Venom Research at Natural Toxins Research Center (NTRC)
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1 Venom Research at Natural Toxins Research Center (NTRC) Dr. John C. Pérez Regents Professor and Director of the NTRC Texas A&M University-Kingsville
2 Snake Venom Research is Important for Numerous Reasons This is how venomous snakes capture their prey Envenomation is a terrifying experience that can cause death The same molecules which cause death can be used to save lives For example, the molecules that cause the animal to hemorrhage can be used to study strokes, heart attacks, cancer
3 Venomous Snakes Venomous snakes capture their prey without a struggle Snakes use their venom for immobilizing and digesting their prey
4 Envenomation is Terrifying and Expensive There are 5 million bites with about 125,000 deaths per year worldwide Many lose extremities or function of extremities Medical treatment is a difficult process
5 Treatment is Complicated and Requires Experience Qualitative differences Quantitative differences Volume of venom injected Location of the snakebite Identification of the snake Natural resistance or sensitivity Improper first aid Delay in treatment Effective antivenom
6 Physicians Encounter a Different Clinical Situation with Each Snakebite Patients expect physicians to follow a well defined treatment protocol
7 Venoms Have a Variety of Toxins Elapidae and Hydrophidae venoms Generally low molecular mass powerful neurotoxins Pharmacokinetic activities that allow rapid distribution Generally classified as toxins Viperidae venoms Generally high molecular mass enzymes The enzymes cause edema, hemorrhage, and necrosis Pharmacokinetic activities that allow slower distribution
8 Qualitative and quantitative differences in venom of two C. helleri rattlesnakes Yellow LD 50 : 1.84 mg/kg ED 50 : 84 mg/kg MHD: 0.71µg Neutralization by D. virginiana: No Conc: 540 mg/ml White LD 50 : 2.95 mg/kg ED 50 : 357 mg/kg MHD: 2.1µg Neutralization by D. virginiana: Yes Conc: 135 mg/ml
9 The most effective form of treatment is with antivenom Antivipmyn (Fab 2 H) Crotalus durissus durissus Bothrops asper CroFab (FabO) C. atrox C. adamanteus C. s. scutulatus A. p. piscivorus The goal of immunotherapy is to neutralize and remove toxins before they reach the target organs
10 ANTIBODY STRUCTURE (IgG)
11 PEPSIN CLEAVAGE
12 PAPAIN CLEAVAGE
13 Comparison Between IgG, F(ab) 2, F(ab) Characteristics IgG F(ab) 2 F(ab) Distribution (h) >3 3 1 Elimination(h) > Tissue affinity High Moderate-High Low Complement Rxn Yes No* No Excretion Immune tissues Immune tissues Kidney *Complement activatedthrough the alternate pathway. Chippaux, 1998
14 ED 50 May Not be the best Assay A. c. contortrix LD Two different venoms C. s. scutulatus A LD Low dilution High dilution 1/700 1/10,000 LD50 LD50 ED50 ED50 More antibody required Less antibody required
15 LD 50 on Unprotected and Protected Mice Unprotected LD50 Protected LD50 Antivenom Venom Venom LD 50 Protected LD 50
16 Certain animals have a natural resistance Gray Woodrat (Neotoma micropus)
17 Super Rat The gray woodrat is 140 more resistant to the Western diamondback rattlesnake venom than white mice. The LD 50 for Western diamondback rattlesnake venom in gray wood rat is 1,121 mg/kg body weight. The LD 50 for Western diamondback rattlesnake venom in white laboratory mice is 8 mg/kg body weight A 300 gm woodrat can withstand about 3 ml of C. atrox venom.
18 Super Rat The gray woodrat, Neotoma micropus, has something in its blood that neutralizes the venom
19 Super Rat The molecules in gray woodrat serum must be antihemorrhagic molecules There are hundreds of proteins in rat serum
20 Proteins can be separated according to: Size/Shape Charge - + Hydrophobicity or Function CH 3 -CH 2 -
21 Proteins can be separated according to Size
22 Proteins can be separated according to their charge
23 Proteins can be separated according to their hydrophobicity
24 Antihemorrhagins are not antibodies Antibody Antihemorrhagin Molecular Weight 150,000 60,000 IpH Electrophoresis γ globulin Albumin Peptides 4 1 Precipitation Yes No Neutralization Yes Yes
25 Venom Molecules That Create Medical Emergencies Could Be Important in Biomedical Research
26 Venoms Are Poorly Characterized in the United States About 95% of all studies have been conducted with only six species of snakes Crotalus atrox (Western Diamondback Rattlesnake) C. adamanteus (Eastern Diamondback Rattlesnake) C. viridis (Prairie Rattlesnake) Agkistrodon contortrix contortrix (Southern Copperhead) A. contortrix laticinctus (Broad-banded Copperhead) and A. piscivorus piscivorus (Eastern Cottonmouth)
27 Reasons Why Venoms Are Poorly Characterized in the United States Reliable sources of venom are difficult to find Many of the snakes are difficult to collect or endangered Restricted geographical locations Research support
28 The Importance of venom in biomedical Research Snake venoms have many biomedical applications There are a lot of different types of molecules in single venom There are a lot of different species of venomous snakes
29 Important Molecules in Snake venom Metalloproteinases Disintegrins Prothrombin activators Factor V activators Factor X activators Anticoagulant activities Serine proteinase
30 Integrins Are heterodimers with α & β subunits Have a ligand-binding site, (fibronectin, fibrinogen, laminin, vitronectin,collagen and disintegrins) Each β chain has a region near the N-terminus called the RGD binding region (Arginine, glycine, and aspartic acid) A single β chain can interact with multiple α chains In mammals, there are at least 22 integrins Each integrin receptor binds to one or more matrix glycoproteins
31 Disintegrins & Venoms Disintegrins are potent inhibitors of cell adhesion found in many snake venoms Initially thought to be inhibitors of platelet aggregation It is now known that disintegrins bind to integrins on the surface of many cells including cancer cells Disintegrins are being experimentally used to inhibit cellular function such as cell migration, proliferation, survival, differentiation, aggregation, cell to cell interaction and cell to membrane binding
32 Possible Applications of disintegrins Strokes Heart attacks Cancer Inflammation in immune response Wound healing Angiogensis Development
33 Receptors of Platelet Aggregation
34 Platelet Aggregation
35 Disintegrins can block fibrinogen
36 A thrombotic stroke occurs when a blood clot blocks a vessel Possible Applications?
37 Metalloproteinases Are Essential in Tumor Metastasis Some Disintegrins Have Been Used to Block Metastasis
38 How Can We Screen for Disintegrins in Venom?
39 Sonoclot Analyzer
40 Sonoclot Platelet Function C L O T Tightening Pulling away from surface S I G N A L O Time (minutes)
41 Sonoclot Profiles of Human Blood
42 Impedance Aggregation Whole Human Blood Whole Human Blood & ADP No ATP Impedance ADP Time (Min.)
43 Impedance Aggregation QuickTime and a Animation decompressor are needed to see this picture. Animation
44 Cellular Adhesion Assay T 24 Cell Adhesion Blocking of Cell adhesion with Disintegrin
45 Mouse Mammary Tumor Cells 66.3p Normal BALB/c lung BALB/c lung with tumors 4.0 X10 4 cells by IV tail vein injection Twenty days after injection
46 Agkistrodon contortrix laticinctus Broad-banded Copperhead
47 Agkistrodon contortrix laticinctus (Broad-banded Copperhead) Cation Exchange Chromatography Buffer A: 0.02M Sodium Phosphate, ph 6.2 Buffer B: 0.02M Sodium Phosphate, ph 6.2 with 0.5M NaCl Total protein 30 mg Fraction # Activity: Hemorrhagic Fibrinolytic Gelatinase %Inhibition of Aggregation (11 ohm) %Inhibition of Platelet function (PF 4) na na na na ACT (218) na na na CR (19) na na na
48 Electrophoretic Titration Curve of Fraction 16
49 Fraction 16 from Cation exchange (Agkistrodon contortrix laticinctus) Column: C18 Total Protein: 3.5 mg Solvent A: 0.1%TFA Solvent B: 80%ACN in 0.1%TFA
50 Agkistrodon contortrix laticinctus Fractions 9 &10 Fr. 9 & 10 Impedance Aggregation
51 Agkistrodon contortrix laticinctus Fractions 9 &10 a Fr. 9 Conc: mg/ml ACT: 181 CR: 13 PF: 0.8 b Fr. 10 Conc: 0.04 mg/ml ACT: 173 CR: 18 PF: 0.1
52 Cellular Adhesion Assay T 24 Cell Adhesion Blocking of Cell adhesion with Disintegrin
53 Binding of T24 Human Urinary Bladder Carcinoma Cells to Fibronectin 100µg 10µg 1µg -Cont 5%BSA + Cont Input Wells T24 cells have α v β 3 and α v β 5 integrins on their cells surfaces
54
55 IC 50 of fraction 10 Inhibition of T24 Human Urinary Bladder Carcinoma Cells to Fibronectin by Fraction 10 of A. c. laticinctus Venom % Inhibition Log. (% Inhibition) IC 50 =0.067µg y = Ln(x) R 2 = Total Protein (µg)
56 Summary Disintegrins are important molecules in biomedical research There have been over 90 disintegrins isolated from snake venoms Only nine have been isolated from snakes in the United States A. c. laticinctus venom was fractionated by cation exchange chromatography and fraction 16 (32 min) inhibiting platelet aggregation and function. Fractions 16 was refractionated by reverse phase chromatography (C18), and disintegrin-like fractions, 9 and 10 were collected at a retention time of 20 min Fraction 10 of A. c. laticinctus inhibited adhesion to T24 tumor cell (IC 50 of 0.067µg)
57 Mission of NTRC is: provide reliable sources of venoms and other products, breed venomous snakes that are endangered or difficult to acquire, characterize toxins, develop an internet database, and do research on medically important venom molecules.
58 Venomous Snakes in NTRC Over 400 venomous snakes All snakes have pit tags Venoms are characterized Information on the venoms can be found on NTRC homepage (ntrc.tamuk.edu) Venoms are never pooled Breeding program
59 Web Page
60 Serpentartium Database Individual Snake Records
61 Screen Captures from the More Information Page
62
63 The NTRC Serpentarium Lighting & temperature computer-controlled cages Computers will mimic the natural conditions of the environment in the cages Large breeding cages with computer-controlled environment
64 Advantages of the NTRC Serpentarium Venoms are not pooled Snakes are all pit tagged Same venom can be reordered at a later date Information can be found on the Internet database
65 Advantages of the NTRC Serpentarium 6,300 sq. ft. of dedicated research space Enough room to expand to 660 snakes Proper identification of snakes Venom collection is done in a clean environment
66 Existing Facilities at Texas A&M University-Kingsville A serpentarium with over 400 venomous snakes Computer controlled serpentarium with modern instrumentation Trained lab and technical personnel
67 Future Expansion of the NTRC The center will breed and provide other venoms from snakes in other countries Biologically important venom proteins will be cloned Venoms from other animals such as insects, spiders and scorpions will be studied
68
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