Protein farnesyltransferase-catalyzed isoprenoid transfer to peptide depends on lipid size and shape, not hydrophobicity

Protein farnesyl transferase (FTase) catalyzes transfer of a 15-carbon farnesyl group from farnesyl diphosphate (FPP) to a conserved cysteine in the C-terminal Ca(1)a(2)X motif of a range of proteins, including the oncoprotein H-Ras ("C" refers to the cysteine, "a" to any aliphatic amino acid, and "X" to any amino acid) and the lipid chain interacts with, and forms part of the Ca(1)a(2)X peptide binding site. Previous studies have shown that H-Ras biological function is ablated when it is modified with lipids that are 3-5 orders of magnitude less hydrophobic than FPP. Here, we employed a library of anilinogeranyl diphosphate (AGPP) and phenoxygeranyl diphosphate (PGPP) derivatives with a range of polarities (log P (lipid alcohol) = 0.7-6.8, log P (farnesol) = 6.1) and shapes to examine whether FTase-catalyzed transfer to peptide is dependent on the hydrophobicity of the lipid. Analysis of steady-state transfer kinetics for analogues to dansyl-GCVLS peptide revealed that the efficiency of lipid transfer was highly dependent on both the shape and size, but was independent of the polarity of the analogue. These observations indicate that hydrophobic features of isoprenoids critical for their association with membranes and/or protein receptors are not required for efficient transfer to Ca(1)a(2)X peptides by FTase. Furthermore, the results of these studies indicate that the role played by the farnesyl lipid in the FTase mechanism is primarily structural. To explain these results we propose a model in which the FTase active site stabilizes a membrane interface-like environment.     

Stereochemistry-dependent inhibition of RAS farnesylation by farnesyl phosphonic acids

This investigation compares the effects of three farnesyl pyrophosphate analogs on selected aspects of isoprenoid metabolism. E,E-α-Hydroxyfarnesylphosphonate was prepared by an improved variation on a literature synthesis, which also gave access to the new Z,E-α-hydroxyfarnesyl- and α-hydroxygeranylphosphonates. A striking find is that only E,E-α-hydroxyfarnesylphosphonate induces alteration of RAS processing in intact human-derived leukemia cells and inhibits farnesyl protein transferase in enzyme assays, while the Z,E-α-farnesyl- and geranylphosphonates are inactive. The inhibitory activity of E,E-α-hydroxyfarnesylphosphonate is greater in enzyme than intact cell assays. This active compound does not significantly inhibit geranylgeranyl protein transferase I or squalene synthase, nor does it diminish cholesterol synthesis. These results indicate that the length of the terpenoid chain and olefin stereochemistry allow selective inhibition of critical enzymes of terpenoid metabolism. Discrimination was observed between inhibition of farnesyl protein transferase and squalene synthase by E,E-α-hydroxyfarnesylphosphonate, even though both enzymes utilize farnesyl pyrophosphate as their natural substrate.     

Protein Prenyltransferases and Their Inhibitors: Structural and Functional Characterization

Protein prenylation is a post-translational modification controlling the localization, activity, and protein–protein interactions of small GTPases, including the Ras superfamily. This covalent attachment of either a farnesyl (15 carbon) or a geranylgeranyl (20 carbon) isoprenoid group is catalyzed by four prenyltransferases, namely farnesyltransferase (FTase), geranylgeranyltransferase type I (GGTase-I), Rab geranylgeranyltransferase (GGTase-II), and recently discovered geranylgeranyltransferase type III (GGTase-III). Blocking small GTPase activity, namely inhibiting prenyltransferases, has been proposed as a potential disease treatment method. Inhibitors of prenyltransferase have resulted in substantial therapeutic benefits in various diseases, such as cancer, neurological disorders, and viral and parasitic infections. In this review, we overview the structure of FTase, GGTase-I, GGTase-II, and GGTase-III and summarize the current status of research on their inhibitors.     

Alteration of product specificity of Aeropyrum pernix farnesylgeranyl diphosphate synthase (Fgs) by directed evolution

Directed evolution of the C25 farnesylgeranyl diphosphate synthase of Aeropyrum pernix (Fgs) was carried out by error-prone PCR with an in vivo color complementation screen utilizing carotenoid biosynthetic pathway enzymes. Screening yielded 12 evolved clones with C20 geranylgeranyl diphosphate synthase activity which were isolated and characterized in order to understand better the chain elongation mechanism of this enzyme. Analysis of these mutants revealed three different mechanisms of product chain length specificity. Two mutants (A64T and A64V) have a single mutation at the 8th amino acid upstream of a conserved first aspartate-rich motif (FARM), which is involved in the mechanism for chain elongation reaction of all prenyl diphosphate synthases. One mutant (A135T) carries a single mutation at the 7th amino acid upstream of another conserved region (141GQ142), which was recently found to be another important region controlling chain elongation of a type III C20 geranylgeranyl diphosphate synthase and Escherichia coli C15 farnesyl diphosphate synthase. Finally, one mutant carrying four mutations (V84I, H88R, I177 M and M191V) is of interest. Molecular modeling, site-directed mutagenesis and in vitro assays of this mutant suggest that product chain-length distribution can be also controlled by a structural change provoked by a cooperative interaction of amino acids.     

Synthesis, structure, and biological activity of des-side chain analogues of 1α,25-dihydroxyvitamin D3 with substituents at C18

An improved synthetic route to 1α,25‐dihydroxyvitamin D₃ des‐side chain analogues 2 a and 2 b with substituents at C18 is reported, along with their biological activity. These analogues display significant antiproliferative effects toward MCF‐7 breast cancer cells and prodifferentiation activity toward SW480‐ADH colon cancer cells; they are also characterized by a greatly decreased calcemic profile. The crystal structure of the human vitamin D receptor (hVDR) complexed to one of these analogues, 20(17→18)‐abeo‐1α,25‐dihydroxy‐22‐homo‐21‐norvitamin D₃ ( 2 a ) reveals that the side chain introduced at position C18 adopts the same orientation in the ligand binding pocket as the side chain of 1α,25‐dihydroxyvitamin D₃.     

Azido and Diazarinyl Analogues of Bis‐Tyrphostin as Asymmetrical Inhibitors of Dynamin GTPase

Probing the dynamin binding site : Bis‐tyrphostin ( 1 , Bis‐T), is a potent inhibitor of the phospholipid‐stimulated GTPase activity of dynamin I. Analogues of Bis‐T have significant potential as a biological probes for the dissection of endocytic pathways. Bis‐T‐derived compounds were synthesised and evaluated for their ability to inhibit the GTPase activity of dynamin I. Two analogues ( 23 and 24 ) represent the first asymmetrically substituted Bis‐T analogues to retain dynamin inhibition.

     

Quantitative Determination of Geranyl Diphosphate Levels in Cultured Human Cells

Geranyl diphosphate (GPP), a 10-carbon isoprenoid, is a key intermediate in the isoprenoid biosynthetic pathway. This pathway, in addition to leading to sterol synthesis, results in the synthesis of farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP), which serve as substrates for protein isoprenylation reactions. Basal levels of GPP in mammalian cells previously have been undetectable. Here we present a novel, sensitive, nonradioactive method which allows for measurement of GPP in mammalian cells. This methodology involves extraction of isoprenoids from cultured cells followed by enzymatic conjugation of GPP to a fluorescent dansylated-peptide via farnesyl transferase and quantification with high-performance liquid chromatography (HPLC). The lower limit of detection of GPP is 5 pg, or 0.015 pmol. Basal levels of GPP were determined in three human multiple myeloma cell lines (RPMI-8226, U266, H929). Treatment of cells with inhibitors of the isoprenoid biosynthetic pathway results in marked changes in GPP levels: the HMG-CoA reductase inhibitor lovastatin decreases GPP levels by over 50%, while the FPP synthase inhibitor zoledronic acid increases GPP levels 16- to 107-fold. This method also allows for the simultaneous measurement of GPP, FPP, and GGPP, thus leading to improved understanding of the pathway in a multitude of biological systems. Furthermore, as drugs targeting this pathway are developed, their biological activity can be more directly linked to effects on isoprenoid levels.     

Synthesis and Biological Evaluation of New (−)‐Englerin Analogues

We report the synthesis and biological evaluation of a series of (?)‐englerin A analogues obtained along our previously reported synthetic route based on a stereoselective gold(I) cycloaddition process. This synthetic route is a convenient platform to access analogues with broad structural diversity and has led us to the discovery of unprecedented and easier‐to‐synthesize derivatives with an unsaturation in the cyclopentyl ring between C4 and C5. We also introduce novel analogues in which the original isopropyl motif has been substituted with cyclohexyl, phenyl, and cyclopropyl moieties. The high selectivity and growth‐inhibitory activity shown by these new derivatives in renal cancer cell lines opens new ways toward the final goal of finding effective drugs for the treatment of renal cell carcinoma (RCC).     

A Family of Related Fungal and Bacterial Di- and Sesterterpenes: Studies on Fusaterpenol and Variediene

The absolute configuration of fusaterpenol (GJ1012E) has been revised by an enantioselective deuteration strategy. A bifunctional enzyme with a terpene synthase and a prenyltransferase domain from Aspergillus brasiliensis was characterised as variediene synthase, and the absolute configuration of its product was elucidated. The uniform absolute configurations of these and structurally related di- and sesterterpenes together with a common stereochemical course for the geminal methyl groups of GGPP unravel a similar conformational fold of the substrate in the active sites of the terpene synthases. For variediene, a thermal reaction observed during GC/MS analysis was studied in detail for which a surprising mechanism was uncovered.     

Biosynthesis of sesqui- and diterpenes by the gibberellin producer Fusarium fujikuroi

The fungus Fusarium fujikuroi IMI58289 emits a complex pattern of volatile terpenoids including two major compounds, the sesquiterpene alcohol α-acorenol and the diterpene ent-kaurene. ent-Kaurene is the precursor for the phytohormone gibberellic acid (GA(3)) and is produced from geranylgeranyl diphosphate (GGPP) via ent-copalyl diphosphate by the bifunctional ent-copalyl diphosphate/ent-kaurene synthase (CPS/KS). Several structurally related diterpenes were identified as side products of the CPS/KS. Deletion of the cps/ks gene or the whole GA(3) biosynthetic gene cluster resulted in completely abolished diterpene production. Mutants with deletions of the cytochrome P450 monooxygenase gene P450-4, which is responsible for the three oxidation steps from ent-kaurene to ent-kaurenoic acid en route to GA(3), accumulate diterpene hydrocarbons. Feeding with [6,6,6-(2) H(3)] mevalonolactone gave insights into the stereochemistry of the GGPP cyclisation, which operates with a chair-chair-"antipodal" fold. A rational biosynthetic scheme for all identified sesquiterpenes demonstrated their formation from farnesyl diphosphate (FPP) via three alternative initial cyclisations. Genome sequencing revealed the presence of five putative sesquiterpene synthase genes in the F. fujikuroi genome. The structures of several trace compounds from other classes have been identified as new natural products; these were delineated from their mass spectra and unambiguously assigned by comparison to synthetic references.     

Characterization of the Rv3377c Gene Product,a Type‐B Diterpene Cyclase,from the Mycobacterium tuberculosis H37 Genome

The Rv3377c gene from the Mycobacterium tuberculosis H37 genome is specifically limited to those Mycobacterium species that cause tuberculosis. We have demonstrated that the gene product of Rv3377c is a diterpene cyclase that catalyzes the formation of tuberculosinol from geranylgeranyl diphosphate (GGPP). However, the characteristics of this enzyme had not previously been studied in detail with homogeneously purified enzyme. The purified enzyme catalyzed the synthesis of tuberculosinyl diphosphate from GGPP, but it did not bring about the synthesis of tuberculosinol. Optimal conditions for the highest activity were found to be as follows: pH 7.5, 30 °C, Mg^II (0.1 mM ), and Triton X‐100 (0.1 %). Under these conditions, the kinetic values of K_M and k_cat were determined to be 11.7±1.9 μM for GGPP and 12.7±0.7 min^?1, respectively, whereas the specific activity was 186 nmol min^?1 mg^?1. The enzyme activity was inhibited at substrate concentrations higher than 50 μM . The catalytic activity was strongly inhibited by 15‐aza‐dihydrogeranylgeraniol and 5‐isopropyl‐N,N,N,2‐tetramethyl‐4‐(piperidine‐1‐carbonyloxy)benzenaminium chloride (Amo‐1618). The DXDTT_293–297 motif, corresponding to the DXDDTA motif conserved among terpene cyclases, was mutated in order to investigate its function. The middle D295 was found to be the most crucial entity for the catalysis. D293 and two threonine residues function synergistically to enhance the acidity of D295, possibly through hydrogen‐bonding networks. The Rv3377c enzyme could also react with (14R/S)‐14,15‐oxidoGGPP to generate 3α‐ and 3β‐hydroxytuberculosinyl diphosphate. Conformational analyses were carried out with deuterium‐labeled GGPP and oxidoGGPP. We found that GGPP and (14R)‐oxidoGGPP adopted a chair/chair conformation, but (14S)‐oxidoGGPP adopted a boat/chair conformation. Interestingly, the conformations of oxidoGGPP for the A‐ring formation are the opposite of those of oxidosqualene when it is used as a substrate by squalene cyclases for the biosynthesis of hopene and tetrahymanol. (3R)‐Oxidosqualene is folded in a boat conformation, whereas (3S)‐2,3‐oxidosqualene folds into a chair conformation, for the formation of the A‐rings of the hopene and tetrahymanol skeletons, respectively.     

Synthesis and pharmacological evaluation of 8- and 9-substituted benzolactam-v8 derivatives as potent ligands for protein kinase C, a therapeutic target for Alzheimer's disease

A central element in the pathophysiology of Alzheimer's disease (AD) is the formation of amyloid plaques, which result from abnormal processing of the amyloid precursor protein (APP). The processing of APP is largely provided by three key enzymes, namely the alpha-, beta-, and gamma-secretases. As the latter two contribute to the formation of neurotoxic Abeta fragments while alpha-secretase does not, a decrease in the amyloidogenic products can be brought about either by inhibition of the beta- and gamma-secretases or through the activation of alpha-secretase. It is now known that the activation of protein kinase C (PKC) enhances alpha-secretase activity and therefore represents a possible target for the development of agents urgently needed for the treatment of this devastating neurodegenerative disorder. In the present study, new benzolactam-V8-based PKC activators were synthesized and tested for their binding affinity toward PKCalpha. All compounds tested showed binding values in the nanomolar concentration range. In accordance with previous publications, 9-substitution dramatically increased PKC binding affinity in comparison with the corresponding 8-substituted analogues. In addition to the location of the side chain on the aromatic ring, the binding affinities of these benzolactams were found to depend on the orientation, length, and electronic properties of this appendage. An interesting decrease in binding affinity was found for the 9-thienyl analogue 13, suggesting adverse electronic interactions of the sulfur atom with PKC or parts of the cellular membrane.     

Apolipoprotein E polymorphism: Automated determination of apolipoprotein E2, E3, and E4 isoforms

Apolipoprotein E (apo E) plays an essential role in lipoprotein metabolism, where it is involved in the clearance of chylomicrons and very low density lipoproteins. Apart from some rate variants, apo E exists in three common isoforms (E2, E3, and E4). The different isoforms have not only been associated with different plasma lipid levels but have also been correlated with certain pathological conditions, such as lipid disorders (dysbetalipoproteinemia, hypercholesterolemia), cardiovascular diseases, and Alzheimer’s disease. Here we describe a rapid, automated test for the determination of the most frequent polymorphisms (E2, E3, and E4). This polymerase chain reaction-based test allows the reliable discrimination of all six genotypes. The assay has been developed especially for the nonspecialized routine clinical laboratory by employing an analyzer and chemistry often present in this type of laboratory. Because of its low costs and easy handling, the assay can be performed on a daily basis.     

Semisynthetic Vitamin E Derivatives as Potent Antibacterial Agents against Resistant Gram-Positive Pathogens

New 5-substituted vitamin E derivatives were semisynthesized, and their antibacterial activity against human Gram-positive and Gram-negative pathogens was evaluated. Several vitamin E analogues were active against methicillin-resistant Staphylococcus aureus (MRSA) and/or methicillin-resistant Staphylococcus epidermidis (MRSE); structure-activity relationships (SARs) are discussed. As a result, it is shown that the presence of a carboxylic acid function at the C-5 position and/or at the end of the side chain is crucial for the antibacterial activity. The bactericidal or bacteriostatic action of three compounds against MRSA and MRSE was confirmed in a time-kill kinetics study, and the cytotoxicity on human cells was evaluated. The preliminary mechanism study by confocal microscopy indicated that those vitamin E analogues led to bacterial cell death through membrane disruption.     

Identification and Characterization of the Streptazone E Biosynthetic Gene Cluster in Streptomyces sp. MSC090213JE08

Streptazone derivatives isolated from Streptomyces species are piperidine alkaloids with a cyclopenta[b]pyridine scaffold. Previous studies indicated that these compounds are polyketides, but the biosynthetic enzymes responsible for their synthesis are unknown. Here, we have identified the streptazone E biosynthetic gene cluster in Streptomyces sp. MSC090213JE08, which encodes a modular type I PKS and tailoring enzymes that include an aminotransferase, three oxidoreductases, and two putative cyclases. The functions of the six tailoring enzymes were analyzed by gene disruption, and two putative biosynthetic intermediates that accumulated in particular mutants were structurally elucidated. On the basis of these results, we propose a pathway for the biosynthesis of streptazone E in which the two putative cyclases of the nuclear transport factor 2–like superfamily are responsible for C?C bond formation coupled with epoxide ring opening to give the five‐membered ring of streptazone E.     

Famesylamine: An inhibitor of famesylation and growth ofras-transformed cells

Farnesylamine, an analogue of farnesol, was shown to inhibit growth of PAP2 cells (ras-transformed NIH 3T3 cells) in a dose-dependent manner. This inhibition was overcome by adding farnesol to the culture medium, but not by adding geranylgeraniol, squalene, cholesterol, dolichol, myristic acid or palmitic acid. Farnesylamine inhibited both farnesyl/protein transferase and geranylgeranyl/protein transferase in whole cell extracts and also inhibited the prenylation of proteins, particularlyras p21, in PAP2 cells. Inhibition of prenylation was associated with increased biosynthesis of other products of the mevalonate biosynthetic pathway. These observations suggest that inhibition of the growth of PAP2 cells by farnesylamine may be due to blocking ofras-mediated signal transduction. This offers a means of investigating mechanisms involved inras action and raises the possibility of developing novel strategies for anticancer therapy.     

5‐Stabilized Phosphatidylinositol 3,4,5‐Trisphosphate Analogues Bind Grp1 PH,Inhibit Phosphoinositide Phosphatases,and Block Neutrophil Migration

Metabolically stabilized analogues of PtdIns(3,4,5)P₃ have shown long‐lived agonist activity for cellular events and selective inhibition of lipid phosphatase activity. We describe an efficient asymmetric synthesis of two 5‐phosphatase‐resistant analogues of PtdIns(3,4,5)P₃, the 5‐methylene phosphonate (MP) and 5‐phosphorothioate (PT). Furthermore, we illustrate the biochemical and biological activities of five stabilized PtdIns(3,4,5)P₃ analogues in four contexts. First, the relative binding affinities of the 3‐MP, 3‐PT, 5‐MP, 5‐PT, and 3,4,5‐PT₃ analogues to the Grp1 PH domain are shown, as determined by NMR spectroscopy. Second, the enzymology of the five analogues is explored, showing the relative efficiency of inhibition of SHIP1, SHIP2, and phosphatase and tensin homologue deleted on chromosome 10 (PTEN), as well as the greatly reduced ability of these phosphatases to process these analogues as substrates as compared to PtdIns(3,4,5)P₃. Third, exogenously delivered analogues severely impair complement factor C5a‐mediated polarization and migration of murine neutrophils. Finally, the new analogues show long‐lived agonist activity in mimicking insulin action in sodium transport in A6 cells.     

Exploiting the substrate tolerance of farnesyltransferase for site-selective protein derivatization

The site-selective modification of proteins with a functional group is an important biochemical technique, but covalent attachment of a desired group to a chosen site is complicated by the reactivity of other amino acid side chains, often resulting in undesired side reactions. One potential solution to this problem involves exploiting the activity of protein-modifying enzymes that recognize a defined protein sequence. Protein farnesyltransferase (FTase) covalently attaches an isoprenoid moiety to a cysteine unit in the context of a short C-terminal sequence that can be easily grafted onto recombinant proteins. Here we describe the synthesis of four phosphoisoprenoids functionalized with biotin, azide, or diene groups. These phosphoisoprenoids bound to FTase with affinities comparable to that of the native substrate. With the exception of the biotin-functionalized analogue, all the phosphoisoprenoids generated could be transferred to peptide and protein substrates by FTase. Unlike proteins modified with farnesyl moieties, Ypt7 prenylated with (2E,6E)-8-(azidoacetamido)-3,7-dimethylocta-2,6-dienyl groups did not oligomerize and showed no detectable increase in hydrophobicity. To assess the suitability of the functionalized isoprenoids for protein modifications they were further derivatized, both by Diels-Alder cycloaddition with 6-maleimidohexanoic acid and by Staudinger ligation with a phosphine. We demonstrate that the Staudinger ligation proceeds more rapidly and is more efficient than the Diels-Alder cycloaddition. Our data validate the use of FTase as a protein-modification tool for biochemical and biotechnological applications.     

Insight into C35 terpene Biosyntheses by Nonpathogenic Mycobacterium Species: Functional Analyses of Three Z‐Prenyltransferases and Identification of Dehydroheptaprenylcyclines

Nonpathogenic Mycobacterium species produce rare cyclic C₃₅ terpenes that are biosynthesized by cyclization of Z‐type C₃₅ polyprenyl diphosphate. To provide deeper insight into the biosynthesis of C₃₅ terpenes, we carried out functional analyses of three Z‐prenyltransferase homologues in M. vanbaalenii identified by genomic analysis. Mvan_3822, a novel bifunctional Z‐prenyltransferase, biosynthesizes C₃₅‐heptaprenyl diphosphate as a main product from (E,E)‐farnesyl diphosphate (E,E‐FPP) and (E,E,E)‐geranylgeranyl diphosphate (E,E,E‐GGPP), but produces a C₅₀‐decaprenyl diphosphate from geranyl diphosphate. Mvan_1705 is a novel Z,E,E‐GGPP synthase. In addition, novel cyclic C₃₅ terpenes, (14E)‐ and (14Z)‐dehydroheptaprenylcycline, were identified as minor metabolites in nonpathogenic Mycobacterium cells. C₃₅ terpenes could be biosynthesized by two routes, in which E and Z geometric isomers of heptaprenyl diphosphate are produced from E,E‐FPP and E,E,E‐GGPP, and the prenylreductase responsible for the biosynthesis of C₃₅ terpenes could reduce both E and Z prenyl residues.     

Discovery of Anti-tubercular Analogues of Bedaquiline with Modified A-, B- and C-Ring Subunits

To date, the clinical use of the anti-tubercular therapy bedaquiline has been somewhat limited due to safety concerns. Recent investigations determined that modification of the B- and C-ring units of bedaquiline delivered new diarylquinolines (for example TBAJ-587) with potent anti-tubercular activity yet an improved safety profile due to reduced affinity for the hERG channel. Building on our recent discovery that substitution of the quinoline motif (the A-ring subunit) for C5-aryl pyridine groups within bedaquiline analogues led to retention of anti-tubercular activity, we investigated the concurrent modification of A-, B- and C-ring units within bedaquiline variants. This led to the discovery that 4-trifluoromethoxyphenyl and 4-chlorophenyl pyridyl analogues of TBAJ-587 retained relatively potent anti-tubercular activity and for the 4-chlorophenyl derivative in particular, a significant reduction in hERG inhibition relative to bedaquiline was achieved, demonstrating that modifications of the A-, B- and C-ring units within the bedaquiline structure is a viable strategy for the design of effective, yet safer (and less lipophilic) anti-tubercular compounds.