The inositol pyrophosphate metabolism of Dictyostelium discoideum does not regulate inorganic polyphosphate (polyP) synthesis.

Initial studies on the inositol phosphates metabolism were enabled by the social amoeba Dictyostelium discoideum. The abundant amount of inositol hexakisphosphate (IP6 also known as Phytic acid) present in the amoeba allowed the discovery of the more polar inositol pyrophosphates, IP7 and IP8, possessing one or two high energy phosphoanhydride bonds, respectively. Considering the contemporary growing interest in inositol pyrophosphates, it is surprising that in recent years D. discoideum, has contributed little to our understanding of their metabolism and function. This work fulfils this lacuna, by analysing the ip6k, ppip5k and ip6k-ppip5K amoeba null strains using PAGE, 13C-NMR and CE-MS analysis. Our study reveals an inositol pyrophosphate metabolism more complex than previously thought. The amoeba Ip6k synthesizes the 4/6-IP7 in contrast to the 5-IP7 isomer synthesized by the mammalian homologue. The amoeba Ppip5k synthesizes the same 1/3-IP7 as the mammalian enzyme. In D. discoideum, the ip6k strain possesses residual amounts of IP7. The residual IP7 is also present in the ip6k-ppip5K strain, while the ppip5k single mutant shows a decrease in both IP7 and IP8 levels. This phenotype is in contrast to the increase in IP7 observable in the yeast vip1Δ strain. The presence of IP8 in ppip5k and the presence of IP7 in ip6k-ppip5K indicate the existence of an additional inositol pyrophosphate synthesizing enzyme. Additionally, we investigated the existence of a metabolic relationship between inositol pyrophosphate synthesis and inorganic polyphosphate (polyP) metabolism as observed in yeast. These studies reveal that contrary to the yeast, Ip6k and Ppip5k do not control polyP cellular level in amoeba.

ITPK1 is an InsP6/ADP phosphotransferase that controls phosphate signaling in Arabidopsis.

In plants, phosphate (Pi) homeostasis is regulated by the interaction of PHR transcription factors with stand-alone SPX proteins, which act as sensors for inositol pyrophosphates. In this study, we combined different methods to obtain a comprehensive picture of how inositol (pyro)phosphate metabolism is regulated by Pi and dependent on the inositol phosphate kinase ITPK1. We found that inositol pyrophosphates are more responsive to Pi than lower inositol phosphates, a response conserved across kingdoms. Using the capillary electrophoresis electrospray ionization mass spectrometry (CE-ESI-MS) we could separate different InsP7 isomers in Arabidopsis and rice, and identify 4/6-InsP7 and a PP-InsP4 isomer hitherto not reported in plants. We found that the inositol pyrophosphates 1/3-InsP7, 5-InsP7, and InsP8 increase several fold in shoots after Pi resupply and that tissue-specific accumulation of inositol pyrophosphates relies on ITPK1 activities and MRP5-dependent InsP6 compartmentalization. Notably, ITPK1 is critical for Pi-dependent 5-InsP7 and InsP8 synthesis in planta and its activity regulates Pi starvation responses in a PHR-dependent manner. Furthermore, we demonstrated that ITPK1-mediated conversion of InsP6 to 5-InsP7 requires high ATP concentrations and that Arabidopsis ITPK1 has an ADP phosphotransferase activity to dephosphorylate specifically 5-InsP7 under low ATP. Collectively, our study provides new insights into Pi-dependent changes in nutritional and energetic states with the synthesis of regulatory inositol pyrophosphates.

Identification of Small-Molecule Inhibitors of Human Inositol Hexakisphosphate Kinases by High-Throughput Screening.

Inositol hexakisphosphate kinases (IP6Ks) catalyze pyrophosphorylation of inositol hexakisphosphate (IP6) into inositol 5-diphospho-1,2,3,4,6-pentakisphosphate (IP7), which is involved in numerous areas of cell physiology including glucose homeostasis, blood coagulation, and neurological development. Inhibition of IP6Ks may be effective for the treatment of Type II diabetes, obesity, metabolic complications, thrombosis, and psychiatric disorders. We performed a high-throughput screen (HTS) of 158 410 compounds for IP6K1 inhibitors using a previously developed ADP-Glo Max assay. Of these, 1206 compounds were found to inhibit IP6K1 kinase activity by more than 25%, representing a 0.8% hit rate. Structural clustering analysis of HTS-active compounds, which were confirmed in the dose-response testing using the same kinase assay, revealed diverse clusters that were feasible for future structure-activity relationship (SAR) optimization to potent IP6K inhibitors. Medicinal chemistry SAR efforts in three chemical series identified potent IP6K1 inhibitors which were further validated in an orthogonal LC-MS IP7 analysis. The effects of IP6K1 inhibitors on cellular IP7 levels were further confirmed and were found to correlate with cellular IP6K1 binding measured by a high-throughput cellular thermal shift assay (CETSA).

Inositol pyrophosphates promote the interaction of SPX domains with the coiled-coil motif of PHR transcription factors to regulate plant phosphate homeostasis.

Phosphorus is an essential nutrient taken up by organisms in the form of inorganic phosphate (Pi). Eukaryotes have evolved sophisticated Pi sensing and signaling cascades, enabling them to stably maintain cellular Pi concentrations. Pi homeostasis is regulated by inositol pyrophosphate signaling molecules (PP-InsPs), which are sensed by SPX domain-containing proteins. In plants, PP-InsP-bound SPX receptors inactivate Myb coiled-coil (MYB-CC) Pi starvation response transcription factors (PHRs) by an unknown mechanism. Here we report that a InsP8-SPX complex targets the plant-unique CC domain of PHRs. Crystal structures of the CC domain reveal an unusual four-stranded anti-parallel arrangement. Interface mutations in the CC domain yield monomeric PHR1, which is no longer able to bind DNA with high affinity. Mutation of conserved basic residues located at the surface of the CC domain disrupt interaction with the SPX receptor in vitro and in planta, resulting in constitutive Pi starvation responses. Together, our findings suggest that InsP8 regulates plant Pi homeostasis by controlling the oligomeric state and hence the promoter binding capability of PHRs via their SPX receptors.

Analysis of inositol phosphate metabolism by capillary electrophoresis electrospray ionization mass spectrometry.

The analysis of myo-inositol phosphates (InsPs) and myo-inositol pyrophosphates (PP-InsPs) is a daunting challenge due to the large number of possible isomers, the absence of a chromophore, the high charge density, the low abundance, and the instability of the esters and anhydrides. Given their importance in biology, an analytical approach to follow and understand this complex signaling hub is desirable. Here, capillary electrophoresis (CE) coupled to electrospray ionization mass spectrometry (ESI-MS) is implemented to analyze complex mixtures of InsPs and PP-InsPs with high sensitivity. Stable isotope labeled (SIL) internal standards allow for matrix-independent quantitative assignment. The method is validated in wild-type and knockout mammalian cell lines and in model organisms. SIL-CE-ESI-MS enables the accurate monitoring of InsPs and PP-InsPs arising from compartmentalized cellular synthesis pathways, by feeding cells with either [13C6]-myo-inositol or [13C6]-D-glucose. In doing so, we provide evidence for the existence of unknown inositol synthesis pathways in mammals, highlighting the potential of this method to dissect inositol phosphate metabolism and signalling.

Analysis of metabolically labeled inositol phosphate messengers by NMR.

Inositol phosphates (InsPs) are an important group of eukaryotic messengers and mediate a wide range of processes. To elucidate the biological functions of these molecules, robust techniques to characterize inositol phosphate metabolism at the cellular level are highly sought after. This chapter provides a detailed protocol for the preparation of 13C-labeled myo-inositol, its use for metabolic labeling of mammalian and yeast cells, and the quantitative analysis of intracellular InsP pools from cell extracts using NMR spectroscopy.

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.

Two bifunctional inositol pyrophosphate kinases/phosphatases control plant phosphate homeostasis.

Many eukaryotic proteins regulating phosphate (Pi) homeostasis contain SPX domains that are receptors for inositol pyrophosphates (PP-InsP), suggesting that PP-InsPs may regulate Pi homeostasis. Here we report that deletion of two diphosphoinositol pentakisphosphate kinases VIH1/2 impairs plant growth and leads to constitutive Pi starvation responses. Deletion of phosphate starvation response transcription factors partially rescues vih1 vih2 mutant phenotypes, placing diphosphoinositol pentakisphosphate kinases in plant Pi signal transduction cascades. VIH1/2 are bifunctional enzymes able to generate and break-down PP-InsPs. Mutations in the kinase active site lead to increased Pi levels and constitutive Pi starvation responses. ATP levels change significantly in different Pi growth conditions. ATP-Mg2+ concentrations shift the relative kinase and phosphatase activities of diphosphoinositol pentakisphosphate kinases in vitro. Pi inhibits the phosphatase activity of the enzyme. Thus, VIH1 and VIH2 relay changes in cellular ATP and Pi concentrations to changes in PP-InsP levels, allowing plants to maintain sufficient Pi levels.

Harnessing 13C-labeled myo-inositol to interrogate inositol phosphate messengers by NMR.

Inositol poly- and pyrophosphates (InsPs and PP-InsPs) are an important group of metabolites and mediate a wide range of processes in eukaryotic cells. To elucidate the functions of these molecules, robust techniques for the characterization of inositol phosphate metabolism are required, both at the biochemical and the cellular level. Here, a new tool-set is reported, which employs uniformly 13C-labeled compounds ([13C6]myo-inositol, [13C6]InsP5, [13C6]InsP6, and [13C6]5PP-InsP5), in combination with commonly accessible NMR technology. This approach permitted the detection and quantification of InsPs and PP-InsPs within complex mixtures and at physiological concentrations. Specifically, the enzymatic activity of IP6K1 could be monitored in vitro in real time. Metabolic labeling of mammalian cells with [13C6]myo-inositol enabled the analysis of cellular pools of InsPs and PP-InsPs, and uncovered high concentrations of 5PP-InsP5 in HCT116 cells, especially in response to genetic and pharmacological perturbation. The reported method greatly facilitates the analysis of this otherwise spectroscopically silent group of molecules, and holds great promise to comprehensively analyze inositol-based signaling molecules under normal and pathological conditions.

The Oncolytic Efficacy and in Vivo Pharmacokinetics of [2-(4-Chlorophenyl)quinolin-4-yl](piperidine-2-yl)methanol (Vacquinol-1) Are Governed by Distinct Stereochemical Features.

Glioblastoma remains an incurable brain cancer. Drugs developed in the past 20 years have not improved the prognosis for patients, necessitating the development of new treatments. We have previously reported the therapeutic potential of the quinoline methanol Vacquinol-1 (1) that targets glioblastoma cells and induces cell death by catastrophic vacuolization. Compound 1 is a mixture of four stereoisomers due to the two adjacent stereogenic centers in the molecule, complicating further development in the preclinical setting. This work describes the isolation and characterization of the individual isomers of 1 and shows that these display stereospecific pharmacokinetic and pharmacodynamic features. In addition, we present a stereoselective synthesis of the active isomers, providing a basis for further development of this compound series into a novel experimental therapeutic for glioblastoma.

Organocatalytic entry into 2,6-disubstituted aza-Achmatowicz piperidinones: application to (-)-sedacryptine and its epimer.

Enantiomerically pure 2,6-disubstituted piperidinones were synthesized from furfural involving an organocatalyzed Mannich reaction, aza-Achmatowicz reaction, and an N-acyliminium ion-mediated coupling step. This approach was also successfully applied to a total synthesis of (-)-sedacryptine and one of its epimers.