Dendrotoxin
A
review
Natalya Krispinsky, Pablo
Sisneros, Micheal Smith, and
Terrill Yazzie
Why we chose dendrotoxin: The potential in biological
exploration
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The dendrotoxin is an , like many other xenobiotics, has a
history to it use and discovery and continues to increase
knowledge of K+ channels
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There is also the research into enzymatic characterization of
dendrotoxins that explain how the differences of primary
structure effects on function
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using the toxin to study multiple diseases involving mutant or nonfunctional K+ channels
K+ channel sub-type involved in the disease can be isolated by exposure
to specific dendrotoxin
These slight differences amongst the various dendrotoxins indicates that
each one has a different target among the various sub-types of K+
channels
replacement of amino acid(s) in the protein structure will affect charge,
conformation and action (the way the toxin molecule interacts with the
target channel)
Biological aspects of organisms
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Various snake species have evolved slightly different toxins
Why we chose dendrotoxin, continued
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Also these methods are being used to study
diabetes, Parkinson's disease, schizophrenia,
Alzheimer's
Including cancer
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Cancer research and understanding of the
cell cycle
Anti-cancer effects caused by disruption of cancer
cell proliferation
Cell cycle progression is dependent on cyclins and
cyclin dependent kinases(Cdk)
Dendrotoxin has inhibitory effect on Cdk
Intent of study
 Dendrotoxin
has had 20 years of research
devoted to understanding this molecule,
and interest has declined since. Our
current investigation develops a review of
past literature to present relevant
information on treatment, biology, and
areas of past research to understand
what has been done; while indicating the
areas of improvement to rejuvenate
scientific significance.
What is a toxin?
 From
a pharmacological perspective, a
toxin is a poison, frequently used to refer
specifically to a protein produced by
some higher plants, certain animals and
pathogenic bacteria, which is highly toxic
for other living organism.
Dendrotoxin Origin
 Dendrotoxin
is produced by the
Dendroaspis genus, more commonly
know as mamba's.
 They are primarily found in Africa
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More plentiful in the red region
Some tribes in Africa believe that
drinking the venom can cure disease
and make them more healthy.
Different toxins are found
between speices
 Black
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mamba dendrotoxins
I, K
 Green
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mamba dendrotoxins
Alpha, Beta, and Gamma and delta
Basic structure of dendrotoxins
 57-60
amino acids
 short version = 57, long
= 59 or 60
 cross-linked with 3 disulfide bridges
Bound 1 = (30-51)
 Bound 2= (5-55)
 Bound 3= (14-38)
(Hollecker et al, 1993.)
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Types of dendrotoxins.
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Alpha dendrotoxin
Beta dendrotoxin
Gama dendrotoxin
Delta dendrotoxin
Dendrotoxin I
Dendrotoxin K
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Sequencing alignment of alpha, delta, I, and K (below).
Alpha dendrotoxin
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Has 59 amino acids in a single chain crosslinked by three di-sulfite bridges.
Inhibits a portion of the potassium current in
the hippocampal pyramidal cells of the brain.
It has a lysine residue near the n-terminus
which is important for the role of binding to
the target site.
Chemical synthesized
LD50 is 23 mg/kg intravenously in a mouse.
Beta dendrotoxin
 LD50
currently unavailable
Gama dendrotoxin
 Potassium
currents that are block by this
toxin are calcium dependent.
 LD50 is currently unavaible
Delta Dendrotoxin
 It
is 60 amino acids long
 It has a lysine residue near the n-terminus
which is important for the role of binding
to the target site.
 Has no effect on Inward Rectify Potassium
1 channels and fermentations in the
ROMK1 protein.
 LD50 currently unavailable
Dendrotoxin I
 Chemically
synthesized
 LD50 In mice by intraperitoneal injection is
4mg/kg
 It can antagonize the effects of BetaBungarotoxin
Dendrotoxin K
 High
affinity for Kv family voltage-gatted
K+ channels
 LD50 not avialable
Voltage-Gated K+ channel families: Dendrotoxins affect mainly the
Kv family.
Common K+ channel
EAG
KCNQ
SK
slo
Kv
KCNQ1 KCNQ2 KCNQ3 KCNQ4 KCNQ5
eag
erg
elk
Kv1 Kv2 Kv3 Kv4 Kv5 Kv6 Kv7 Kv8 Kv9
How does dendrotoxin affect the cell cycle?
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Progression through the cell cycle is ordered and directional
Cyclins and Cyclin dependent kinases (Cdk) form heterodimer
and function to control the cycle progression
Cdk's are the catalytic subunit and cyclin is non-functional
without it
Cyclin D-Cdk4 is the first heterodimer formed and starts
process of G1 to S progression
Cells exposed to dendrotoxin showed an increase of Cdk
inhibitors
Cell cycle progression is halted so cells cannot replicate
Dendrotoxins effect on the
hypocampus
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Suppress a transient, voltage-sensitive, of K channels which
control the outward current in hippocampal CA3 cells.
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The transient current initiated by depolarization exerts a shortlived hyperpolarizing influence and reduces excitability.
The conductance increase associated with channel opening
also reduces excitability.
Mimic the action of 4Aminopyridine, but is 2000 time more
effective in molar terms.
Enhancement of synaptic transition in the hippocampus.
Convulsive activity has been seen in the hippocampus.
This is due to inhibitions of the I A which increased cell excitability.
(Halliwell et al, 1986.)
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Dendrotoxin Facts
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Humans have long feared the Black Mamba, Dendroaspis polylepis, the
Eastern Green Mamba, Dendroaspis angusticeps, and the Western Green
Mamba, Dendroaspis viridis.
Considered to be one of the world’s most venomous land snakes. Is the fastest
(10-12 mph).
In 1960’s death rate nearly 100% (polylepis). Unknown for other 2 spp.
Estimated to be high (50-70%).
Stories and legends primarily the reason it is feared and hunted
(Wikipedia.org).
Use in Research: Dendrotoxin proteins isolated 20 years ago.
Blocks various potassium channels.
Structural analogues of dendrotoxins have helped define the molecular
recognition properties of different types of K+ channels,
Radiolabelled dendrotoxins have also been useful in helping to discover toxins
from other sources that bind to K+ channels.
Because dendrotoxins are useful markers of subtypes of K+ channels in vivo,
dendrotoxins are widely used as probes for studying the function of K+
channels in physiology and pathophysiology (Harvey, 2001).
Routes of Administration
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Subdermal via snake bite. Once bitten, severe neurotoxicity ensues.
Potassium-blockade effect supressed by raising the [Magnesium] or by
lowering the temperature (Wu, 1989).
Neurological, respiratory, and cardiovascular symptoms rapidly begin to
manifest, usually within ten minutes or less.
Common symptoms are rapid onset of dizziness, drowsiness, coughing or
difficulty breathing, convulsions, and an erratic heartbeat.
Other common symptoms are neuromuscular symptoms, shock, loss of
consciousness, hypotension, pallor, ataxia, excessive salivation (oral secretions
may become profuse and thick), limb paralysis, nausea and vomiting, ptosis,
fever, and very severe abdominal pain. Edema is typically minimal.
In cases where the victim has received larger amounts of venom than
average (over 200 mg +) or the venom is delivered right into a vein, death
can result within as little as 10 minutes from respiratory or cardiac arrest.
Death is due to suffocation resulting from paralysis of the respiratory muscles
(Strydom, 1972).
First Aid
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In the event of a suspected or confirmed mamba bite, the following steps are
recommended.
(1) Carefully separate the snake from the victim and others. Killing the snake and bringing it to
the hospital is not necessary, although positive identification is helpful to the medical staff.
(2) A lymphatic/venous constriction bandage (pressure/immobilization) should be applied
immediately. Splinting and wrapping a crepe bandage about the limb as described in Figure 2 is
recommended.
(3) Transport the victim to a medical facility as quickly as possible. Incisions, suction to the
wound, and ice are not recommended. Appropriate measures include quieting and reassuring
the
victim, immobilizing the afflicted limb, and removing constricting jewelry. Rest the bitten
extremity at a level at or below the victim's heart.
(4) Do not remove the constricting band or crepe bandage until the victim has reached the
hospital and is receiving antivenin treatment.
(5) Medical facilities in the U.S. and in South Africa should have access to SAIMR polyvalent
antiserum. This antivenin may be ordered from: The South African Institute for Medical
Research, P.O. Box 1038, Johannesburg 2000, South Africa.
Additional notes on the
treatment of Mamba bites
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Stay calm!
Receive medical intervention!
Ensure inspection for multiple bites, polylepis is notorious for biting multiple
times.
First aid treatment includes lymphatic retardation with immobilization and
pressure wrap.
Medical management requires the intravenous administration of mambaspecific anti-venom.
Anti-venom is hydrocortisone and polyvalent anti-venom.
If allergic reaction takes place then glucocorticoids, epinephrine, and
antihistamines are the mainstay for treating the reactions.
Oral prednisolone is given when serum sickness occurs.
Moderate symptoms do not require anti-venom; symptoms that do occur are
treated accrodingly (some cases not a full dose of venom is administered).
If treatment occurs later than it should permanent paralysis could ensue
(Hodgson, 1996).
Medical Management
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(1) Establish the identity of the offending snake.
(2) Locate fang marks and areas of swelling and pain. Recall that the mamba
is known to strike repeatedly and that multiple bites are common.
(3) If a crepe bandage and splint have not been applied, apply immediately.
(4) Secure 16-20 vials of SAIMR polyvalent antiserum regardless of the
perceived severity of envenomation, age, or body weight of victim. There is
no outside time limit for antivenin administration .
(5) Admit the patient to an emergency department or other intensive care
setting. If not already instituted, intubation or respiratory assistance may be
necessary if respiratory distress becomes marked. Begin a peripheral IV
infusion of lactated Ringer's solution at a rate sufficient to maintain a brisk
urine output.
(6) Draw blood from an uninvolved extremity and submit it for the tests listed in
. Obtain a urine specimen for urinalysis.
(7) Observe the patient. Envenomation is diagnosed by the presence of pain
or inflammation or by systemic or neurological signs summarized in Tables 1
and 2. Absence of fang marks does not preclude envenomation. Conversely,
presence of fang marks does not confirm envenomation since dry bites may
occur. Grading envenomation in mamba bites is not useful. Any evidence of
mamba envenomation is treated as diagnostic of severe envenomation.
Narcotics are contraindicated since they depress respiration and alter mental
status. Benzodiazepines may be used in low doses to calm the patient
(Hodgson, 1996).
Medical Management Continued
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(8) If there are no symptoms of envenomation during a I-hour observation period,
remove crepe bandage and observe carefully. If any changes occur, assume
envenomation. Otherwise observe the patient carefully for at least 24 h.
(9) Begin antivenin therapy if mamba envenomation is diagnosed. Even if the
patient was bitten several hours before, antivenin can reverse much of the venom's
effects. Reconstitute 6 vials of SAIMR polyvalent antiserum in lactated Ringer's
solution at room temperature. Vigorously shake the vials to insure maximum
dissolution. Transfer the solution to an IV piggyback set up with a volumetric
regulator.
(10) A precautionary test dose of antivenin may be administered to signal the need
for antianaphylaxis therapy. One ml of antiserum solution diluted in 9 ml of Ringer's
solution slowly delivered via IV may be used to elicit an allergic response. Skin tests
are not advised due to unreliability and needless delays in therapy. However,
glucocorticoids and epinephrine should be kept available whether or not a test
dose is negative. Antivenin infusion should be judiciously slowed if an allergic
reaction becomes evident at any stage of therapy.
(11) Infuse the diluted antivenin slowly at first, then increase to 1 vial every 7-10 min
(0.7-1.0 ml/min). The crepe bandage should be removed slowly after the second vial
of antivenin has been delivered.
(12) Should any manifestations of allergy/anaphylaxis present themselves (e.g.
cough, dyspnea, bronchoconstriction, urticaria, itching, increased oral secretions,
hypotension), reduce or discontinue antivenin infusion and treat the patient with
epinephrine, glucocorticoids, or antihistamines. When the patient has stabilized,
resume antivenin administration at a rate tolerated by the patient (Hodgson, 1996).
Medical Management Continued
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(13) Administer an initial course of 6 vials of SAIMR polyvalent antiserum. Then reassess the patient's condition.
Stable pulse and blood pressure; Absence or amelioration of allergic reactions; EKG normal; Decrease in pain at
wound; Absence of systemic evidence of envenomation; Absence of muscle fasciculations; Decrease in ptosis
and other neurologic signs.
(14) The key to subsequent therapy lies in the titration of antivenin against the signs and symptoms of
envenomation. Symptoms that are not useful in determining the need for further antivenin therapy include
circumoral tingling and slight depressions in serum potassium. These symptoms need not be corrected unless the
patient becomes symptomatic.
(15) Additional antivenin should be administered in three vial doses. The low neutralizing capacity of a single vial
precludes the usefulness of more artful titration. Urine output should be maintained by aggressive hydration and
the use of diuretics as necessary.
(16) Respiratory obstruction and failure are the greatest immediate concerns. Make sure that adequate suction is
available and operative at the bedside. If any sign of oropharyngeal paralysis or impaired swallowing exists
nothing should be given orally. The patient should be kept on his side, head down. Intubation may be necessary
to protect the airway.
(17) Neostigmine provides an additional treatment option for neuromuscular junction postsynaptic blockade
when used in conjunction with antivenin or as a second line alternative if antivenin is not available. Atropine is
given to prevent overexcition of the autonomic nervous system.
(18) Tetanus toxoid vaccination should be current. Prophylactic antibiotics are not required.
(19) Patients who receive antivenin should be observed in an intensive care setting for 12-24 h. They may be
discharged the following day if stable.
(20) Patients who receive larger doses of antivenin might develop a cutaneous allergic reaction. This usually
appears as urticaria 7-21 days after treatment. Oral antihistamines or corticosteroids may be prescribed to treat
the reaction (Hodgson, 1996).
Dendrotoxin as a research tool
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Many diseases involve mutant or non-functional K+
channels
There are multiple channel types, and different
varieties of dendrotoxin affect different channels
This allows researchers to isolate which specific
channel is affected by the disease
Dendrotoxin – Alzheimers Research
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Abnormal K+ channel identified in brain
cells of Alzheimer's patients
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Specific channels either not present or
unresponsive to inhibition
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Possibly due to structural change in the
binding site
Current Research- Cancer
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Dendrotoxin-k suppresses tumor
growth
Research done in vivo
Adenocarcinoma A549 cells – human
lung cancer
Voltage-gated K+ (Kv) channels
overexpressed by cancer cells
Kv 1.1 involved in cell cycle
progression- G1 – S
Selective knockdown of Kv1 inhibits
cell proliferation
Current Research Cancer - Results
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Tumor volume significantly reduced when
treated with dendrotoxin
A549 proliferation reduced after treatment
with DTX-k
Expression of cyclin dependent kinase
inhibitors (CDKI) increase- p21, p27,p15
CDK’s (Cyclin D1-3) involved in regulation of
G1 progression
Inhibition of CDK’s stop cell cycle
Research into pancreatic ß-cells
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β-cells secrete insulin
K+ channels repolarize β-cell action potential
Dendrotoxin used to identify specific K+ channel
mutated in mice with megencephaly (enlarged
brain) – a condition in which K+ channels are
known to be mutated
Breast Cancer Research
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Multiple diseases- Kv1 channels
changed in function or localization
Research done in vitro
Treatment with 1 , 10 nM DTX
Cell proliferation reduced by 20%, 30%
conclusion
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There is still a considerable amount of work
that can be done in the area of biology to
increase the knowledge of dendrotoxins.
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More scientific characteristics surrounding the
Beta-dendrotoxin, Gamma-dendrotoxin, and
dendrotoxin K still need to be addressed (ex,
LD50 is unknown).
Investigative efforts in these areas can help
increase knowledge in cancer research by
developing model binding sites that have a
high affinity for cancer cells
Literature Cited
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Harvey, AL. 2001. Twenty Years of Dendrotoxins. Toxicon. 2001 Jan;39(1):15-26.
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