Craig H. Kuder

Structural analogues of schweinfurthin F: Probing the steric, electronic, and hydrophobic properties of the D-ring substructure

The natural tetracyclic schweinfurthins are potent and selective inhibitors of cell growth in the National Cancer Institutes 60-cell line screen. An interest in determination of their cellular or molecular target has inspired our efforts to prepare both the natural products and analogues. In this paper, chemical synthesis of analogues modified in different olefinic positions, and preliminary results from studies of their biological activity, are reported.

Synthesis and biological activity of a fluorescent schweinfurthin analogue

Most of the natural schweinfurthins are potent and selective inhibitors of cell growth as measured by the National Cancer Institutes 60-cell line screen. Due to the limited supply of these natural products, we have initiated a program aimed at their synthesis. To date, this effort has led to the preparation of three natural schweinfurthins and more than 40 analogues, and assays on these compounds have afforded some understanding of structure-activity relationships in this family. Further development of schweinfurthins as chemotherapeutic agents would benefit from characterization of their mechanism(s) of action. This perspective led to development of a fluorescent schweinfurthin analogue that retains the differential activity of the natural products, and yet has properties that facilitate its visualization within cells.

Pleiotropic effects of a schweinfurthin on isoprenoid homeostasis.

The schweinfurthins, a family of natural products derived from the isoprenoid biosynthetic pathway (IBP), have marked growth inhibitory activity. However, the biochemical basis for the schweinfurthins cellular effects has remained ill-defined. Here, the effects of the synthetic schweinfurthin, 3-deoxyschweinfurthin (3dSB) on multiple aspects of isoprenoid homeostasis are explored. Cytotoxicity assays demonstrate a synergistic interaction between 3dSB and the HMG-CoA reductase inhibitor lovastatin but not with other IBP inhibitors in a variety of human cancer cell lines. The cytotoxic effects of 3dSB were enhanced in cells incubated in lipid-depleted serum. 3dSB was found to enhance the lovastatin-induced decrease in protein prenylation. In addition, 3dSB decreases intracellular farnesyl pyrophosphate and geranylgeranyl pyrophosphate levels in both established cell lines and primary cells. To determine whether 3dSB alters the regulation of expression of genes involved in isoprenoid homeostasis, real-time PCR studies were performed in human cell lines cultured in either lipid-replete or -deplete conditions. These studies demonstrate that 3dSB abrogates lovastatin-induced upregulation of sterol regulatory element-containing genes and lovastatin-induced downregulation of ABCA1. In aggregate, these studies are the first to demonstrate that a schweinfurthin exerts pleiotropic effects on isoprenoid homeostasis.

Functional Evaluation of a Fluorescent Schweinfurthin: Mechanism of Cytotoxicity and Intracellular Quantification

Schweinfurthins are potent inhibitors of cancer cell growth, especially against human central nervous system tumor lines such as SF-295 cells. However, the mechanisms through which these compounds impede cell growth are not fully understood. In an effort to understand the basis for the effects of schweinfurthins, we present a fluorescent schweinfurthin, 3-deoxyschweinfurthin B-like p-nitro-bis-stilbene (3dSB-PNBS), which displays biological activity similar to that of 3-deoxyschweinfurthin B (3dSB). These two schweinfurthins retain the unique differential activity of the natural schweinfurthins, as evidenced by the spindle-like morphological changes induced in SF-295 cells and the unaltered appearance of human lung carcinoma A549 cells. We demonstrate that incubation with 3dSB or 3dSB-PNBS results in cleavage of poly-ADP-ribose polymerase (PARP) and caspase-9, both markers of apoptosis. Coincubation of 3dSB or 3dSB-PNBS with the caspase-9 inhibitor (Z)-Leu-Glu(O-methyl)-His-Asp(O-methyl)-fluoromethylketone prevents PARP cleavage. Therapeutic agents that induce apoptosis often activate cellular stress pathways. A marker for multiple stress pathways is the phosphorylation of eukaryotic initiation factor 2α, which is phosphorylated in response to 3dSB and 3dSB-PNBS treatment. Glucose-regulated protein 78 and protein disulfide isomerase, both endoplasmic reticulum chaperones, are up-regulated with schweinfurthin exposure. Using the fluorescent properties of 3dSB-PNBS and dimethoxyphenyl-p-nitro-bis-stilbene (DMP-PNBS), a control compound, we show that the intracellular levels of 3dSB-PNBS are higher than those of Rhodamine 123 or DMP-PNBS in SF-295 and A549 cells.

Biologically active biotin derivatives of schweinfurthin F.

As a prelude to efforts to identify schweinfurthin binding proteins, an ester conjugate and an amide conjugate of schweinfurthin F and biotin have been prepared by chemical synthesis. These compounds maintain activity in SF-295 cells comparable to the parent system, and display the lower potency in A549 cells that is a characteristic of the schweinfurthin pattern of activity.

3-Deoxyschweinfurthin B Lowers Cholesterol Levels by Decreasing Synthesis and Increasing Export in Cultured Cancer Cell Lines

AbstractThe schweinfurthins have potent antiproliferative activity in multiple glioblastoma multiforme (GBM) cell lines; however, the mechanism by which growth is impeded is not fully understood. Previously, we demonstrated that the schweinfurthins reduce the level of key isoprenoid intermediates in the cholesterol biosynthetic pathway. Herein, we describe the effects of the schweinfurthins on cholesterol homeostasis. Intracellular cholesterol levels are greatly reduced in cells incubated with 3-deoxyschweinfurthin B (3dSB), an analog of the natural product schweinfurthin B. Decreased cholesterol levels are due to decreased cholesterol synthesis and increased cholesterol efflux; both of these cellular actions can be influenced by liver X-receptor (LXR) activation. The effects of 3dSB on ATP-binding cassette transporter 1 levels and other LXR targets are similar to that of 25-hydroxycholesterol, an LXR agonist. Unlike 25-hydroxycholesterol, 3dSB does not act as a direct agonist for LXR α or β. These data suggest that cholesterol homeostasis plays a significant role in the growth inhibitory activity of the schweinfurthins and may elucidate a mechanism that can be targeted in human cancers such as GBM.

In vitro studies in a myelogenous leukemia cell line suggest an organized binding of geranylgeranyl diphosphate synthase inhibitors

A small set of isoprenoid bisphosphonates ethers has been tested in the K562 chronic myelogenous leukemia cell line to determine their impact on isoprenoid biosynthesis. Five of these compounds inhibit geranylgeranyl diphosphate synthase (GGDPS) with IC50 values below 1 μM in enzyme assays, but in cells their apparent activity is more varied. In particular, the isomeric C-geranyl-O-prenyl and C-prenyl-O-geranyl bisphosphonates are quite different in their activity with the former consistently demonstrating greater impairment of geranylgeranylation in cells but the latter showing greater impact in the enzyme assays with GGDPS. Together, these findings suggest an organized binding of these inhibitors in the two hydrophobic channels of the geranylgeranyl diphosphate synthase enzyme.

Quantitative determination of isopentenyl diphosphate in cultured mammalian cells

Isopentenyl diphosphate (IPP), an intermediate of the isoprenoid biosynthetic pathway (IBP), has several important biological functions, yet a method to determine its basal level has not been described. Here, we describe a nonradioactive and sensitive analytical method to isolate and specifically quantify IPP from cultured mammalian cells. This method applies an enzymatic coupling reaction to determine intracellular concentrations of IPP. In this reaction, geranylgeranyl diphosphate synthase catalyzes the formation of geranylgeranyl diphosphate (GGPP) from IPP and farnesyl diphosphate (FPP). Subsequently, geranylgeranyl protein transferase I conjugates GGPP with a fluorescently labeled peptide. The geranylgeranylated peptide can be quantified by high-performance liquid chromatography (HPLC) with a fluorescence detector, thereby allowing for IPP quantification. The detection lower limit of the fluorescence-labeled geranylgeranyl peptide is approximately 5 pg (~0.017 pmol). This method was used to examine the effects of IBP inhibitors such as lovastatin and zoledronate on intracellular levels of IPP. Inhibition of hydroxymethylglutaryl coenzyme A reductase (HMGCR) by lovastatin (50 nM) decreases IPP levels by 78% and 53% in K562 and MCF-7 cells, respectively. Whereas zoledronic acid (10 μM) increased IPP levels 12.6-fold when compared with untreated cells in the K562 cell line, an astonishing 960-fold increase was observed in the MCF-7 cells.

Abstract 4032: Targeting the isoprenoid biosynthetic pathway in a murine model of metastatic prostate cancer

The isoprenoid biosynthetic pathway (IBP) provides substrates for the post-translational modification of key proteins in regulating malignant cell properties, including proliferation, migration, and invasion. Several studies have found broad inhibitors of the IBP, such as statins, to significantly reduce the risk of advanced prostate cancer (PCa), potentially through their induced-reduction of geranylgeranylated proteins. However, such inhibition of the entire IBP is limited by side effects resulting from depletion of all isoprenoids and their products. We have developed a novel compound, geranylgeraniol bisphosphonate (GGOHBP), selectively targeting geranylgeranyl diphosphate synthase (GGDPS) and reducing the post-translational modification of geranylgeranylated proteins. Luciferase-expressing human-derived 22Rv1 PCa cells were injected into the left ventricle of 20 mice using bioluminescence imaging (BLI) to visualize tumor development. After three weeks 18/20 mice had developed tumors and daily subcutaneous (SQ) injections of 1.5mg/kg GGOHBP were given for one month. All 18 mice developed mandible tumors, 13 developed adrenal gland tumors, 4 developed intestinal tumors, 2 developed femoral tumors, 2 developed liver tumors, and 1 developed a tumor in an unidentified location in the chest cavity. Ex vivo photon count analyses revealed an 80% (p=0.1458) reduction in adrenal gland tumors of the treated mice as compared to vehicle-treated controls. This corresponded to a 54% (p=0.0007) reduction in total adrenal gland tumor weight in the treated as compared to the control mice. Western blot analysis of the adrenal gland tumors and one intestinal tumor showed a reduction in Rap1A geranylgeranylation in the treated mice and not the control mice, while non-tumorous tissues showed no Rap1A alteration. A follow-up study of non-tumor bearing mice given SQ injections of 1.25 or 1.5 mg/kg GGOHBP daily did not reveal alterations in Rap1A geranylgeranylation in the adrenal glands, kidneys, liver, or heart. Our findings detail a novel compound capable of preferentially altering the IBP in the adrenal gland tumors of a murine model of metastatic PCa. Future work defining the mechanism behind this focal effect and the adrenal gland isoprenoid environment will allow compound optimization and further understanding of the IBP9s role in PCa metastasis. Citation Format: Jacqueline E. Reilly, Jeffrey D. Neighbors, Nadine Bannick, Michael D. Henry, Craig H. Kuder, Raymond J. Hohl. Targeting the isoprenoid biosynthetic pathway in a murine model of metastatic prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4032. doi:10.1158/1538-7445.AM2014-4032