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