Andrew J. Wiemer

The intermediate enzymes of isoprenoid metabolism as anticancer targets.

Inhibitors of isoprenoid biosynthesis are widely used to treat human disease including statins and nitrogenous bisphosphonates. Due to the importance of core human isoprenoid biosynthesis for diverse cellular processes related to cancer cell growth and metastasis, inhibition of this pathway may produce beneficial anticancer consequences. For example, ras oncogenes are well known; ras proteins are overexpressed in many human cancers, and these proteins must be isoprenylated to function. The rho proteins are important for regulating cell motility, and also must be isoprenylated. This has drawn significant attention to inhibitors of protein prenyl transferases. In addition to the reactions that are targeted in current clinical applications, there are other enzymes that have not been studied as extensively. Inhibition of these enzymes, from mevalonate kinase to geranylgeranyl diphosphate synthase, could be attractive as a single agent therapy or in combination with current agents for treatment of cancers in which isoprenylated proteins have been implicated. While detailed in vivo data for many of these putative targets is lacking, there have been several breakthroughs in recent years that could facilitate further studies. In particular, compounds that specifically inhibit some of the downstream isoprenoid biosynthesis enzymes have been developed and their effects in cancer models are emerging. This review will discuss current knowledge of these lesser known isoprenoid pathway enzymes, identify trends in the development of their small molecule inhibitors, and describe the applications and effects of these compounds in cancer models.

Prodrugs of Phosphonates and Phosphates: Crossing the Membrane Barrier

A substantial portion of metabolism involves transformation of phosphate esters, including pathways leading to nucleotides and oligonucleotides, carbohydrates, isoprenoids and steroids, and phosphorylated proteins. Because the natural substrates bear one or more negative charges, drugs that target these enzymes generally must be charged as well, but small charged molecules can have difficulty traversing the cell membrane by means other than endocytosis. The resulting dichotomy has stimulated a great deal of effort to develop effective prodrugs, compounds that carry little or no charge to enable them to transit biological membranes, but able to release the parent drug once inside the target cell. This chapter presents recent studies on advances in prodrug forms, along with representative examples of their application to marketed and developmental drugs.

Pivaloyloxymethyl-modified isoprenoid bisphosphonates display enhanced inhibition of cellular geranylgeranylation.

Nitrogenous bisphosphonate inhibitors of farnesyl disphosphate synthase have been used clinically for treatment of bone disease. Because many of their effects may be mediated by depletion of geranylgeranyl diphosphate, our group has sought compounds that do this more directly through inhibition of geranylgeranyl diphosphate synthase and we have discovered a number of isoprenoid-containing bisphosphonates that selectively inhibit this enzyme. These compounds have a high negative charge at physiological pH which is necessary for inhibition of the enzyme but may limit their ability to enter cells. Therefore, chemical modifications that mask this charge may enhance their cellular potency. We now have synthesized novel pivaloyloxymethyl-modified isoprenoid bisphosphonates and investigated their ability to inhibit protein geranylgeranylation within cells. We have found that addition of pivaloyloxymethyl moieties to isoprenoid bisphosphonates increases their potency towards cellular geranylgeranylation even though this modification decreases their in vitro inhibition of geranylgeranyl diphosphate synthase. Pivaloyloxymethyl modifications more effectively increase the cellular activity of the more polar isoprenoid bisphosphonates. These results reveal structural relationships between in vitro and cellular activity which may serve as the basis for future development of more potent and/or drug-like inhibitors of geranylgeranyl diphosphate synthase.

Inhibition of Geranylgeranyl Diphosphate Synthase Induces Apoptosis through Multiple Mechanisms and Displays Synergy with Inhibition of Other Isoprenoid Biosynthetic Enzymes

Inhibitors of isoprenoid synthesis are widely used for treatment of human diseases, including hypercholesterolemia and osteoporosis, and they have the potential to be useful for treatment of cancer. Statin drugs inhibit the enzyme HMG-CoA reductase, whereas nitrogenous bisphosphonates have more recently been shown to inhibit farnesyl disphosphate synthase. In addition, our laboratory has recently developed several potent and specific bisphosphonate inhibitors of geranylgeranyl diphosphate synthase, including digeranyl bisphosphonate. Because all three enzymes fall in the same biosynthetic pathway and many of the biological effects are due to depletion of downstream products, we hypothesized that simultaneous inhibition of these enzymes would result in synergistic growth inhibition. In this study, we show that inhibition of geranylgeranyl diphosphate synthase induces apoptosis in K562 leukemia cells. This induction of apoptosis is in part dependent upon both geranylgeranyl diphosphate depletion and accumulation of farnesyl diphosphate. Combinations of either lovastatin or zoledronate with digeranyl bisphosphonate synergistically inhibited growth and induced apoptosis. These combinations also potently inhibited cellular geranylgeranylation. These results support the potential for combinations of multiple inhibitors of isoprene biosynthesis to inhibit cancer cell growth or metastasis at clinically achievable concentrations.

Contact-Dependent T Cell Activation and T Cell Stopping Require Talin1

T cell–APC contact initiates T cell activation and is maintained by the integrin LFA-1. Talin1, an LFA-1 regulator, localizes to the immune synapse (IS) with unknown roles in T cell activation. In this study, we show that talin1-deficient T cells have defects in contact-dependent T cell stopping and proliferation. Although talin1-deficient T cells did not form stable interactions with APCs, transient contacts were sufficient to induce signaling. In contrast to prior models, LFA-1 polarized to T cell–APC contacts in talin1-deficient T cells, but vinculin and F-actin polarization at the IS was impaired. These results indicate that T cell proliferation requires sustained, talin1-mediated T cell–APC interactions and that talin1 is necessary for F-actin polarization and the stability of the IS.

Quantitative determination of farnesyl and geranylgeranyl diphosphate levels in mammalian tissue

Farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP) are branch point intermediates of isoprenoid biosynthesis. Inhibitors of isoprenoid biosynthesis, such as the statins and bisphosphonates, are widely used therapeutic agents. However, little is known about the degree to which they alter levels of upstream and downstream isoprenoids, including FPP and GGPP. Therefore, we developed a method to isolate and quantify FPP and GGPP from mammalian tissues. Tissues from mice were collected, snap frozen in liquid nitrogen, and stored at -80 degrees C. FPP and GGPP were isolated by a combined homogenization and extraction procedure and were purified with a C18 solid phase extraction column. Farnesyl protein transferase (FTase) or geranylgeranyl protein transferase I (GGTase I) were used to conjugate FPP and GGPP with fluorescent dansylated peptides. FPP and GGPP were quantified by high-performance liquid chromatography (HPLC). The respective concentrations of FPP and GGPP are as follows: 0.355+/-0.030 and 0.827+/-0.082 units of nmol/g wet tissues in brain, 0.320+/-0.019 and 0.293+/-0.035 units of nmol/g wet tissues in kidney, 0.326+/-0.064 and 0.213+/-0.029 units of nmol/g wet tissues in liver, and 0.364+/-0.015 and 0.349+/-0.023 units of nmol/g wet tissues in heart (means+/-SEM). This method allows for determination of FPP and GGPP concentrations in any tissue type and is sensitive enough to detect changes following treatment with inhibitors of isoprenoid biosynthesis.

Geranylgeranyl diphosphate synthase: an emerging therapeutic target.

Proteins modified post‐translationally by geranylgeranylation have been implicated in numerous cellular processes related to human disease. In recent years, the study of protein geranylgeranylation has advanced tremendously in both cellular and animal models. The advances in our understanding of the biological roles of geranylgeranylated proteins have been paralleled by advances in the medicinal chemistry of geranylgeranylation inhibitors such as those that target geranylgeranyl transferases I and II and geranylgeranyl diphosphate synthase (GGDPS). Although these findings provide the rationale for further development of geranylgeranylation as a therapeutic target, more advanced studies on the efficacy of this approach in various disease models will be required to support translation to clinical studies. This article attempts to describe the advances in (and the challenges of) validation of GGDPS as a novel therapeutic target and assesses the advantages of targeting GGDPS relative to other enzymes involved in geranylgeranylation.

Cyclopropene Cycloadditions with Annulated Furans: Total Synthesis of (+)- and (-)-Frondosin B and (+)-Frondosin A

The asymmetric total syntheses of the natural products (+)- and (-)-frondosin B and (+)-frondosin A are reported based on a diastereoselective cycloaddition between tetrabromocyclopropene and an annulated furan to provide a highly functionalized common building block. The bridged bicyclic intermediate could be stereo- and chemoselectively manipulated to produce the two structurally distinct members of the frondosins. Both syntheses feature regioselective palladium-coupling reactions and an unprecedented phosphine-mediated ether bridge cleavage. Surprisingly, the planned enantioselective synthesis of frondosin B led to the opposite epimer of the natural product, suggesting an unusual late stage stereoinversion at C8. Frondosin A, but not frondosin B, was shown to have selective antiproliferative activity against several B-cell lines.

Calpain inhibition impairs TNF-α-mediated neutrophil adhesion, arrest and oxidative burst

Proinflammatory cytokines, such as tumor necrosis factor alpha (TNF-alpha), are increased in many chronic inflammatory disorders, including rheumatoid arthritis, and contribute to recruitment of neutrophils into areas of inflammation. TNF-alpha induces a stop signal that promotes neutrophil firm adhesion and inhibits neutrophil polarization and chemotaxis. Calpain is a calcium-dependent protease that mediates cytoskeletal reorganization during cell migration. Here, we show that calpain inhibition impairs TNF-alpha-induced neutrophil firm adhesion to fibrinogen-coated surfaces and the formation of vinculin-containing focal complexes. Calpain inhibition induces random migration in TNF-alpha-stimulated cells and prevents the generation of reactive oxygen species, but does not alter TNF-alpha-mediated activation of p38 MAPK and ERK MAPK. These findings suggest that the TNF-alpha-induced neutrophil arrest requires the activity of calpain independent of p38 MAPK and ERK signaling seen after TNF-alpha stimulation. Together, our data suggest that therapeutic inhibition of calpain may be beneficial for limiting TNF-alpha-induced inflammatory responses.

Isoprenoid Metabolism as a Therapeutic Target in Gram-Negative Pathogens

Gram-negative Enterobacteria include a variety of human pathogens, perhaps most notably E. coli, Salmonella, Shigella, Yersinia, and Proteus. While there are treatment options for the diseases caused by these organisms, multi-drug resistance is often a problem and development of novel antibiotics has lagged over recent years. In humans, the isoprenoid biosynthetic pathway has become a subject of intense research for therapeutic modulation of human enzymes in diseases including hypercholesterolemia, osteoporosis, and cancer. In bacteria, isoprenoid metabolism is arguably just as important, giving rise to components that are essential for electron transport and cell wall biosynthesis. Blocking these biosynthetic processes, either with the antibiotic fosmidomycin or by gene knockout strategies, has demonstrated the necessity of isoprenoid biosynthesis for bacterial growth. In this review, current knowledge of the biochemical pathways involved in farnesyl diphosphate metabolism in Enterobacteria, efforts to develop inhibitors of the involved enzymes, and insights from inhibitors of human isoprenoid metabolism that may be relevant for future studies of antibiotics that target these key enzymes, are described.