From Fragments to Actin: The Bistramide A Story March 12 th, 2010
Act I: The Protagonist Synthesis of Bistramide A Gillis, Wang, Davis, Fujii, Bromann
Bistramide A Biological Activity Cell Cycle Regulation Protein Kinase C activator Sodium Channel Blocker H N H H H N H Synthetic Interest No previous syntheses C-37 stereochemistry unknown Spiroketal formation 31 22 37 H NH 2 R 13 H H 18 NH 2 Fragment B H Fragment A Fragment C
Syntheses of Bistramide A Crimmins, JACS 2006 Panek, rg. Lett. 2007 Panek, JC 2009 (synthesis and biology of all 36 stereoisomers) Yadav, rg. Lett. 2007
Kozmin Synthesis N H H H N H H H H 31 22 NR 2 H P P P NR 2 31 22 37 H NH 2 R 13 H H 18 NH 2 Fragment B H P P R R P NR 2 Fragment A Fragment C Statsuk, A. V.; Liu, D.; Kozmin, S. A. Synthesis of Bistramide A. J. Am. Chem. Soc. 2004, 126, 9546-9547
Fragment A
Fragment B 1. B(Ipc) 2 BocHN H 2. 2 C(), PPTS 2 3. RuCl 3, NaI 4 4. 3M HCl, EtAc 5. FmocSu, dioxane-h 2 H H 25%, 5 steps Fragment B NHFmoc
Fragment C
NH 2 H Fragment A N H H H H NHFmoc Fragment B H PyBP, DMF H N Bistramide A Final Coupling 85% Confirmed stereochemistry N H H H 1. Et 2 HN, DMF 2. DMF, 20 o C, 65% N Fragment C NHFmoc From the outset, our objective was to design a flexible and convergent strategy to bistramide A which would enable efficient assembly of both diastereomers at C 37 for direct comparison of the two synthetic samples with the natural product.
Act II: The Conflict Actin is the primary cellular receptor of Bistramide A Dailey, Gray, Ballmer, Davis, Cherney, Lee
Does Bistramide A Act on Protein Kinase C?
Effects on Actin Cytoskeleton
Identifying Bistramide A Binding Protein
Isothermal Titration Calorimetry Single experiment gives complete thermodynamic parameters of reaction Injection syringe Contains excess of ligand K calculated by fitting the data to binding curves ΔG calculated from K, ΔS calculated from ΔG and ΔH h H injection total macromolecule macromolecule bound total Raw Data n h k 1 H total mmacromolec ule G RT ln K G H T S Contains macromolecule Integrated Heats Images adapted from Microcal Inc.: http://www.microcal.com/technology/itc.asp Seelig, J. Biochim. Biophys. Acta 1997, 1331, 103.
In vitro Fluorescence Studies pyrenyl G-actin polymerization (9 mm) fast G-actin slow bisa F-actin Bistramide A = bisa = actin = pyrenyl-actin pyrenyl Mg 2+ F-actin depolymerization slow F-actin fast bisa G-actin Statsuk, A.V.; et al. Nat. Chem. Biol. 2005, 1, 383.
Affinity Chromatography Stationary phase consists of a biospecific ligand The sample (commonly a cell lysate) is applied and the target of interest is bound, while other biomolecules pass through Any remaining impurities are eliminated during the wash. The target of interest is eluted, usually by changing ph or using a sodium decyl sulfate (SDS) solution. Identifying the ligand-binding protein: -Protein is digested into peptide fragments -Mass spec can be used to identify peptides, comparision to databases -Western blots are also used -digested protein subjected to gel electrophoresis -antibody binds to protein of interest and this interaction is detected by chemiluminescence, fluorescence, or by using dyes Silver-stained western blot. Lane 1: cell lysate, Lanes 2-6: washes with lysis buffer, Lane 7: wash with elution buffer
Act III: The Foreshadowing Structure of Bistramide A-Actin Complex at 1.35A Resolution Anderson, Knapp, Uno, Li, Dick
X-Ray Crystallography The 1914 Nobel Prize in Physics was awarded to Max von Laue (left) for his discovery that crystals diffract X-rays. ne year later, the Prize was awarded to Sir William Henry Bragg (center) and William Lawrence Bragg for analyzing crystal structures by X-ray d i f f r a c t i o n. X-ray diffraction pattern of calf DNA (Franklin, R.E.; Gosling, R.G. Nature 1953, 171, 740-741
Bistramide A crystallized with actin
Amino Acid Contacts
Act IV: Finale The dual mode of action of Bistramide A entails severing of filamentous actin and covalent protein modification Palacios, Wilcox, Woerly, Klubnick, Maryum, Struble
Binding to and Severing of Actin Amide, Spiroketal, and Enone are Required Bistramide A K d =7.0 nm GI 50 = 20.4 nm MALDI: covalent bond to actin K d = 43 nm GI 50 = 15.1 um MALDI: no covalent bond to actin K d =70 nm GI 50 = 87.1 nm MALDI: covalent bond to actin No binding or activity K d = 319 nm GI 50 = 11.2 um MALDI: no covalent bond to actin Rizvi, S.; Rock, R.; Kozmin, S. A. Proc. Natl. Acad. Sci. USA. 2008, 105, 4088-4092.
Total Internal Reflectance Fluorescence Microscopy Based Upon Snell s Law of Reflectance i r n 1 n 2 Θ i = angle of incidence Θ r = angle of reflectance n 1 = higher refractive index medium n 2 = lower refractive index medium Snell s Law is given by: n 1 sinθ i = n 2 sinθ r n 1 n 2 c The angle at which TIR is achieved is termed the critical angle and is given by Θ c = arcsin(n 2 /n 1 ) The limited depth of the wave excites fewer fluorophores and leads to vastly superior signal to noise www.olympusamerica.com/seg_section/.../amlabtirfm.pdf
Depolymerization of Actin in Cells Enone Increases Potency Bistramide A Actin depolymerization Limited actin depolymerization Actin depolymerization Limited actin depolymerization Actin depolymerization No activity Limited actin depolymerization Rizvi, S.; Rock, R.; Kozmin, S. A. Proc. Natl. Acad. Sci. USA. 2008, 105, 4088-4092.
The Afterword