Venom Research at Natural Toxins Research Center (NTRC) Dr. John C. Pérez Regents Professor and Director of the NTRC Texas A&M University-Kingsville
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
Venomous Snakes Venomous snakes capture their prey without a struggle Snakes use their venom for immobilizing and digesting their prey
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
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
Physicians Encounter a Different Clinical Situation with Each Snakebite Patients expect physicians to follow a well defined treatment protocol
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
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
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
ANTIBODY STRUCTURE (IgG)
PEPSIN CLEAVAGE
PAPAIN CLEAVAGE
Comparison Between IgG, F(ab) 2, F(ab) Characteristics IgG F(ab) 2 F(ab) Distribution (h) >3 3 1 Elimination(h) >100 60 10 Tissue affinity High Moderate-High Low Complement Rxn Yes No* No Excretion Immune tissues Immune tissues Kidney *Complement activatedthrough the alternate pathway. Chippaux, 1998
ED 50 May Not be the best Assay A. c. contortrix LD 50 5.2 Two different venoms C. s. scutulatus A LD 50 0.47 Low dilution High dilution 1/700 1/10,000 LD50 LD50 ED50 ED50 More antibody required Less antibody required
LD 50 on Unprotected and Protected Mice Unprotected LD50 Protected LD50 Antivenom Venom Venom LD 50 Protected LD 50
Certain animals have a natural resistance Gray Woodrat (Neotoma micropus)
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.
Super Rat The gray woodrat, Neotoma micropus, has something in its blood that neutralizes the venom
Super Rat The molecules in gray woodrat serum must be antihemorrhagic molecules There are hundreds of proteins in rat serum
Proteins can be separated according to: Size/Shape Charge - + Hydrophobicity or Function CH 3 -CH 2 -
Proteins can be separated according to Size
Proteins can be separated according to their charge
Proteins can be separated according to their hydrophobicity
Antihemorrhagins are not antibodies Antibody Antihemorrhagin Molecular Weight 150,000 60,000 IpH 6.6-8.6 4.2 Electrophoresis γ globulin Albumin Peptides 4 1 Precipitation Yes No Neutralization Yes Yes
Venom Molecules That Create Medical Emergencies Could Be Important in Biomedical Research
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)
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
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
Important Molecules in Snake venom Metalloproteinases Disintegrins Prothrombin activators Factor V activators Factor X activators Anticoagulant activities Serine proteinase
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
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
Possible Applications of disintegrins Strokes Heart attacks Cancer Inflammation in immune response Wound healing Angiogensis Development
Receptors of Platelet Aggregation
Platelet Aggregation
Disintegrins can block fibrinogen
A thrombotic stroke occurs when a blood clot blocks a vessel Possible Applications?
Metalloproteinases Are Essential in Tumor Metastasis Some Disintegrins Have Been Used to Block Metastasis
How Can We Screen for Disintegrins in Venom?
Sonoclot Analyzer
Sonoclot Platelet Function C L O T 100 80 60 Tightening Pulling away from surface S I G N A L 40 20 0 O 5 10 15 Time (minutes)
Sonoclot Profiles of Human Blood
Impedance Aggregation Whole Human Blood Whole Human Blood & ADP No ATP Impedance ADP 2 4 6 8 10 12 14 Time (Min.)
Impedance Aggregation QuickTime and a Animation decompressor are needed to see this picture. Animation
Cellular Adhesion Assay T 24 Cell Adhesion Blocking of Cell adhesion with Disintegrin
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
Agkistrodon contortrix laticinctus 011-065-018 Broad-banded Copperhead
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: 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 Hemorrhagic - + + + + + - + + + + - - - - + + + + + + Fibrinolytic - + + - - - - - - - - - - - - - + + + - - Gelatinase - - + - - - - - - - + - - - - - - - + - - %Inhibition of Aggregation (11 ohm) %Inhibition of Platelet function (PF 4) 0 45 9 0 0 0 0 27 18 0 36 0 82 36 0 91 0 0 9 91 82 0 0 0 0 0 50 50 75 na 25 0 0 na 0 50 75 na na 75 75 100 ACT (218) 214 171 195 233 224 170 177 189 na 176 213 248 na 209 183 225 na 452 325 237 160 CR (19) 14 16 21 15 17 24 21 15 na 18 21 14 na 13 15 10 na 3 5 14 16
Electrophoretic Titration Curve of Fraction 16
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
Agkistrodon contortrix laticinctus Fractions 9 &10 Fr. 9 & 10 Impedance Aggregation
Agkistrodon contortrix laticinctus Fractions 9 &10 a Fr. 9 Conc: 0.037 mg/ml ACT: 181 CR: 13 PF: 0.8 b Fr. 10 Conc: 0.04 mg/ml ACT: 173 CR: 18 PF: 0.1
Cellular Adhesion Assay T 24 Cell Adhesion Blocking of Cell adhesion with Disintegrin
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
1 10 100
IC 50 of fraction 10 Inhibition of T24 Human Urinary Bladder Carcinoma Cells to Fibronectin by Fraction 10 of A. c. laticinctus Venom 120 100 99 100 89 80 60 72 % Inhibition Log. (% Inhibition) 40 38 IC 50 =0.067µg y = 14.285Ln(x) + 87.679 R 2 = 0.8633 20 26 28 25 21 31 0 0 0 0.15 0.3 0.45 0.6 0.75 0.9 1.05 1.2 1.35 1.5 1.65 1.8 1.95 2.1-20 Total Protein (µg)
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)
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.
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
Web Page http://ntri.tamuk.edu
Serpentartium Database Individual Snake Records
Screen Captures from the More Information Page
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
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
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
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
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