Photolysis of Caged Inositol Pyrophosphate InsP8 Directly Modulates Intracellular Ca2+ Oscillations and Controls C2AB Domain Localization.

Inositol pyrophosphates constitute a family of hyperphosphorylated signaling molecules involved in the regulation of glucose uptake and insulin sensitivity. While our understanding of the biological roles of inositol heptaphosphates (PP-InsP5) has greatly improved, the functions of the inositol octaphosphates ((PP)2-InsP4) have remained unclear. Here we present the synthesis of two enantiomeric cell-permeant and photocaged (PP)2-InsP4 derivatives and apply them to study the functions in living ß-cells. Photorelease of the naturally occurring isomer 1,5-(PP)2-InsP4 led to an immediate and concentration-dependent reduction of intracellular calcium oscillations, while other caged inositol pyrophosphates (3,5-(PP)2-InsP4, 5-PP-InsP5, 1-PP-InsP5, 3-PP-InsP5) showed no immediate effect. Furthermore, uncaging of 1,5-(PP)2-InsP4 but not 3,5-(PP)2-InsP4 induced translocation of the C2AB domain of granuphilin from the plasma membrane to the cytosol. Granuphilin is involved in membrane docking of secretory vesicles. This suggests that 1,5-(PP)2-InsP4 impacts ß-cell activity by regulating granule localization and/or priming and calcium signaling in concert.

d-chiro-Inositol Ribophostin: A Highly Potent Agonist of d-myo-Inositol 1,4,5-Trisphosphate Receptors: Synthesis and Biological Activities.

Analogues of the Ca2+-releasing intracellular messenger d-myo-inositol 1,4,5-trisphosphate [1, Ins(1,4,5)P3] are important synthetic targets. Replacement of the α-glucopyranosyl motif in the natural product mimic adenophostin 2 by d-chiro-inositol in d-chiro-inositol adenophostin 4 increased the potency. Similar modification of the non-nucleotide Ins(1,4,5)P3 mimic ribophostin 6 may increase the activity. d-chiro-Inositol ribophostin 10 was synthesized by coupling as building blocks suitably protected ribose 12 with l-(+)-3-O-trifluoromethylsulfonyl-6-O-p-methoxybenzyl-1,2:4,5-di-O-isopropylidene-myo-inositol 11. Separable diastereoisomeric 3-O-camphanate esters of (±)-6-O-p-methoxy-benzyl-1,2:4,5-di-O-isopropylidene-myo-inositol allowed the preparation of 11. Selective trans-isopropylidene deprotection in coupled 13, then monobenzylation gave separable regioisomers 15 and 16. p-Methoxybenzyl group deprotection of 16, phosphitylation/oxidation, then deprotection afforded 10, which was a full agonist in Ca2+-release assays; its potency and binding affinity for Ins(1,4,5)P3R were similar to those of adenophostin. Both 4 and 10 elicited a store-operated Ca2+ current ICRAC in patch-clamped cells, unlike Ins(1,4,5)P3 consistent with resistance to metabolism. d-chiro-Inositol ribophostin is the most potent small-molecule Ins(1,4,5)P3 receptor agonist without a nucleobase yet synthesized.

Scalable Chemoenzymatic Synthesis of Inositol Pyrophosphates.

The inositol pyrophosphates (PP-InsPs) are an important group of cellular messengers that influence a broad range of biological processes. To elucidate the functions of these high-energy metabolites at the biochemical level, access to the purified molecules is required. Here, a robust and scalable strategy for the synthesis of various PP-InsPs [5PP-InsP5, 1PP-InsP5, and 1,5(PP)2-InsP4] is reported, relying on the highly active inositol hexakisphosphate kinase A from Entamoeba histolytica and the kinase domain of human diphosphoinositol pentakisphosphate kinase 2. A facile purification procedure using precipitation with Mg2+ ions and an optional strong anion exchange chromatography on an FPLC system afforded PP-InsPs in high purity. Furthermore, the newly developed protocol could be applied to simplify the synthesis of radiolabeled 5PP-InsP5-ß32P, which is a valuable tool for studying protein pyrophosphorylation. The chemoenzymatic method for obtaining PP-InsPs is readily amenable to both chemists and biologists and will thus foster future research on the multiple signaling functions of PP-InsP molecules.

Perdeuterated and 13C-enriched myo-inositol for DNP assisted monitoring of enzymatic phosphorylation by inositol-3-kinase.

The synthesis of perdeuterated and 13C enriched myo-inositol is presented. Myo-inositol and its derivatives are of interest as substrates for enzymes producing phosphorylated species with regulatory functions in many organisms. Its utility in monitoring real-time phosphorylation by myo-inositol-3-kinase is illustrated using dynamic nuclear polarization (DNP) to enhance NMR observation.

A clue to unprecedented strategy to HIV eradication: "Lock-in and apoptosis".

Despite the development of antiretroviral therapy against HIV, eradication of the virus from the body, as a means to a cure, remains in progress. A "kick and kill" strategy proposes "kick" of the latent HIV to an active HIV to eventually be "killed". Latency-reverting agents that can perform the "kick" function are under development and have shown promise. Management of the infected cells not to produce virions after the "kick" step is important to this strategy. Here we show that a newly synthesized compound, L-HIPPO, captures the HIV-1 protein Pr55Gag and intercepts its function to translocate the virus from the cytoplasm to the plasma membrane leading to virion budding. The infecting virus thus "locked-in" subsequently induces apoptosis of the host cells. This "lock-in and apoptosis" approach performed by our novel compound in HIV-infected cells provides a means to bridge the gap between the "kick" and "kill" steps of this eradication strategy. By building upon previous progress in latency reverting agents, our compound appears to provide a promising step toward the goal of HIV eradication from the body.

Cellular delivery and photochemical release of a caged inositol-pyrophosphate induces PH-domain translocation in cellulo.

Inositol pyrophosphates, such as diphospho-myo-inositol pentakisphosphates (InsP7), are an important family of signalling molecules, implicated in many cellular processes and therapeutic indications including insulin secretion, glucose homeostasis and weight gain. To understand their cellular functions, chemical tools such as photocaged analogues for their real-time modulation in cells are required. Here we describe a concise, modular synthesis of InsP7 and caged InsP7. The caged molecule is stable and releases InsP7 only on irradiation. While photocaged InsP7 does not enter cells, its cellular uptake is achieved using nanoparticles formed by association with a guanidinium-rich molecular transporter. This novel synthesis and unprecedented polyphosphate delivery strategy enable the first studies required to understand InsP7 signalling in cells with controlled spatiotemporal resolution. It is shown herein that cytoplasmic photouncaging of InsP7 leads to translocation of the PH-domain of Akt, an important signalling-node kinase involved in glucose homeostasis, from the membrane into the cytoplasm.

Synthesis of inositol phosphate-based competitive antagonists of inositol 1,4,5-trisphosphate receptors.

Inositol 1,4,5-trisphosphate receptors (IP3Rs) are intracellular Ca(2+) channels that are widely expressed in animal cells, where they mediate the release of Ca(2+) from intracellular stores evoked by extracellular stimuli. A diverse array of synthetic agonists of IP3Rs has defined structure-activity relationships, but existing antagonists have severe limitations. We combined analyses of Ca(2+) release with equilibrium competition binding to IP3R to show that (1,3,4,6)IP4 is a full agonist of IP3R1 with lower affinity than (1,4,5)IP3. Systematic manipulation of this meso-compound via a versatile synthetic scheme provided a family of dimeric analogs of 2-O-butyryl-(1,3,4,6)IP4 and (1,3,4,5,6)IP5 that compete with (1,4,5)IP3 for binding to IP3R without evoking Ca(2+) release. These novel analogs are the first inositol phosphate-based competitive antagonists of IP3Rs with affinities comparable to that of the only commonly used competitive antagonist, heparin, the utility of which is limited by off-target effects.

The "Other" Inositols and Their Phosphates: Synthesis, Biology, and Medicine (with Recent Advances in myo-Inositol Chemistry).

Cell signaling via inositol phosphates, in particular via the second messenger myo-inositol 1,4,5-trisphosphate, and phosphoinositides comprises a huge field of biology. Of the nine 1,2,3,4,5,6-cyclohexanehexol isomers, myo-inositol is pre-eminent, with "other" inositols (cis-, epi-, allo-, muco-, neo-, L-chiro-, D-chiro-, and scyllo-) and derivatives rarer or thought not to exist in nature. However, neo- and d-chiro-inositol hexakisphosphates were recently revealed in both terrestrial and aquatic ecosystems, thus highlighting the paucity of knowledge of the origins and potential biological functions of such stereoisomers, a prevalent group of environmental organic phosphates, and their parent inositols. Some "other" inositols are medically relevant, for example, scyllo-inositol (neurodegenerative diseases) and d-chiro-inositol (diabetes). It is timely to consider exploration of the roles and applications of the "other" isomers and their derivatives, likely by exploiting techniques now well developed for the myo series.

Prometabolites of 5-Diphospho-myo-inositol Pentakisphosphate.

Diphospho-myo-inositol phosphates (PP-InsP(y)) are an important class of cellular messengers. Thus far, no method for the transport of PP-InsP(y) into living cells is available. Owing to their high negative charge density, PP-InsP(y) will not cross the cell membrane. A strategy to circumvent this issue involves the generation of precursors in which the negative charges are masked with biolabile groups. A PP-InsP(y) prometabolite would require twelve to thirteen biolabile groups, which need to be cleaved by cellular enzymes to release the parent molecules. Such densely modified prometabolites of phosphate esters and anhydrides have never been reported to date. This study discloses the synthesis of such agents and an analysis of their metabolism in tissue homogenates by gel electrophoresis. The acetoxybenzyl-protected system is capable of releasing 5-PP-InsP5 in mammalian cell/tissue homogenates within a few minutes and can be used to release 5-PP-InsP5 inside cells. These molecules will serve as a platform for the development of fundamental tools required to study PP-InsP(y) physiology.

New paradigms for the chiral synthesis of inositol phosphates.

Paradigms found: Inositol phosphates are biomolecules found ubiquitously in eukaryotes, in which they play a number of vital biological roles. Their enantioselective synthesis has recently received a boost from two complementary phosphorylation methods that could change the way they are synthesised, and hopefully provide invaluable chemical biology tools to further our understanding of this large family.

Synthesis and in vitro anticancer activity evaluation of novel bioreversible phosphate inositol derivatives.

The chemistry and biology of phosphorylated inositols have become intense areas of research during the last two decades due to their involvement in various cellular signaling processes. However, the metabolic instability by phosphatases or kinases and poor penetration make it difficult to become a drug used in the clinic. The bioreversible protection technique can enhance membrane penetration characteristics and increase the stability of phosphorylated inositols against enzymatic degradation and is applied widely in drug discovery and development. In this paper, we described the design and synthesis of 22 bioreversible phosphotriester inositols, along with the initial antitumor activity results. Most compounds exhibited significant cytotoxic activity against human cancer cell lines A549, MDA-MB-231 and HeLa, but lower cellular toxicity on normal cell MCF10A in comparison with Cisplatin. These compounds can be used as probes to study the mechanism of intracellular signal transduction mediated by phosphate inositol or as leads of phosphate inositol drugs in the clinic.

Design and synthesis of lipid-coupled inositol 1,2,3,4,5,6-hexakisphosphate derivatives exhibiting high-affinity binding for the HIV-1 MA domain.

The precursor of Gag protein (Pr55(Gag)) of human immunodeficiency virus, the principal structural component required for virus assembly, is known to bind d-myo-phosphatidylinositol 4,5-bisphosphate (PIP2). The N-terminus of Pr55(Gag), the MA domain, plays a critical role in the binding of Pr55(Gag) to the plasma membrane. Herein, we designed and synthesized myo-phosphatidylinositol 2,3,4,5,6-pentakisphosphate (PIP5) derivatives comprising highly phosphorylated inositol and variously modified diacylglycerol to examine the MA-binding properties. The inositol moiety was synthesized starting with myo-inositol and assembled with a hydrophobic glycerol moiety through a phosphate linkage. The Kd value for MA-binding of the PIP5 derivative 2 (Kd = 0.25 µM) was the lowest (i.e., highest affinity) of all derivatives, i.e., 70-fold lower than the Kd for the PIP2 derivative 1 (Kd = 16.9 µM) and 100-fold lower than the Kd for IP6 (Kd = 25.7 µM), suggesting the possibility that the PIP5 derivative blocks Pr55(Gag) membrane binding by competing with PIP2 in MA-binding.

Synthetic inositol phosphate analogs reveal that PPIP5K2 has a surface-mounted substrate capture site that is a target for drug discovery.

Diphosphoinositol pentakisphosphate kinase 2 (PPIP5K2) is one of the mammalian PPIP5K isoforms responsible for synthesis of diphosphoinositol polyphosphates (inositol pyrophosphates; PP-InsPs), regulatory molecules that function at the interface of cell signaling and organismic homeostasis. The development of drugs that inhibit PPIP5K2 could have both experimental and therapeutic applications. Here, we describe a synthetic strategy for producing naturally occurring 5-PP-InsP4, as well as several inositol polyphosphate analogs, and we study their interactions with PPIP5K2 using biochemical and structural approaches. These experiments uncover an additional ligand-binding site on the surface of PPIP5K2, adjacent to the catalytic pocket. This site facilitates substrate capture from the bulk phase, prior to transfer into the catalytic pocket. In addition to demonstrating a "catch-and-pass" reaction mechanism in a small molecule kinase, we demonstrate that binding of our analogs to the substrate capture site inhibits PPIP5K2. This work suggests that the substrate-binding site offers new opportunities for targeted drug design.

Cellular internalisation of an inositol phosphate visualised by using fluorescent InsP5.

When applied extracellularly, myo-inositol hexakisphosphate (InsP6 ) and myo-inositol pentakisphosphate (InsP5 ) can inhibit the growth and proliferation of tumour cells. There is debate about whether these effects result from interactions of InsP6 and InsP5 with intracellular or extracellular targets. We synthesised FAM-InsP5 , a fluorescent conjugate of InsP5 that allows direct visualisation of its interaction with cells. FAM-InsP5 was internalised by H1229 tumour cells, a finding that supports earlier reports that externally applied inositol phosphates can-perhaps surprisingly-enter into cells. Close examination of the process of FAM-InsP5 uptake suggests a mechanism of non-receptor-mediated endocytosis, which is blocked at 4 °C and probably involves interaction of the ligand with the glycocalyx. However, our results are difficult to reconcile with antiproliferative mechanisms that require direct interactions of externally applied InsP5 or InsP6 with cytosolic proteins, because internalised FAM-InsP5 appears in lysosomes and apparently does not enter the cytoplasm. Studies using FAM-InsP5 are less difficult and time-consuming than experiments using InsP5 or InsP6 , a factor that allowed us to analyse cellular uptake across a range of human cell types, identifying strong cell-specific differences.

A fully synthetic and biochemically validated phosphatidyl inositol-3-phosphate hapten via asymmetric synthesis and native chemical ligation.

We report the synthesis and biochemical validation of a phosphatidyl inositol-3 phosphate (PI3P) immunogen. The inositol stereochemistry was secured through peptide-catalyzed asymmetric phosphorylation catalysis, and the subsequent incorporation of a cysteine residue was achieved by native chemical ligation (NCL). Conjugation of the PI3P hapten to maleimide-activated keyhole limpet hemocyanin (KLH) provided a PI3P immunogen, which was successfully used to generate selective PI3P antibodies. The incorporation of a sulfhydryl nucleophile into a phosphoinositide hapten demonstrates a general strategy to reliably access phosphoinositide immunogens.

Desymmetrization of 4,6-diprotected myo-inositol.

The asymmetric desymmetrization of 4,6-diprotected myo-inositol derivatives was achieved by using a bifunctional, readily available nucleophilic catalyst. The orthogonally protected products were obtained in 80-99% yield with 90-99% ee. Such structures serve as potential enantiopure building blocks for the synthesis of myo-inositol phosphates.

Synthesis and antitumor activity of inositol phosphotriester analogues.

Inositol phosphates, as important second messengers of signal transduction, regulate many biological functions. However, cell penetration and phospholipase stability could be two main issues faced by inositol phosphate analogues used as lead compounds for drug discovery. Inositol phosphotriester analogues could be more beneficial to diffuse across plasma membrane. In this paper, we describe the design and synthesis of a series of inositol phosphotriester analogues based on phosphatidylinositol, along with the initial antitumor activity analysis. Several compounds exhibited good cytotoxic activity against human cancer cell lines A549, HepG2, MDA-MB-231 and HeLa, especially compound 33 was cytotoxic against all the four cancer cell lines with good IC(50) values.

Design and synthesis of biotinylated inositol 1,3,4,5-tetrakisphosphate targeting Grp1 pleckstrin homology domain.

A bifunctional molecule containing biotin and d-myo-inositol 1,3,4,5-tetrakisphosphate was synthesized. This molecule was designed on the basis of X-ray structure of the complex of d-myo-inositol 1,3,4,5-tetrakisphosphates, Ins(1,3,4,5)P(4), and Grp1 PH (general receptor of phosphoinositides pleckstrin homology) domain for the application to the widely employed biotin-avidin techniques. The building block of inositol moiety was synthesized starting with myo-inositol and assembled with the biotin-linker moiety through a phosphate linkage. The equilibrium dissociation constant K(D) of biotinylated Ins(1,3,4,5)P(4) binding of original Grp1 PH domain was 0.14 µM in pull-down analysis, which was comparable to that of unmodified Ins(1,3,4,5)P(4). Furthermore, biotinylated Ins(1,3,4,5)P(4) had an ability to distinguish Grp1 PH domain from PLCδ(1) PH domain. Thus, biotinylated Ins(1,3,4,5)P(4) retained the binding affinity and selectivity of original Grp1 PH domain, and realized the intracellular Ins(1,3,4,5)P(4) despite a tethering at the 1-phosphate group of inositol.

Desymmetrization of 2,4,5,6-tetra-O-benzyl-D-myo-inositol for the synthesis of mycothiol.

An efficient chemical synthesis of mycothiol involving the regioselective ketopinyl desymmetrization of 2,4,5,6-tetrabenzylated D-myo-inositol as the key step is described. Together with a highly α-stereoselective D-glucosaminylation, the whole procedure was accomplished in eight steps with an overall yield of 40%.