PFIs block p-MLC downstream of Rho signaling in MDA-MB-231 cells.

<p>MDA-MB-231 cells were treated with 100 µM AGOH and 20 µM AFOH for 3 days, plated on collagen I coated dishes, and then treated with 100 nM LPA for 5 min. Cell lysates were immunoblotted with p-MLC. Total MLC-2 and β-actin serve as the loading controls.</p

AGOH inhibits MDA-MB-231 cells invasion.

<p>(A) MDA-MB-231 cells were treated with AGOH at the indicated concentration and then assessed for Matrigel invasion toward the combination of 5 ng/ml EGF and 250 ng/ml insulin. (B) Cells treated with AGOH for 3 days were harvested in RIPA buffer, immunoblotted with polyAG-antibody. Asterisk (*) symbolizes a p value <0.05. These results are representative from at least three separate experiments.</p

AFOH blocks RhoA and RhoC activation in response to LPA or EGF.

<p>MDA-MB-231 cells (A, C, E and F) or MDA-MB-468 cells (B and D) were treated with AFOH at the indicated concentration for 3 days, plated on collagen I coated dishes in the presence of AFOH and then treated with 100 nM LPA (A and E, quantified in C and F) or 5 ng/ml EGF (B, quantified in D) for 5 min prior to harvesting for RhoA (A–D) or RhoC (E, F) activity assays. Rho activity assays are representative from at least three separate experiments.</p

Use of Synthetic Isoprenoids to Target Protein Prenylation and Rho GTPases in Breast Cancer Invasion

<div><p>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 <i><u>P</u></i>renyl <i><u>F</u></i>unction <i><u>I</u></i>nhibitors (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.</p></div

AFOH blocks 3D invasive growth of MDA-MB-231 cells.

<p>Cells were seeded in growth factor reduced Matrigel, treated with DMSO (A, B) or 5 µM AFOH (C, D) for 10 days. Then phase contrast images were taken from randomly chosen fields (A, C). Colonies grown in Matrigel were smeared onto slides, immunostained for F-actin (red) and nuclei (blue), and then imaged by confocal microscopy (B, D). The representative images from three experiments are shown.</p

Prenyl Function Inhibitors.

<p>Structure relationship between natural isoprenoids (A) and lead PFIs (B).</p

AGOH blocks RhoA and RhoC activation in response to LPA.

<p>MDA-MB-231 cells (A and E) or MDA-MB-468 cells (B) were treated with 100 µM AGOH for 3 days, plated on collagen I coated dishes in the presence of PFI and then treated with 100 nM LPA or 5 ng/ml EGF as indicated for 5 min before harvesting for RhoA (A and B, quantified in C and D) or RhoC (E, quantified in F) activity assay. Rho activity assays are representative from at least three separate experiments.</p

Farnesyl Diphosphate Analogues with Aryl Moieties Are Efficient Alternate Substrates for Protein Farnesyltransferase

Farnesylation is an important post-translational modification essential for the proper localization and function of many proteins. Transfer of the farnesyl group from farnesyl diphosphate (FPP) to proteins is catalyzed by protein farnesyltransferase (FTase). We employed a library of FPP analogues with a range of aryl groups substituting for individual isoprene moieties to examine some of the structural and electronic properties of the transfer of an analogue to the peptide catalyzed by FTase. Analysis of steady-state kinetics for modification of peptide substrates revealed that the multiple-turnover activity depends on the analogue structure. Analogues in which the first isoprene is replaced with a benzyl group and an analogue in which each isoprene is replaced with an aryl group are good substrates. In sharp contrast with the steady-state reaction, the single-turnover rate constant for dansyl-GCVLS alkylation was found to be the same for all analogues, despite the increased chemical reactivity of the benzyl analogues and the increased steric bulk of other analogues. However, the single-turnover rate constant for alkylation does depend on the Ca₁a₂X peptide sequence. These results suggest that the isoprenoid transition-state conformation is preferred over the inactive E·FPP·Ca₁a₂X ternary complex conformation. Furthermore, these data suggest that the farnesyl binding site in the exit groove may be significantly more selective for the farnesyl diphosphate substrate than the active site binding pocket and therefore might be a useful site for the design of novel inhibitors

Formation of a Novel Macrocyclic Alkaloid from the Unnatural Farnesyl Diphosphate Analogue Anilinogeranyl Diphosphate by 5‑Epi-Aristolochene Synthase

As part of an effort to identify substrate analogs suitable for helping to resolve structural features important for terpene synthases, the inhibition of 5-epi-aristolochene biosynthesis from farnesyl diphosphate (FPP) by the tobacco 5-epi-aristolochene synthase incubated with anilinogeranyl diphosphate (AGPP) was examined. The apparent noncompetitive nature of the inhibition supported further assessment of how AGPP might be bound to crystallographic forms of the enzyme. Surprisingly, the bound form of the inhibitor appeared to have undergone a cyclization event consistent with the native mechanism associated with FPP catalysis. Biocatalytic formation of a novel 13-membered macrocyclic paracyclophane alkaloid was confirmed by high-resolution GC-MS and NMR analysis. This work provides insights into new biosynthetic means for generating novel, functionally diversified, medium-sized terpene alkaloids