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Evaluation of alkyne-modified isoprenoids as chemical reporters of protein prenylation.

ABSTRACT: Protein prenyltransferases catalyze the attachment of C15 (farnesyl) and C20 (geranylgeranyl) groups to proteins at specific sequences localized at or near the C-termini of specific proteins. Determination of the specific protein prenyltransferase substrates affected by the inhibition of these enzymes is critical for enhancing knowledge of the mechanism of such potential drugs. Here, we investigate the utility of alkyne-containing isoprenoid analogs for chemical proteomics experiments by showing that these compounds readily penetrate mammalian cells in culture and become incorporated into proteins that are normally prenylated. Derivatization via Cu(I) catalyzed click reaction with a fluorescent azide reagent allows the proteins to be visualized and their relative levels to be analyzed. Simultaneous treatment of cells with these probes and inhibitors of prenylation reveals decreases in the levels of some but not all of the labeled proteins. Two-dimensional electrophoretic separation of these labeled proteins followed by mass spectrometric analysis allowed several labeled proteins to be unambiguously identified. Docking experiments and density functional theory calculations suggest that the substrate specificity of protein farnesyl transferase may vary depending on whether azide- or alkyne-based isoprenoid analogs is employed. These results demonstrate the utility of alkyne-containing analogs for chemical proteomic applications.

SUBMITTER: DeGraw AJ

PROVIDER: S-EPMC3058306 | biostudies-literature | 2010 Dec

REPOSITORIES: biostudies-literature

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Evaluation of alkyne-modified isoprenoids as chemical reporters of protein prenylation.

DeGraw Amanda J AJ Palsuledesai Charuta C Ochocki Joshua D JD Dozier Jonathan K JK Lenevich Stepan S Rashidian Mohammad M Distefano Mark D MD

Chemical biology & drug design 20101011 6

Protein prenyltransferases catalyze the attachment of C15 (farnesyl) and C20 (geranylgeranyl) groups to proteins at specific sequences localized at or near the C-termini of specific proteins. Determination of the specific protein prenyltransferase substrates affected by the inhibition of these enzymes is critical for enhancing knowledge of the mechanism of such potential drugs. Here, we investigate the utility of alkyne-containing isoprenoid analogs for chemical proteomics experiments by showing ...[more]

PMID: 21040496

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Project description:Statin treatment of hypercholesterolemia is accompanied also with depletion of the mevalonate intermediates, including farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP) necessary for proper function of small GTPases. These include Ras proteins, prevalently mutated in pancreatic cancer. In our study, we evaluated the effect of three key intermediates of the mevalonate pathway on GFP-K-Ras protein localization and the gene expression profile in pancreatic cancer cells after exposure to individual statins.These effects were tested on MiaPaCa-2 human pancreatic cancer cells carrying a K-Ras activating mutation (G12C) after exposure to individual statins (20 ?M). The effect of statins (atorvastatin, lovastatin, simvastatin, fluvastatin, cerivastatin, rosuvastatin, and pitavastatin) and mevalonate intermediates on GFP-K-Ras protein translocation was analyzed using fluorescence microscopy. The changes in gene expression induced in MiaPaCa-2 cells treated with simvastatin, FPP, GGPP, and their combinations with simvastatin were examined by whole genome DNA microarray analysis.All tested statins efficiently inhibited K-Ras protein trafficking from cytoplasm to the cell membrane of the MiaPaCa-2 cells. The inhibitory effect of statins on GFP-K-Ras protein trafficking was partially prevented by addition of any of the mevalonate pathway's intermediates tested. Expressions of genes involved in metabolic and signaling pathways modulated by simvastatin treatment was normalized by the concurrent addition of FPP or GGPP. K-Ras protein trafficking within the pancreatic cancer cells is effectively inhibited by the majority of statins; the inhibition is eliminated by isoprenoid intermediates of the mevalonate pathway.Our data indicate that the anticancer effects of statins observed in numerous studies to a large extent are mediated through isoprenoid intermediates of the mevalonate pathway, as they influence expression of genes involved in multiple intracellular pathways.

Project description:Dysregulation of Ras and Rho family small GTPases drives the invasion and metastasis of multiple cancers. For their biological functions, these GTPases require proper subcellular localization to cellular membranes, which is regulated by a series of post-translational modifications that result in either farnesylation or geranylgeranylation of the C-terminal CAAX motif. This concept provided the rationale for targeting farnesyltransferase (FTase) and geranylgeranyltransferases (GGTase) for cancer treatment. However, the resulting prenyl transferase inhibitors have not performed well in the clinic due to issues with alternative prenylation and toxicity. As an alternative, we have developed a unique class of potential anti-cancer therapeutics called Prenyl Function Inhibitors (PFIs), which are farnesol or geranyl-geraniol analogs that act as alternate substrates for FTase or GGTase. Here, we test the ability of our lead PFIs, anilinogeraniol (AGOH) and anilinofarnesol (AFOH), to block the invasion of breast cancer cells. We found that AGOH treatment effectively decreased invasion of MDA-MB-231 cells in a two-dimensional (2D) invasion assay at 100 µM while it blocked invasive growth in three-dimensional (3D) culture model at as little as 20 µM. Notably, the effect of AGOH on 3D invasive growth was phenocopied by electroporation of cells with C3 exotransferase. To determine if RhoA and RhoC were direct targets of AGOH, we performed Rho activity assays in MDA-MB-231 and MDA-MB-468 cells and found that AGOH blocked RhoA and RhoC activation in response to LPA and EGF stimulation. Notably, the geranylgeraniol analog AFOH was more potent than AGOH in inhibiting RhoA and RhoC activation and invasive growth. Interestingly, neither AGOH nor AFOH impacted 3D growth of MCF10A cells. Collectively, this study demonstrates that AGOH and AFOH dramatically inhibit breast cancer invasion, at least in part by blocking Rho function, thus, suggesting that targeting prenylation by using PFIs may offer a promising mechanism for treatment of invasive breast cancer.

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Evaluation of alkyne-modified isoprenoids as chemical reporters of protein prenylation.

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Evaluation of alkyne-modified isoprenoids as chemical reporters of protein prenylation.

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