Carolyn Weinbaum

GGTase-I deficiency reduces tumor formation and improves survival in mice with K-RAS-induced lung cancer.

Protein geranylgeranyltransferase type I (GGTase-I) is responsible for the posttranslational lipidation of CAAX proteins such as RHOA, RAC1, and cell division cycle 42 (CDC42). Inhibition of GGTase-I has been suggested as a strategy to treat cancer and a host of other diseases. Although several GGTase-I inhibitors (GGTIs) have been synthesized, they have very different properties, and the effects of GGTIs and GGTase-I deficiency are unclear. One concern is that inhibiting GGTase-I might lead to severe toxicity. In this study, we determined the effects of GGTase-I deficiency on cell viability and K-RAS-induced cancer development in mice. Inactivating the gene for the critical beta subunit of GGTase-I eliminated GGTase-I activity, disrupted the actin cytoskeleton, reduced cell migration, and blocked the proliferation of fibroblasts expressing oncogenic K-RAS. Moreover, the absence of GGTase-I activity reduced lung tumor formation, eliminated myeloproliferative phenotypes, and increased survival of mice in which expression of oncogenic K-RAS was switched on in lung cells and myeloid cells. Interestingly, several cell types remained viable in the absence of GGTase-I, and myelopoiesis appeared to function normally. These findings suggest that inhibiting GGTase-I may be a useful strategy to treat K-RAS-induced malignancies.

Zoledronic acid treatment impairs protein geranyl-geranylation for biological effects in prostatic cells

BackgroundNitrogen-containing bisphosphonates (N-BPs) have been designed to inhibit osteoclast-mediated bone resorption. However, it is now accepted that part of their anti-tumor activities is related to interference with the mevalonate pathway.MethodsWe investigated the effects of zoledronic acid (ZOL), on cell proliferation and protein isoprenylation in two tumoral (LnCAP, PC-3,), and one normal established (PNT1-A) prostatic cell line. To assess if inhibition of geranyl-geranylation by ZOL impairs the biological activity of RhoA GTPase, we studied the LPA-induced formation of stress fibers. The inhibitory effect of ZOL on geranyl geranyl transferase I was checked biochemically. Activity of ZOL on cholesterol biosynthesis was determined by measuring the incorporation of 14C mevalonate in cholesterol.ResultsZOL induced dose-dependent inhibition of proliferation of all the three cell lines although it appeared more efficient on the untransformed PNT1A. Whatever the cell line, 20 μM ZOL-induced inhibition was reversed by geranyl-geraniol (GGOH) but neither by farnesol nor mevalonate. After 48 hours treatment of cells with 20 μM ZOL, geranyl-geranylation of Rap1A was abolished whereas farnesylation of HDJ-2 was unaffected. Inhibition of Rap1A geranyl-geranylation by ZOL was rescued by GGOH and not by FOH. Indeed, as observed with treatment by a geranyl-geranyl transferase inhibitor, treatment of PNT1-A cells with 20 μM ZOL prevented the LPA-induced formation of stress fibers. We checked that in vitro ZOL did not inhibit geranyl-geranyl-transferase I. ZOL strongly inhibited cholesterol biosynthesis up to 24 hours but at 48 hours 90% of this biosynthesis was rescued.ConclusionAlthough zoledronic acid is currently the most efficient bisphosphonate in metastatic prostate cancer management, its mechanism of action in prostatic cells remains unclear. We suggest in this work that although in first intention ZOL inhibits FPPsynthase its main biological actitivity is directed against protein Geranylgeranylation.

A Novel Protein Geranylgeranyltransferase-I Inhibitor with High Potency, Selectivity, and Cellular Activity

Inhibiting protein prenylation is an attractive means to modulate cellular processes controlled by a variety of signaling proteins, including oncogenic proteins such as Ras and Rho GTPases. The largest class of prenylated proteins contain a so-called CaaX motif at their carboxyl termini and are subject to a maturation process initiated by the attachment of an isoprenoid lipid by either protein farnesyltransferase (FTase) or protein geranylgeranyltransferase type I (GGTase-I). Inhibitors of FTase, termed FTIs, have been the subject of intensive development in the past decade and have shown efficacy in clinical trials. Although GGTase-I inhibitors (GGTIs) have received less attention, accumulating evidence suggests GGTIs may augment therapies using FTIs and could be useful to treat a myriad of additional disease states. Here we describe the characterization of a selective, highly potent, and cell-active GGTase-I inhibitor, GGTI-DU40. Kinetic analysis revealed that inhibition by GGTI-DU40 is competitive with the protein substrate and uncompetitive with the isoprenoid substrate; the Ki for the inhibition is 0.8 nm. GGTI-DU40 is highly selective for GGTase-Iboth in vitro and in living cells. Studies indicate GGTI-DU40 blocks prenylation of a number of geranylgeranylated CaaX proteins. Treatment of MDA-MB-231 breast cancer cells with GGTI-DU40 inhibited thrombin-induced cell rounding via a process that involves inhibition of Rho proteins without significantly effecting parallel mobilization of calcium via Gβγ. These studies establish GGTI-DU40 as a prime tool for interrogating biologies associated with protein geranylgeranylation and define a novel structure for this emerging class of experimental therapeutics.

Dissociation of Rac1(GDP)·RhoGDI Complexes by the Cooperative Action of Anionic Liposomes Containing Phosphatidylinositol 3,4,5-Trisphosphate, Rac Guanine Nucleotide Exchange Factor, and GTP

Rac plays a pivotal role in the assembly of the superoxide-generating NADPH oxidase of phagocytes. In resting cells, Rac is found in the cytosol in complex with Rho GDP dissociation inhibitor (RhoGDI). NADPH oxidase assembly involves dissociation of the Rac·RhoGDI complex and translocation of Rac to the membrane. We reported that liposomes containing high concentrations of monovalent anionic phospholipids cause Rac·RhoGDI complex dissociation ( Ugolev, Y., Molshanski-Mor, S., Weinbaum, C., and Pick, E. (2006) J. Biol. Chem. 281, 19204-19219 ). We now designed an in vitro model mimicking membrane phospholipid remodeling during phagocyte stimulation in vivo. We showed that liposomes of “resting cell membrane” composition (less than 20 mol % monovalent anionic phospholipids), supplemented with 1 mol % of polyvalent anionic phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) in conjunction with constitutively active forms of the guanine nucleotide exchange factors (GEFs) for Rac, Trio, or Tiam1 and a non-hydrolyzable GTP analogue, cause dissociation of Rac1(GDP)·RhoGDI complexes, GDP to GTP exchange on Rac1, and binding of Rac1(GTP) to the liposomes. Complexes were not dissociated in the absence of GEF and GTP, and optimal dissociation required the presence of PtdIns(3,4,5)P3 in the liposomes. Dissociation of Rac1(GDP)·RhoGDI complexes was correlated with the affinity of particular GEF constructs, via the N-terminal pleckstrin homology domain, for PtdIns(3,4,5)P3 and involved GEF-mediated GDP to GTP exchange on Rac1. Phagocyte membranes enriched in PtdIns(3,4,5)P3 responded by NADPH oxidase activation upon exposure in vitro to Rac1(GDP)·RhoGDI complexes, p67phox, GTP, and Rac GEF constructs with affinity for PtdIns(3,4,5)P3 at a level superior to that of native membranes.

Liposomes Comprising Anionic but Not Neutral Phospholipids Cause Dissociation of Rac(1 or 2)·RhoGDI Complexes and Support Amphiphile-independent NADPH Oxidase Activation by Such Complexes

Activation of the phagocyte NADPH oxidase involves the assembly of a membrane-localized cytochrome b559 with the cytosolic components p47phox, p67phox, p40phox, and the GTPase Rac (1 or 2). In resting phagocytes, Rac is found in the cytosol as a prenylated protein in the GDP-bound form, associated with the Rho GDP dissociation inhibitor (RhoGDI). In the process of NADPH oxidase activation, Rac is dissociated from RhoGDI and translocates to the membrane, in concert with the other cytosolic components. The mechanism responsible for dissociation of Rac from RhoGDI is poorly understood. We generated Rac(1 or 2)·RhoGDI complexes in vitro from recombinant Rac(1 or 2), prenylated enzymatically, and recombinant RhoGDI, and purified these by anion exchange chromatography. Exposing Rac(1 or 2)(GDP)·RhoGDI complexes to liposomes containing four different anionic phospholipids caused the dissociation of Rac(1 or 2)(GDP) from RhoGDI and its binding to the anionic liposomes. Rac2(GDP)·RhoGDI complexes were more resistant to dissociation, reflecting the lesser positive charge of Rac2. Liposomes consisting of neutral phospholipid did not cause dissociation of Rac(1 or 2)·RhoGDI complexes. Rac1 exchanged to the hydrolysis-resistant GTP analogue, GMPPNP, associated with RhoGDI with lower affinity than Rac1(GDP) and Rac1(GMPPNP)·RhoGDI complexes were more readily dissociated by anionic liposomes. Rac1(GMPPNP)·RhoGDI complexes elicited NADPH oxidase activation in native phagocyte membrane liposomes in the presence of p67phox, without the need for an anionic amphiphile, as activator. Both Rac1(GDP)·RhoGDI and Rac1(GMPPNP)·RhoGDI complexes elicited amphiphile-independent, p67phox-dependent NADPH oxidase activation in phagocyte membrane liposomes enriched in anionic phospholipids but not in membrane liposomes enriched in neutral phospholipids.

The Guanine Nucleotide Exchange Factor Trio Activates the Phagocyte NADPH Oxidase in the Absence of GDP to GTP Exchange on Rac “THE EMPEROR'S NEW CLOTHES”

The superoxide-generating NADPH oxidase complex of phagocytes consists of a membrane-associated flavocytochrome b 559 and four cytosolic components as follows: p47 phox , p67 phox , p40 phox , and the small GTPase Rac (1 or 2). Activation of the oxidase is the result of assembly of the cytosolic components with cytochrome b 559 and can be mimicked in vitro by mixtures of membrane and cytosolic components exposed to an anionic amphiphile, serving as activator. We reported that prenylation of Rac1 endows it with the ability to support oxidase activation in conjunction with p67 phox but in the absence of amphiphile and p47 phox . We now show the following 6 points. 1) The Rac guanine nucleotide exchange factor Trio markedly potentiates oxidase activation by prenylated Rac1-GDP. 2) This occurs in the absence of exogenous GTP or any other source of GTP generation, demonstrating that the effect of Trio does not involve GDP to GTP exchange on Rac1. 3) Trio does not potentiate oxidase activation by prenylated Rac1-GTP, by nonprenylated Rac1-GDP in the presence or absence of amphiphile, and by a prenylated [p67 phox -Rac1] chimera in GDP-bound form. 4) Rac1 mutants defective in the ability to bind Trio or to respond to Trio by nucleotide exchange fail to respond to Trio by enhanced oxidase activation. 5) A Trio mutant with conserved Rac1-binding ability but lacking nucleotide exchange activity fails to enhance oxidase activation. 6) The effect of Trio is mimicked by displacement of Mg2+ from Rac1-GDP. These results reveal the existence of a novel mechanism of Rac activation by a guanine nucleotide exchange factor and suggest that the induction by Trio of a conformational change in Rac1, in the absence of nucleotide exchange, is sufficient for enhancing its effector function.

Synthesis and evaluation of GGPP geometric isomers: divergent substrate specificities of FTase and GGTase I

A stereocontrolled synthetic route has been used to prepare two of the geometric isomers of all-trans-GGPP. Neither of these isomers is effective substrates for mammalian GGTase I, but 3 is a potent inhibitor of this enzyme (IC(50)=100 nM). Surprisingly, both compounds are effective substrates for mammalian FTase.

Grignard-mediated synthesis and preliminary biological evaluation of novel 3-substituted farnesyl diphosphate analogues.

A series of substituents was installed at the 3 position of farnesyl diphosphate through a copper-cyanide mediated coupling of a vinyl triflate with various Grignard reagents. These novel FPP mimetics were then evaluated as inhibitors of or substrates for mammalian protein farnesyl transferase. The IC50 values for these compounds range from 18 to 10,100 nm, with the 3-isopropenyl analogue being one of the most potent FPP-mimetic mFTase inhibitors yet synthesized.