1. Medicinal Properties of Venom Components
Literature Seminar-Kelsey Mayer
Animal images from Google images

2. Venom
Venom is typically characterized by its ability to impair the vital functions of organisms.
This ability is due to interactions with physiologically important molecular targets.
Venomous species have coevolved with their prey such that venom components are highly efficient and selective in their toxicity.
Venom can be isolated from a variety of animals such as snakes, spiders, scorpions, bees and wasps, marine snails and even mammals.
Venom is a natural toxin that is injected or secreted from venom glands. Many venom components are related to natural toxins found in other organisms
Leading to paralysis and possibly death
From Google images
Estrada, G., et al. Nat. Prod. Rep. 2007, 24,
Nelson, L. Nature, 2004, 429,
Sher, E., et al. Biochimie. 2000, 82,

3. Venom
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.
However, when the venom
components of these animals
is isolated and examined they
are found to have some
remarkable medicinal properties.
Those that do not die frequently suffer severe necrosis
Severe necrosis from a snake bite
Nelson, L. Nature, 2004, 429,
Kasturiratne A., et al. PLoS Med. 2008, 5, 218.

4. 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.
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.
Typically venom components are isolated through milking or by isolation from the dead animal. The venom is removed from the animal dissolved in solvent and purified by iterative HPLC through fractionation of the components and subsequent purifications. The individual components are then testing in live animals or cell based assays to determine activity. It takes multiple steps to determine a given components target and activity.
Absorbance at 214 nm
% concentration of acetonitrile
Lucio, A. D., et al. Protein Peptide Lett. 2008, 15,

5. Venom
Because of the difficulties in isolating and characterizing venom components, new active components are being discovered regularly.
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:
Treat cancer and interact with platelet aggregation.
Alleviate chronic pain
Treat neurodegenerative disorders.
Any one of the subtypes of venom components that I talk about today could be discussed in enough detail to fill an hour talk, my intent is to discuss a few exemplary venom components while giving you an idea of the huge diversity and biological importance of these components.
First I am going to talk about a component of snake venom called disintegrins.
Uemura, D., et al. Pure appl. Chem. 2009, 81,

6. RGD site
Disintegrins
Disintegrins are peptides that are isolated from snake venom
These peptides vary in size from ,000 Da.
They are disulfide rich.
Can be classified into several catagories,
They are either mono- or dimeric.
They are classified as either RGD- containing or non-RGD containing. (Arg-Gly-Asp)
PDB 1FVL
Flavoridin: PDB code 1FVL
Swenson, S., et al. Curr. Pharm. Design. 2007, 13,

7. Integrin/Disintegrin Interaction
RGD-containing disintegrins are effective antagonists of several of the subtypes of proteins known as Integrins.
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.
RGD binding site
Integrin Protein
integrin-mediated signaling regulates gene expression, cell growth, differentiation and survival (angiogenisis)
ECM proteins fibronectin and vitronectin) or cell surface immunoglobulin proteins
Cell Membrane
Francavilla, C. et al. Semin. Cancer Biol. 2009, 19, 298–309.
Yeh, C. H., et al. Blood. 1998, 92,

8. 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.
Disintegrins prevent metastasis through this type of inhibition.
Zhou, Q., et al. Breast Cancer Res. Treat. 2000, 61,

9. Medicinal properties of Disintegrins
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.
Inhibition of platelet aggregation and cellular adhesion occurs through competitive inhibition at the RGD binding site of integrins.
The mechanism of angiogenesis inhibition is not fully understood. Though it is known that this occurs through interactions with the αVβ3 integrin subtype.
αIIbβ3 αVβ3 αVβ5
Dana-farber/harvard cancer center
Micrograph of rat muscle that shows blood vessels growing toward a sarcoma tumor.
Zhou, Q., et al. Breast Cancer Res. Treat. 2000, 61,

10. Angiogenesis
Angiogenesis is the process through which new blood cells form from pre-existing blood cells.
Angiogenesis is a leading factor in cancer proliferation
Method by which tumor cells are provided with oxygen and nutrients.
Shorten to bullet points
essential for wound healing, reproduction and development.
It has been found that the growth of new blood vessels is required for tumor growth and metastasis and it is through new blood vessels tumor cells enter the circulatory system.
Francavilla, C. et al. Semin. Cancer Biol. 2009, 19, 298–309.

11. Disintegrins and Cancer
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.
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.
Accutin-anti-angio in vitro and in vivo (chick chorioallantoic membrane)
Swenson, S., et al. Curr. Pharm. Design. 2007, 13,

12. Inhibition of endothelial cell adhesion by Accutin
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.
It is a monomeric peptide, ~5000 kDa
From Google images
Yeh, C. H., et al. Blood. 1998, 92,

13. Accutin inhibition of Angiogenesis
Accutin was shown to effectively
inhibit angiogenesis in vivo
through the use of a chick
embryo model.
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.
Control
Accutin at 10 μM
the effect of accutin on angiogenesis in vivo was evaluated by using the in vivo model chick chorioallantoic
membrane (CAM)
of chick embryo CAM assay (Fig 3). Upon dissection of the
CAM of 12-day-old chick embryo, the spontaneous angiogenesis
in CAM was clearly observed (Fig 3A). After its topical
application for 48 hours, accutin inhibited the spontaneous
angiogenesis in a dose-dependent manner (Fig 3C through F).
Yeh, C. H., et al. Blood. 1998, 92,

14. Liposomal Delivery of Contortrostatin
Contortrostatin is an RGD-containing disintegrin isolated from the venom of the southern copperhead snake (Agkistrodon contortrix contortrix).
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
Swenson, S., et al. Mol. Cancer Ther. 2004, 3,

15. Contortrostatin Inhibition of Breast Cancer Progression
Analysis of contortrostatin has indicated that it has significant affects on inhibition of tumor growth and metastasis.
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%.
Contortrostatin inhibits angiogenesis through interaction with the integrins α5β3, αvβ3 and αvβ5.
orthotopic xenograft nude (MDA-MB-435)
Swenson, S., et al. Mol. Cancer Ther. 2004, 3,

16. Liposomal Delivery of Contortrostatin
Researchers studied the efficacy of contortrostatin encapsulated within a liposome.
Liposome encapsulation allows contortrostatin to be delivered intravenously by IV and delivered effectively to the tumor site.
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.
A clinically relevant method for delivery of contortrostatin was developed
Contortrostatin
Interestingly.
The liposomal contortrostatin effectively inhibited tumor cell growth and was delivered effectively to the tumor site.
No immune response (explain further)
Also, the in vivo half life contortrostatin was found to be increased through encapsulating into the liposome.
Increased from 0.5 hours to 19 hours.
From Google images
Swenson, S., et al. Mol. Cancer Ther. 2004, 3,

17. Liposomal Delivery of Contortrostatin
Intravenously administered LCN was compared to CN that had been directly injected into the tumor.
Found that the LCN was an effective inhibiter of tumor cell growth when injected intravenously.
Swenson, S., et al. Mol. Cancer Ther. 2004, 3,

18. Structural Features
While the absolute structure of contortrostatin is not known it is believed to be similar to the heterodimeric RGD-containing disintegrin acostatin.
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.

19. Structural Features
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
Moiseeva, N. et al. Acta Cryst. 2008, D64, 466–470.

20. Structural Features RGD site Flexible Loop Acostatin: PDB code 3C05
Moiseeva, N. et al. Acta Cryst. 2008, D64, 466–470.

21. Conotoxins-A Novel Treatment for Chronic Pain
Conotoxins are small peptides that are isolated form marine cone snails.
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.
There are conotoxins that that are highly selective ligands for the subtypes of voltage-gated calcium, potassium and sodium channels, blockers of neuronal and muscle subtypes of nicotinic acetylcholine receptors and target G-protein coupled receptors. This list of targets is still expanding as more conotoxin structures are determined.
ω-MVIIA: PDB code 1TT3
Han, T. S., et al. Curr. Pharm. Design. 2008, 14,

22. Conotoxins-Familial Divisions
Conotoxins share few distinct features.
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
Han, T. S., et al. Curr. Pharm. Design. 2008, 14,

23. Clinical Studies of Conotoxins
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
Pre-clinical
Han, T. S., et al. Curr. Pharm. Design. 2008, 14,

24. α-Conotoxins Inhibit Nicotinic Acetylcholine Receptors
Research indicates that α-Conotoxins are effective antagonists of nicotinic acetylcholine receptors (nAChRs).
There are several native ligands for nAChRs, including nicotine and acetylcholine. These bind at the interfaces of two subunits of the receptor.
Native ligands…. Nicotine, acetylcholine, choline.
Binds in cleft between two subunits.
acetylcholine
nicotine
Han, T. S., et al. Curr. Pharm. Design. 2008, 14,

25. Structural Features of α-Conotoxins
α-CTx
Primary sequence
IC50 (nM) α3β2
PIA
RDPCCSNPVCTVHNPQIC
74.20
MII
GCCSNPACHLEHSNLC
2.20
GIC
GCCSHPACAGNNQHIC
1.10
OmIA
GCCSHPACNVNNPHICG
11.00
PnIA
GCCSLPPCALNNPKYC
9.56
OmIA: PDB code 2GCZ
Researchers have found several important common structural features that give alpha conotoxins their affinity.
Most α-conotoxins contain an Asp or His in position 5 and 12.
The Cys are highly conserved gives secondary structure.
Pro6 initiates the helix.
There is a Pro in position 6, this is typically the beginning residue of a helix. IC5041 nm
Luo, S., et al. Biochemistry. 1999, 38,
Talley, T. T., et al. J. Biol. Chem. 2006, 281,
Turner, M., et al. Bioorgan. Med. Chem. 2009, 17,

26. Structural Features of α-Conotoxins
The common hydrophobic and hydrophilic strips are responsible for the binding affinity with the pertinent subtypes of nAChRs.
It is believed that hydrophobic interactions are the predominant factor for ligand binding to NAChRs
Amphiphilic peptide.
OmIA: PDB code 2GCZ
Talley, T. T., et al. J. Biol. Chem. 2006, 281,
Turner, M., et al. Bioorgan. Med. Chem. 2009, 17,

27. α-Conotoxin Binding
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 α- conotoxins.
a-Ctx is buried deep within the ligand-binding site and interacts with
residues on both faces of adjacent subunits. The toxin itself does not change conformation
Trp145, Tyr186, Cys188, Cys189 and Tyr193, Val146, Ser148, Glu151 Glu191
Asp162, Ser164 Thr34 and Arg77. Val106 Met114 and Ile116
Blue = subunits of receptor
Green = bound conotoxin
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,
Tomizawa, M., et al. Biochemistry , 46,
Turner, M., et al. Bioorgan. Med. Chem. 2009, 17,

28. α-Conotoxin Binding PDB code 2C9T
Hydrophobic interactions Protein Ile and Met, peptide Leu and Ala
Ser H-bond?
Celie, P. H. N., et al. Nat. Struct. Mol. Biol. 2005, 12,

29. α-Conotoxin binding PDB code 2C9T
Celie, P. H. N., et al. Nat. Struct. Mol. Biol. 2005, 12,

30. Acylpolyamines Isolated From Spider Venom
Types of polyamines are commonly found in almost every prokaryotic and eukaryotic cell type.
Acylpolyamines have been shown to interact with ionotropic glutamate receptors, nicotinic acetylcholine receptors and other types of ligand gated ion channels.
These acylpolyamines are largely responsible for the spider’s ability to paralyze prey.
The first structure of an acylpolyamine
isolated from spider venom was
determined in 1986 from argiope lobata.
Paralysis is related to there ability to interact with receptors and is what makes the good targets for treat of pain, and neurodegenerative diseases.
From Google images
Estrada, G., et al. Nat. Prod. Rep. 2007, 24,

31. Ionotropic Glutamate Receptors
Ionotropic glutamate receptors (iGluRs) are ligand-gated ion channels that mediate excitatory synaptic transmission for vertebrates and are crucial for normal brain function.
Problems with iGluRs leads to disorders such as Ischemia related to stroke, and neurodegenerative disorders. iGluRs are considered important drug targets for these disorders.
One example of an iGluR inhibitor that has made it through clinical trials is memantine, which is used in the treatment of Alzheimer's.
It has been difficult thus far to develop selective and clinically effective inhibitors for iGluRs.
Such as Alzheimer's, Parkinson’s and Hodgkin's disease
Estrada, G., et al. Nat. Prod. Rep. 2007, 24,
Mayer, M. L. et al. Annu. Rev. Physiol. 2004, 66,

32. Subtypes of Ionotropic Glutamate Receptors
There are 3 subtypes of iGluRs.
N-methyl-D-aspartate, (NMDA)
α-Amino-3-hydroxy-5-methylisoxazole-
4-propionic acid hydrate (AMPA)
Kainate
Acylpolyamines are antagonists of iGluRs
Acylpolyamines have a high affinity and are highly selective for iGluRs.
However, they are not as selective for the subtypes of iGluRs.
NMDA
AMPA
The kainate and AMPA subtypes are commonly referred as non-NMDA receptors.
Kainate
Estrada, G., et al. Nat. Prod. Rep. 2007, 24,
Mayer, M. L. et al. Annu. Rev. Physiol. 2004, 66,

33. Function of Ionotropic Glutamate Receptors
In NMDA receptors Mg2+
prevents influx of Ca2+ unless cell voltage increases resulting in release of Mg2+
NMDA receptor
Non-NMDA receptor
In non-NMDA receptors binding of an agonist results in the influx of Ca2+
Images from
Mayer, M. L. et al. Annu. Rev. Physiol. 2004, 66,

34. General Structure of Acylpolyamines Isolated from Spider Venom
All polyamines isolated from spider venom share certain structural similarities.
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,

35. General Synthesis of Acylpolyamines
Solid phase synthesis allows for late stage alterations to provide other structures (diversification)
Wang, F., et al. Org. Lett. 2000, 2,

36. Acylpolyamines Isolated From Spider Venom
Derivatives of PhTX-433 were made that are able to selectively inhibit one type of iGluRs.
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,
Mellor, I. R., et al. Neuropharmacology. 2003, 44,

37. Derivative Analysis of Acylpolyamines
Derivatives of PhTX-433 were made that are able to selectively inhibit one type of iGluRs.
In the series of analogs the polyamine core length was varied while the total length of the acylpolyamine constant.
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)
PhTX-83 (m = 8, n = 3)
PhTX-92 (m = 9, n = 2)
Loss of amino group from phTX-433 to PhTX-deriv
Kromann, H., et al. J. Med. Chem. 2002, 45,

38. Antagonist effect on AMPA and Kainate receptors, -80 mV
Derivatives of PhTX-433
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)
Compounds
AMPA subtype 1
AMPA subtype 2
Kainate
PhTX-433
0.022 ± 0.003
> 5
0.015 ± 0.004
PhTX-56 (m = 5, n = 6) ±
5 ± 3
2 ± 1
PhTX-83 (m = 8, n = 3)
0.07 ± 0.02
> 10
0.25 ± 0.02
It is possible to develop a polyamine that is more selective for the sub-types of the non-NMDA glutamate receptors
Alteration of the polyamine core lead to increased selectivity and binding affinity.
38, and 92 have poor antagonist effects on GLuR1 and GluR2 however 47 has strong antagonistic effects on GluR5(Q)
92 has the best selectivity between the 3 types of receptors. 83 is mostly selective for GluR1
Kromann, H., et al. J. Med. Chem. 2002, 45,

39. Derivative Analysis of Acylpolyamines
Argiotoxin-636 derivatives
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.
Evaluated by measuring the inhibitory activity on mammalian cells expressing either the AMPA receptor GluR1 or the NMDA receptor subunits NR1 and NR2A.
In 2009 another attempt was made at derivatizing acylpolyamines to improve selectivity between the subgroups of iGluRs.
Nelson, J. K., et al. Angew. Chem. In. Ed. 2009, 48,

40. Derivatives of Argiotoxin-636
IC50 [nm]
Compound X Y W Z
AMPA
NMDA
Selectivity (IC50AMPA / IC50NMDA) 1 NH
NH2
77 ± 14
10 ± 1 8 4
CH2
78 ± 12
842 ± 117
0.09 7
454 ± 27
14 ± 1 32
All data collected in triplicate
Need to mention that changing guanidine group was detrimental to activity.
Interestingly replacement of the NH group at X produces selectivity for The NMDA receptors while replacement of the NH group at Y produces selectivity for the AMPA receptors.
Nelson, J. K., et al. Angew. Chem. In. Ed. 2009, 48,

41. Conclusions
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.
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.
Venom components contain a wide variety of compounds from small molecules to proteins and many of this can be utilized for their physiological properties.

42. Future Directions
There are still components of venom being identified.
Recently several different types of sulfated nucleosides were identified in spider venom.
Studies indicate that these components
Block kainate receptors and weakly block
L-type calcium channels
Many venom components have not been structurally and physiologically characterized.
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,

43. Acknowledgements
Professor Samuel Gellman Dr. Pil Seok Chae Dr. Brendan Mowery Dr. Kim Kaufman Li Guo
Matt Windsor
Jonathan Zhang
Mike Giuliano
Holly Haase
Brooke Richardson
Jay Steinkruger
Aaron Almeida
Lisa Johnson
Brain Parker
Stacy Maynard
David Mortenson
Younghee Shin
Teresa Beary
Joe Grim
Aaron McCoy