Transcript Medicinal properties of Venom Components

Medicinal Properties of Venom
Components
Literature Seminar-Kelsey Mayer
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Animal images from Google images
Venom
Venom is typically characterized by its ability to impair the vital
functions of organisms.
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This ability is due to interactions with physiologically important
molecular targets.
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Venomous species have coevolved with their prey such that venom
components are highly efficient and selective in their toxicity.
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Venom can be isolated from a variety of animals such as snakes, spiders,
scorpions, bees and wasps, marine snails and even mammals.
From Google images
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Estrada, G., et al. Nat. Prod. Rep. 2007, 24, 145-161.
Nelson, L. Nature, 2004, 429, 798-799.
Sher, E., et al. Biochimie. 2000, 82, 927-936.
Venom
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Venomous species are generally considered to be
dangerous and undesirable animals. In fact ~ 20,000
people die from snake bites yearly, ~30 die from cone
snail stings and ~20 from spider bites.
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However, when the venom
components of these animals
is isolated and examined they
are found to have some
remarkable medicinal properties.
Severe necrosis from a snake bite
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Nelson, L. Nature, 2004, 429, 798-799.
Kasturiratne A., et al. PLoS Med. 2008, 5, 218.
Venom
Venom samples are extremely complex and contain a wide
variety of components from organic acids to large proteins.
This makes separation and structure/sequence determination
difficult.
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Absorbance at 214 nm
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This can require extensive purification and characterization through
the use of NMR, mass spectrometry and Edman sequencing. In many
cases, with small molecule components the stereochemistry can only
be determined through the synthesis of analogs.
% concentration of acetonitrile
Lucio, A. D., et al. Protein Peptide Lett. 2008, 15, 700-708.
Venom
Because of the difficulties in isolating and characterizing
venom components, new active components are being
discovered regularly.
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Many of these components have remarkable physiological
properties and have been examined for their ability to
contribute to human health and well-being.
There are venom components that may be able to:
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Treat cancer and interact with platelet aggregation.
Alleviate chronic pain
Treat neurodegenerative disorders.
Uemura, D., et al. Pure appl. Chem. 2009, 81, 1093-1111.
RGD site
Disintegrins
Disintegrins are peptides that are
isolated from snake venom
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These peptides vary in size from
5000-14,000 Da.
They are disulfide rich.
Can be classified into several
catagories,
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They are either mono- or dimeric.
They are classified as either RGDcontaining or non-RGD containing.
(Arg-Gly-Asp)
Flavoridin: PDB code 1FVL
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Swenson, S., et al. Curr. Pharm. Design. 2007, 13, 2860-2871.
Integrin/Disintegrin Interaction
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RGD-containing disintegrins are effective antagonists of several
of the subtypes of proteins known as Integrins.
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Integrins are proteins on the cell
surface that mediate attachment
between the cell and the tissues
around it, such as the extracellular
matrix (ECM)
Integrins are heterodimers that
contain an α and β subunit that are
non-covalently associated.
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RGD binding site
Integrin Protein
Cell Membrane
Francavilla, C. et al. Semin. Cancer Biol. 2009, 19, 298–309.
Yeh, C. H., et al. Blood. 1998, 92, 3268-3276.
Disintegrin Binding Through the RGD
Binding Site
Several integrins bind to extracellular matrix proteins
through the RGD sequence that these proteins contain.
RGD-containing disintegrins are able to competitively
inhibit binding to these extracellular matrix proteins via
the RGD binding site on the α subunit of the integrin
protein.
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Zhou, Q., et al. Breast Cancer Res. Treat. 2000, 61, 249-260.
Medicinal properties of Disintegrins
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Disintegrins have been shown to prevent tumor cell growth through
inhibition of angiogenesis, prevent tumor metastasis through inhibition of
cellular adhesion and inhibit platelet aggregation.
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Inhibition of platelet aggregation and cellular adhesion occurs through
competitive inhibition at the RGD binding site of integrins.
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The mechanism of angiogenesis inhibition is not fully understood. Though it is
known that this occurs through interactions with the αVβ3 integrin subtype.
Micrograph of rat muscle that
shows blood vessels growing
toward a sarcoma tumor.
www.dfhcc.harvard.edu
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Zhou, Q., et al. Breast Cancer Res. Treat. 2000, 61, 249-260.
Angiogenesis
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Angiogenesis is the process
through which new blood
cells form from pre-existing
blood cells.
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Angiogenesis is a leading
factor in cancer proliferation
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Method by which tumor cells
are provided with oxygen
and nutrients.
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Francavilla, C. et al. Semin. Cancer Biol. 2009, 19, 298–309.
Disintegrins and Cancer
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Within the last 10 years a large group of disintegrins have
been found to inhibit angiogenesis as well as prevent
adhesion of tumor cells to the extracellular matrix.
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The disintegrin accutin has been found to inhibit angiogenesis.
The disintegrin salmosin exhibited anti-metastatic effects in
vivo.
The disintegrin contortrostatin inhibits tumor growth and
angiogenesis.
Swenson, S., et al. Curr. Pharm. Design. 2007, 13, 2860-2871.
Inhibition of endothelial cell adhesion by
Accutin
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Accutin is a RGD-containing disintegrin isolated from the
snake Agkistrodon acutus.
It has been shown to inhibit endothelial cell adhesion in vitro
and in vivo.
Research indicates that this occurs primarily through an
antagonistic interaction with the integrin subtype αvβ3.
From Google images
Yeh, C. H., et al. Blood. 1998, 92, 3268-3276.
Accutin inhibition of Angiogenesis
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Accutin was shown to effectively
inhibit angiogenesis in vivo
through the use of a chick
embryo model.
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In the control case the chick
embryo was dissected and
observed by microscope
indicating large amounts of
angiogenesis.
In the accutin example the chick
embryo was coated with a 10 μM
solution of accutin and observed
after 48 hours of incubation.
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Control
Accutin at 10 μM
Yeh, C. H., et al. Blood. 1998, 92, 3268-3276.
Liposomal Delivery of Contortrostatin
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Contortrostatin is an RGD-containing disintegrin isolated
from the venom of the southern copperhead snake
(Agkistrodon contortrix contortrix).
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It is homodimeric, each monomeric unit is made up of 65
amino acid residues and each contains one RGD site which are
placed at the tip of a flexible loop.
From Google images
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Swenson, S., et al. Mol. Cancer Ther. 2004, 3, 499-511.
Contortrostatin Inhibition of Breast Cancer
Progression
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Analysis of contortrostatin has indicated that it has
significant affects on inhibition of tumor growth and
metastasis.
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Injection of contortrostatin daily into a tumor mass of human
breast cancer cells in a mouse model indicated that it
significantly inhibited tumor growth and reduced metastasis by
65%.
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Contortrostatin inhibits angiogenesis through interaction with
the integrins α5β3, αvβ3 and αvβ5.
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Swenson, S., et al. Mol. Cancer Ther. 2004, 3, 499-511.
Liposomal Delivery of Contortrostatin
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Researchers studied the efficacy of contortrostatin
encapsulated within a liposome.
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Liposome encapsulation allows contortrostatin to be delivered
intravenously by IV and delivered effectively to the tumor site.
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The encapsulated contortrostatin does not elicit an immune
response and increases the in vivo half life of contortrostatin
from 0.5 hours to 19 hours.
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From Google images
Swenson, S., et al. Mol. Cancer Ther. 2004, 3, 499-511.
Liposomal Delivery of Contortrostatin
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Intravenously administered LCN was compared to CN that
had been directly injected into the tumor.
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Found that the LCN was an effective inhibiter of tumor cell growth
when injected intravenously.
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Swenson, S., et al. Mol. Cancer Ther. 2004, 3, 499-511.
Structural Features
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While the absolute structure of contortrostatin is not
known it is believed to be similar to the heterodimeric
RGD-containing disintegrin acostatin.
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Acostatin is also isolated from Agkistrodon contortrix
contortrix, similar to contortrostatin it has ~ 65 amino acid
residues and one RGD sequence per monomeric unit.
Moiseeva, N. et al. Acta Cryst. 2008, D64, 466–470.
Structural Features
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In acostatin and most disintegrin dimers the two units orient such that
the RGD sequences are opposite each other. This occurs through the
disulfide linkage at the n-terminus.
The loop containing the RGD sequence is flexible.
The secondary structure of disintegrins is observed in large part because
of the highly conserved cysteine residues which form the disulfides bond
that yield the disintegrin’s secondary structure.
Acostatin: PDB code 3C05
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Moiseeva, N. et al. Acta Cryst. 2008, D64, 466–470.
RGD site
Structural Features
Flexible Loop
Acostatin: PDB code 3C05
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Moiseeva, N. et al. Acta Cryst. 2008, D64, 466–470.
Conotoxins-A Novel Treatment for Chronic
Pain
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Conotoxins are small peptides that are isolated form
marine cone snails.
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Each cone snail species produces ~100 different conotoxins
and there are over 500 different species of cone snails.
The spectrum of ion channels and receptors that are targeted
by conotoxins is vast.
ω-MVIIA: PDB code 1TT3
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Han, T. S., et al. Curr. Pharm. Design. 2008, 14, 2462-2479.
Conotoxins-Familial Divisions
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Conotoxins share few distinct features.
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They are rich in disulfide bonds as well as post-translation
modifications. The placement of the disulfide bonds is conserved
across a family of conotoxins but there are few similarities in the
rest of the primary sequence or the post-translational
modifications.
Red = 4 residues
Purple = 7 residues
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Han, T. S., et al. Curr. Pharm. Design. 2008, 14, 2462-2479.
Clinical Studies of Conotoxins
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Out of the few hundred identified conotoxins, 1 is
currently on the market for the treatment of chronic pain,
6 have made it to clinical trials, and 4 are in pre-clinical
trials.
Conopeptide
Indication
Molecular target
Clinical stage
ω-MVIIA
Intractable pain
N-type calcium
channels/antagonist
Phase IV
(market, Elan)
Conantokin-G
Intractable
epilepsy
NMDA
receptor/antagonist
Phase I
Α-Vc1.1
Neuropathic pain
nAChR/antagonist
Phase II
CGX-1204
Muscle relaxer
nAChR/antagonist
Pre-clinical
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Han, T. S., et al. Curr. Pharm. Design. 2008, 14, 2462-2479.
α-Conotoxins Inhibit Nicotinic Acetylcholine
Receptors
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Research indicates that α-Conotoxins are effective
antagonists of nicotinic acetylcholine receptors
(nAChRs).
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There are several native ligands for nAChRs, including
nicotine and acetylcholine. These bind at the interfaces of
two subunits of the receptor.
acetylcholine
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nicotine
Han, T. S., et al. Curr. Pharm. Design. 2008, 14, 2462-2479.
Structural Features of α-Conotoxins
α-CTx
IC50 (nM) α3β2
Primary sequence
123 5
8
12
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PIA
RDPCCSNPVCTVHNPQIC
74.20
MII
GCCSNPACHLEHSNLC
2.20
GIC
GCCSHPACAGNNQHIC
1.10
OmIA
GCCSHPACNVNNPHICG
11.00
PnIA
GCCSLPPCALNNPKYC
9.56
OmIA: PDB code 2GCZ
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Luo, S., et al. Biochemistry. 1999, 38, 14542-14548.
Talley, T. T., et al. J. Biol. Chem. 2006, 281, 24678-24686.
Turner, M., et al. Bioorgan. Med. Chem. 2009, 17, 5894-5899.
Structural Features of α-Conotoxins
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The common hydrophobic and
hydrophilic strips are
responsible for the binding
affinity with the pertinent
subtypes of nAChRs.
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It is believed that hydrophobic
interactions are the predominant
factor for ligand binding to
NAChRs
OmIA: PDB code 2GCZ
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Talley, T. T., et al. J. Biol. Chem. 2006, 281, 24678-24686.
Turner, M., et al. Bioorgan. Med. Chem. 2009, 17, 5894-5899.
α-Conotoxin Binding
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The amphiphilic α-helix, His
and Asn residues at positions
5 and 12 and the disulfides
bonds are all significant
contributors to the binding
selectivity for the αconotoxins.
Blue = subunits of receptor
Green = bound conotoxin
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Acetylcholine binding proteins, a homopentamer
homolog of nAChR, used to model conotoxin binding to
nAChR: PDB code 2C9T
Celie, P. H. N., et al. Nat. Struct. Mol. Biol. 2005, 12, 582-588.
Tomizawa, M., et al. Biochemistry. 2007, 46, 8798-8806.
Turner, M., et al. Bioorgan. Med. Chem. 2009, 17, 5894-5899.
α-Conotoxin Binding
PDB code 2C9T
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Celie, P. H. N., et al. Nat. Struct. Mol. Biol. 2005, 12, 582-588.
α-Conotoxin binding
PDB code 2C9T
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Celie, P. H. N., et al. Nat. Struct. Mol. Biol. 2005, 12, 582-588.
Acylpolyamines Isolated From Spider Venom
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Types of polyamines are commonly found in almost every
prokaryotic and eukaryotic cell type.
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Acylpolyamines have been shown to interact with ionotropic glutamate
receptors, nicotinic acetylcholine receptors and other types of ligand
gated ion channels.
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These acylpolyamines are largely responsible for the spider’s ability to
paralyze prey.
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The first structure of an acylpolyamine
isolated from spider venom was
determined in 1986 from argiope lobata.
From Google images
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Estrada, G., et al. Nat. Prod. Rep. 2007, 24, 145-161.
Ionotropic Glutamate Receptors
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Ionotropic glutamate receptors (iGluRs) are ligand-gated ion
channels that mediate excitatory synaptic transmission for
vertebrates and are crucial for normal brain function.
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Problems with iGluRs leads to disorders such as Ischemia related
to stroke, and neurodegenerative disorders. iGluRs are considered
important drug targets for these disorders.
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One example of an iGluR inhibitor that has made it through clinical
trials is memantine, which is used in the treatment of Alzheimer's.
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Estrada, G., et al. Nat. Prod. Rep. 2007, 24, 145-161.
Mayer, M. L. et al. Annu. Rev. Physiol. 2004, 66, 161-181.
Subtypes of Ionotropic Glutamate Receptors
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There are 3 subtypes of iGluRs.
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N-methyl-D-aspartate, (NMDA)
α-Amino-3-hydroxy-5-methylisoxazole4-propionic acid hydrate (AMPA)
Kainate
NMD
A
AMPA
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Acylpolyamines are antagonists of
iGluRs
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Acylpolyamines have a high affinity and are
highly selective for iGluRs.
However, they are not as selective for the
subtypes of iGluRs.
Estrada, G., et al. Nat. Prod. Rep. 2007, 24, 145-161.
Mayer, M. L. et al. Annu. Rev. Physiol. 2004, 66, 161-181.
Kainate
Function of Ionotropic Glutamate Receptors
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In NMDA receptors Mg2+
prevents influx of Ca2+ unless
cell voltage increases resulting
in release of Mg2+
NMDA receptor
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Non-NMDA receptor
In non-NMDA
receptors binding of
an agonist results in
the influx of Ca2+
Images from
www.standford.edu
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Mayer, M. L. et al. Annu. Rev. Physiol. 2004, 66, 161-181.
General Structure of Acylpolyamines
Isolated from Spider Venom
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All polyamines isolated from spider venom share certain
structural similarities.
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An aromatic moiety at one end with a primary amino or
guanidine group at the other end.
A lipophilic core that attaches directly to the aromatic moiety
through an amide or amino acid linker.
Estrada, G., et al. Nat. Prod. Rep. 2007, 24, 145-161.
General Synthesis of Acylpolyamines
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Wang, F., et al. Org. Lett. 2000, 2, 1581-1583.
Acylpolyamines Isolated From Spider Venom
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Derivatives of PhTX-433 were made that are able to
selectively inhibit one type of iGluRs.
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In the series of analogs the polyamine core length was varied
while the total length of the acylpolyamine constant.
Kromann, H., et al. J. Med. Chem. 2002, 45, 5745-5754.
Mellor, I. R., et al. Neuropharmacology. 2003, 44, 70-80.
Derivative Analysis of Acylpolyamines
Derivatives of PhTX-433 were made that are able to
selectively inhibit one type of iGluRs.
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In the series of analogs the polyamine core length was varied while the
total length of the acylpolyamine constant.
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PhTX-38 (m = 3, n = 8)
PhTX-47 (m = 4, n = 7)
PhTX-56 (m = 5, n = 6)
PhTX-65 (m = 6, n = 5)
PhTX-74 (m = 7, n = 4)
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PhTX-83 (m = 8, n = 3)
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PhTX-92 (m = 9, n = 2)
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Kromann, H., et al. J. Med. Chem. 2002, 45, 5745-5754.
Derivatives of PhTX-433
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The PhTX-433 derivatives were tested using two electrode
voltage clamped mammalian cells expressing one of three subunits
of non-NMDA receptors
Antagonist effect on AMPA and Kainate receptors, -80 mV
Ki (μM)
AMPA
Compounds
AMPA subtype 1
Kainate
subtype 2
PhTX-433
>5
0.015 ± 0.004
0.022 ± 0.003
PhTX-56 (m = 5, n = 6)
0.0033 ± 0.0008
5±3
2±1
PhTX-83 (m = 8, n = 3)
0.07 ± 0.02
> 10
0.25 ± 0.02
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Kromann, H., et al. J. Med. Chem. 2002, 45, 5745-5754.
Derivative Analysis of Acylpolyamines
Argiotoxin-636 derivatives
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X = NH or CH2
Y = NH or CH2
W = NH2 or CH3
Z = NH or O
All possible variations were synthesized and tested for
potency and selectivity.
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Evaluated by measuring the inhibitory activity on mammalian cells
expressing either the AMPA receptor GluR1 or the NMDA
receptor subunits NR1 and NR2A.
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Nelson, J. K., et al. Angew. Chem. In. Ed. 2009, 48, 3087-3091.
Derivatives of Argiotoxin-636
IC50 [nm]
Compound
X
Y
W
Z
AMPA
NMDA
Selectivity (IC50AMPA / IC50NMDA)
1
NH NH NH2 NH
77 ± 14
10 ± 1
8
4
NH CH2 NH2 NH
78 ± 12
842 ± 117
0.09
7
CH2 NH NH2 NH 454 ± 27
14 ± 1
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All data collected in triplicate
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Nelson, J. K., et al. Angew. Chem. In. Ed. 2009, 48, 3087-3091.
Conclusions
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Venom components contain a wide variety of compounds
from small molecules to proteins and many of these can be
utilized for their physiological properties.
In many cases, acquiring the desired venom component can be
difficult as synthesis can be complex and only small quantities
can be isolated from the animal.
Despite these difficulties there are a multitude of benefits that
can result from venom components.
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Components have been identified that interact with a multitude of
receptors and ion channels.
These components have been examined for their ability to treat
cancer, chronic pain and neurodegenerative disorders.
Future Directions
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There are still components of venom being identified.
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Recently several different types of sulfated nucleosides were
identified in spider venom.
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Many venom components have not been structurally and
physiologically characterized.
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Studies indicate that these components
Block kainate receptors and weakly block
L-type calcium channels
There are still a huge number of discoveries to be made within this field.
Taggi, A. E., et al. J. Am. Chem. Soc. 2004, 126, 10364-10369.
Acknowledgements
Professor Samuel Gellman
Matt Windsor
Jay Steinkruger
Dr. Pil Seok Chae
Jonathan Zhang
Aaron Almeida
Dr. Brendan Mowery
Mike Giuliano
Lisa Johnson
Dr. Kim Kaufman
Holly Haase
Li Guo
Brooke Richardson
Brain Parker
Stacy Maynard
David Mortenson
Younghee Shin
Teresa Beary
Joe Grim
Aaron McCoy
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