Synthetic Probes of Protein Prenylation: Covalent modification by isoprenoid lipids (prenylation) is a critical post-translational event for many proteins involved in cellular signaling. The discovery that the members of the Ras family of protooncogenes are modified by the farnesyl isoprenoid (farnesylation), and that prenylation is required for the oncogenic forms of these proteins to express their transforming potential, has led to intense investigation of protein-farnesyltransferase (FTase) inhibitors (FTIs) as promising cancer chemotherapeutic agents. However, recent developments have made it clear that the mechanism of FTI action is unexpectedly complex, although it involves inhibition of FTase. In addition, the contribution of isoprenoid lipids to the overall biology of Ras is incompletely understood. Therefore, a key to applying FTase-based pharmacological intervention is a thorough understanding of the in vivo farnesylation pathways. Knowledge of the substrate specifities for FTase, and the cellular function of the prenyl moiety are critical to improving the design of future FTIs. The central hypothesis of this study is that the prenyl group plays an active role in directing both post- translational processing and cellular membrane localization of prenylated proteins. An important corollary to this hypothesis is that modifications to the prenyl structure may lead to significant, biologically relevant effects on the activity of the unnaturally prenylated protein. We have synthesized FPP analogs that are transferred to oncogenic Ras but fail to support transformation. These molecules are leads for a unique class of potential anti-cancer therapeutics we term RFIs (Ras function inhibitors).
The specific aims of this project are: 1) specifically substituted unnatural analogs of farnesyl pyrophosphate and the homologous isoprenoid geranylgeranyl pyrophosphate will be synthesized in a combinatorial scheme; 2) these compounds will be screened as substrates or inhibitors of FTase and the closely related enzyme geranylgeranyltransferase I; 3) building upon promising preliminary results in this area, an in vivo isoprenoid structure-function relationship will be established by replacing the H-Ras farnesyl group with a select subset of the analogs available from the studies described in specific aim 1and 2 and analyzing their biological functions following microinjection intoXenopus oocytes. The results of these experiments will provide further insight into the mechanisms of Ras processing and transformation, a greater understanding of the specific functions of the H-Ras farnesyl group in vivo, and fruitful directions for improvements in FTIs as well as novel transferable analogs which might act as RFIs.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
3R01GM066152-04S1
Application #
7364926
Study Section
Bio-Organic and Natural Products Chemistry Study Section (BNP)
Program Officer
Fabian, Miles
Project Start
2002-05-01
Project End
2008-04-30
Budget Start
2005-05-01
Budget End
2008-04-30
Support Year
4
Fiscal Year
2007
Total Cost
$97,667
Indirect Cost
Name
University of Kentucky
Department
Biochemistry
Type
Schools of Medicine
DUNS #
939017877
City
Lexington
State
KY
Country
United States
Zip Code
40506
Rush, Jeffrey S; Subramanian, Thangaiah; Subramanian, Karunai Leela et al. (2015) Novel Citronellyl-Based Photoprobes Designed to Identify ER Proteins Interacting with Dolichyl Phosphate in Yeast and Mammalian Cells. Curr Chem Biol 9:123-141
Rising, Kathleen A; Crenshaw, Charisse M; Koo, Hyun Jo et al. (2015) Formation of a Novel Macrocyclic Alkaloid from the Unnatural Farnesyl Diphosphate Analogue Anilinogeranyl Diphosphate by 5-Epi-Aristolochene Synthase. ACS Chem Biol 10:1729-36
Subramanian, Thangaiah; Ren, Hongmei; Subramanian, Karunai Leela et al. (2014) Design and synthesis of non-hydrolyzable homoisoprenoid ?-monofluorophosphonate inhibitors of PPAPDC family integral membrane lipid phosphatases. Bioorg Med Chem Lett 24:4414-4417
Degagné, Emilie; Pandurangan, Ashok; Bandhuvula, Padmavathi et al. (2014) Sphingosine-1-phosphate lyase downregulation promotes colon carcinogenesis through STAT3-activated microRNAs. J Clin Invest 124:5368-84
Srinivasan, Kamalakkannan; Subramanian, Thangaiah; Spielmann, H Peter et al. (2014) Identification of a farnesol analog as a Ras function inhibitor using both an in vivo Ras activation sensor and a phenotypic screening approach. Mol Cell Biochem 387:177-86
Chen, Min; Knifley, Teresa; Subramanian, Thangaiah et al. (2014) Use of synthetic isoprenoids to target protein prenylation and Rho GTPases in breast cancer invasion. PLoS One 9:e89892
Onono, Fredrick; Subramanian, Thangaiah; Sunkara, Manjula et al. (2013) Efficient use of exogenous isoprenols for protein isoprenylation by MDA-MB-231 cells is regulated independently of the mevalonate pathway. J Biol Chem 288:27444-55
Subramanian, Thangaiah; Subramanian, Karunai Leela; Sunkara, Manjula et al. (2013) Syntheses of deuterium labeled prenyldiphosphate and prenylcysteine analogues for in vivo mass spectrometric quantification. J Labelled Comp Radiopharm 56:370-5
Subramanian, Thangaiah; Pais, June E; Liu, Suxia et al. (2012) Farnesyl diphosphate analogues with aryl moieties are efficient alternate substrates for protein farnesyltransferase. Biochemistry 51:8307-19
Borowsky, Alexander D; Bandhuvula, Padmavathi; Kumar, Ashok et al. (2012) Sphingosine-1-phosphate lyase expression in embryonic and adult murine tissues. J Lipid Res 53:1920-31

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