Presentation on theme: "Medicinal Properties of Venom Components"— Presentation transcript:
1 Medicinal Properties of Venom Components Literature Seminar-Kelsey MayerAnimal images from Google images
2 VenomVenom 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 organismsLeading to paralysis and possibly deathFrom Google imagesEstrada, G., et al. Nat. Prod. Rep. 2007, 24,Nelson, L. Nature, 2004, 429,Sher, E., et al. Biochimie. 2000, 82,
3 VenomVenomous 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 venomcomponents of these animalsis isolated and examined theyare found to have someremarkable medicinal properties.Those that do not die frequently suffer severe necrosisSevere necrosis from a snake biteNelson, L. Nature, 2004, 429,Kasturiratne A., et al. PLoS Med. 2008, 5, 218.
4 VenomVenom 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 acetonitrileLucio, A. D., et al. Protein Peptide Lett. 2008, 15,
5 VenomBecause 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 painTreat 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 siteDisintegrinsDisintegrins are peptides that are isolated from snake venomThese 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 1FVLFlavoridin: PDB code 1FVLSwenson, 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 cellsurface that mediate attachmentbetween the cell and the tissuesaround it, such as the extracellularmatrix (ECM)Integrins are heterodimers thatcontain an α and β subunit that arenon-covalently associated.RGD binding siteIntegrin Proteinintegrin-mediated signaling regulates gene expression, cell growth, differentiation and survival (angiogenisis)ECM proteins fibronectin and vitronectin) or cell surface immunoglobulin proteinsCell MembraneFrancavilla, 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 αVβ5Dana-farber/harvard cancer centerMicrograph of rat muscle that shows blood vessels growing toward a sarcoma tumor.Zhou, Q., et al. Breast Cancer Res. Treat. 2000, 61,
10 AngiogenesisAngiogenesis is the process through which new blood cells form from pre-existing blood cells.Angiogenesis is a leading factor in cancer proliferationMethod by which tumor cells are provided with oxygen and nutrients.Shorten to bullet pointsessential 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 kDaFrom Google imagesYeh, C. H., et al. Blood. 1998, 92,
13 Accutin inhibition of Angiogenesis Accutin was shown to effectivelyinhibit angiogenesis in vivothrough the use of a chickembryo model.In the control case the chickembryo was dissected andobserved by microscopeindicating large amounts ofangiogenesis.In the accutin example the chickembryo was coated with a 10 μMsolution of accutin and observedafter 48 hours of incubation.ControlAccutin at 10 μMthe effect of accutin on angiogenesis in vivo was evaluated by using the in vivo model chick chorioallantoicmembrane (CAM)of chick embryo CAM assay (Fig 3). Upon dissection of theCAM of 12-day-old chick embryo, the spontaneous angiogenesisin CAM was clearly observed (Fig 3A). After its topicalapplication for 48 hours, accutin inhibited the spontaneousangiogenesis 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 imagesSwenson, 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 developedContortrostatinInterestingly.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 imagesSwenson, 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 FeaturesWhile 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 FeaturesIn 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 3C05Moiseeva, 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 1TT3Han, 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 residuesPurple = 7 residuesHan, 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 stageω-MVIIAIntractable painN-type calcium channels/antagonistPhase IV (market, Elan)Conantokin-GIntractable epilepsyNMDA receptor/antagonistPhase IΑ-Vc1.1Neuropathic painnAChR/antagonistPhase IICGX-1204Muscle relaxerPre-clinicalHan, 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.acetylcholinenicotineHan, T. S., et al. Curr. Pharm. Design. 2008, 14,
25 Structural Features of α-Conotoxins α-CTxPrimary sequenceIC50 (nM) α3β2PIARDPCCSNPVCTVHNPQIC74.20MIIGCCSNPACHLEHSNLC2.20GICGCCSHPACAGNNQHIC1.10OmIAGCCSHPACNVNNPHICG11.00PnIAGCCSLPPCALNNPKYC9.56OmIA: PDB code 2GCZResearchers 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 nmLuo, 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 NAChRsAmphiphilic peptide.OmIA: PDB code 2GCZTalley, T. T., et al. J. Biol. Chem. 2006, 281,Turner, M., et al. Bioorgan. Med. Chem. 2009, 17,
27 α-Conotoxin BindingThe 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 withresidues on both faces of adjacent subunits. The toxin itself does not change conformationTrp145, Tyr186, Cys188, Cys189 and Tyr193, Val146, Ser148, Glu151 Glu191Asp162, Ser164 Thr34 and Arg77. Val106 Met114 and Ile116Blue = subunits of receptorGreen = bound conotoxinAcetylcholine binding proteins, a homopentamer homolog of nAChR, used to model conotoxin binding to nAChR: PDB code 2C9TCelie, 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 AlaSer 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 acylpolyamineisolated from spider venom wasdetermined 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 imagesEstrada, 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 diseaseEstrada, 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)KainateAcylpolyamines are antagonists of iGluRsAcylpolyamines have a high affinity and are highly selective for iGluRs.However, they are not as selective for the subtypes of iGluRs.NMDAAMPAThe kainate and AMPA subtypes are commonly referred as non-NMDA receptors.KainateEstrada, 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 receptorNon-NMDA receptorIn non-NMDA receptors binding of an agonist results in the influx of Ca2+Images fromMayer, 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-derivKromann, H., et al. J. Med. Chem. 2002, 45,
38 Antagonist effect on AMPA and Kainate receptors, -80 mV Derivatives of PhTX-433The PhTX-433 derivatives were tested using two electrode voltage clamped mammalian cells expressing one of three subunits of non-NMDA receptorsAntagonist effect on AMPA and Kainate receptors, -80 mVKi (μM)CompoundsAMPA subtype 1AMPA subtype 2KainatePhTX-4330.022 ± 0.003> 50.015 ± 0.004PhTX-56 (m = 5, n = 6)±5 ± 32 ± 1PhTX-83 (m = 8, n = 3)0.07 ± 0.02> 100.25 ± 0.02It is possible to develop a polyamine that is more selective for the sub-types of the non-NMDA glutamate receptorsAlteration 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 GluR1Kromann, H., et al. J. Med. Chem. 2002, 45,
39 Derivative Analysis of Acylpolyamines Argiotoxin-636 derivativesX = NH or CH2Y = NH or CH2W = NH2 or CH3Z = NH or OAll 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]CompoundXYWZAMPANMDASelectivity (IC50AMPA / IC50NMDA)1NHNH277 ± 1410 ± 184CH278 ± 12842 ± 1170.097454 ± 2714 ± 132All data collected in triplicateNeed 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 ConclusionsVenom 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 DirectionsThere are still components of venom being identified.Recently several different types of sulfated nucleosides were identified in spider venom.Studies indicate that these componentsBlock kainate receptors and weakly blockL-type calcium channelsMany 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 AcknowledgementsProfessor Samuel Gellman Dr. Pil Seok Chae Dr. Brendan Mowery Dr. Kim Kaufman Li GuoMatt WindsorJonathan ZhangMike GiulianoHolly HaaseBrooke RichardsonJay SteinkrugerAaron AlmeidaLisa JohnsonBrain ParkerStacy MaynardDavid MortensonYounghee ShinTeresa BearyJoe GrimAaron McCoy