reu final presentation - august 2, 2013
TRANSCRIPT
Use of Synthetic Isoprenoids to Target Cancer Metastasis through
Protein Isoprenylation
Ryan D. Turnewitsch – Marietta College
NSF-REU in the Biochemical Sciences
Laboratory of H. Peter Spielmann
Background
H-Ras, 2º Structure
• Ras Superfamily Proteins – Ras and Rho
• Need to be prenylated for physiological function
• Molecular Switches (GDP-bound “off” to GTP-bound “on”)
• Ras and Rho common oncoproteins
• Isoprenylation
• Addition of isoprenoid to C-terminal Cysteine (Ca1a2X) peptide
• Natural isoprenoids – Farnesyl Diphosphate and Geranylgeranyl Diphosphate
Gysin et al., Genes and Cancer, Vol. 2, 2011Troutman et al., Bioconjugate, Vol. 16, 2005Subramanian et al., ChemBioChem, Vol. 9, 2008
Protein linked to Isoprenoids
Isoprenylation• Isoprenoid already present
within or introduced into cell
• Isoprenol kinases convert isoprenol to Isoprenoid-diphosphate
• Isoprenoid-diphosphates are utilized by protein prenyl transferases (FTase or GGTase-1) to modify
protein substrates
• “Farnesylation” or “Geranylgeranylation”
Spielmann et al., Powerpoint, 2013Subramanian et al., ChemBioChem, Vol. 9, 2008
Farnesyl Transferase Inhibitors (FTIs)
Spielmann et al., Powerpoint, 2013
• Used to block farnesylation and inactivate protein
• Proteins underwent geranylgeranylation instead
• GGTase inhibitors too toxic
Our Strategy – Alternative Substrates
AFOH AGOH
Spielmann et al., Powerpoint, 2013
• Provide the prenyl transferases with an alternative molecule to which they can bind instead of binding to farnesyl DPP or geranylgeranyl DPP
• These molecules inhibit the protein’s function
• Molecules are Anilinogeraniol (AGOH) and Anilinofarnesol (AFOH)
A. Natural Isoprenols
B. Synthetic Isoprenols
Stuctural relationship between natural isoprenoids and synthetic isoprenoids
Note: Aromatic ring acceptable isostere for terminal isoprene
FOH - FarnesylationGGOH - Geranylgeranylation
AGOH - FarnesylationAFOH - Geranylgeranylation
Subramanian et al., ChemBioChem, Vol. 9, 2008Chen et al., Publication pending, 2013
OP
OP
O-
O O
O- O-
HN
OHHN
SHN
Ras
Kinases
FTase
Alternative SubstrateA. Isoprenylation of Farnesol B. Isoprenylation of AGOH
Spielmann et al., Powerpoint, 2013Subramanian et al., ChemBioChem, Vol. 9, 2008Kareem et al., Journal of Organic Chemistry, Vol. 65, 2000
My Work: Synthesis of AGOH and AFOH
• Our two synthetic isoprenoid analogs• Analogs are alternative substrates for prenyl transferases• Alcohol precursors of Isoprenoid-diphosphates
Synthetic Scheme - AGOH
Geranyl Acetate
T-butyl OOHSeO2
Salicylic AcidMnO2
(CH2Cl2)
AnilineAcetic AcidNaBH-(OAc)3
(C2H4Cl2)
K2CO3
H2O
(MeOH) AGOH
Kareem et al., Journal of Organic Chemistry, Vol. 65, 2000
Synthetic Scheme - AFOH
Farnesyl Acetate Farnesyl Benzoate
Farnesol
Benzoyl chlorideDMAP
Et3N
(CH2Cl2)
Acetic AnhydridePyridineHCl
(CH2Cl2)
Subsequent Steps Identical to AGOH Synthesis
• Oxidation of terminal methylene• Reductive Amination• Saponification of Acetate
Kareem et al., Journal of Organic Chemistry, Vol. 65, 2000Mori et al., Organic Syntheses, Vol. 81, 2005Kulkarni et al., Organic Syntheses, Vol. 83, 2006
AFOH
• Maximum Tolerated Dosage ExperimentsAGOH and AFOH injected into mice and serum samples analyzed using HPLC to determine a MTD within mice
• Drug Concentration ExperimentsArea under the curve quantified from serum samples to determine the rate of compound’s dissipation over time
• Metastasis Experiments Using determined dosage schedule, in vivo effects of AGOH and AFOH on aggressive cancer metastasis analyzed within mice
Future of Study
ReferencesChehade, Kareem A. H. , Douglas A. Andres, Hiromi Morimoto, and H. Peter Spielmann. "Design and Synthesis of a Transferable Farnesyl Pyrophosphate Analogue to Ras by Protein Farnesyltransferase." Journal of Organic Chemisty 65 (2000): 3027-3033. Print. Chehade, Kareem A. H. , Katarzyna Kiegiel, Richard J. Isaacs, Jennifer S. Pickett, Katherine E. Bowers, Carol A. Fierke, Douglas A. Andres, and H. Peter Spielmann. "Photoaffinity Analogues of Farneyl Pyrophosphate Transferable by Protein Farneyl Transferase." Journal of the American Chemical Society 124 (2002): 8206-8219. Print. Chen, Min, Teresa Knifely, Thangaiah Subramanian, H. Peter Spielmann, and Kathleen O'Connor. "Use of Unnatrual Isoprenoids to targey protein prenylation and Rho GTPases in breast cancer invasion." Publication Pending (2013) Gysin, Stephan , Megan Salt, Amy Young, and Frank McCormick . "Therapeutic Strategies for Targeting Ras Proteins." Genes and Cancer 2.3 (2011): 359-372. Print.
Kulkarni, Amol A. , and Steven T. Diver. "2-Substituted-1,3-Cyclohexadienes by Intermolecular, Methylene-Free Tandem Enyne Metathesis." Organic Syntheses 83 (2006): 200-208. Print. Mori, Miwako, Keisuke Tonogaki, and Atsushi Kinoshita. "Synthesis of 1,3-Dienes from Alkynes and Ethylene: Acetic Acid 2-Methylene-3-Phenethylbut-3-Enyl Ester." Organic Syntheses 81 (2005): 1-13. Print. Subramanian, Thangaiah, Suxia Liu, Jerry M. Troutman, Douglas A. Andres, and H. Peter Spielmann. "Protein Farnesyltransferase-Catalyzed Isoprenoid Transfer to Peptide Depends on Lipid Size and Shape, not
Hydrophobicity." ChemBioChem 9 (2008): 2872-2882. Print. Troutman, Jerry M., Michael J. Roberts, Douglas A. Andres, and H. Peter Spielmann. "Tools to Analyze Protein Farnesylation in Cells." Bioconjugate chemistry 16 (2005): 1209-1217. Print.
Acknowledgements• Dr. Peter Spielmann• Brittney Metts• Dr. Kathy O’Connor• Teresa Knifely
• Dr. Trevor Creamer• Dr. Rebecca Dutch • Stacy Webb
• Dr. Kevin Pate• Dr. Suzanne George
• University of Kentucky Department of Molecular and Cellular Biochemistry