HDL mimetic CER-001 targets atherosclerotic plaques in patients.

BACKGROUND AND AIMS: Infusion of high-density lipoprotein (HDL) mimetics aimed at reducing atherosclerotic burden has led to equivocal results, which may relate in part to the inability of HDL mimetics to adequately reach atherosclerotic lesions in humans. This study evaluated delivery of recombinant human apolipoprotein A-I (apoA-I) containing HDL mimetic CER-001 in carotid plaques in patients. METHODS: CER-001 was radiolabeled with the long-lived positron emitter zirconium-89 ((89)Zr) to enable positron emission tomography with computed tomography (PET/CT) imaging. Eight patients with atherosclerotic carotid artery disease (>50% stenosis) received a single infusion of unlabeled CER-001 (3 mg/kg), co-administered with 10 mg of (89)Zr-labeled CER-001 (18 MBq). Serial PET/CT imaging and contrast enhanced-magnetic resonance imaging (CE-MRI) were performed to evaluate targeted delivery of CER-001. RESULTS: One hour after infusion, mean plasma apoA-I levels increased by 9.9 mg/dL (p = 0.026), with a concomitant relative increase in the plasma cholesterol efflux capacity of 13.8% (p < 0.001). Using serial PET/CT imaging, we showed that arterial uptake of CER-001 expressed as target-to-background ratio (TBRmax) increased significantly 24 h after infusion, and remained increased up to 48 h (TBRmax t = 10 min: 0.98; t = 24 h: 1.14 (p = 0.001); t = 48 h: 1.12 (p = 0.007)). TBRmax was higher in plaque compared with non-plaque segments (1.18 vs. 1.05; p < 0.001). Plaque TBRmax correlated with local plaque contrast enhancement (r = 0.56; p = 0.019) as assessed by CE-MRI. CONCLUSIONS: Infusion of HDL mimetic CER-001 increases plasma apoA-I concentration and plasma cholesterol efflux capacity. Our data support the concept that CER-001 targets plaque regions in patients, which correlates with plaque contrast enhancement. These clinical findings may also guide future nanomedicine development using HDL particles for drug delivery in atherosclerosis. CLINICAL TRIAL REGISTRATION: Netherlands Trial Registry - NTR5178. http://www.trialregister.nl/trialreg/admin/rctview.asp?TC=5178.

HDL and CER-001 Inverse-Dose Dependent Inhibition of Atherosclerotic Plaque Formation in apoE-/- Mice: Evidence of ABCA1 Down-Regulation.

OBJECTIVE: CER-001 is a novel engineered HDL-mimetic comprised of recombinant human apoA-I and charged phospholipids that was designed to mimic the beneficial properties of nascent pre-ß HDL. In this study, we have evaluated the dose-dependent regulation of ABCA1 expression in vitro and in vivo in the presence of CER-001 and native HDL (HDL3). METHODS AND RESULTS: CER-001 induced cholesterol efflux from J774 macrophages in a dose-dependent manner similar to natural HDL. A strong down-regulation of the ATP-binding cassette A1 (ABCA1) transporter mRNA (- 50%) as well as the ABCA1 membrane protein expression (- 50%) was observed at higher doses of CER-001 and HDL3 compared to non-lipidated apoA-I. In vivo, in an apoE-/- mouse "flow cessation model," in which the left carotid artery was ligatured to induce local inflammation, the inhibition of atherosclerotic plaque burden progression in response to a dose-range of every-other-day CER-001 or HDL in the presence of a high-fat diet for two weeks was assessed. We observed a U-shaped dose-response curve: inhibition of the plaque total cholesterol content increased with increasing doses of CER-001 or HDL3 up to a maximum inhibition (- 51%) at 5 mg/kg; however, as the dose was increased above this threshold, a progressively less pronounced inhibition of progression was observed, reaching a complete absence of inhibition of progression at doses of 20 mg/kg and over. ABCA1 protein expression in the same atherosclerotic plaque was decreased by-45% and-68% at 50 mg/kg for CER-001 and HDL respectively. Conversely, a-12% and 0% decrease in ABCA1 protein expression was observed at the 5 mg/kg dose for CER-001 and HDL respectively. CONCLUSIONS: These data demonstrate that high doses of HDL and CER-001 are less effective at slowing progression of atherosclerotic plaque in apoE-/- mice compared to lower doses, following a U-shaped dose-response curve. A potential mechanism for this phenomenon is supported by the observation that high doses of HDL and CER-001 induce a rapid and strong down-regulation of ABCA1 both in vitro and in vivo. In conclusion, maximally efficient HDL- or CER-001-mediated cholesterol removal from atherosclerotic plaque is achieved by maximizing macrophage-mediated efflux from the plaque while minimizing dose-dependent down-regulation of ABCA1 expression. These observations may help define the optimal dose of HDL mimetics for testing in clinical trials of atherosclerotic burden regression.

P2Y13 receptor regulates HDL metabolism and atherosclerosis in vivo.

High-density lipoprotein (HDL) is known to protect against atherosclerosis by promoting the reverse cholesterol transport. A new pathway for the regulation of HDL-cholesterol (HDL-c) removal involving F1-ATPase and P2Y13 receptor (P2Y13R) was described in vitro, and recently in mice. However, the physiological role of F1-ATPase/P2Y13R pathway in the modulation of vascular pathology i.e. in the development of atherosclerotic plaques is still unknown. We designed a specific novel agonist (CT1007900) of the P2Y13R that caused stimulation of bile acid secretion associated with an increased uptake of HDL-c in the liver after single dosing in mice. Repeated dose administration in mice, for 2 weeks, stimulated the apoA-I synthesis and formation of small HDL particles. Plasma samples from the agonist-treated mice had high efflux capacity for mobilization of cholesterol in vitro compared to placebo group. In apoE-/- mice this agonist induced a decrease of atherosclerotic plaques in aortas and carotids. The specificity of P2Y13R pathway in those mice was assessed using adenovirus encoding P2Y13R-shRNA. These results demonstrate that P2Y13R plays a pivotal role in the HDL metabolism and could also be a useful therapeutic agent to decrease atherosclerosis. In this study, the up-regulation of HDL-c metabolism via activation of the P2Y13R using agonists could promote reverse cholesterol transport and promote inhibition of atherosclerosis progression in mice.

CER-001, a HDL-mimetic, stimulates the reverse lipid transport and atherosclerosis regression in high cholesterol diet-fed LDL-receptor deficient mice.

OBJECTIVE: CER-001 is a novel engineered HDL-mimetic comprised of recombinant human apoA-I and phospholipids that was designed to mimic the beneficial properties of nascent pre-ß HDL. In this study, we have evaluated the capacity of CER-001 to perform reverse lipid transport in single dose studies as well as to regress atherosclerosis in LDLr(-/-) mice after short-term multiple-dose infusions. APPROACH AND RESULTS: CER-001 induced cholesterol efflux from macrophages and exhibited anti-inflammatory response similar to natural HDL. Studies with HUVEC demonstrated CER-001 at a concentration of 500 µg/mL completely suppressed the secretion of cytokines IL-6, IL-8, GM-CSF and MCP-1. Following infusion of CER-001 (10mg/kg) in C57Bl/6J mice, we observed a transient increase in the mobilization of unesterified cholesterol in HDL particles containing recombinant human apoA-I. Finally we show that cholesterol elimination was stimulated in CER-001 treated animals as demonstrated by the increased cholesterol concentration in liver and feces. In a familial hypercholesterolemia mouse model (LDL-receptor deficient mice), the infusion of CER-001 caused 17% and 32% reductions in plaque size, 17% and 23% reductions in lipid content after 5 and 10 doses given every 2 days, respectively. Also, there was an 80% reduction in macrophage content in the plaque following 5 doses, and decreased VCAM-1 expression by 16% and 22% in the plaque following 5 and 10 intravenous doses of CER-001, respectively. CONCLUSION: These data demonstrate that CER-001 rapidly enhances reverse lipid transport in the mouse, reducing vascular inflammation and promoting regression of diet-induced atherosclerosis in LDLr(-/-) mice upon a short-term multiple dose treatment.

Rab1a and Rab5a preferentially bind to binary lipid compositions with higher stored curvature elastic energy.

Rab proteins are a large family of GTP-binding proteins that regulate cellular membrane traffic and organelle identity. Rab proteins cycle between association with membranes and binding to RabGDI. Bound on membranes, each Rab has a very specific cellular location and it is this remarkable degree of specificity with which Rab GTPases recognize distinct subsets of intracellular membranes that forms the basis of their ability to act as key cellular regulators, determining the recruitment of downstream effectors to the correct membrane at the correct time. The molecular mechanisms controlling Rab localization remain poorly understood. Here, we present a fluorescence-based assay to investigate Rab GTPase membrane extraction and delivery by RabGDI. Using EGFP-Rab fusion proteins the amount of Rab:GDI complex obtained by GDI extraction of Rab proteins from HEK293 membranes could be determined, enabling control of complex concentration. Subsequent partitioning of the Rab GTPases into vesicles made up of artificial binary lipid mixtures showed for the first time, that the composition of the target membrane plays a key role in the localization of Rab proteins by sensing the stored curvature elastic energy in the membrane.

Synthesis, stereochemistry and SAR of a series of minodronate analogues as RGGT inhibitors.

Phosphonocarboxylate (PC) analogues of bisphosphonates are of interest due to their selective inhibition of a key enzyme in the mevalonate pathway, Rab geranylgeranyl transferase (RGGT). The dextrarotatory enantiomer of 2-hydroxy-3-(imidazo[1,2-a]pyridin-3-yl)-2-phosphonopropanoic acid (3-IPEHPC, 1) is the most potent PC-type RGGT inhibitor thus far identified. The absolute configuration of (+)-1 in the active site complex has remained unknown due to difficulties in obtaining RGGT inhibitor complex crystals suitable for X-ray diffraction analysis. However, we have now succeeded in crystallizing (-)-1 and here report its absolute configuration (AC) obtained by X-ray crystallography, thus also defining the AC of (+)-1. An Autodock Vina 1.1 computer modeling study of (+)-1 in the active site of modified RGGT binding GGPP (3DSV) identifies stereochemistry-dependent interactions that could account for the potency of (+)-1 and supports the hypothesis that this type of inhibitor binds at the TAG tunnel, inhibiting the second geranylgeranylation step. We also report a convenient (31)P NMR method to determine enantiomeric excess of 1 and its pyridyl analogue 2, using α- and ß-cyclodextrins as chiral solvating agents, and describe the synthesis of a small series of 1 α-X (X = H, F, Cl, Br; 7a-d) analogues to assess the contribution of the α-OH group to activity at enzyme and cellular levels. The IC(50) of 1 was 5-10× lower than 7a-d, and the LED for inhibition of Rab11 prenylation in vitro was 2-8× lower than for 7a-d. However, in a viability reduction assay with J774 cells, 1 and 7b had similar IC(50) values, ~10× lower than those of 7a and 7c-d.

The gunmetal mouse reveals Rab geranylgeranyl transferase to be the major molecular target of phosphonocarboxylate analogues of bisphosphonates.

The described ability of phosphonocarboxylate analogues of bisphosphonates (BPs) to inhibit Rab geranylgeranyl transferase (RGGT) is thought to be the mechanism underlying their cellular effects, including their ability to reduce macrophage cell viability and to inhibit osteoclast-mediated resorption. However, until now the possibility that at least some of the effects of these drugs may be mediated through other targets has not been excluded. Since RGGT is the most distal enzyme in the process of Rab prenylation, it has not proved possible to confirm the mechanism underlying the effects of these drugs by adding back downstream intermediates of the mevalonate pathway, the approach used to demonstrate that bisphosphonates act through this pathway. We now confirm that RGGT is the major pharmacological target of phosphonocarboxylates by using several alternative approaches. Firstly, analysis of several different phosphonocarboxylate drugs demonstrates a very good correlation between the ability of these drugs to inhibit RGGT with their ability to: (a) reduce macrophage cell viability; (b) induce apoptosis; and (c) induce vacuolation in rabbit osteoclasts. Secondly, we have found that cells from the gunmetal (gm/gm) mouse, which bear a homozygous mutation in RGGT that results in ~80% reduced activity of this enzyme compared to wild-type or heterozygous mice, are more sensitive to the effects of active phosphonocarboxylates (including reducing macrophage cell viability, inhibiting osteoclast formation and inhibiting fluid-phase endocytosis), confirming that these effects are mediated through inhibition of RGGT. In conclusion, these data demonstrate that all of the pharmacological effects of phosphonocarboxylates found thus far appear to be mediated through the specific inhibition of RGGT, highlighting the potential therapeutic value of this class of drugs.

Synthesis, chiral high performance liquid chromatographic resolution and enantiospecific activity of a potent new geranylgeranyl transferase inhibitor, 2-hydroxy-3-imidazo[1,2-a]pyridin-3-yl-2-phosphonopropionic acid.

3-(3-Pyridyl)-2-hydroxy-2-phosphonopropanoic acid (3-PEHPC, 1) is a phosphonocarboxylate (PC) analogue of 2-(3-pyridyl)-1-hydroxyethylidenebis(phosphonic acid) (risedronic acid, 2), an osteoporosis drug that decreases bone resorption by inhibiting farnesyl pyrophosphate synthase (FPPS) in osteoclasts, preventing protein prenylation. 1 has lower bone affinity than 2 and weakly inhibits Rab geranylgeranyl transferase (RGGT), selectively preventing prenylation of Rab GTPases. We report here the synthesis and biological studies of 2-hydroxy-3-imidazo[1,2-a]pyridin-3-yl-2-phosphonopropionic acid (3-IPEHPC, 3), the PC analogue of minodronic acid 4. Like 1, 3 selectively inhibited Rab11 vs. Rap 1A prenylation in J774 cells, and decreased cell viability, but was 33-60x more active in these assays. After resolving 3 by chiral HPLC (>98% ee), we found that (+)-3-E1 was much more potent than (-)-3-E2 in an isolated RGGT inhibition assay, approximately 17x more potent (LED 3 microM) than (-)-3-E2 in inhibiting Rab prenylation in J774 cells and >26x more active in the cell viability assay. The enantiomers of 1 exhibited a 4-fold or smaller potency difference in the RGGT and prenylation inhibition assays.

Single choroideremia gene in nonmammalian vertebrates explains early embryonic lethality of the zebrafish model of choroideremia.

PURPOSE: Mutations of the CHM gene underlie the X-linked chorioretinal degeneration choroideremia (CHM). The affected gene product, Rab Escort Protein (REP)1, mediates the posttranslational prenyl modification of Rab GTPases. In patients with CHM, the related REP2 partially compensates for the loss of function of REP1. The objective of this investigation was to study the natural history of disease in a zebrafish model of CHM. METHODS: Zebrafish chm(-/-) were bred and subjected to extensive histologic analysis and TUNEL assays, and cellular extracts were used for immunoblot and in vitro prenylation assays. A detailed evolutionary analysis was performed on the REP family. RESULTS: The retina of chm(-/-) zebrafish develops normally for the first 4 days postfertilization (dpf) but that catastrophic multilayer degeneration synchronous with severe multisystem disease follows. Mean survival time is 4.8 dpf. At the onset of generalized disease, a significant reduction in rep expression levels and activity, with unprenylated rabs accumulating in the cytosol was demonstrated. Extensive bioinformatic analysis of the REP family of proteins revealed a single rep isoform in fish and other nonmammalian vertebrates and invertebrates that is similar to mammalian REP1. CONCLUSIONS: REP1 appears to be the ancestral gene in the family, whereas the intronless REP2 gene is restricted to the mammalian lineage. The results of this study propose that in chm(-/-) zebrafish, maternally derived rep allows initial successful development of the embryo, but its gradual loss leads to multisystem disease and invariably to lethality. In its current form, the chm(-/-) zebrafish has limited usefulness.

Phosphonocarboxylates inhibit the second geranylgeranyl addition by Rab geranylgeranyl transferase.

Rab geranylgeranyl transferase (RGGT) catalyzes the post-translational geranylgeranyl (GG) modification of (usually) two C-terminal cysteines in Rab GTPases. Here we studied the mechanism of the Rab geranylgeranylation reaction by bisphosphonate analogs in which one phosphonate group is replaced by a carboxylate (phosphonocarboxylate, PC). The phosphonocarboxylates used were 3-PEHPC, which was previously reported, and 2-hydroxy-3-imidazo[1,2-a]pyridin-3-yl-2-phosphonopropionic acid ((+)-3-IPEHPC), a >25-fold more potent related compound as measured by both IC50 and Ki.(+)-3-IPEHPC behaves as a mixed-type inhibitor with respect to GG pyrophosphate (GGPP) and an uncompetitive inhibitor with respect to Rab substrates. We propose that phosphonocarboxylates prevent only the second GG transfer onto Rabs based on the following evidence. First, geranylgeranylation of Rab proteins ending with a single cysteine motif such as CAAX, is not affected by the inhibitors, either in vitro or in vivo. Second, the addition of an -AAX sequence onto Rab-CC proteins protects the substrate from inhibition by the inhibitors. Third, we demonstrate directly that in the presence of (+)-3-IPEHPC, Rab-CC and Rab-CXC proteins are modified by only a single GG addition. The presence of (+)-3-IPEHPC resulted in a preference for the Rab N-terminal cysteine to be modified first, suggesting an order of cysteine geranylgeranylation in RGGT catalysis. Our results further suggest that the inhibitor binds to a site distinct from the GGPP-binding site on RGGT. We suggest that phosphonocarboxylate inhibitors bind to a GG-cysteine binding site adjacent to the active site, which is necessary to align the mono-GG-Rab for the second GG addition. These inhibitors may represent a novel therapeutic approach in Rab-mediated diseases.

Rab geranylgeranylation occurs preferentially via the pre-formed REP-RGGT complex and is regulated by geranylgeranyl pyrophosphate.

Prenylation (or geranylgeranylation) of Rab GTPases is catalysed by RGGT (Rab geranylgeranyl transferase) and requires REP (Rab escort protein). In the classical pathway, REP associates first with unprenylated Rab, which is then prenylated by RGGT. In the alternative pathway, REP associates first with RGGT; this complex then binds and prenylates Rab proteins. In the present paper we show that REP mutants defective in RGGT binding (REP1 F282L and REP1 F282L/V290F) are unable to compete with wild-type REP in the prenylation reaction in vitro. When over-expressed in cells, REP wild-type and mutants are unable to form stable cytosolic complexes with endogenous unprenylated Rabs. These results suggest that the alternative pathway may predominate in vivo. We also extend previous suggestions that GGPP (geranylgeranyl pyrophosphate) acts as an allosteric regulator of the prenylation reaction. We observed that REP-RGGT complexes are formed in vivo and are unstable in the absence of intracellular GGPP. RGGT increases the ability of REP to extract endogenous prenylated Rabs from membranes in vitro by stabilizing a soluble REP-RGGT-Rab-GG (geranylgeranylated Rab) complex. This effect is regulated by GGPP, which promotes the dissociation of RGGT and REP-Rab-GG to allow delivery of prenylated Rabs to membranes.

Rab3GEP is the non-redundant guanine nucleotide exchange factor for Rab27a in melanocytes.

Rab GTPases regulate discrete steps in vesicular transport pathways. Rabs require activation by specific guanine nucleotide exchange factors (GEFs) that stimulate the exchange of GDP for GTP. Rab27a controls motility and regulated exocytosis of secretory granules and related organelles. In melanocytes, Rab27a regulates peripheral transport of mature melanosomes by recruiting melanophilin and myosin Va. Here, we studied the activation of Rab27a in melanocytes. We identify Rab3GEP, previously isolated as a GEF for Rab3a, as the non-redundant Rab27a GEF. Similar to Rab27a-deficient ashen melanocytes, Rab3GEP-depleted cells show both clustering of melanosomes in the perinuclear area and loss of the Rab27a effector Mlph. Consistent with a role as an activator, levels of Rab27a-GTP are decreased in cells lacking Rab3GEP. Recombinant Rab3GEP exhibits guanine nucleotide exchange activity against Rab27a and Rab27b in vitro, in addition to its previously documented activity against Rab3. Our results indicate promiscuity in Rab GEF action and suggest that members of related but functionally distinct Rab subfamilies can be controlled by common activators.

Synthesis and biological evaluation of alpha-halogenated bisphosphonate and phosphonocarboxylate analogues of risedronate.

Alpha-halogenated analogues of the anti-resorptive bisphosphonate risedronate (5, Ris) and its phosphonocarboxylate cognate (7, 3-PEHPC) were synthesized and compared with 5, 7, and the corresponding desoxy analogues in bone mineral affinity and mevalonate pathway inhibition assays. The Ris (5e-h) and 3-PEHPC (7e-h) analogues had decreased bone mineral affinity, confirming that the alpha-OH group in 5 and 7 enhances bone affinity. The 5 alpha-halo-analogues potently inhibited farnesyl pyrophosphate synthase (FPPS) with IC50 values from 16 (alpha-F) to 340 (alpha-Br) nM (5, 6 nM). In contrast, 7 alpha-halo-analogues were ineffective versus FPPS (IC50 > 600 microM), but inhibited Rab geranylgeranyl transferase (RGGT) (IC50 = 16-35 microM) similarly to 7 itself (IC50 = 24 microM). The alpha-F analogue 7e was 1-2 times as active as 7 in J774 cell viability and Rab11 prenylation inhibition assays.

Time-dependent inhibition of isoprenylcysteine carboxyl methyltransferase by indole-based small molecules.

Isoprenylcysteine carboxyl methyltransferase (Icmt) catalyzes the methylation of the C-terminal prenylcysteine found on prenylated proteins. Numerous studies have shown that the methylation step is important for the correct localization and function of many prenylated proteins, most notably GTPases in the Ras superfamily. We recently reported identification of a small molecule derived from an indole core as a potent, cell-active inhibitor of Icmt whose potency was increased upon preincubation with the enzyme [Winter-Vann, A. M., Baron, R. A., et al. (2005) Proc. Natl. Acad. Sci. U.S.A. 102 (12), 4336-41]. In the study presented here, we performed a kinetic characterization of this time-dependent inhibition of Icmt by 2-[5-(3-methylphenyl)-1-octyl-1H-indol-3-yl]acetamide (cysmethynil). These analyses revealed that cysmethynil is a competitive inhibitor with respect to the isoprenylated cysteine substrate and a noncompetitive inhibitor with respect to AdoMet, the methyl donor in the reaction. The Ki of cysmethynil for Icmt, which represents the dissociation constant of the initial complex with the enzyme, was 2.39 +/- 0.02 microM, and the Ki*, which is the overall dissociation constant of the inhibitor for the final complex, was 0.14 +/- 0.01 microM. The first-order rate constant for the conversion of the initial enzyme-inhibitor complex to the final high-affinity complex was 0.87 +/- 0.06 min-1, and that for the reverse process was 0.053 +/- 0.003 min-1; the latter rate constant corresponds to a half-life for the high-affinity complex of 15 min. Structure-activity relationships of a number of closely related indole compounds revealed that the hydrophobicity of the substituent on the nitrogen of the indole core was responsible for the manifestation of time-dependent inhibition. These findings markedly enhance our understanding of the mechanism of inhibition of Icmt by this indole class of compounds and should facilitate ongoing efforts to assess the potential of targeting this enzyme in anticancer drug design.

Quantitative structure-activity relationship (QSAR) of indoloacetamides as inhibitors of human isoprenylcysteine carboxyl methyltransferase.

A QSAR is developed for the isoprenylcysteine carboxyl methyltransferase (ICMT) inhibitory activities of a series of indoloacetamides (n=72) that are structurally related to cysmethynil, a selective ICMT inhibitor. Multivariate analytical tools (principal component analysis (PCA) and projection to latent structures (PLS)), multi-linear regression (MLR) and comparative molecular field analysis (CoMFA) are used to develop a suitably predictive model for the purpose of optimizing and identifying members with more potent inhibitory activity. The resulting model shows that good activity is determined largely by the characteristics of the substituent attached to the indole nitrogen, which should be a lipophilic residue with fairly wide dimensions. In contrast, the substituted phenyl ring attached to the indole ring must be of limited dimensions and lipophilicity.

Rab GTPases containing a CAAX motif are processed post-geranylgeranylation by proteolysis and methylation.

Post-translational modification by protein prenylation is required for membrane targeting and biological function of monomeric GTPases. Ras and Rho proteins possess a C-terminal CAAX motif (C is cysteine, A is usually an aliphatic residue, and X is any amino acid), in which the cysteine is prenylated, followed by proteolytic cleavage of the AAX peptide and carboxyl methylation by the Rce1 CAAX protease and Icmt methyltransferase, respectively. Rab GTPases usually undergo double geranylgeranylation within CC or CXC motifs. However, very little is known about processing and membrane targeting of Rabs that naturally contain a CAAX motif. We show here that a variety of Rab-CAAX proteins undergo carboxyl methylation, both in vitro and in vivo, with one exception. Rab38(CAKS) is not methylated in vivo, presumably because of the inhibitory action of the lysine residue within the AAX motif for cleavage by Rce1. Unlike farnesylated Ras proteins, we observed no targeting defects of overexpressed Rab-CAAX proteins in cells deficient in Rce1 or Icmt, as reported for geranylgeranylated Rho proteins. However, endogenous geranylgeranylated non-methylated Rab-CAAX and Rab-CXC proteins were significantly redistributed to the cytosol at steady-state levels and redistribution correlates with higher affinity of RabGDI for non-methylated Rabs in Icmt-deficient cells. Our data suggest a role for methylation in Rab function by regulating the cycle of Rab membrane recruitment and retrieval. Our findings also imply that those Rabs that undergo post-prenylation processing follow an indirect targeting pathway requiring initial endoplasmic reticulum membrane association prior to specific organelle targeting.

Syntheses of functionalized biotin N-1' derivatives: new tools for the control of gene expression with small molecules.

The exceptionally high affinity of biotin toward avidin and streptavidin is at the basis of (strept)avidin-biotin biotechnology, which has numerous applications in life sciences. Recent biotin developments for in vivo and in vitro acylation of selective targeted protein and intein-mediated site specific protein biotinylation require the free biotin carboxyl function to covalently bind with the targeted protein. However, recently this carboxylic function has been used to substitute biotin with numerous ligands and flags. In the present work, we propose the N-1' labeling possibilities of biotin, keeping the valeric chain free. We describe liquid and solid-phase syntheses of functionalized biotin N-1' derivatives. Although the N-1' modification involves a two-log decrease in affinity, in vitro these molecules kept their high avidin affinity (around 10(-12) M) and the in vivo acylation ability of new biotin derivatives.

Thematic review series: lipid posttranslational modifications. geranylgeranylation of Rab GTPases.

Rab GTPases require special machinery for protein prenylation, which include Rab escort protein (REP) and Rab geranylgeranyl transferase (RGGT). The current model of Rab geranylgeranylation proposes that REP binds Rab and presents it to RGGT. After geranylgeranylation of Rab C-terminal cysteines, REP delivers the prenylated protein to membranes. The REP-like protein Rab GDP dissociation inhibitor (RabGDI) then recycles the prenylated Rab between the membrane and the cytosol. The recent solution of crystal structures of the Rab prenylation machinery has helped to refine this model and provided further insights. The hydrophobic prenyl binding pocket of RGGT and geranylgeranyl transferase type-I (GGT-I) differs from that of farnesyl transferase (FT). A bulky tryptophan residue in FT restricts the size of the pocket, whereas in RGGT and GGT-I, this position is occupied by smaller residues. A highly conserved phenylalanine in REP, which is absent in RabGDI, is critical for the formation of the REP:RGGT complex. Finally, a geranylgeranyl binding site conserved in REP and RabGDI has been identified within helical domain II. The postprenylation events, including the specific targeting of Rabs to target membranes and the requirement for single versus double geranylgeranylation by different Rabs, remain obscure and should be the subject of future studies.

A small-molecule inhibitor of isoprenylcysteine carboxyl methyltransferase with antitumor activity in cancer cells.

Many key regulatory proteins, including members of the Ras family of GTPases, are modified at their C terminus by a process termed prenylation. This processing is initiated by the addition of an isoprenoid lipid, and the proteins are further modified by a proteolytic event and methylation of the C-terminal prenylcysteine. Although the biological consequences of prenylation have been characterized extensively, the contributions of prenylcysteine methylation to the functions of the modified proteins are not well understood. This reaction is catalyzed by the enzyme isoprenylcysteine carboxyl methyltransferase (Icmt). Recent genetic disruption studies have provided strong evidence that blocking Icmt activity has profound consequences on oncogenic transformation. Here, we report the identification of a selective small-molecule inhibitor of Icmt, 2-[5-(3-methylphenyl)-1-octyl-1H-indol-3-yl]acetamide (cysmethynil). Cysmethynil treatment results in inhibition of cell growth in an Icmt-dependent fashion, demonstrating mechanism-based activity of the compound. Treatment of cancer cells with cysmethynil results in mislocalization of Ras and impaired epidermal growth factor signaling. In a human colon cancer cell line, cysmethynil treatment blocks anchorage-independent growth, and this effect is reversed by overexpression of Icmt. These findings provide a compelling rationale for development of Icmt inhibitors as another approach to anticancer drug development.