Note: Descriptions are shown in the official language in which they were submitted.
WO94121278 21~ 8 0 ~ 1 PCT~S9~/02750
DESCRIPTION
POTASSIUM CHANNEL BLOCKING COMPOUNDS AND THEIR USE
Field of the Invention
This invention relates to inhibitors of the
transient outward potassium channel and other potassium
channels.
Background of the Invention
The following is a description of relevant art,
none of which is admitted to be prior art to the claims.
Potassium (K+) channels are membrane-spanning
proteins that allow the selective movement of K~ into or
out of cells in response to changes in membrane potential,
or in response to activation by cations and/or ligands.
The primary role of K+channels is maintenance of the
resting membrane potential; another role concerns their
contribution to repolarization of action potentials in
excitable cells. Potassium channels represent a diverse
group of ion channel proteins, and several toxins have
been described that act primarily (if not exclusively) by
blocking one or more specific K+channels (Rudy, "Diversity
and Ubiquity of K Channels", 25 Neuroscience 729, 1988).
Cardiac cells are characterized by a remarkable variety of
different K+ channel subtypes. Several K+ channels types
are opened in response to depolarization of the membrane
during an action potential, and the currents carried by
these different channels sum to cause repolar~zation of
the membrane to the resting potential. One of these chan-
nel types, the transient outward Kt channel, conducts acurrent (I~o) which activates very rapidly (within l-l0
WO94/21278 PCT~S94/02750
2lS 8 0S 1 2
milliseconds) upon membrane depolarization and then decays
(inactivates) rapidly (10-200 milliseconds). I,~ contri-
butes most significantly to initial repolarization of the
cardiac action potential, and is blo~ed by several non-
specific pharmacological compounds such as aminopyridinesand tedisamil, a class III antiarr~ythmic agent (Dukes et
al., "Tedisamil Blocks the Transient and Delayed Rectifier
K+ Currents in Mammalian Cardiac and Glial Cells", 254 J.
Pharmacol. Exp. Ther. 560, 1990). Blockage of Ilo leads to
prolongation of the cardiac action potential. I~o has been
recorded from human cardiac cells as described, for
example, in Escande et al., "Two Types of Transient
Outward Currents in Adult Human Atrial Cells", 252 Am. J.
Physiol. H142, 1987. Lengthening of cardiac action poten-
tials (and hence refractoriness) is said to be one mecha-
nism for suppression of reentrant atrial and ventricular
arrhythmias (Lynch et al., "Therapeutic Potential of
Modulating Potassium Currents in the Diseased Myocardium",
6 FASEB J. 2952, 1992). Currently available class III
antiarrhythmic agents, most of which act by blocking a
separate subtype of K+ channels known as delayed rectifier
K+channels, may cause excessive prolongation of cardiac
action potentials that can lead to the development of a
ventricular arrhythmia, known as torsades de pointes
(Sanguinetti, "Modulation of Potassium Channels by Antiar-
rhythmic and Antihypertensive Drugs", 19 HyPertension 228,
1992).
The opening of voltage-dependent K+ channels is
also the mechanism by which repolarization of the cell
membrane occurs during the very short action potential
characteristic of central neurons. Transient outward K+
currents (referred to as I~ in neurons) play a role in this
process. Dendrotoxin, a toxin derived from snakes,
selectively blocks the delayed non-inactivating K+ current
in dorsal root ganglion neurons (Penner et al.,
"Dendrotoxin: A Selective Blocker of a Non-inactivating
Potassium Current in Guinea-pig Dorsal Root Ganglion
WO94/21278 215 8 Q 51 PCT~S94/02750
Neurones", 407 Pfluqers Arch. 365, 1986), and also blocks
the transient outward K+ current (IA) in hippocampal slices
(Halliwell et al., "Central Action of Dendrotoxin:
Selective Reduction of a Transient K Conductance in
Hippocampus and Binding to Localized Acceptors", 83 Proc.
Natl. Acad. Sci. USA 493, 1986). Prolongation of action
potential duration in neurons by dendrotoxin results in an
enhanced release of neurotransmitters (Harvey and
Anderson, "Dendrotoxins: Snake Toxins That Block Potassium
Channels and Facilitate Neurotransmitter Release", 31
Pharmac. Ther 33, 1985). It has been suggested that fur-
ther work in this field may confirm this as a pharmaco-
logical approach to the treatment of cognitive disorders
such as Alzheimer's disease (Lavretsky and Jarvik, "A
Group of Potassium-channel Blockers- Acetylcholine
Releasers: New Potentials for Alzheimer Disease? A
Review", 12 J. Clinical Psychopharm. 110, 1992).
Summary of the Invention
This invention generally concerns specific and
potent novel inhibitors or blocking agents of the tran-
sient outward potassium channel, for example, currents
referred to as I,~ or IA in cardiac cells and in neurons,
respectively. The invention also features novel poly-
peptides isolated from spider venom, or their equivalent,
which are active at one or more potassium channels, e.g.,
the transient outward potassium channel.
Specifically, examples are provided of the novel
activity of polypeptide toxins isolated from the venom of
the spiders Heteropoda venatoria, and Olios fasciculatus
These peptides, referred to simply herein as Compounds 1,
2 and 3 are examples of specific and potent blockers of
voltage-dependent transient outward K+ channels, which
block the corresponding whole-cell current (I~o) of cardiac
cells. As such, these agents, fragments th~reof, or
compounds discovered using a ligand ~inding assay (or its
equivalent) employing these toxins, or their equivalent,
are useful in the treatment of cardiac arrhythmias and
WO94/21278 PCT~S94/027~0
2~ S8~51
have utility in the treatment of disorders of learning and
memory, (such as Alzheimer's disease), Parkinson's
disease, multiple sclerosis, schizophrenia, epilepsy,
stroke and muscle spasticity. ~
In general, useful K~ channel inhibiting poly-
peptides of the type described ànd claimed herein can be
isolated from the venom of the spiders Heteropoda
venatoria and Olios fasciculatus. Other polypeptides (or
their equivalent) with similar or homologous amino acid
(or other compound or monomer) sequences that block
potassium channels can also be isolated. Applicant
believes it is the first to determine the existence of
such K+ channel blocking activity in toxins derived from
spider venom, and thus demonstrates that it is useful and
productive to screen for other such polypeptides in spider
venom. This invention also concerns methods of using
these polypeptides to screen for other agents acting at a
common site (i.e., the transient outward potassium
channel) as active agents.
Chemical agents that selectively block I~
channels in central neurons, and thereby cause enhanced
release of neurotransmitters, are believed by applicant to
be useful in the treatment of Alzheimer's disease and
other neural disorders. Similarly, agents which selec-
tively block I~o channels in cardiac cells are believed by
applicant to be useful in treatment of cardiac arrhyth-
mias. Thus, these polypeptides and related compounds or
agents can be used for the treatment of cardiac arrhyth-
mias, Alzheimer's disease, Parkinson's disease, multiple
sclerosis, schizophrenia, epilepsy, stroke and muscle
spasticity.
Thus, in a first aspect, the invention features
speci~ic potent transient outward potassium channel inhi-
bitors, blockers or antagonists.
The phrase "transient outward potassium channel"
is a well recognized phrase which defines a specific sub-
type of potassium channels within a variety of cells,
WO94/21278 21 S ~ 0 51 PCT~S94/02750
e.q., as characterized by the currents Ilv and IA noted
above, in cardiac and neural cells, respectively.
The term "inhibitor" is used to mean agents
which reduce current conducted by transient outward
potassium channels. In the art, terms such as "blocker"
and "antagonist" have been used interchangeably with the
term "inhibitor".
By "specific" is meant that the blockage of
transient outward K+ channels is half-maximal (IC~)at or
below lO0 nM, and that no effect on other K+ channels (such
as delayed rectifier, inward rectifier, acetylcholine-
activated or ATP-inhibited K+ channels), Na+ or Ca2+
channels occurs at concentrations at least lO-fold greater
than the IC~ for transient outward K~ channels.
By "potent" is meant that a given transient out-
ward K+ channel is blocked by 50% at a concentration of an
inhibitor less than lO0 nM, more preferably less than lO
nM, and even more preferably less than l nM.
In preferred embodiments, these agents are poly-
peptides or are derived from polypeptides present inspider venom. While specific examples of such polypep-
tides are provided herein, these examples are not limiting
in this invention and those of ordinary skill in the art
will recognize that other polypeptides can be readily iden-
tified within various spider venoms, including but notlimited to those described herein. In addition, those of
ordinary skill in the art will recognize that equivalent
polypeptides can be synthetically formed using standard
procedures. Specific portions of those peptides which are
active in blocking, inhibiting, or antagonizing a selected
transient outward potassium channel can be readily iden-
tified using standard screening procedures. For example,
specific peptide fragments can be synthesized, or produced
from intact polypeptides using various peptidases, and
those fragments assayed for inhibitory activity in assays,
as described below. Those fragments which are active as
PCT~S94/02750
WO94121278 21~5 ~
-
inhibitors are useful in this invention in various screen-
ing assays, and in therapeutic applications.
In addition, analogues o~uteins of such poly-
peptides can be readily synthesi~éd. These may contain
modifications in the amino acid s~equence in regions which
do not affect the inhibitory or blocking activity of the
original polypeptide. In more conserved regions of the
inhibitor, amino acids may be substituted such that the
activity of the inhibitor is not significantly altered,
for example, by substitution of small amino acids, such as
glycine, for other small amino acids, such as valine, or
positively or negatively charged amino acids for similarly
charged amino acids.
The term "derived" simply indicates that com-
pounds can be synthesized based upon the general structureof polypeptides identified in spider venom. Such deriva-
tization is performed by methods well recognized in the
art, specific examples of which are generally provided
above. Preferably, the term "derived~' includes analogues,
muteins and fragments, as described above, which have a
desired modulatory activity of a polypeptide toxin des-
cribed herein.
The above inventions are exemplified by poly-
peptides found in the venom of the spiders Heteropoda
venatoria and Olios fasciculatus. Three specific poly-
peptides of this invention and the fractions in which they
are present according to this invention include Heteropoda
venatoria peptide Compound l (SEQ. ID. NO. l), Heteropoda
venatoria peptide Compound 2 (SEQ. ID. NO. 2), and Olios
fasciculatus peptide Compound 3 ( SEQ. ID NO. 3). In one
example, polypeptides of this invention block transient
outward K+ channels in cardiac and neural cells. This
invention includes polypeptides which have substantially
the same amino acid sequence, and substantially, the same
K+ current blocking activity as the polypeptides Heteropoda
venatoria peptides Compound l and Compound 2, and Olios
fasciculatus peptide Compound 3.
WO94/21278 ~1 5 8 0 5 1 PCT~S94/02750
In a second aspect, the invention features a
method for screening for a transient outward potassium
channel active agent by contacting a transient outward
potassium channel with a known specific transient outward
potassium channel inhibitor (such as those described
above) and a potential transient outward potassium channel
active agent, and detecting inhibition of binding of the
known inhibitor by the potential active agent. Inhibition
of binding is an indication of a useful transient outward
potassium channel active agent. Such active agents can be
readily screened to determine their specificity.
An "active agent" is a compound that either
increases (if it acts as an agonist) or decreases (if it
acts as an inhibitor) current through a transient outward
potassium channel.
In a related aspect, the invention features a
method for screening spider venom for a useful K+ channel
active agent as exemplified by methods described herein.
Such venom is screened to determine fractions which con-
tain the desired activity.
By "K+ channel active agent" is meant a compound
that either increases or inhibits any other type of K+
channel such as delayed rectifier, inward rectifier, Ca2+-
activated or ATP-sensitive K+ channels.
In preferred embodiments, the screening method
involves use of transient outward potassium channels from
cardiac or neural tissue.
Also within the scope of this invention is a
method for identifying compounds that bind to the tran-
sient outward K+channel, preferably at the same site as
that bound by the Heteropoda venatoria peptides Compound
l and Compound 2, and Olios fasciculatus peptide Compound
3.
In a third aspect, the invention f,eatures a
method for treatment of a disease or condition in which a
therapeutically useful result is achieved by modulating a
transient outward potassium channel activity, by adminis-
WO94/21278 21 S 8 ~ ~ ~ PCT~S94/02750
tering to the organism a therapeutically effective amountof a specific transient outward potassium channel in~ibi-
tor, or a polypeptide (or its equivalent) from spider
venom. Specific diseases to be treated include (but are
not limited to) those listed above~.-
By "modulating" is mea~t a decrease of transientK+ channel activity. For example, by blocking the pore of
the channel, or by changing- the voltage dependance of
channel gating.
Treatment involves the steps of first iden-
tifying a patient (human or non-human) that suffers from
a disease or condition by standard clinical methodology
and then providing such a patient with a therapeutically
available composition of the present invention.
By "therapeutically effective" is meant an
amount that relieves (to some extent) one or more symptoms
of the disease or condition in the patient. Additionally,
by "therapeutically effective" is meant an amount that
returns to normal, either partially or completely,
physiological or biochemical parameters associated with or
causative of the disease or condition. Generally, it is
an amount between about l nmole/kg and l ~mole/kg of the
molecule, dependent on its ECs~ and on the age, size, and
disease associated with the patient.
In a further aspect, the invention features a
pharmaceutically acceptable composition including a
specific transient outward potassium channel inhibitor or
spider venom polypeptide (or its equivalent).
In yet a further aspect, the invention features
a polypeptide (or analogues thereof) obtainable from a
spider venom, that is an inhibitor of potassium channel
activity. The invention also features unique fragments of
such a polypeptide. Such analogues, as defined above, are
not themselves obtained from the venom but are ~erived by
analysis of an isolated polypeptide (as exemplified
herein) or can be obtained by screening other venoms.
WO94/21278 215 8 0 51 PCT~S941027~0
The term "unique fragments" refers to portions
that find no identical counterpart in known sequences as
of the date of filing this application. These fragments
can be identified easily by an analysis of polypeptide
data bases existing as of the date of filing to detect
counterparts.
By "pharmaceutically acceptable composition" is
meant a therapeutically effective amount of a compound of
the present invention in a pharmaceutically acceptable
carrier, i.e., a formulation to which the compound can be
added to dissolve or otherwise facilitate administration
of the compound. Examples of pharmaceutically acceptable
carriers include water, saline, and physiologically buf-
fered saline. Such a pharmaceutical composition is pro-
vided in a suitable dose. Such compositions are generallythose which are approved for use in treatment of a speci-
fied disorder by the FDA or its equivalent in non-U.S.
countries.
By "disease or conditionl' is meant those
diseases listed above and related diseases concerning
cardiac or neural cells.
Also within the scope of the invention is the
use of such compounds as insecticidal agents. The
compounds described herein are believed by Applicant to
possess significant insecticidal action, perhaps through
block of a channel that is structurally similar to the
transient outward K+ channel of mammalian heart muscle.
Spiders are known to produce venoms that contain a variety
of toxins with potent insecticidal activities (Quistad,
G.B., et al. "Insecticidal activity of spider (Araneae),
centipede (Chilopoda), scorpion (Scorpionidae), and snake
(Serpentes) venoms", 85 Journal Economic EntomoloqY 33,
1992). These toxins have evolved in response to
evolutionary pressures to produce effective tqxins that
effectively and rapidly kill or paralyse prey insects
(Jackson, H. and P.N.R. Usherwood, "Spider toxins as tools
WO94/Z1278 ~ PCT~S94/02750
for dissecting elements of excitary amino acid
transmission", 11 Trends in Neurosciences 278, 1988).
Other features and advantages of the invention
will be apparent from the followl ~ description of the
preferred embodiments thereof, and from the claims.
Description of the Preferred Embodiments
The drawings will first briefly be described.
Drawinqs
Fig. 1 is a chromatograph of Heteropoda
venatoria venom (120 ~l) fractioned on a Vydac C18
reversed-phase HPLC column (10 x 250 mm) equilibrated in
80~ A/20% B.. Elution with a linear gradient from 76%
A/24% B to 65% A/35% B over 44 min. (A=0.1% TFA (aq),
B=0.1~ TFA in CH~CN). The gradient was begun 5 minutes
after injection of the venom and interrupted at 39 min to
take the column to 50% B over 3 minutes to elute the final
peaks. The absorbance of the eluant was monitored at 220
nm. The fractions (#1 - 8, and the end fraction) are
identified by number at the bottom of the absorbance
traces.
Fig. 2 is a chromatogram of peptide Compounds 1
and 2 fractionated on a cation-exchange column. (Panel a)
For peptide Compound 1 the column was developed with a
linear gradient from 0-0.32 M NaCl in 50 mM sodium
acetate, pH 4.0 in 32 min followed by a linear gradient
from 0.32-1 M NaCl in 50 mM sodium acetate, pH 4.0 in 5
min. (Panel b) For peptide Compound 2 the column was
developed with a linear gradient from 0-0.3 M NaCl in 50
mM sodium acetate, pH 4.0 in 3 min followed by a linear
gradient from 0.3-1 M NaCl in 50 mM sodium acetate, pH 4.0
in 35 min. Elution was at 1 ml/min and the effluent was
monitored at 280 nm. Fractions were collected as noted on
the chromatogram.
Fig. 3 is a chromatogram of Olios fasciculatus
venom (108 ~l) fractionated on a Vydac C-18 reversed phase
column (300 A, lo x 250 mm). Five minutes after injection
of the sample, the column was developed with a linear
WO94121278 21 5 8 ~ 1 PCT~S94/02750
gradient from 20-45% acetonitrile/O.l~ TFA in 75 minutes.
At 50 minutes, the column was taken to 100%
acetonitrile/0.1% TFA over 7 min. The flow rate was 3.0
ml/min and the effluent was monitored at 220 nm.
Fractions were collected as noted on the chromatogram.
Fig. 4 is a chromatogram of peptide Compound 3
purified by cation-exchange chromatography. Five minutes
after injection of the sample, the column was developed
with a linear gradient from 0.25-l M NaCl in 50 mM sodium
acetate buffer, pH 4.0 in 75 min. Elution was at l
ml/minute and the effluent was monitored at 280 nm.
Fractions were collected as noted on the chromatogram.
The following is a detailed description of the
methods and tests by which useful compounds of this inven-
tion can be discovered and utilized for treatment of,e.q., cardiac arrhythmias and disorders of memory and
learning. A key method is the means by which compounds,
both synthetic and natural products, can be rapidly
screened with radioligand binding techniques (or their
equivalent) to identify those which modify binding at the
site on the transient outward K+ channel bound by Compound
l, Compound 2, or Compound 3. Screening for K+ channel
blockers from spider venom can be performed in a similar
manner. In addition, useful derivatives or analogues or
muteins of such polypeptides can be readily screened using
this methodology.
Additional testing will include whole-cell re-
cording of ionic currents in cardiac and neural cells to
confirm that agents which compete with radiolabeled Com-
pound l, Compound 2, or Compound 3 binding act as specific
active agents of transient outward K+currents, and are
without significant effect on other channel types.
The desired properties of agents identified by
means outlined in this invention include: ,
l) Specific and potent block of a potassium
channel, e.q., cardiac or neural transient outward K+
channels. Specific block implies that the agent will not
12
have demonstrable effects on other ionic channels or
receptors at concentrations in vitro that block I10 or IA at
dosages that prove to be of therapeutic utility in the
treatment of cardiac arrhythmias or disorders of learning
and memory, or the other diseases listed above.
2) Lack of significant side effects at thera-
peutic doses, including excessive prolongation of QT
interval of the electrocardiogram, bradycardia, hyper-
excitability or seizures.
Isolation of Spider Venom Toxins
The following is a non-limiting example of
methods by which inhibitory spider toxins of this inven-
tion may be isolated. Those in the art will recognize
that equivalent methods can be used to isolate and iden-
tify other polypeptides (or their equivalent) having
useful activity in this invention. Equivalent compounds
are those referred to herein as analogues, muteins and
derivatives which are identified by methods described
herein as having useful activity of one or more K+
channels. Those in the art will recognize that once one
useful K+ channel active agent is identified and sequenced
it can be chemically synthesized in totality or as frag-
ments, and modifications in the sequence made, as
described below. In addition, such fragments or the poly-
peptide itself may be used to screen for other such active
agents which are equivalent in their activity to the
original polypeptide.
Venom is obtained from the spiders Heteropoda
Venatoria and Olios fasciculatus through the process of
milking by electrical stimulation according to standard
methods well known to those skilled in the art. It is
preferred that the method employed is one which safeguards
against contamination of the whole venom by abdominal
regurgitant or hemolymph. Such methods are well known to
those skilled in the art. The whole venom so obtained is
stored in a frozen state at about -78° C until used for
purification as described below. Purification of the
13
constituents from the whole venom is accomplished by
reversed-phase high performance liquid chromatography
(HPLC) on a variety of preparative and semi-preparative
columns such as C-4 and C-18 Vydac columns (Rainin
Instrument Co. Inc., Mack Road, Woburn, Massachusetts
01801). Peak detection is carried out monochromatically
at 220 nm. Further analysis of the fractions can be
accomplished with, for example, polychrome UV data col-
lected with a Waters 990 diode array detector (Millipore
Corporation, Waters Chromatography Division, 34 Maple
Street, Milford, Massachusetts 01757). The fractions from
the columns are collected by know methods such as through
the use of a fraction collector and an ISCO 2159 peak
detector (ISCO, 4700 Superior, Lincoln, Nebraska, 68504).
The fraction are collected in appropriately sized vessels
such as sterile polyethylene laboratory ware. Concentra-
tion of the fractions is then accomplished by lyophiliza-
tion from the eluant followed by lyophilization from
water. Purity of the resulting constituent fractions then
can be determined by chromatographic analysis using a
different type of column than the system used in the final
purification of the fractions.
Peptide Sequencing
The polypeptides of the invention, e.g., identi-
fied as described herein, can be sequenced according to
known methods. A general strategy for determining the
primary structure includes, for example, the following
steps: 1) Reduction and S-pyridylation of disulfide-
bridged cysteine residues to enhance substrate suscepti-
bility to enzymatic attack; 2) Controlled cleavage of the
peptide through single or multi-step enzymatic digestion;
3) Isolation and purification of peptide fragments via
reversed-phase high performance liquid chromatography
(HPLC); 4) Characterization of peptide fragments through
N-terminal sequencing and ion-spray mass spectrometry.
S-pyridylethylation of cysteine residues of the
polypeptides under study can be performed, for example, in
WO94/21278 215 8 ~51 PCT~S94/02750 ~
14
solution followed by amino acid sequencing of the polypep-
tides. One such procedure for S-pyridylethylation can be
accomplished as described below.
About 1 to 10 ~g of polypeptide is dissolved or
diluted in up to 50 ~l of a buffer prepared by mixing 1
part TrisHCl, pH 8.5, containing 4 mM EDTA and 3 parts 8M
guanidineHCl. 2.5 ~l of 10% aqueous 2-mercaptoethanol is
added and the mixture is incubated at room temperature in
the dark under argon for two hours. After incubation,
lo 2 ~l of 4-vinylpyridine tfresh reagent stored under argon
at - 20C) is added and the mixture is incubated for
another two hours at room temperature in the dark under
argon. The mixture is then desalted, preferably by chro-
matography on a short reversed-phase column. The re-
covered alkylated polypeptide is then sequenced according
to known methods.
Alternatively, the polypeptide can be sequenced
after in situ reduction and S-pyridylethylation as
described ïn Kruft et al., 193 Anal. Biochem. 306 (1991).
20Given the benefits of the disclosure herein with
respect to the peptides Compound 1 and Compound 2 from the
venom of Heteropoda venatoria, and peptide Compound 3 of
Olios fasciculatus it is now possible to obtain other
peptides by methods other than through isolation/purifica-
tion from whole venom. The polypeptides of this invention
can be produced using recombinant DNA techniques through
the cloning of a coding sequence for said polypeptides or
portions thereof. For example, hybridization probes which
take advantage of the now known amino acid sequence infor-
mation of said polypeptides can be employed according to
methods well known to those skilled in the art to clone a
coding sequence for the entire polypeptide. A combination
of recombinant DNA techniques and in vitro protein syn-
thesis can also be employed to produce the polypçptides of
this invention. Such in vitro protein synthesis methods
include, but are not limited to, use of an ABI 430A solid
phase peptide synthesizer (Applied Biosystems, Inc., 850
WO9~/21278 21 5~ O~,1 PCT~S94/02750
Lincoln Center Drive, Foster City, California 94404)
employing standard Merrifield chemistry or other solid
phase chemistries well known to those skilled in the art.
Equivalent Peptides
It is well known in the art that certain amino
acid substitutions can be made in polypeptides which do
not affect, or do not substantially affect, the function
of said polypeptides. The exact substitutions which are
possible vary from polypeptide to polypeptide. Determina-
tion of permissible substitutions is accomplished acsord-
ing to procedures well known to those skilled in the art.
Thus, all polypeptides having substantially the same amino
acid sequence and substantially the same K+ channel
blocking activity are within the scope of this invention.
l~ Bioloqical ActivitY
The polypeptides or fragments thereof are useful
in the treatment of cardiac arrhythmias or in disorders of
memory and learning such as Alzheimer's disease and those
other diseases noted above. When used for such indica-
tions, the peptides and fragments are formulated accordingto standard formulation methods known in the art, such as
those disclosed in Remington's Pharmaceutical Sciences
(latest edition, Mack Publishing Company, Easton, PA).
The nature of the formulation will depend on the route of
administration and the dosage required. Optimization of
the dosage for a particular indication can be accomplished
using standard optimization techniques as is generally
practiced for peptide medicaments.
In general, administration by injection is pre-
ferred, either intravenous, intramuscular, subcutaneous orintraperitoneal. For injection, the peptide or fragments
are formulated in liquid medium, such as Ringer's solu-
tion, Hank's solution, or other forms of physiological
saline. Formulations may also involve lyophiliz,ed prepar-
ations which can be reconstituted for administration.Alternative means of providing the active compounds of the
invention to the subject include transmucosal and trans-
W O 94nl278 ~15 8 ~ 5 I PCTrUS94/02750
16
dermal administration, wherein the formulation includes apermeation enhancer, such as a detergent, as well as addi-
tional excipients. Properly formulated, oral administra-
tion is also within the scope of th`e~invention.
Screeninq Assay Employinq Peptid ~ ComPounds 1, 2 and 3
In addition, the p~ptides and biologically
active fragments thereof are useful in screening assays to
assess the ability of small molecules or other candidate
drugs to inhibit the binding of Compounds 1, 2 or 3 to car-
diac or neural transient outward K+channels. Describedherein below is a suitable assay for competitive binding
in which the Compound 1, 2 or 3 of the invention are
useful. For use in this assay, generally, the polypep-
tides Compound 1, 2 or 3 (or an active fragment) is sup-
plied in radiolabeled form and the ability of thecandidate compound to compete with radiolabeled Compound
1, 2 or 3 is assessed.
The following examples are intended to illus-
trate but not to limit the invention.
ExamPle 1: Heteropoda venatoria Venom Fractions
Approximately 900 ~1 of Heteropoda venatoria
venom was fractionated by diluting 75-120 ~1 aliquots of
the crude venom to 1 ml with A and applying the diluted
fractions to a Vydac C18 column (10 x 250 mm) equilibrated
in 20% B. (A=0.1% TFA (aq); B=0.1% TFA in CH3CN). After
3 min, the gradient was changed to 24% B over 1 min and at
5 min, a linear gradient from 24~ to 35~ B over 44 min was
begun. The flow rate was 3.5 ml/min and the effluent was
detected at 220 nm (Fig. 1). The following fractions were
collected: fraction 1 (peaks eluting between 5 and 16
minutes), fraction 2 (peaks eluting between 16 and 19
minutes), fraction 3 (peaks eluting between 19 and 23.5
minutes), fraction 4 (peaks eluting between 23.5 and 26.5
minutes), fraction 5 (peaks eluting between 26.,5 and 29.5
minutes), fraction 6 (peaks eluting between 29.5 and 33
minutes), fraction 7 (peaks eluting between 33 and 37
minutes), fraction 8 (peaks eluting between 37 and 39
W094121278 21~ 8 0 ~1 PCT~S94/02750
minutes), and the end fraction (peaks eluting between 39
and 46 minutes). At 39 min, after the majority of the
venom components had eluted, the column was taken to 50
B over 3 min. When no further peaks eluted (~7 minutes)
the column was returned to 20~ B over 4 min and
equilibrated for the next chromatography. Like fractions
from the 8 chromatographic runs were combined and
lyophilized. The major peaks of fractions 6 and 7
correspond to compounds l and 2, respectively described in
Examples 2 and 4 below.
ExamPle 2: Heteropoda Peptide Compound l
Crude Heterop~da venatoria venom (-50 ~l) was
applied to a reversed phase HPLC column (Vydac, C-18 300
A, 22 x 250 nm) and was operated using a biphasic linear
gradient program from 80% A and 20% B to 65~ A and 35% B
over 60 minutes (A = 0.1% trifluoroacetic acid (TFA), B =
acetonitrile) with detection at 220 nm and a flow rate of
15 ml/min. The desired fraction was collected from 43 to
44 min. Pooled fractions from individual runs were con-
centrated by lyophilization.
The structure of peptide Compound l was deter-
mined and verified by the following methods. PTC amino
acid analysis was carried out on l-lO nmols in triplicate
using the Waters Pico-Tag system. N-terminal sequencing
was carried out on a pulse-liquid sequenator (ABI) on both
native and reduced/pyridylethylated peptide. Mass spectral
analysis was obtained from a SCI-EX API III ion spray mass
spectrometer.
A pyridylethylated derivative of Compound
A suitable for N-terminal sequencing was generated in situ
according to the method of Kruft et al., 193 Anal.
Biochem. 306 (l99l).
The data taken together affirm the st~ucture of
peptide Compound l as shown below.
WO94/21278 215 ~ 1 PCT~S94/02750
18
SEQ. ID. NO. 1:
Asp Asp Cys Gly Lys Leu Phe Ser Gly Cys Asp Thr Asn Ala
1 5 10
Asp Cys Cys Glu Gly Tyr Val Cys Arg Leu Trp Cys Lys Leu
~-' 25
Asp Trp ~ ~
:
30 residues, 6 cysteines, 3 disulfide bonds.
Calculated mass = 3412.86 (amide).
Observed mass = 3412.70 (ion spray m.s.).
Estimated pl = 3.76.
Example 3: Heteropoda Peptide Compound 1
Peptide Compound l was also further purified by
cation-exchange chromatography on a HEMA-IEC BIO SB column
(10 ~m, 4.6 x 150 cm; Alltech Associates, Deerfield, IL
60015). The lyophilized material containing peptide
Compound 1 from the reversed-phase chromatography was
dissolved in three ml of 50 mM sodium acetate, pH 4.0 and
chromatographed in three equal portions as follows. One
ml was loaded onto the HEMA-IEC BIO SB column equilibrated
in 50 mM sodium acetate, pH 4Ø After 5 min, the column
was developed with a linear gradient from 0-0.32 M NaCl in
50 mM sodium acetate, pH 4.0 in 32 min followed by a
linear gradient from 0.32-1 M NaCl in 50 mM sodium
acetate, pH 4.0 in 5 min (Fig. 2a). After 10 min, the
column was returned to the starting conditions in 5 min
and equilibrated for subsequent chromatographies. Elution
was at 1 ml/min and the effluent was monitored at 280 nm.
Fractions were collected as noted on the chromatogram.
The remaining two ml of crude Compound 1 was
chromatographed as described above and like fractions from
the three chromatographies were combined.
The major absorbance peak, which eluted from the
cation-exchange column between 26.5 and 29 min, was
desalted on a Vydac C-18 reversed-phase column (10 x 250
mm, 300 A). The pooled fraction (-10 ml) was loaded onto
W094121278 215 8 0 ~1 PCT~S94/02750
19
the reversed-phase column equilibrated in 20~
acetonitrile/0.1% TFA. After 10 min, the column was
developed with a linear gradient from 20-35%
acetonitrile/0.1~ TFA in 30 min at a flow rate of 3.5
ml/min and the effluent was monitored at 220 nm. The
fraction eluting between 35.5 and 38 min was lyophilized
to give 641 ~g of purified peptide Compound 1. The
observed mass of this peptide was 3412.72 (electrospray
ionization).
ExamPle 4: Heteropoda Peptide Compound 2
Crude Heteropoda venatoria venom (~50 ~l) was
applied to a reversed-phase HPLC column (Vydac, C-18, 300
A, 22x250 mm) and was operated using a biphasic linear
gradient program from 80% A and 20% B to 65% A and 35% B
over 60 min (A = 0.1% trifluoroacetic acid (TFA), B =
acetonitrile) with detection at 220 nm and a flow rate of
15 ml/min. The desired fraction was collected from 46 to
48.5 min. Pooled fractions from individual runs were
concentrated by lyophilization.
The material from the fractionation above,
derived from 50 ~l of crude venom, was applied to a
reversed-phase HPLC column (Vydac, C-18, 300 A, 22 x
250 mm) and was operated using an isocratic program of 75%
A and 25% B + (A = 0.1~ TFA, B = acetonitrile) with detec-
tion at 220 nm and a flow rate of 3.5 ml/min. The desired
fraction was collected from 55 to 68 min. Pooled like
fractions from individual runs were concentrated by
lyophilization.
The structure of peptide Compound 2 was deter-
mined and verified by the following methods. PTC amino
acid analysis was carried out on 1-10 nmols in triplicate
using the Waters Pico-Tag system. N-terminal sequencing
was carried out on a pulse-liquid sequenator (AB~) on both
native and reduced/pyridylethylated peptide. Mass spec-
tral analysis was obtained from a SCI-EX API III ion spray
mass spectrometer.
WO94/21278 2 1 ~ 8 0~1 PCT~S94/02750
A pyridylethylated derivative of Compound 2
suitable for N-terminal sequencing was generated in situ
according to the method of Kruft et al., 193 Anal.
Biochem. 306 (1991). ~
The data taken together affirm the structure of
peptide Compound 2 as shown belo~.
SEQ. ID. NO. 2:
Glu Cys Gly Thr Leu Phe Ser Gly Cys Ser Thr His Ala Asp
1 5 10
Cys Cys Glu Gly Phe Ile Cys Lys Leu Trp Cys Arg Tyr Glu
15 20 25
Arg Thr Trp
31 residues, 6 cysteines, 3 disulfide bonds.
Calculated mass = 3599.05 (amide).
Observed mass = 3599.38 (ion spray m.s.)
Estimated pl = 5.41.
Example 5: Heteropoda PePtide Compound 2
Peptide Compound 2 was also purified by cation-
exchange chromatography on a HEMA-IEC BIO SB column (10
~m, 4.6 x 150 cm). The lyophilized material containing
peptide Compound 2 from the initial reversed-phase
chromatography was dissolved in three ml of 50 mM sodium
acetate, pH 4.0 and chromatographed in three equal
portions as follows. One ml was loaded onto the HEMA-IEC
BIO SB column equilibrated in 50 mM sodium acetate, pH
4Ø After 5 min, the column was developed with a linear
gradient from 0-0.3 M NaC1 in 50 mM sodium acetate, pH 4.0
in 3 min followed by a linear gradient from 0.3-1 M NaCl
in 50 mM sodium acetate, pH 4.0 in 35 min (Fig. 2b).
After 5 min the column was returned to the starting
conditions over 10 min and equilibrated for subsequent
chromatographies. Elution was at 1 ml/min, and the
effluent was monitored at 280 nm. Fractions were
collected as noted on the chromatogram. The remaining two
ml of crude Compound 2 was chromatographed as described
W094/21278 21 5 8 0 ~1 PCT~S94/02750
above and like fractions from the three chromatographies
were combined.
The major absorbance peak, which eluted from the
cation-exchange column between 30 and 34 min, was desalted
in two portions on a Vydac C-18 reversed-phase column (lO
x 250 mm, 300 A). The column was equilibrated in 25%
acetonitrile/0.1% TFA and eluted with the starting solvent
for 10 min, followed by a linear gradient from 25-35%
acetonitrile/0.1% TFA in 20 min at a flow rate of 3.5
ml/min. The effluent was monitored at 220 nm and peptide
Compound 2 eluted as a single peak from 26.5 to 29 min.
The remaining pool from the cation-exchange column was
then desalted and like fractions were combined. This pool
was lyophilized to give 1.88 mg of purified peptide
Compound 2. The observed mass of this peptide was 3599.52
(electrospray ionization).
Example 6: Fractionation of Olios fasciculatus venom
Approximately 108 ~l of Olios fasciculatus venom
was fractionated by diluting the whole venom with 1.5 ml
of 20% acetonitrile/0.1% TFA and loading the sample on to
a Vydac C-18 column (300 ~, 10 X 250 mm) equilibrated in
the same buffer. Five minutes after injection of the
sample, the column was developed with a linear gradient
from 20-45% acetonitrile/0.1% TFA in 75 min (Fig. 3). At
50 min, after the majority of the venom components had
eluted, the column was taken to 100% acetonitrile/0.1~ TFA
over 7 min. The flow rate was 3.0 ml/min and the effluent
was monitored at 220 nm. Fractions were collected as
noted on the chromatogram. The fraction (#21) containing
peptide Compound 3, which eluted between 40 and 42 min,
was lyophilized and the residue dissolved in 2 ml of 50 mM
sodium acetate, 0.25 M NaCl, pH 4Ø
Example 7: Olios fasciculatus Compound 3
Peptide Compound 3 was further purified by
cation exchange chromatography on a HEMA-IEC BI0 SB column
W094/21278 215 ~ PCT~S94/02750
(10 ~m, 4.6 x 150 cm, from Alltech Associates, Deerfield,
IL 60015). The solution containing peptide Compound 3
(1.5 ml) from the r~versed-phase~chromatography was loaded
onto the HEMA-IEC BIO SB colu~ equilibrated in the same
buffer. After 5 minutes, th~tcolumn was developed with a
linear gradient from 0.25-1 M NaCl in 50 mM sodium acetate
buffer, pH 4.0 in 75 min (Fig 4). Elution was at 1 ml/min
and the effluent was monitored at 280 nm. Fractions were
collected as noted on the chromatogram.
10 The major peak of material (fraction #4), which
eluted from the cation-exchange column between 27 and 32
min, was desalted on a Vydac C-18 reversed-phase column
(10 x 250 mm, 300 A). The fraction (-4.5 ml) was loaded
onto the reversed-phase column equilibrated in 0.1~TFA.
After 3 min, the column was developed with a linear
gradient from 0-15% iso-propanol/0.1% TFA in 3 min
followed by a linear gradient from 15-30~ iso-
propanol/0.1% TFA in 30 min and from 30-50% iso-
propanol/0.1% TFA in 5 min. Elution was at 1.0 ml/min and
the effluent was monitored at 220 nm. The fraction
eluting between 43 and 45 min was lyophilized to give 70
~g of purified peptide Compound 3. The observed mass of
this peptide was 3786.64 (electrospray ionization).
N-terminal sequence analysis was obtained for
reduced, derivatized peptide Compound 3. The sequence is
as follows:
SEQ. ID. NO. 3:
Asp Asp Cys Ala Gly Trp Met Glu Ser Cys Ser Ser Lys
1 5 10
Pro Cys Cys Ala Gly Arg Lys Cys Phe Ser Glu Trp Tyr
15 20 25
Cys Lys Leu Val Val Asp Gln Asn
34 residues, 6 cysteines, 3 disulfide bonds.
Observed mass = 3786.64 (ion spray m.s.)
WO9~/21278 21 5~ 0 51 PCT~S941027~0
There is low confidence in the identity of amino acids 33
and 34.
,
Example 8: K+ Channel Blockinq Activity of Heteropoda
venatoria and Olios fasciculatus Peptide
Fractions and Compounds 1 2 and 3
The ability of the peptide fractions and
Compounds 1, 2 and 3 of this invention to block transient
outward K+ channels is demonstrated by the following
procedure.
Rat ventricular myocytes were isolated according
to the procedure described previously (Kamp et al.,
'IVoltage- and Use-dependent Modulation of Cardiac Calcium
Channels by the Dihydropyridine (+)-202-791", 64 Circ.
15 Res. 338, 1989). The method involves retrograde perfusion
of an excised rat heart with a solution containing
collagenase and protease to enzymatically digest the
entire heart so as to isolate individual cardiac myocytes
suitable for use in standard voltage clamp experiments.
Whole-cell currents are recorded from isolated myocytes
using the voltage clamp techniques described in detail
elsewhere (Hamill et al., "Improved Patch Clamp Techniques
for High-resolution Current Recording from Cells and
Cell-free Membrane Patches", 391 Pfluqers Arch. 85, 1981).
Cells are placed in a 0.5 ml recording chamber and bathed
in a buffered solution of the following composition (in
mM): NaCl, 132; MgCl2, 1.2; CaCl2, 1.8; KCl, 4; HEPES, 10;
glucose, lO; pH = 7.4. In most experiments in which K-
currents were recorded, Ca2+ current was blocked by
omission of CaCl2 and addition of 1 mM co7+ to this
solution. The myocytes are voltage clamped using a
commercially available patch clamp amplifier (Axon
Instruments Axopatch lD), and data acquisition and
analysis is performed using a personal compute,r. Cells
were clamped at a potential of -60 mV. Test potentials
(500 msec duration) were applied to potentials ranging
from -40 to +30 mV. Using these techniques several K~
-
WO9~/21278 215 8 0 5 1 PCT~S94/02750
24
currents can be recorded in these cells, including an
inward rectifier K+ current; a rapidly activating,
non-inactivating delayed rectifler K+ current; and a
voltage-dependent transient out~d K+ current (I~o)~ The
dried fraction residues of veno~m fractions 1-8 and the end
fraction prepared as describëd in Example 1 were each
dissolved in 1 ml of water. A 10 ~l sample of each was
then diluted with 3 ml of the buffered solution to test
for effects on cardiac K+ currents. Under these con-
ditions, peptide fractions 2-9 blocked I,c, in a voltage-
dependent manner. Block was complete at a test potential
of -10 mV, with block reduced to 30 to 70% of control
values at a test potential of +30 mV. The predominant
peptides of fractions 6 (Compound 1) and 7 (Compound 2)
were isolated and purified as described in Examples 3 and
5 above. Compound 1 blocked I,~ in a voltage-dependent
manner, with greater block occuring at less depolarized
test potentials. This was quantified by determining the
concentration required to inhibit I,~ by 50% (IC~) and the
maximum block of this current at three different test
potentials. The maximum block of I,l, at test potentials of
-10 mV, +20 mV and +50 mV was 100%, 79% and 69%,
respectively. The IC~ for block of I~o was 16 nM at -10 mV,
nM at +20 mV, and 138 nM at +50 mV (n = 4-6).
Consistent with block of I~o~ Compound 1 (30 nM) prolonged
action potential duration, measured at 90% repolarization,
of isolated rat ventricular myocytes by 34+5% (n=5).
Selective prolongation of action potential duration
represents class III antiarrhythmic activity (Vaughan
Williams, "Delayed ventricular repolarization as an
antiarrhythmic principle", 6 Eur. Heart J. 145, 1985).
The effects of Compound 1 or 2 on other cardiac currents
was determined to assess their specificity. At
concentrations of 0.2 - 1.0 ~M, Compound 1 or Çompound 2
did not affect the following cardiac currents as measured
using standard whole cell-voltage clamp techniques:
WO94/2127821 5 8 0 51 PCT~S94/02750
-ultrarapid delayed rectifier K- current (I~) in rat
ventricular myocytes
- -slow delayed rectifier K current (I~,) in guinea pig
ventricular myocytes
-inward rectfier Kt current (I~,) in rat and guinea pig
ventricular myocytes
-sodium current ( IN.) in rat ventricular myocytes
-L-type Ca2+ current ( ICJ.L) in rat and guinea pig
ventricular myocytes
lOIn an isolated human ventricular myocyte,
Compound l (0.2 ~M) also blocked I,o but not I~w, similar to
the findings in rat ventricular myocytes.
Compound 3 had similar activity, blocking I,o
completely at test potentials < 0 mV at l ~M, while having
no effect on either delayed rectifier or inward rectifier
K+currents. The effects of Compound l and Compound 2
(l ~M) were substantially reversed upon washout of the
toxins.
Compound l or 2 was also tested for activity on
a number of other Kr channels recorded from isolated non-
cardiac cells. At 0.2 - l.0 ~M, Compound l or 2 had no
effect on:
-rapid delayed rectifier K~ currents (I~) of rat neural
cells (Purkinje neurons, cerebellar granule cells,
hippocampal pyramidal cells, sympathetic ganglion cells),
GH3 pituitary cells, or rabbit osteoclasts.
-transient outward current (IA) of rat cerebellar
granule cells or sympathetic ganglion cells.
-a cloned channel (Kvl.4) expressed in xenopus oocytes.
Thus, Compounds l and 2 were shown to be quite
specific for one type of channel (a voltage-activated,
transient outward K current) in cardiac myocytes. The
only other toxin reported to inhibit a transient outward
K current (in neural cells) is dendrotoxin (Haliwell et
al., "Central action of dendrotoxin: Selective reduction
of a transient K conductance in hippocampus and binding to
localized acceptors", 83 Proc. Natl. Acad. Sci. USA 493,
WO 94121278 2 ¦ 5 8 o 5 1 PCT/US9~/027~0
26
1986). However, we have shown that dendrotoxin (2 ,uM) has
no effect on rat cardiac Ito. Therefore, Compounds 1, 2 and
3 are the first toxins described that specifically block
cardiac I~o~ `
Example 9: Neural effects of Compound 1 and 2
The ability of Compounds 1 and 2 of this
invention to affect neural activity is demonstrated by
their electrophysiological effects on hippocampal slices.
10Male Sprague-Dawley rats (100-200 g) were
sacrificed by decapitation. The brain was removed from
the cranium, immediately placed in cold (4-6C), oxygenated
(95% 2/5% C02) artificial cerebrospinal fluid (aCSF)
consisting of (in mM) NaCl, 126; KCl, 2.5; NaH2P04, 1.24;
15MgS04, 1.3; CaCl2, 2.4; NaHC03, 26; glucose, 11, and slices
of hippocampus were prepared as described previously
(Mueller et al., "Arylamine spider toxins antagonize NMDA
receptor-mediated synaptic transmission in rat hippocampal
slices", 9 Synapse. 244, 1991). Slices were maintained in
20 a reservoir of 200 ml of oxygenated aCSF at room
temperature. Following a 1 hr recovery period, a single
slice was transferred to a small volume (~300 ,ul)
recording chamber. Small platinum weights were placed on
the slice to increase the stability of the recording. The
25 slice was covered with aCSF and a superfusion system
maintained the flow of fresh, oxygenated aCSF at 2 ml/min.
The slice was held submerged in the flow of aCSF so that
potential problems with access of drugs into the slice
were minimized. The temperature in the recording chamber
30 was held at 33C for extracellular field potential
recording. Bipolar concentric stimulating electrodes were
placed under visual guidance in the stratum radiatum near
the border of CA1-CA2. To evoke synaptic responses,
monophasic 50 ,usec pulses of 3-50 V were delivelred to the
35 slice every 30 sec while testing the response until
potentials of maximal amplitude were obtained from a
particular recording site. The voltage was then set so as
W094/21278 ~1 5 8 ~ ~ ~ PCT~S9410275~
to evoke a half-maximal response. Recording was done with
2-3 MW glass microelectrodes filled with 0.9% NaCl, which
were also placed under visual guidance. Synaptic
responses were recorded from the CAl pyramidal cell layer
(population spike) or from stratum radiatum (field
excitatory postsynaptic potential (EPSP) and afferent
volley (AV)), digitized, and entered into an IBM PC-based
data acquisition and storage system. Toxins were made up
in phosphate-buffered saline (PBS: NaCl, 140 mM; KCl, 2.5
mM; KH2P04, 1.5 mM; Na2HP04, 8.1 mM; pH 7.4) at lO0-lO00
times the desired final concentration, and then trans-
ferred to the reservoir syringe so as to achieve, via
dilution and mixing, the desired final concentration. All
drugs and toxins were applied by superfusion for 30 min,
at which time the response amplitude had generally
plateaued. All waveforms were digitized and stored on
disk. Extracellularly recorded response amplitudes were
averaged over a 5 min period just prior to drug
application (control, pre-drug) and again over a 5 min
period following drug application (25-30 min post-drug).
Compound l produced a sustained increase in the
population spike amplitude which did not recover during
washout with fresh aCSF. The mean response to 500 nM
Compound l was a 35 + 9% increase (mean + S.E.M., n = 5
slices). In two of these slices, the amplitude of the
simultaneously recorded field EPSP was increased by an
average of 16~, while the afferent volley was unchanged.
Similar results were obtained using purified
Compound 2, which produced an increase in the population
spike amplitude of 25 + 7~ (mean + S.E.M., n = 9 slices)
when applied at a final concentration of l ~M. In two of
these slices, the amplitude of the simultaneously recorded
field EPSP was increased by an average of 12%, while the
afferent volley was unchanged.
Taken together, these results demonstrate that
Compounds l and 2 produce long-term increases in synaptic
transmission at the Schaffer collateral-CAl pyramidal cell
WO94/21278 PCT~S94/02750
~8~s~
synapse. These data cannot distinguish between a pre- and
post-synaptic site of action,~or can they point clearly
to a mechanism of action. ~uch increases in synaptic
transmission could be due to blockade of voltage-sensitive
potassium channels.
Many K~ channel blocking agents can cause
seizures when injected intravenously (i.v.), or when
administered by intracerebroventricular (i.c.v.)
injection. For example, dendrotoxin causes convulsions
and death in mice when injected i.c.v. at 0.008 ~g/g,
equivalent to about 0.24 ~g/mouse (Schweitz, H. et al.,
"Purification and pharmacological characterization of
peptide toxins from the black mamba (Dendroaspis
polylepis) venom", 28 Toxicon 847, 1990). In contrast,
Compound 2 did not cause convulsions or seizures in
audiogenic seizure-prone mice injected i.c.v. with 1 ~g
(n=3) or 2 ~g (n=l) of Compound 2. After i.v. injection
at doses of 10, 15 or 24 ~g (n=1 each dose), Compound 2
caused a transient ataxia in mice, but convulsions were
not observed.
Example 10: Other K+ Channel Blockinq Toxins
Compounds 1, 2 and 3 represent the first
reported examples of toxins isolated from spider venoms
that block specific K+ channels. Venoms from species of
spiders other than Heteropoda venatoria and Olios
fasciculatus may contain structurally unrelated toxins
(peptides and nonpeptides) that potently block Ito or other
types of K+channels in mammalian cells.
Several other toxins isolated from venoms of
invertebrate and vertebrate venoms have been well
characterized. For example, two toxins have been isolated
from venom of the bee Apis mellifera that block K+
channels. Apamin blocks a low conductance Ca~+lactivated
K+channel, whereas MCD (mast cell degranulating) peptide
blocks a non-inactivating delayed rectifier K+ channel
(strong, ~Potassium Channel Toxins", 46 Pharmac. Ther.
2~S8051
WO94121278 PCT~S9~/027~0
137, l99O). Other K+ channel specific blocking toxins have
been isolated from venoms produced by scorpions and
snakes. For example, venom from the scorpion Leiurus
quinquestriatus contains at least two toxins, charybdo-
toxin and leiurotoxin that block high conductance, and lowconductance Ca2+-activated K+ channels, respectively
(Strong, "Potassium Channel Toxins", 46 Pharm. Ther. 137,
1990). Toxins that block non-inactivating delayed
rectifier K+ channels of neurons have also been isolated
from the venoms of mamba snakes (Harvey and Anderson,
"Dendrotoxins: Snake Toxins That Block Potassium Channels
and Facilitate Neurotransmitter Release", 31 Pharmac.
Ther. 33, 1985). Dendrotoxin from the green mamba snake
(Dendroaspis angusticeps) and Toxin 1 from the black mamba
(D. polylepis) both share considerable sequence homology
with ~-bungarotoxin, an inhibitory presynaptic neurotoxin
isolated from venom of Bungarus multlcinctus that also
blocks the same type of K+ channel (Moczydlowski et al.,
"An Emerging Pharmacology of Peptide Toxins Targeted
Against Potassium Channels", 105 J. Membrane Biol. 95,
1988). The above noted toxins are reported to have
effects beyond block of non- inactivating delayed
rectifier K+ channels. For example, ~-bungarotoxin also
exhibits phospholipase A2 activity (Moczydlowski et al.)
and dendrotoxin also blocks sodium current and a slow
inactivating transient K+current in hippocampal neurons
(Li and McArdle, "Dendrotoxin Inhibits Sodium and
Transient Potassium Currents in Murine Hippocampal
Neurons", 64 BioPhys. J. A198, 1993).
The above-noted toxins have been useful in
defining the role of specific K+ channels in the physiology
of normal cells and cells of diseased tissues. However,
there are several K+ channels known for which no highly
specific and potent modulators have been discovered.
Spider venoms represent an untapped source for the
discovery of such novel channel ligands.
WO91/21278 2~ 8a5 ~ PCT~S94/02750
The existence of K+ channel-specific toxins in
spider venoms is examined by testing the effects of whole
venoms, venom fractions separated by standard HPLC
methodology, and isolated toxin~s--on K+ currents measured
using standard whole-cell .~oltage clamp recording
techniques on isolated mammalian cardiac and neural cells
as described in Example 8 above.
Example ll: Method for Screeninq Compounds that Bind to
Compound l/Compound 2/ComPound 3 Site on
the Transient Outward K+ Channel in Neural
Tissue
Compound l,2 or 3 or related peptides are
labeled with i~sI by procedures known in the art
(lactoperoxidase, Bolton-Hunter, chloramine T, etc.).
Candidate compounds acting at the Compound l/Compound 2/
Compound 3 binding site are assessed by determining their
ability to displace specific binding of ['25I]Compound l,
[~2sI]Compound 2, or ['~I]Compound 3 or related peptides
labeled with l2~I using techniques described below.
The following assay can be utilized as a high
throughput assay to screen product libraries (e.q.,
natural product libraries and compound files at major
pharmaceutical companies) to identify new classes of
compounds with activity at the Compound l/Compound 2
/Compound 3 binding site on the Il~, channel. These new
classes of compounds are then utilized as chemical lead
structures for a drug development program targeting the
Compound l/Compound 2/Compound 3 binding site on the
neural Ilo channel. The compounds identified by this assay
offer a novel therapeutic approach to disorders of
learning and memory such as Alzheimer's disease, and those
other diseases listed above. It is important to
demonstrate that a peptide retains its biologica,l activity
if it is to be used in a quantitative binding assay.
Iodinated ('1'I) Compound l, Compound 2, and Compound 3
retain their normal activity with regard to block of
W094/21278 215 8 0 51 PCT~S94/02750
cardiac I~o~ For example, l27I-Compound 1 blocked I~o of rat
ventricular myocytes in a voltage-dependent manner, with
approximate IC~'s of 25 ~lM at -10 mV, 70 nM at +20 mV, and
150 nM at +50 mV.
Rat brain membranes are prepared according to
the method of Williams et al. ("Effects of Polyamines on
the Binding of [3H]MK-801 to the NMDA Receptor:
Pharmacological Evidence for the Existence of a Polyamine
Recognition Site", 36 Molec. Pharmacol. 575, 1989) as
follows: Male Sprague-Dawley rats (Simonsen Laboratories)
weighing 100-200 g are sacrificed by decapitation. The
brains from 20 rats (minus cerebellum and brainstem) are
homogenized at 4 C with a glass/Teflon homogenizer in 300
ml 0.32 M sucrose containing 5 mM K-EDTA (pH 7.0). The
homogenate is centrifuged for lO minutes at 1,000 x g and
the supernatant removed and centrifuged at 30,000 x g for
30 minutes. The resulting pellet is resuspended in 250 ml
5 mM K-EDTA (pH 7.0) stirred on ice for 15 minutes, and
then centrifuged at 30,000 x g for 30 minutes. The pellet
is resuspended in 90 ml 5 mM K-EDTA (pH 7.0), and 15-ml
aliquots are layered over discontinuous sucrose gradients
of 0.9 M and 1.2 M sucrose (10 ml each). The gradients
are centrifuged at 95,000 x g for 90 minutes, and the
synaptic plasma membrane (SPM) fraction at the 0.9 M/1.2
M sucrose interface collected. Membranes are washed by
resuspension in 500 ml 5 mM K-EDTA (pH 7.0), incubated at
32 C for 30 minutes, and centrifuged at 100,000 x g for 30
minutes. The wash procedure, including the 30 minutes
incubation, is repeated three times. The final pellet is
resuspended in 60 ml 5 mM K-EDTA (pH 7.0) and stored in
aliquots at -80 C. To perform a binding assay with
['2sI]Compound 1, 2 or 3, aliquots of synaptic plasma
membranes (SPMs) are thawed, washed once by incubation at
32~C for 30 minutes, and centrifuged at lOO,OOO,x g for 30
minutes. SPMs are resuspended in buffer A (20 mM K-HEPES,
1 mM K-EDTA, pH 7.0). The [l~sI]Compound 1, 2 or 3 is added
to this reaction mixture. Binding assays are carried out
WO94/21278 PCT~S94/02750
21S 8~5 ~ 32
in polypropylene test tubes. The final incubation volume
is 200 ~l. Nonspecific binding:is determined in the
presence of 100 ~M nonradioactiye~ Compound 1, 2 or 3.
Triplicate samples are incuba~tëd at 32 C for 2 hours.
Assays are terminated by the addition of lO ml of ice-cold
buffer A, followed by filtration over glass-fiber filters
(Schleicher & Schuell No. 30). The filters are washed
with another 10 ml of buffer A, and radioactivity is
determined by gamma counting for læsI.
In order to validate the above assay, the
following experiments are also performed:
(a) The amount of nonspecific binding of the
[~2sI]Compound 1, 2 or 3 to the filters is determined by
passing 200 ~l of buffer A containing 100 nM [~2sI]Compound
1, 2 or 3 through the glass-fiber filters. The filters
are washed with another 10 ml of buffer A, and
radioactivity bound to the filters is determined by
scintillation counting. If a significant amount of
nonspecific binding of the [I~sI]Compound 1,2 or 3 occurs,
then filters are prewashed with unlabeled Compound 1, 2 or
3 to limit this binding. If high nonspecific binding
remains a problem, assays will be terminated by
centrifugation rather than by filtration, and the amount
of radioactivity in the pellet will be determined by
scintillation counting.
(b) A saturation curve is constructed by
resuspending SPMs in buffer A. The assay buffer (200 ~l)
contains 75 ~g of protein. Nine concentrations of
['25I]Compound 1, 2 or 3 are used, ranging from 10 nM to 100
~M in half-log units. A saturation curve is constructed
from the data, and an apparent K" value and B",~ value
determined by Scatchard analysis (Scatchard, "The
Attractions of Proteins for Small Molecules and Ions", 51
Ann. N.Y. Acad. Sci. 660, 1949). The cooper~tivity of
binding of the [l2sI]Compound 1, 2 or 3 is determined by the
construction of a Hill plot (Hill, "A New Mathematical
Treatment of Changes of Ionic Concentrations in Muscle and
~ 215~051
WO94/21278 PCT~S94/02750
Nerve Under the Action of EIectric Currents, With a Theory
to Their Mode of Excitation", 40 J. Phvsiol. l90, l9lO).
(c) The dependence of binding on protein
(receptor) concentration is determined by resuspending
SPMs in buffer A. The assay buffer (200 ~l) contains a
concentration of [~2sI]Compound l, 2 or 3 equal to its K~
value and increasing concentrations of protein. The
specific binding of ['2sI]Compound l, 2 or 3 should be
linearly related to the amount of protein (receptor)
present.
(d) The time course of ligand-receptor binding
is determined by resuspending SPMs in buffer A. The assay
buffer (300 ~l) contains a concentration of ['~5I]Compound
l, 2 or 3 equal to its K~ value and lO0 ~g of protein.
Triplicate samples are incubated at 32 C for varying
lengths of time; the time at which equilibrium is reached
is determined, and this time point is routinely used in
all subsequent assays.
(e) The pharmacology of the binding site can be
analyzed by competition experiments. In such experiments,
the concentration of [~2sI]Compound l, 2 or 3 and the amount
of protein are kept constant, while the concentration of
test (competing) drug is varied. This assay allows for
the determination of an ICs(, and an apparent K~ for the
competing drug (Cheng and Prusoff, IlRelationship Between
the Inhibition Constant (Kj) and the Concentration of
Inhibitor Which Causes 50 Percent Inhibition (ICs(,) of an
Enzymatic Reaction", 22 J. Biochem. Pharmacol. 3099,
1973). The cooperativity of binding of the competing drug
is determined by Hill plot analysis.
- Specific binding of the ['25I]Compound l, 2 or 3
represents binding to a novel site on the I,o channel. As
such, peptides related to Compound l, 2 or 3 should
compete with the binding of ['25I]Compound l, 2~or 3 in a
competitive fashion, and their potencies in this assay
should correlate with their inhibitory potencies in a
functional assay of I~o block (e. q., inhibition of I~o in
-
WO94/21278 PCT~S9~/02750
2~58~5l
34
isolated neural or cardiac cells). Conversely, compounds
which have activity at the other sites on the Ito channel
should not displace ['2sI]CompQund l, 2 or 3 binding in a
competitive manner. Rather,~c~mplex allosteric modulation
of ['25I]Compound 1, 2 or ~! binding, indicative of non-
competitive interactions~,~might be expected to occur.
(f) Studies to estimate the dissociation
kinetics are performed by measuring the binding of
['25I]Compound 1, 2 or 3 after it is allowed to come to
equilibrium (see (d) above), and a large excess of
nonradioactive competing drug is added to the reaction
mixture. Binding of the ['25I]Compound l, 2 or 3 is then
assayed at various time intervals. With this assay, the
association and dissociation rates of binding of the
['25I]Compound l, 2 or 3 are determined (Titeler, "Multiple
Dopamine Receptors: Receptor Binding Studies in Dopamine
Pharmacology", Marcel Dekker, Inc., New York, 1983).
Additional experiments involve varying the reaction
temperature (20 C to 37 C) in order to understand the
temperature dependence of these parameters.
Example 12: Method for Screeninq Compounds that Bind to
Compound l/Com~ound 2/Compound 3 Site on
the Transient Outward K+ Channel in Cardiac
25Tissue
Compound 1, Compound 2, Compound 3 and related
peptides are labeled with l25I by procedures known in the
art (lactoperoxidase, Bolton-Hunter, chloramine T, etc.).
Candidate compounds acting at the Compound l/Compound 2/
Compound 3 binding site are assessed by determining their
ability to displace specific binding of ['25I]Compound l,
['2sI]Compound 2, ['~I]Compound 3 or related peptides labeled
with l25I using techniques described below.
The following assay can be utilized ,as a high
throughput assay to screen product libraries (e.q.,
natural product libraries and compound files at major
`pharmaceutical companies) to identify new classes of
W094121278 215 8 0 51 PCT~S94/02750
compounds with activity at the Compound l/Compound 2/
Compound 3 binding site on the cardiac Ito channel. These
new classes of compounds are then utilized as chemical
lead structures for a drug development program targeting
the Compound 1/Compound 2 binding/Compound 3 site on the
cardiac Ito channel. The compounds identified by this assay
offer a novel therapeutic approach to the treatment of
reentrant supraventricular and ventricular cardiac
arrhythmias.
Cardiac sarcolemmal vesicles are prepared
according to the method of Doyle et al. ("Saxitoxin
binding and "Fast" Sodium Channel Inhibition in Sheep
Heart Plasma Membrane", 249 Am. J. Physiol. H328, 1985)
and Jones and Besch ("Isolation of Canine Cardiac
Sarcolemmal Vesicles", 5 Methods in PharmacoloqY 1, 1984),
as modified by Kamp and Miller ("Voltage-dependent
Nitrendipine Binding to Cardiac Sarcolemmal Vesicles", 32
Mol. Pharmacol. 278, 1987). Cardiac sarcolemmal vesicles
are prepared from fresh bovine or other suitable mammalian
heart tissue at 0-4 C. The heart is cut into 1 cm3 pieces,
and converted into a paste with a meat grinder. The paste
was homogenized in 4-times its volume of 0.75 M choline Cl
buffered to pH 7.4 with 30 mM N-2-hydroxyethylpipera-
zine-N'-2- ethanesulfonic acid (HEPES)-15 mM Tris. The
homogenization was carried out twice for 30 seconds in 300
ml polypropylene centrifuge jars with a Tekmar T185 shaft.
This and all other buffers include the proteinase inhibi-
tors: 0.2 mM phenylmethylsulfonyl fluoride, 1 mM EGTA,
and 1 mM dithiothreitol. The resultant homogenate is
centrifuged for 20 minutes at 27,000 x g in the GSA rotor
of a Sorvall centrifuge. The supernatant is discarded,
and the pellet is resuspended in 10 mM HEPES-5 mM Tris, pH
7.4 and recentrifuged as before. The pellet from this
centrifugation is resuspended in 10 mM HEPES,-Tris and
homogenized three times for 30 seconds each with the T185
shaft of the Tekmar at a setting of 5. The resulting
homogenate is centrifuged for 20 minutes in the GSA rotor
WO94121278 2 ~ 5 8 ~ ~ ~ PCT~S94/02750 ~
36
at 14,000 x g. The supernatant is then centrifuged in the
GSA rotor at 27,500 x g for 70 minutes.
After the preliminary centrifugations the
membranes are suspended in 50% s~rose, 150 mM KCl, 100 mM
TrisCl, and 5 mM Na pyrophos~hate. These vesicles are
loaded onto the bottom of~ a four step discontinuous
gradient with additional steps at 30%, 21.5%, and 9.5%
sucrose. This gradient is centrifuged at 1.5 hour at
193,000 x g in a Beckman 50.2Ti rotor. The pellicle at
the 9.5%-21.5% sucrose interface is enriched in surface
sarcolemma. This pellicle is collected and diluted into
a buffer containing 150 mM KCl, 0.8 mM MgS04 and 10 mM
TrisCl (pH=7.4 @ 22 C), then centrifuged for 35 minutes at
193,000 x g. The resulting pellet is resuspended in
loading buffer and centrifuged once again. The final
pellet is resuspended in loading buffer to a final protein
concentration of 2 mg protein/ml, frozen in liquid N2 and
stored at -70 C until use.
Membrane vesicles (20-40 ~g protein) are loaded
with 150 mM KCl and diluted 50-fold into 1 ml of binding
buffer containing 150 mM KCl. The vesicles are
preincubated 5 minutes at 37 C and then incubated for an
additional time required for the attainment of
equilibration conditions (exact time determined by
preliminary experiments) in the presence of varying
concentrations of [~2sI]Compound 1, 2 or 3 (1 nM-l ~M). The
binding reaction is terminated by addition of 4 ml of ice
cold binding buffer and then rapid filtration over Whatman
GF/C filters followed by three additional 4 ml washes with
ice cold binding buffer. The radioactivity associated
with the filters is determined using standard gamma
counting techniques. Specific ['2sI]Compound 1, 2 or 3
binding is defined as total binding minus binding measured
in the presence of 1-10 ~M cold Compound 1, 2 o~ 3.
The above assay is validated using the
procedures outlined in paragraphs (a) - (f) of Example 8.
-
WO94121278 ~ PCT~S91/02750
ExamPle 13: Rçcombinant Receptor Bindinq AssaY
The following is one example of a rapid
screening assay for useful compounds of this invention.
In this assay, a cDNA or gene clone encoding the Ito channel
binding site (receptor) from a suitable organism such as
a human is obtained using standard procedures. Such
receptors have been cloned and are known in the art.
Distinct fragments of the clone are expressed in an
appropriate expression vector to produce the smallest
polypeptide(s) obtainable from the receptor which retain
the ability to bind Compound l, 2 or 3. In this way, the
polypeptide(s) which includes the novel Compound l/
Compound 2/Compound 3 receptor for these compounds can be
iden~ified. Such experiments can be facilitated by
utilizing a stably transfected mammalian cell line (e.q.,
HEK 293 cells) expressing the I,~ channel.
Alternatively, the Compound l/Compound 2/
Compound 3 receptor can be chemically reacted with
chemically modified Compound l, 2 or 3 in such a way that
amino acid residues of the Compound l/Compound 2/Compound
3 peptide receptor which contact (or are adjacent to) the
selected compound are modified and thereby identifiable.
The fragment(s) of the Compound l/Compound 2/Compound 3
receptor containing those amino acids which are determined
to interact with Compound l, 2 or 3 and are sufficient for
binding to said molecules, can then be recombinantly
expressed, as described above, using a standard expression
vector(s).
The recombinant polypeptide(s) having the
desired binding properties can be bound to a solid phase
support using standard chemical procedures. This solid
phase, or affinity matrix, may then be contacted with
Compound l, 2 or 3 to demonstrate that those compounds
can bind to the column, and to identify conditions by
which the compounds may be removed from the solid phase.
This procedure may then be repeated using a large library
of compounds to determine those compounds which are able
-
WO94/21Z78 2 ~ S 8 0 5 ~ PCT~S94/02750 ~
38
to bind to the affinity matrix, and then can be released
in a manner similar to Compound l, 2 or 3. However,
alternative binding and release conditions may be utilized
in order to obtain compounds ~ca~able of binding under
conditions distinct from th ~è used for Compound l/
Compound 2/Compound 3 pept~idè binding (e.q., conditions
which better mimic physiological conditions encountered,
especially in pathological states). Those compounds which
do bind can thus be selected from a very large collection
of compounds present in a liquid medium or extract.
Once compounds able to bind to the Compound l/
Compound 2/Compound 3 binding polypeptide(s) described
above are identified, those compounds can then be readily
tested in the various assays described above to determine
whether they, or simple derivatives thereof, are useful
compounds for therapeutic treatment of cardiac and
neurological disorders described above.
In an alternate method, native Compound l, 2 or
3 receptor can be bound to a column or other solid phase
support. Those compounds which are not competed off by
reagents which bind other sites on the receptor can then
be identified. Such compounds define novel binding sites
on the receptor. Compounds which are competed off by
other known compounds, thus bind to known sites, or bind
to novel sites which overlap known binding sites.
Regardless, such compounds may be structurally distinct
from known compounds and thus may define novel chemical
classes of agonists or antagonist which may be useful as
therapeutics.
Formulation and Administration
As demonstrated herein, useful compounds of this
invention may be used to treat neurological diseases or
disorders. While these compounds will typically be used
in therapy for human patients, they may be used to treat
similar or identical diseases in other vertebrates such as
other primates, farm animals such as swine, cattle and
poultry, and sports animals and pets such as horses, dogs
~ WO94/21278 215 8 0 ~1 PCT~S94/02750
and cats.
In therapeutic and/or diagnostic applications,
the compounds of the invention can be formulated for a
variety of modes of administration, including systemic and
topical or localized administration. Techniques and
formulations generally may be found in Remington ~5
Pharmaceutical Sciences, Mack Publishing Co., Easton PA.
For systemic administration, oral administration
is preferred. Alternatively, injection may be used, e.q.,
intramuscular, intravenous, intraperitoneal, and
subcutaneous. For injection, the compounds of the
invention are formulated in liquid solutions, preferably
in physiologically compatible buffers such as Hank's
solution or Ringer's solution. Alternatively, the com-
pounds of the invention are formulated in one or moreexcipients (e.q., propylene glycol) that are generally
accepted as safe as defined by USP standards. In addi-
tion, the compounds may be formulated in solid form and
redissolved or suspended immediately prior to use.
Lyophilized forms are also included.
Systemic administration can also be by
transmucosal or transdermal means, or the compounds can be
administered orally. For transmucosal or transdermal
administration, penetrants appropriate to the barrier to
be permeated are used in the formulation. Such penetrants
are generally known in the art, and include, for example,
for transmucosal administration, bile salts and fusidic
acid derivatives. In addition, detergents may be used to
facilitate permeation. Transmucosal administration may be
through nasal sprays, for example, or using suppositories.
For oral administration, the compounds are formulated into
conventional oral administration forms such as capsules,
tablets and tonics.
For topical administration, the compounds of the
invention are formulated into ointments, salves, gels, or
creams, as is generally known in the art.
The amounts of various compounds of this
WO94/21278 PCT~S94/02750 ~
21~805~
invention which must be administered can be determined by
standard procedure. Generally it is an amount between
about 1 and 50 mg/kg of the animal to be treated.
Other embodiments-~ are within the following
5 claims. ~ ~
~ WO94/21278 215 8 0 51 PCT~S94/02750
41
"Sequence Listing"
(1) GENERAL INFORMATION:
5 (i) APPLICANT: Michael C. Sanguinetti
Alan Mueller
(ii) TITLE OF INVENTION: POTASSIUM CHANNEL BLOCKING
COMPOUNDS AND THEIR USE
(iii) NUMBER OF SEQUENCES: 3
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Lyon & Lyon
(B) STREET: 611 West Sixth Street
(C) CITY: Los Angeles
20 (D) STATE: California
(E) COUNTRY: USA
(F) ZIP: 90017
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: 3.5" Diskette, 1.44 Mb storage
(B) COMPUTER: IBM Compatible
(C) OPERATING SYSTEM: IBM MS-DOS (Version 5.0)
(D) SOFTWARE: WordPerfect (Version 5.1)
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
Prior applications total,
including application
described below:
(A) APPLICATION NUMBER: 08/033,388
(B) FILING DATE: 03/18/93
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: WARBURG, RICHARD J.
(B) REGISTRATION NUMBER: 32,327
(C) REFERENCE/DOCKET NUMBER: 206/093
WO94/21278 21 S 8 0 5 ~ PCT~S94/02750 ~
42
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (213) 48-1600
(B) TELEFAX: (213) 955-0440
(C) TELEX: 67-3510
(2) INFORMATION FOR SEQ ID NO - 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
Asp Asp Cys Gly Lys Leu Phe Ser Gly Cys Asp Thr Asn Ala
1 5 10
Asp Cys Cys Glu Gly Tyr Val Cys Arg Leu Trp Cys Lys Leu
15 20 25
Asp Trp
- 30
(2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
Glu Cys Gly Thr Leu Phe Ser Gly Cys Ser Thr His Ala Asp
1 5 10
Cys Cys Glu Gly Phe Ile Cys Lys Leu Trp Cys Arg Tyr Glu
15 20 25
Arg Thr Trp
(3) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 34
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
~ 215~051
WO94/21278 PCT~S94/02750
43
(ii) SEQUENCE DESCRIPTION: SEQ ID NO:3:
Asp Asp Cys Ala Gly Trp Met Glu Ser Cys Ser Ser Lys Pro
l 5 l0
Cys Cys Ala Gly Arg Lys Cys Phe Ser Glu Trp Tyr Cys Lys
Leu Val Val Asp Gln Asn
