Richard A. Gibbs

Geranylgeranyl Switching Regulates

Rab GTPases, key regulators of membrane targeting and fusion, require the covalent attachment of geranylgeranyl lipids to their C terminus for function. To elucidate the role of lipid in Rab recycling, we have determined the crystal structure of Rab guanine nucleotide dissociation inhibitor (alphaGDI) in complex with a geranylgeranyl (GG) ligand (H(2)N-Cys-(S-GG)-OMe). The lipid is bound beneath the Rab binding platform in a shallow hydrophobic groove. Mutation of the binding pocket in the brain-specific alphaGDI leads to mental retardation. Strikingly, lipid binding acts through a conserved allosteric switching mechanism to promote release of the GDI-Rab[GDP] complex from the membrane.

Non-Peptidic Prenyltransferase Inhibitors: Diverse Structural Classes and Surprising Anti-Cancer Mechanisms

The development of farnesyltransferase inhibitors (FTIs) has been one of the most active areas of anticancer drug development for the past ten years. This review presents a general overview of the developments in this area, along with a critical appraisal of the anticancer activity of FTIs. A historical survey of the protein prenylation field is given, in particular to emphasize the key role played by the Ras oncoprotein in driving the discovery of prenyltransferase enzymes. The different classes of prenylated proteins will be described along with the biochemical characteristics of the key drug target--farnesyltransferase (FTase). Numerous potent farnesyltransferase inhibitors have been developed. The FTIs developed can be separated into three different categories, based on their origin and/or mechanism of action: a) natural products; b) peptidomimetics and other CAAX-competitive inhibitors; c) farnesyl pyrophosphate (FPP) mimetics or analogs and other FPP-competitive inhibitors. Along with a survey of newer FTIs in each class, the development of several representative, potent compounds will be discussed in depth as we discuss the potential advantages and liabilities of each class. Particular emphasis is given to the discovery of new, more potent FPP-competitive FTIs of several diverse structural classes. Testing of different FTIs for their ability to block the growth of various cancer cell types in animal models will be discussed. There are a number of key differences between these compounds and traditional cytotoxic cancer chemotherapeutic agents, with surprising exceptions to their expected modes of action. As some FTIs have entered human clinical trials, answers may soon become available to key mechanistic questions concerning the extent and nature of their antitumor growth properties.

A NOVEL GERANYLGERANYL TRANSFERASE INHIBITOR IN COMBINATION WITH LOVASTATIN INHIBITS PROLIFERATION AND INDUCES AUTOPHAGY IN STS-26T MPNST CELLS

Prenylation inhibitors have gained increasing attention as potential therapeutics for cancer. Initial work focused on inhibitors of farnesylation, but more recently geranylgeranyl transferase inhibitors (GGTIs) have begun to be evaluated for their potential antitumor activity in vitro and in vivo. In this study, we have developed a nonpeptidomimetic GGTI, termed GGTI-2Z [(5-nitrofuran-2-yl)methyl-(2Z,6E,10E)-3,7,11,15-tetramethylhexadeca-2,6,10,14-tetraenyl 4-chlorobutyl(methyl)phosphoramidate], which in combination with lovastatin inhibits geranylgeranyl transferase I (GGTase I) and GGTase II/RabGGTase, without affecting farnesylation. The combination treatment results in a G0/G1 arrest and synergistic inhibition of proliferation of cultured STS-26T malignant peripheral nerve sheath tumor cells. We also show that the antiproliferative activity of drugs in combination occurs in the context of autophagy. The combination treatment also induces autophagy in the MCF10.DCIS model of human breast ductal carcinoma in situ and in 1c1c7 murine hepatoma cells, where it also reduces proliferation. At the same time, there is no detectable toxicity in normal immortalized Schwann cells. These studies establish GGTI-2Z as a novel geranylgeranyl pyrophosphate derivative that may work through a new mechanism involving the induction of autophagy and, in combination with lovastatin, may serve as a valuable paradigm for developing more effective strategies in this class of antitumor therapeutics.

The isoprenoid substrate specificity of isoprenylcysteine carboxylmethyltransferase: development of novel inhibitors.

Isoprenylcysteine carboxylmethyltransferase (Icmt) is an integral membrane protein localized to the endoplasmic reticulum of eukaryotic cells that catalyzes the post-translational α-carboxylmethylesterification of CAAX motif proteins, including the oncoprotein Ras. Prior to methylation, these protein substrates all contain an isoprenylcysteine residue at the C terminus. In this study, we developed a variety of substrates and inhibitors of Icmt that vary in the isoprene moiety in order to gain information about the nature of the lipophilic substrate binding site. These isoprenoid-modified analogs of the minimal Icmt substrate N-acetyl-S-farnesyl-l-cysteine (AFC) were synthesized from newly and previously prepared farnesol analogs. Using both yeast and human Icmt enzymes, these compounds were found to vary widely in their ability to act as substrates, supporting the isoprenoid moiety as a key substrate recognition element for Icmt. Compound 3 is a competitive inhibitor of overexpressed yeast Icmt (KI = 17.1 ± 1.7 μm). Compound 4 shows a mix of competitive and uncompetitive inhibition for both the yeast and the human Icmt proteins (yeast KIC = 35.4 ± 3.4 μm, KIU = 614.4 ± 148 μm; human KIC = 119.3 ± 18.1 μm, KIU = 377.2 ± 42.5 μm). These data further suggest that differences in substrate specificity exist between the human and yeast enzymes. Biological studies suggest that inhibition of Icmt results in Ras mislocalization and loss of cellular transformation ability, making Icmt an attractive and novel anticancer target. Further elaboration of the lead compounds synthesized and assayed here may lead to clinically useful higher potency inhibitors.

Context-dependent substrate recognition by protein farnesyltransferase

Prenylation is a posttranslational modification whereby C-terminal lipidation leads to protein localization to membranes. A C-terminal Ca(1)a(2)X sequence has been proposed as the recognition motif for two prenylation enzymes, protein farnesyltransferase (FTase) and protein geranylgeranyltransferase type I. To define the parameters involved in recognition of the a(2) residue, we performed structure-activity analysis which indicates that FTase discriminates between peptide substrates based on both the hydrophobicity and steric volume of the side chain at the a(2) position. For nonpolar side chains, the dependence of the reactivity on side chain volume at this position forms a pyramidal pattern with a maximal activity near the steric volume of valine. This discrimination occurs at a step in the kinetic mechanism that is at or before the farnesylation step. Furthermore, a(2) selectivity is also affected by the identity of the adjacent X residue, leading to context-dependent substrate recognition. Context-dependent a(2) selectivity suggests that FTase recognizes the sequence downstream of the conserved cysteine as a set of two or three cooperative, interconnected recognition elements as opposed to three independent amino acids. These findings expand the pool of proposed FTase substrates in cells. A better understanding of the molecular recognition of substrates performed by FTase will aid in both designing new FTase inhibitors as therapeutic agents and characterizing proteins involved in prenylation-dependent cellular pathways.

Detection of nonsterol isoprenoids by HPLC–MS/MS

Isoprenoids constitute an important class of biomolecules that participate in many different cellular processes. Most available detection methods allow the identification of only one or two specific nonsterol isoprenoid intermediates following radioactive or fluorescent labeling. We here report a rapid, nonradioactive, and sensitive procedure for the simultaneous detection and quantification of the eight main nonsterol intermediates of the isoprenoid biosynthesis pathway by means of tandem mass spectrometry. Intermediates were analyzed by HPLC-MS/MS in the multiple reaction monitoring mode using a silica-based C(18) HPLC column. For quantification, their stable isotope-labeled analogs were used as internal standards. HepG2 cells were used to validate the method. Mevalonate, phosphomevalonate, and the six subsequent isoprenoid pyrophosphates were readily determined with detection limits ranging from 0.03 to 1.0mumol/L. The intra- and interassay variations for HepG2 cell homogenates supplemented with isoprenoid intermediates were 3.6-10.9 and 4.4-11.9%, respectively. Under normal culturing conditions, isoprenoid intermediates in HepG2 cells were below detection limits. However, incubation of the cells with pamidronate, an inhibitor of farnesyl pyrophosphate synthase, resulted in increased levels of mevalonate, isopentenyl pyrophosphate/dimethylallyl pyrophosphate, and geranyl pyrophosphate. This method will be suitable for measuring profiles of isoprenoid intermediates in cells with compromised isoprenoid biosynthesis and for determining the specificity of potential inhibitors of the pathway.

Coupling of isoprenoid triflates with organoboron nucleophiles: Synthesis of all-trans-geranylgeraniol

Abstract The Suzuki coupling reaction has been used to introduce a methyl group derived from commercially available methaneboronic acid into a vinyl triflate. This has led to a concise synthesis of all- trans -geranylgeraniol, with the key step being the Pd-catalyzed, silver-mediated methylation of 9 to give ethyl geranylgeranoate 10 . This coupling protocol has also been used to produce the novel GGPP analog 3-phenyl-3-desmethylgeranylgeranyl diphosphate ( 12 ).

Induction of Apoptosis in Neurofibromatosis Type 1 Malignant Peripheral Nerve Sheath Tumor Cell Lines by a Combination of Novel Farnesyl Transferase Inhibitors and Lovastatin

Neurofibromatosis type 1 (NF1) is a genetic disorder that is driven by the loss of neurofibromin (Nf) protein function. Nf contains a Ras-GTPase-activating protein domain, which directly regulates Ras signaling. Numerous clinical manifestations are associated with the loss of Nf and increased Ras activity. Ras proteins must be prenylated to traffic and functionally localize with target membranes. Hence, Ras is a potential therapeutic target for treating NF1. We have tested the efficacy of two novel farnesyl transferase inhibitors (FTIs), 1 and 2, alone or in combination with lovastatin, on two NF1 malignant peripheral nerve sheath tumor (MPNST) cell lines, NF90-8 and ST88-14. Single treatments of 1, 2, or lovastatin had no effect on Ras prenylation or MPNST cell proliferation. However, low micromolar combinations of 1 or 2 with lovastatin (FTI/lovastatin) reduced Ras prenylation in both MPNST cell lines. Furthermore, this FTI/lovastatin combination treatment reduced cell proliferation and induced an apoptotic response as shown by morphological analysis, procaspase-3/-7 activation, loss of mitochondrial membrane potential, and accumulation of cells with sub-G1 DNA content. Little to no detectable toxicity was observed in normal rat Schwann cells following FTI/lovastatin combination treatment. These data support the hypothesis that combination FTI plus lovastatin therapy may be a potential treatment for NF1 MPNSTs.

Coupling of Isoprenoid Triflates with Organoboron Nucleophiles: Synthesis and Biological Evaluation of Geranylgeranyl Diphosphate Analogues

The Suzuki coupling reaction has been used to introduce a methyl group derived from commercially available methylboronic acid into a vinyl triflate. This has led to a concise synthesis of all-trans-geranylgeraniol, with the key step being the palladium-catalyzed, silver-mediated methylation of triflate to give ethyl geranylgeranoate. This coupling protocol has also been used to produce the novel geranylgeranyl diphosphate (GGPP) analogue 3-phenyl-3-desmethylgeranylgeranyl diphosphate (3-PhGGPP, ). Our previously developed organocuprate coupling protocol has been used to introduce the cyclopropyl and tert-butyl moieties into the 3-position of vinyl triflate. The four GGPP analogues 3-vinyl-3-desmethylgeranylgeranyl diphosphate (3-vGGPP, ), 3-cyclopropyl-3-desmethylgeranylgeranyl diphosphate (3-cpGGPP, ), 3-tert-butyl-3-desmethyl-geranylgeranyl diphosphate (3-tbGGPP, ), and were then evaluated as potential inhibitors of recombinant yeast protein-geranylgeranyl transferase I (PGGTase I). The potential mechanism-based inhibitors 3-vGGPP and 3-cpGGPP did not exhibit time-dependent inactivation of PGGTase I. Instead, both analogues were alternative substrates, in accord with the interaction of the corresponding farnesyl analogues 3-vFPP and 3-cpFPP with PFTase. The tert-butyl and phenyl analogues were not substrates, but were instead competitive inhibitors of PGGTase I. Note that all four of the GGPP analogues were bound less tightly by the enzyme than the natural substrate, in contrast to the behavior of the 3-substituted FPP analogues.

Novel limonene phosphonate and farnesyl diphosphate analogues: design, synthesis, and evaluation as potential protein-farnesyl transferase inhibitors

Limonene and its metabolite perillyl alcohol are naturally-occurring isoprenoids that block the growth of cancer cells both in vitro and in vivo. This cytostatic effect appears to be due, at least in part, to the fact that these compounds are weak yet selective and non-toxic inhibitors of protein prenylation. Protein-farnesyl transferase (FTase), the enzyme responsible for protein farnesylation, has become a key target for the rational design of cancer chemotherapeutic agents. Therefore, several alpha-hydroxyphosphonate derivatives of limonene were designed and synthesized as potentially more potent FTase inhibitors. A noteworthy feature of the synthesis was the use of trimethylsilyl triflate as a mild, neutral deprotection method for the preparation of sensitive phosphonates from the corresponding tert-butyl phosphonate esters. Evaluation of these compounds demonstrates that they are exceptionally poor FTase inhibitors in vitro (IC50 > or = 3 mM) and they have no effect on protein farnesylation in cells. In contrast, farnesyl phosphonyl(methyl)phosphinate, a diphosphate-modified derivative of the natural substrate farnesyl diphosphate, is a very potent FTase inhibitor in vitro (Ki=23 nM).