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

Medicinal Properties of Venom Components

Literature Seminar-Kelsey Mayer

Animal images from Google images1

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.

Sher, E., et al. Biochimie. 2000, 82, 927-936.Nelson, L. Nature, 2004, 429, 798-799.

Estrada, G., et al. Nat. Prod. Rep. 2007, 24, 145-161.From Google images

2

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 theyare found to have some remarkable medicinal properties.

Kasturiratne A., et al. PLoS Med. 2008, 5, 218.

Severe necrosis from a snake bite

Nelson, L. Nature, 2004, 429, 798-799.3

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.

Lucio, A. D., et al. Protein Peptide Lett. 2008, 15, 700-708.

Ab

sorb

an

ce a

t 214

nm

% concentration of acetonitrile

4

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.

Uemura, D., et al. Pure appl. Chem. 2009, 81, 1093-1111.

5

Disintegrins Disintegrins are peptides

that are isolated from snake venom These peptides vary in size

from 5000-14,000 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)

Swenson, S., et al. Curr. Pharm. Design. 2007, 13, 2860-2871.

Flavoridin: PDB code 1FVL

RGD site

6

Integrin/Disintegrin Interaction RGD-containing disintegrins are effective

antagonists of several of the subtypes of proteins known as Integrins.

Integrins are proteins on the cellsurface that mediate attachmentbetween the cell and the tissues around it, such as the extracellular matrix (ECM)

Integrins are heterodimers thatcontain an α and β subunit that arenon-covalently associated.

Yeh, C. H., et al. Blood. 1998, 92, 3268-3276.

RGD binding site

Francavilla, C. et al. Semin. Cancer Biol. 2009, 19, 298–309.

Integrin Protein

Cell Membrane

7

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.

Zhou, Q., et al. Breast Cancer Res. Treat. 2000, 61, 249-260.

8

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.

Micrograph of rat muscle that shows blood vessels growing toward a sarcoma tumor. www.dfhcc.harvard.edu

Zhou, Q., et al. Breast Cancer Res. Treat. 2000, 61, 249-260.

9

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.

Francavilla, C. et al. Semin. Cancer Biol. 2009, 19, 298–309.

10

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.

Swenson, S., et al. Curr. Pharm. Design. 2007, 13, 2860-2871.11

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.

From Google images

Yeh, C. H., et al. Blood. 1998, 92, 3268-3276.

12

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 andobserved by microscope indicating large amounts of angiogenesis.

In the accutin example the chick embryo was coated with a 10 μMsolution of accutin and observed after 48 hours of incubation.

Accutin at 10 μM

Control

Yeh, C. H., et al. Blood. 1998, 92, 3268-3276.

13

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.

Swenson, S., et al. Mol. Cancer Ther. 2004, 3, 499-511.

From Google images

14

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.

Swenson, S., et al. Mol. Cancer Ther. 2004, 3, 499-511.

15

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.

From Google images Swenson, S., et al. Mol. Cancer Ther. 2004, 3, 499-511.

16

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, 499-511.

17

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, 466–470.18

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


Structural FeaturesRGD site

Flexible Loop

Acostatin: PDB code 3C05


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.

Conotoxins-A Novel Treatment for Chronic Pain

Han, T. S., et al. Curr. Pharm. Design. 2008, 14, 2462-2479.

ω-MVIIA: PDB code 1TT3

21

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 residuesPurple = 7 residues


22

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 nAChR/antagonist Pre-clinicalHan, T. S., et al. Curr. Pharm. Design. 2008, 14, 2462-2479.

23

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.

acetylcholine nicotine


24

Structural Features of α-Conotoxins α-CTx Primary sequence IC50 (nM) α3β2

123 5 8 12 16

PIA RDPCCSNPVCTVHNPQIC 74.20

MII GCCSNPACHLEHSNLC 2.20

GIC GCCSHPACAGNNQHIC 1.10

OmIA GCCSHPACNVNNPHICG 11.00

PnIA GCCSLPPCALNNPKYC 9.56

Turner, M., et al. Bioorgan. Med. Chem. 2009, 17, 5894-5899.

Talley, T. T., et al. J. Biol. Chem. 2006, 281, 24678-24686.

OmIA: PDB code 2GCZ

Luo, S., et al. Biochemistry. 1999, 38, 14542-14548.25

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

OmIA: PDB code 2GCZ

Turner, M., et al. Bioorgan. Med. Chem. 2009, 17, 5894-5899.

Talley, T. T., et al. J. Biol. Chem. 2006, 281, 24678-24686.

26

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.

Tomizawa, M., et al. Biochemistry. 2007, 46, 8798-8806.

Acetylcholine binding proteins, a homopentamer homolog of nAChR, used to model conotoxin binding to nAChR: PDB code 2C9T

Blue = subunits of receptorGreen = bound conotoxin

Celie, P. H. N., et al. Nat. Struct. Mol. Biol. 2005, 12, 582-588.Turner, M., et al. Bioorgan. Med. Chem. 2009, 17, 5894-5899.

27

α-Conotoxin Binding

PDB code 2C9T

Celie, P. H. N., et al. Nat. Struct. Mol. Biol. 2005, 12, 582-588.

28

α-Conotoxin binding

PDB code 2C9T

Celie, P. H. N., et al. Nat. Struct. Mol. Biol. 2005, 12, 582-588.

29

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.

From Google images

Estrada, G., et al. Nat. Prod. Rep. 2007, 24, 145-161.30

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.

Mayer, M. L. et al. Annu. Rev. Physiol. 2004, 66, 161-181.


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

Kainate



Function of Ionotropic Glutamate Receptors In NMDA receptors Mg2+

prevents influx of Ca2+ unless cell voltage increases resulting in release of Mg2+

In non-NMDA receptors binding of an agonist results in the influx of Ca2+

NMDA receptor Non-NMDA receptor

Images from www.standford.edu


33

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.


General Synthesis of Acylpolyamines

Wang, F., et al. Org. Lett. 2000, 2, 1581-1583.

35

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.

Mellor, I. R., et al. Neuropharmacology. 2003, 44, 70-80.

Kromann, H., et al. J. Med. Chem. 2002, 45, 5745-5754.

36

Derivative Analysis of Acylpolyamines

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)

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.


37

Derivatives of PhTX-433

Antagonist effect on AMPA and Kainate receptors, -80 mVKi (μM)

Compounds

AMPA subtype 1AMPA

subtype 2Kainate

PhTX-433 0.022 ± 0.003 > 5 0.015 ± 0.004 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

The PhTX-433 derivatives were tested using two electrode voltage clamped mammalian cells expressing one of three subunits of non-NMDA receptors


38

Derivative Analysis of Acylpolyamines

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.

X = NH or CH2

Y = NH or CH2

W = NH2 or CH3

Z = NH or OArgiotoxin-636 derivatives

Nelson, J. K., et al. Angew. Chem. In. Ed. 2009, 48, 3087-3091.

39

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 ± 12842 ± 117

0.09

7 CH2 NH NH2 NH454 ±

2714 ± 1 32

All data collected in triplicate

Nelson, J. K., et al. Angew. Chem. In. Ed. 2009, 48, 3087-3091.

40

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.

41

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, 10364-10369.

42

AcknowledgementsProfessor Samuel Gellman

Dr. Pil Seok Chae

Dr. Brendan Mowery

Dr. Kim Kaufman

Li Guo

Jay Steinkruger

Aaron Almeida

Lisa Johnson

Brain Parker

Stacy Maynard

David Mortenson

Younghee Shin

Teresa Beary

Joe Grim

Aaron McCoy

Matt Windsor

Jonathan Zhang

Mike Giuliano

Holly Haase

Brooke Richardson

43

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

Documents

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