Cys Val Cys Asn Lys
1 5 10 15
Thr Cys Gly
(2) INFORMATION FOR SEQ ID N0:4s
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 amino acids
(B) TYPE: amino acid --
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:
Gly Cys Cys Gly Ser Tyr Xaa Asn Ala Ala Cys His Xaa Cys Ser Cys
1 5 10 15
Lys Asp Arg Xaa Ser Tyr Cys Gly Gln
20 25
(2) INFORMATION FOR SEQ ID N0:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:5:
Gly Cys Cys Gly Ser Tyr Xaa Asn Ala Ala Cys His Pro Cys Ser Cys
1 5 10 15
Lys Asp Arg Xaa Ser Tyr Cys G1y Gln
20 25
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARAGTERZSTICS:
(A) LENGTH: 18 amino acids
(8) TYPE: amino acid
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
CA 02420184 2003-03-12
-45--
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:
Cys Cys Gly Val Xaa Asn Ala Ala Cys His Xaa Cys Val Cys Lys Asn
1 5 10 15
Thr Cys
(2) INFORMATION FOR SEQ ID N0:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 46 amino acids
(8) TYPE: amino acid
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:7:
Gly Xaa Ser Phe Cys Lys Ala Asp Glu Lys Xaa Cys Glu Tyr His Ala
1 5 10 15
Asp Cys Cys Asn Cys Cys Leu Ser Gly Ile Cys Ala Xaa Ser Thr Asn
20 25 30
Trp Ile Leu Pro Gly Cys Ser Thr Ser Ser Phe Phe Lys Ile
35 40 45
(2) INFORMATION FOR SEQ ID N0:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID No:8:
Gly Cys Cys Ser His Pro Ala Cys Ser Gly Lys Tyr Gln Xaa Tyr Cys
1 S 10 15
Arg ?caa Ser
{2) INFOR?SATION FOR 5EQ ID N0:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
CA 02420184 2003-03-12
-46-
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:9:
His Xaa Xaa Cys Cys Leu Tyr Gly Lys Cys Arg Arg Tyr Xaa Gly Cys
1 5 10 15
Ser Ser Ala Ser Cys Cys Gln
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
{ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
Cys Lys Thr Tyr Ser Lys Tyr Cys Xaa Ala Asp Ser Xaa Cys Cys Thr
1 5 10 15
Xaa Gln Cys Val Arg Ser Tyr Cys Thr Leu Phe
20 25
(2) INFORMATION FOR SEQ ID NO: I1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
Ser Thr Ser Cys Met Glu Ala Gly Ser Tyr Cys Gly Ser Thr Thr Arg
1 5 10 15
Ile Cys Cys Gly Tyr Cys Ala Tyr Phe Gly Lys Lys Cys Ile Asp Tyr
20 25 30
Pro Ser Asn
(2) INFORMATION FOR SEQ ID N0:12:
CA 02420184 2003-03-12
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:12:
Gly Glu Xaa Xaa STal Ala Lys Met Ala Ala Xaa Leu Ala Arg Xaa Asn
1 S 10 15
Zle Ala Lys Gly Cys Lys Val Asn Cys Tyr Pro
20 25
(2) INFORMATION FOR SEQ ID N0:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:13:
Glu Ser Glu Glu Gly Gly Ser Asn Ala Thr Lys Lys Pro Tyr Ile Leu
1 5 10 15
