Presentation is loading. Please wait.

Presentation is loading. Please wait.

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

Similar presentations


Presentation on theme: "Medicinal Properties of Venom Components Literature Seminar-Kelsey Mayer Animal images from Google images 1."— Presentation transcript:

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

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. Sher, E., et al. Biochimie. 2000, 82, Nelson, L. Nature, 2004, 429, Estrada, G., et al. Nat. Prod. Rep. 2007, 24, From Google images 2

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. Kasturiratne A., et al. PLoS Med. 2008, 5, 218. Severe necrosis from a snake bite Nelson, L. Nature, 2004, 429,

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. Lucio, A. D., et al. Protein Peptide Lett. 2008, 15, Absorbance at 214 nm % concentration of acetonitrile 4

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. Uemura, D., et al. Pure appl. Chem. 2009, 81,

6 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) Swenson, S., et al. Curr. Pharm. Design. 2007, 13, Flavoridin: PDB code 1FVL RGD site 6

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. Yeh, C. H., et al. Blood. 1998, 92, RGD binding site Francavilla, C. et al. Semin. Cancer Biol. 2009, 19, 298–309. Integrin Protein Cell Membrane 7

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. 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 α V β 3 integrin subtype. 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. Francavilla, C. et al. Semin. Cancer Biol. 2009, 19, 298–

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. 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 α v β 3. 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. Accutin at 10 μM Control 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. Swenson, S., et al. Mol. Cancer Ther. 2004, 3, From Google images 14

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 α 5 β 3, α v β 3 and α v β 5. 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. 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–

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–

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

21  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, ω-MVIIA: PDB code 1TT3 21

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. ConopeptideIndicationMolecular targetClinical stage ω-MVIIA Intractable painN-type calcium channels/antagonist Phase IV (market, Elan) Conantokin-GIntractable epilepsy NMDA receptor/antagonist Phase I Α-Vc1.1 Neuropathic painnAChR/antagonistPhase II CGX-1204Muscle relaxernAChR/antagonistPre-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. acetylcholinenicotine Han, T. S., et al. Curr. Pharm. Design. 2008, 14,

25 Structural Features of α- Conotoxins α-CTx Primary sequence IC 50 (nM) α 3 β PIA RDPCCSNPVCTVHNPQIC MII GCCSNPACHLEHSNLC 2.20 GIC GCCSHPACAGNNQHIC 1.10 OmIA GCCSHPACNVNNPHICG PnIA GCCSLPPCALNNPKYC 9.56 Turner, M., et al. Bioorgan. Med. Chem. 2009, 17, Talley, T. T., et al. J. Biol. Chem. 2006, 281, OmIA: PDB code 2GCZ Luo, S., et al. Biochemistry. 1999, 38,

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 OmIA: PDB code 2GCZ Turner, M., et al. Bioorgan. Med. Chem. 2009, 17, Talley, T. T., et al. J. Biol. Chem. 2006, 281,

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. Tomizawa, M., et al. Biochemistry. 2007, 46, Acetylcholine binding proteins, a homopentamer homolog of nAChR, used to model conotoxin binding to nAChR: PDB code 2C9T Blue = subunits of receptor Green = bound conotoxin Celie, P. H. N., et al. Nat. Struct. Mol. Biol. 2005, 12, Turner, M., et al. Bioorgan. Med. Chem. 2009, 17,

28 α- Conotoxin Binding PDB code 2C9T 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. 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. Mayer, M. L. et al. Annu. Rev. Physiol. 2004, 66, Estrada, G., et al. Nat. Prod. Rep. 2007, 24,

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. NMD A AMPA Kainate Mayer, M. L. et al. Annu. Rev. Physiol. 2004, 66, Estrada, G., et al. Nat. Prod. Rep. 2007, 24,

33 Function of Ionotropic Glutamate Receptors  In NMDA receptors Mg 2+ prevents influx of Ca 2+ unless cell voltage increases resulting in release of Mg 2+  In non-NMDA receptors binding of an agonist results in the influx of Ca 2+ NMDA receptor Non-NMDA receptor 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 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. Mellor, I. R., et al. Neuropharmacology. 2003, 44, Kromann, H., et al. J. Med. Chem. 2002, 45,

37 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. Kromann, H., et al. J. Med. Chem. 2002, 45,

38 Derivatives of PhTX-433 Antagonist effect on AMPA and Kainate receptors, -80 mV K i ( μ M) CompoundsAMPA subtype 1 AMPA subtype 2 Kainate PhTX ± > ± PhTX-56 (m = 5, n = 6) ± ± 32 ± 1 PhTX-83 (m = 8, n = 3) 0.07 ± 0.02 > ± 0.02  The PhTX-433 derivatives were tested using two electrode voltage clamped mammalian cells expressing one of three subunits of non-NMDA receptors Kromann, H., et al. J. Med. Chem. 2002, 45,

39 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 CH 2 Y = NH or CH 2 W = NH 2 or CH 3 Z = NH or O Argiotoxin-636 derivatives Nelson, J. K., et al. Angew. Chem. In. Ed. 2009, 48,

40 Derivatives of Argiotoxin-636 IC 50 [nm] CompoundXYWZAMPANMDASelectivity (IC 50 AMPA / IC 50 NMDA) 1NH NH 2 NH77 ± 1410 ± 18 4NHCH 2 NH 2 NH78 ± ± CH 2 NHNH 2 NH454 ± 2714 ± 132 All data collected in triplicate 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. 41

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 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


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

Similar presentations


Ads by Google