Phosphatidylinositol Analogues Research Articles

Cotreatment with a novel phosphoinositide analogue inhibitor and carmustine enhances chemotherapeutic efficacy by attenuating AKT activity in gliomas

Heightened activity of the AKT signaling pathway is prominent in malignant gliomas and has been suggested to play a role in treatment resistance. Selective targeting of AKT, therefore, may increase chemosensitivity. Recently, a novel class of AKT-selective inhibitors has been described, including SH-6, a phosphatidylinositol analogue. The effects of SH-6 on AKT signaling were tested in glioma cells, and the putative role of AKT signaling in chemoresistance was tested by attenuating AKT signaling pharmacologically and genetically. The initial characterization of SH-6 included treatment of glioma cells with increasing doses of SH-6 (0.30-30 microM) and examining the effects on AKT signaling proteins by Western blot analyses and in kinase assays with immunoprecipitated AKT1. Dose-response studies with SH-6 administered to glioma cell lines were performed using a luminescent cell-viability assay (0.1-30 microM). Studies examining the effect of carmustine, either alone or in combination with either the phosphatidylinositol 3-kinase inhibitor LY294002 or SH-6, were performed by cell viability assays and clonogenic survival assays. The effect of carmustine on AKT activity as a response to treatment also was examined. Caspase assays were used to examine the potential role of apoptosis in SH-6/ carmustine -elicited cell death. Finally, the induction of a dominant-negative AKT1 transgene was used in combination with carmustine to demonstrate the role of AKT1 in carmustine chemoresistance. Serum-stimulated phosphorylation of AKT1 was inhibited by SH-6 at doses > or =10 microM (>70% decrease in Threonine 308 and Serine 473 phosphorylation of AKT1). In adenosine triphosphate assays, 72 hours of treatment with SH-6 led to 50% lethal doses near 10 microM for 2 cell lines tested. SH-6 enhancement of carmustine-mediated cell death led to synergistic increases in Caspase 3/Capsase 7 activity, implicating apoptosis as the cell death mechanism. In clonogenic assays, SH-6 cotreatment with carmustine significantly decreased the number of colonies at 10 microM (P < .05) compared with carmustine alone. No decrease was observed in cells that were treated with SH-6 alone (10 microM). LY294002 (10 microM) was also able to enhance the effects of carmustine significantly in both cell lines. In the current study, the authors characterized the efficacy of a new class of adjuvant chemotherapeutics that show promise in enhancing the efficacy of standard chemotherapy regimens in gliomas.

Streamlined Synthesis of Phosphatidylinositol (PI), PI3P, PI3,5P2, and Deoxygenated Analogues as Potential Biological Probes

Highly direct total syntheses of phosphatidylinositol (PI), phosphatidylinositol-3-phosphate (PI3P), phosphatidylinositol-3,5-bisphosphate (PI3,5P2), and a range of deoxygenated versions are reported. Each synthesis is carried out to deliver the target in optically pure form. The key step for each synthesis is a catalytic asymmetric phosphorylation reaction that affects desymmetrization of an appropriate myo-inositol precursor. Elaboration to each target compound is then carried out employing a diversity-oriented strategy from the common precursors. In addition to three natural products, several additional streamlined total syntheses of deoxygenated PI analogues are reported. These syntheses set the stage for high-precision biological investigations of polar headgroup/biological target interactions of these membrane-associated signaling molecules.

Synthesis and Biological Activity of Phospholipase C-Resistant Analogues of Phosphatidylinositol 4,5-bisphosphate

The membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) is an important regulator in cell physiology. Hydrolysis of PtdIns(4,5)P2 by phospholipase C (PLC) releases two second messengers, Ins(1,4,5)P3 and diacylglycerol. To dissect the effects of PtdIns(4,5)P2 from those resulting from PLC-generated signals, a metabolically stabilized analogue of PtdIns(4,5)P2 was required. Two analogues were designed in which the scissile O-P bond was replaced with a C-P bond that could not be hydrolyzed by PLC activity. Herein we describe the asymmetric total synthesis of the first metabolically stabilized phospholipase C-resistant analogues of PtdIns(4,5)P2. The key transformation was a Pd(0)-catalyzed coupling of a H-phosphite with a vinyl bromide to form the desired C-P linkage. The phosphonate analogues of PtdIns(4,5)P2 were found to be effective in restoring the sensitivity of the TRPM4 channel to Ca2+ activation.

Activation of Akt (protein kinase B) stimulates metaphase I to metaphase II transition in bovine oocytes

In somatic cells, the serine/threonine kinase Akt (or protein kinase B) was shown to contribute to processes linked to cellular growth, cell survival and cell cycle regulation. In contrast to these findings, the function of Akt during the meiosis of mammalian oocytes remains to be investigated. We analysed the phosphorylation pattern and the activity of Akt during meiotic maturation (transition from prophase I to metaphase II) of bovine oocytes. The oocytes were matured in vitro (IVM) for 0, 10 and 24 h to reach the germinal vesicle (GV), metaphase I (M I) and metaphase II (M II) stages respectively. The abundance and phosphorylation pattern of Akt was revealed by Western blotting using total Akt or phosphoso-Akt-specific antibodies. The activity of this particular kinase was determined by an in vitro kinase assay. Furthermore, functional properties were analysed by cultivating oocytes in the presence of the Akt inhibitor SH6. The results showed that the overall abundance of Akt did not change significantly during IVM. On the other hand, Akt became phosphorylated at Thr 308 and Ser 473, reaching its maximum at the M I phase. In the GV and M II stages, only low basal phosphorylation levels were observed on both sides. This phosphorylation profile corresponded strictly to the activity of the kinase. The cultivation of oocytes in the presence of the phosphatidylinositol analogue SH6 for 24 h showed that, with higher concentrations, up to 65% of the oocytes were arrested in the M I stage. This result indicated that Akt is involved in the M I/M II transition during the meiotic maturation of bovine oocytes. The physiological aspects of the Akt function will be discussed.

Identification of N10-Substituted Phenoxazines as Potent and Specific Inhibitors of Akt Signaling

A series of 30 N10-substituted phenoxazines were synthesized and screened as potential inhibitors of Akt. In cellular assays at 5 mum, 17 compounds inhibited insulin-like growth factor 1 (IGF-I)-stimulated phosphorylation of Akt (Ser-473) by at least 50% but did not inhibit IGF-I-stimulated phosphorylation of Erk-1/2 (Thr-202/Tyr-204). Substitutions at the 2-position (Cl or CF3) did not alter inhibitory activity, whereas N10-substitutions with derivatives having acetyl (20B) or morpholino (12B) side chain lost activity compared with propyl or butyl substituents (7B and 14B). Inhibition of Akt phosphorylation was associated with the inhibition of IGF-I stimulation of the mammalian target of rapamycin phosphorylation (Ser-2448 and Ser-2481), phosphorylation of p70 S6 kinase (Thr-389), and ribosomal protein S6 (Ser-235/236) in Rh1, Rh18, and Rh30 cell lines. The two most potent compounds 10-[4'-(N-diethylamino)butyl]-2-chlorophenoxazine (10B) and 10-[4'-[(beta-hydroxyethyl)piperazino]butyl]-2-chlorophenoxazine (15B) (in vitro, IC50 approximately 1-2 microM) were studied further. Inhibition of Akt phosphorylation correlated with inhibition of its kinase activity as determined in vitro after immunoprecipitation. Akt inhibitory phenoxazines did not inhibit the activity of recombinant phosphatidylinositol 3'-kinase, PDK1, or SGK1 but potently inhibited the kinase activity of recombinant Akt and Akt deltaPH, a mutant lacking the pleckstrin homology domain. Akt inhibitory phenoxazines blocked IGF-I-stimulated nuclear translocation of Akt in Rh1 cells and suppressed growth of Rh1, Rh18, and Rh30 cells (IC50 2-5 microM), whereas "inactive" derivatives were > or = 10-fold less potent inhibitors of cell growth. In contrast to rapamycin analogs, Akt inhibitory phenoxazines induced significant levels of apoptosis under serum-containing culture conditions at concentrations of agent consistent with Akt inhibition. Thus, the cellular responses to phenoxazine inhibitors of Akt appear qualitatively different from the rapamycin analogs. Modeling studies suggest inhibitory phenoxazines may bind in the ATP-binding site, although ATP competition studies were unable to distinguish between competitive and noncompetitive inhibition.

Phospholipase D2 stimulates integrin-mediated adhesion via phosphatidylinositol 4-phosphate 5-kinase Iγb

Cellular adhesion can be regulated by, as yet, poorly defined intracellular signalling events. Phospholipase D enzymes generate the messenger lipid phosphatidate and here we demonstrate that suppression of this reaction inhibits cellular adhesion. This effect was reversed by the addition of cell-permeable analogues of either phosphatidate or phosphatidylinositol 4,5-bisphosphate. By contrast, neither diacylglycerol nor lysophosphatidic acid were able to reverse this effect suggesting that phosphatidate itself acts directly on a target protein(s) to regulate adhesion rather than as the result of its conversion to either of these metabolite lipids. Antibodies that block beta1 and beta2 integrin-substrate interactions inhibited adhesion stimulated by both phosphatidate and phosphatidylinositol 4,5-bisphosphate indicating that these lipids regulate beta1 and beta2 integrin-mediated adhesion. In vivo, these lipids can be generated by phospholipase D2 and phosphatidylinositol 4-phosphate 5-kinase Igamma b, respectively, and over-expression of catalytically-functional forms of these enzymes dose-dependently stimulated adhesion while siRNA depletion of PLD2 levels inhibited adhesion. Furthermore the ability of over-expressed phospholipase D2 to stimulate adhesion was inhibited by a dominant-negative version of phosphatidylinositol 4-phosphate 5-kinase Igamma b. Consistent with this, phosphatidylinositol 4-phosphate 5-kinase Igamma b-mediated adhesion was dependent upon phospholipase D2's product, phosphatidate indicating that phosphatidylinositol 4-phosphate 5-kinase Igamma b is downstream of, and necessary for, phospholipase D2's regulation of adhesion. It is likely that this phospholipase D2-generated phosphatidate directly stimulates phosphatidylinositol 4-phosphate 5-kinase Igamma b to generate phosphatidylinositol 4,5-bisphosphate as this mechanism has previously been demonstrated in vitro. Thus, our data indicates that during the initial stages of adhesion, phospholipase D2-derived phosphatidate stimulates phosphatidylinositol 4-phosphate 5-kinase Igamma b to generate phosphatidylinositol 4,5-bisphosphate and that consequently this inositol phospholipid promotes adhesion through its regulation of cell-surface integrins.

The Akt/PKB Family of Protein Kinases: A Review of Small Molecule Inhibitors and Progress Towards Target Validation

This article describes recent advances in the development and biological evaluation of small molecule inhibitors for the serine/threonine kinase Akt (PKB). Akt plays a pivotal role in cell survival and proliferation through a number of downstream effectors. Recent studies indicate that unregulated activation of the PI3K/Akt pathway is a prominent feature of many human cancers and Akt is over-expressed or activated in all major cancers. Akt is considered an attractive target for chemotherapy and it has been postulated that inhibition of Akt alone or in combination with standard cancer chemotherapeutics will reduce the apoptotic threshold and preferentially kill cancer cells. The development of specific and potent inhibitors will allow this hypothesis to be tested in animals. The majority of small molecule inhibitors in this nascent field are classic ATP-competitive inhibitors which provide little specificity. Phosphatidylinositol (PI) analogs have been reported to inhibit Akt, but these inhibitors may also have specificity problems with respect to other PH domain containing proteins and may have poor bioavailability. None of the inhibitors in these classes have been reported to have Akt isozyme specificity. Recently, novel allosteric inhibitors have been reported which are pleckstrin homology domain dependent and exhibit Akt isozyme selectivity. Inhibitors in this class may have sufficient potency and specificity to test for tumor efficacy in animal models and recently reported preliminary experiments are reviewed.

Synthesis of Antitumor Phosphatidylinositol Analogues from Glucose by the Use of Ring‐Closing Olefin Metathesis.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Synthesis of anti-tumour phosphatidylinositol analogues from glucose by the use of ring-closing olefin metathesis.

A divergent strategy is described for synthesis of the novel phosphatidylinositols 1-3. The synthetic approach commences from benzyl-protected methyl 6-iodo-6-deoxy-alpha-D-glucopyranoside, which undergoes zinc-mediated reductive fragmentation followed by vinyl Grignard addition and ring-closing metathesis to afford the key conduritol B intermediate 7. This can trifurcate to form three different benzyl-protected myo-inositol headgroups 4-6, which after phosphorylation and attachment of the glycerolipid part give phosphatidylinositols 1-3. Preliminary biological testing against human colon adenocarcinoma cells reveals that analogues 1-3 are significant anti-tumour agents.

Novel PI analogues selectively block activation of the pro-survival serine/threonine kinase Akt.

The synthesis from l-quebrachitol of a series of 3-deoxygenated ether lipid-type phosphatidylinositol (PI) analogues is reported, that selectively block activation of Akt and downstream substrates without affecting activation of the upstream kinase, PDK-1, or other kinases downstream of ras such as MAPK in H157 and H1703 lung cancer cells that have high levels of constitutively active Akt. The 2-hydroxyl in these compounds was deleted or alkylated with the intent to preclude metabolic degradation of these compounds by PI-specific phospholipase C (PI-PLC). PI analogues with phosphate linkers are more effective than those with carbonate linkers. Specific inhibition of Akt by these compounds validates ligand design targeted to the PH domains of crucial signaling proteins, thus providing a unique class of possible cancer therapeutics.

Synthesis and biological evaluation of a PtdIns(4,5)P2 and a phosphatidic acid affinity matrixDedicated to the memory of the late Professor Roy Gigg.

Analogues of dipalmitoyl phosphatidic acid (PA), dilauroyl PA and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] were synthesised and immobilised onto a solid support, Affi-Gel 10. Using them as affinity matrices, a number of known proteins as well as a set of novel proteins were found to bind specifically to PA.

3-(Hydroxymethyl)-bearing phosphatidylinositol ether lipid analogues and carbonate surrogates block PI3-K, Akt, and cancer cell growth.

Phosphatidylinositol 3-kinase (PI3-K) phosphorylates the 3-position of phosphatidylinositol to give rise to three signaling phospholipids. Binding of the pleckstrin homology (PH) domain of Akt to membrane PI(3)P's causes the translocation of Akt to the plasma membrane bringing it into contact with membrane-bound Akt kinase (PDK1 and 2), which phosphorylates and activates Akt. Akt inhibits apoptosis by phosphorylating Bad, thus promoting its binding to and blockade of the activity of the cell survival factor Bcl-x. Herein we present the synthesis and biological activity of several novel phosphatidylinositol analogues and demonstrate the ability of the carbonate group to function as a surrogate for the phosphate moiety. Due to a combination of their PI3-K and Akt inhibitory activities, the PI analogues 2, 3, and 5 proved to be good inhibitors of the growth of various cancer cell lines with IC(50) values in the 1-10 microM range. The enhanced Akt inhibitory activity of the axial hydroxymethyl-bearing analogue 5 compared to its equatorial counterpart 6 is rationalized based upon postulated differences in the H-bonding patterns of these compounds in complex with a homology modeling generated structure of the PH domain of Akt. This work represents the first attempt to examine the effects of 3-modified PI analogues on these two crucial cell signaling proteins, PI3-K and Akt, in an effort to better understand their cell growth inhibitory properties.

Phosphorylation of Unnatural Phosphatidylinositols with Phosphatidylinositol 3-Kinase

Phosphatidylinositol analogs (PI C2–PI C18) having a series of saturated fatty acid (C2–C18) at sn-2 position were synthesized and subjected to the phosphorylation reaction with phosphatidylinositol 3-kinase (PI 3-kinase). The reactivity of PI C8 with PI 3-kinase turned out to be comparable to that of natural PI, although PI C18 was not phosphorylated under the same condition. The chirality of sn-2 center was not responsible for the phosphorylation reaction.

Synthesis of deoxy phosphatidylinositol analogues and phosphonate isosters of Ins(1,4,5)P 3

The synthesis of phosphatidylinositol analogues, 6-deoxy Ins 1-(1,2-di- O-palmitoyl- sn-glycero)phosphate and 4,5-bisphosphate derivatives is presented. Two series of phosphonate isosters, 6-deoxy Ins(1)-butylphosphonate and 6-deoxy Ins(1)- C-methylenephosphonate as well as its 4,5-bisphosphate analogue were also prepared. All phosphoinositide analogues were obtained from cyclohexanone polyol derived from the d-galactose. Modification of charge distribution at position 1 of PtdIns and InsP derivatives, by replacement of a POH group by an alkyl substitution or a PC bond, resistant to cleavage by lipases, could induce inhibition of activity at further strategic enzymatic levels of the inositide cascade.

Identification of a Novel Catalytic Triad with Dual Functions in Enzymatic Cleavage of the P−O Bond

We report identification of a novel Arg-Asp-His catalytic triad that serves dual functionssphosphate activation and leaving group protonationsto catalyze the P-O bond cleavage for phosphatidylinositol-specific phospholipase C (PI-PLC) from B. thuringiensis. PI-PLC cleaves the P-O bond of phosphatidylinositol (PI) to form inositol 1,2-cyclic phosphate (IcP), which is then slowly hydrolyzed to inositol 1-phosphate.1 X-ray structure2 along with site-directed mutagenesis2,3 led to the mechanism in Figure 1A, which involves three elements: His32 as a general base assisted by Asp274, His82 as a general acid, and Arg69, which activates the phosphate group. We recently added two additional aspects to the mechanism: that Arg69 activates the phosphate by hydrogen bonding to the pro-S oxygen at the transition state,4,5 and that Asp33 and His82 function intimately together as a “composite general acid” (Figure 1B).6,7 We now further show that the functions of Asp33 and Arg69 are also inseparable, which led us to propose the mechanism involving the novel catalytic triad (Figure 1C). Our conclusions were achieved by rigorously cross-examining the bridging and nonbridging “thio-effects” (kO/kS ratios)6-9 of PI-PLC and its mutants with PI analogues shown in Figure 2. Both types of thio-effects are usually >1 in enzymatic reactions since sulfur weakens potential hydrogen bonding10 and also introduces possible steric11 and charge8 differences. The usefulness of the nonbridging thio-effect lies mainly in the comparison of the enzyme activity toward Rpand Sp-isomers. Bridging thioeffects are more difficult to interpret since they are directly related to the bond being cleaved, and P-S bonds are much weaker than P-O bonds (i.e. kO/kS is ,1 in chemical reactions).12 The maximal activities of WT, D33N, and D33A toward substrate analogues are listed in Table 1. While the rates depend on the detergent used to disperse the substrate (see footnote b), all data in Table 1 were obtained under the same conditions with dihexanoyl lecithin as a detergent. Furthermore, the analogues

Synthesis of diacylglycerol analogs of phosphatidylinositol 3,4,5-trisphosphate

Phosphatidylinositol 3,4,5-trisphosphate analogs with saturated diacylglycerol substructure have been designed, focusing on their feactivity with PIP3 5-phosphatase. Dephosphorylation of native PIP3 was competitively inhibited in the presence of synthetic PIP3 C2 and PIP3 C4, respectively.

Synthesis of fluorescent phosphatidylinositols using a novel inositol H-phosphonate

Abstract Coupling of 1,2-diradyl-sn-glycerol 5 with the novel inositol H-phosphonate derivative, 6-O-benzyl-2,3:4,5-di-O-isopropylidene-myo-inositol H-phosphonate (3), gave fluorescent analogs of phosphatidylinositol (PtdIns, 1) and PtdIns(4,5)-bisphosphate (PtdIns(4,5)P2, 2). Unlike the corresponding phosphoramidate, 3 was stable at −20 °C for several months, making it a useful intermediate for the synthesis of myo-inositol phospholipids.

Probing the phosphoinositide 4,5-bisphosphate binding site of human profilin I.

Profilin is a widely and highly expressed 14 kDa protein that binds actin monomers, poly(L-proline) and polyphosphoinositol lipids. It participates in regulating actin-filament dynamics that are essential for many types of cell motility. We sought to investigate the site of interaction of profilin with phosphoinositides. Human profilin I was covalently modified using three tritium-labeled 4-benzoyldihydrocinnamoyl (BZDC)-containing photoaffinity analogs of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2). The P-1-tethered D-myoinositol 1,4,5-trisphosphate (Ins(1,4,5)P3) modified profilin I efficiently and specifically; the covalent labeling could be displaced by co-incubation with an excess of PtdIns(4,5)P2 but not with Ins(1,4,5)P3. The acyl-modified PtdIns(4,5)P2 analog showed little protein labeling even at very low concentrations, whereas the head-group-modified PtdIns(4,5)P2 phosphotriester-labeled monomeric and oligomeric profilin. Mass spectroscopic analyses of CNBr digests of [3H]BZDC-Ins(1,4,5)P3-modified recombinant profilin suggested that modification was in the amino-terminal helical CNBr fragment. Edman degradation confirmed Ala1 of profilin I (residue 4 of the recombinant protein) was modified. Molecular models show a minimum energy conformation in which the hydrophobic region of the ligand contacts the amino-terminal helix whereas the 4,5-bisphosphate interacts with Arg135 and Arg136 of the carboxy-terminal helix. The PtdIns(4,5)P2-binding site of profilin I includes a bisphosphate interaction with a base-rich motif in the carboxy-terminal helix and contact between the lipid moiety of PtdIns(4,5)P2 and a hydrophobic region of the aminoterminal helix of profilin. This is the first direct evidence for a site of interaction of the lipid moiety of a phosphoinositide bisphosphate analog with profilin.

ChemInform Abstract: Synthesis of Phosphorothioate Analogues of Phosphatidylinositol 3,4,5‐ Trisphosphate.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Structural features of ether lipids in the archaeobacterial thermophilesPyrococcus furiosus,Methanopyrus kandleri,Methanothermus fervidus, andSulfolobus acidocaldarius

The ether lipids of several thermophilic archaea (archaeobacteria) were compared by negative-ion fast atom bombardment mass spectrometry. The major polar lipids in extracts of Pyrococcus furiosus were assigned as archaeol lipids (phosphatidylglycerol diether, m/z 805; phosphatidylinositol diether, m/z 893; and diglycosyl diether, m/z 975) and caldarchaeol lipids (diglycosyl phosphatidylglycerol tetraether, m/z 1778; and diglycosyl phosphatidylinositol tetraether, m/z 1866). The polar lipids of Methanopyrus kandleri were primarily glycolipids consisting of a series of archaeol lipids with one to six hexose units, composed primarily of mannose (mannose:glucose 9:1); phospholipids consisting of archaeol lipids (phosphatidylinositol diether; and a novel phosphatidylcholine diether, m/z 802.7), and phosphoglycolipids as minor caldarchaeol lipids (primarily diglycosyl phosphatidylglycerol tetraether). Methanothermus fervidus extracts contained archaeol lipids (phosphatidylinositol diether; diglycosyl diether; and acetyldiglycosyl diether, m/z 1016), and caldarchaeol lipids (glycosyl phosphatidylinositol tetraether, m/z 1704; diglycosyl phosphatidylinositol tetraether; and acetyldiglycosyl phosphatidylinositol tetraether, m/z 1907). Acetylation of a sugar residue occurred commonly in this thermophile and increased as cells entered the stationary growth phase. Lipid extracts of Sulfolobus acidocaldarius contained detectable amounts of archaeol and hydroxyarchaeol analogs of phosphatidylinositol, phosphatidylglycerol, and phosphatidylethanolamine lipids, in addition to the dominant caldarchaeol lipids already reported. All four thermophiles contained both archaeol and caldarchaeol lipids and phosphoinositol head groups, but no single structural entity uniquely separated their lipids from those found previously in mesophilic archaea. By contrast, extremely halophilic archaea appear to be distinguished from the thermophilic archaea by the presence of a major phosphatidylglyceromethylphosphate lipid.Key words: ether lipids, mass spectrometry, hyperthermophiles, extreme halophiles, Archaea.