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Inositol pyrophosphates (PP-InsPs) are nutrient messengers whose cellular levels are precisely regulated. Diphosphoinositol pentakisphosphate kinases (PPIP5Ks) generate the active signaling molecule 1,5-InsP8. PPIP5Ks harbor phosphatase domains that hydrolyze PP-InsPs. Plant and Fungi Atypical Dual Specificity Phosphatases (PFA-DSPs) and NUDIX phosphatases (NUDTs) are also involved in PP-InsP degradation. Here, we analyze the relative contributions of the three different phosphatase families to plant PP-InsP catabolism. We report the biochemical characterization of inositol pyrophosphate phosphatases from Arabidopsis and Marchantia polymorpha. Overexpression of different PFA-DSP and NUDT enzymes affects PP-InsP levels and leads to stunted growth phenotypes in Arabidopsis. nudt17/18/21 knock-out mutants have altered PP-InsP pools and gene expression patterns, but no apparent growth defects. In contrast, Marchantia polymorpha Mppfa-dsp1ge, Mpnudt1ge and Mpvip1ge mutants display severe growth and developmental phenotypes and associated changes in cellular PP-InsP levels. Analysis of Mppfa-dsp1geand Mpvip1ge mutants supports a role for PP-InsPs in Marchantia phosphate signaling, and additional functions in nitrate homeostasis and cell wall biogenesis. Simultaneous elimination of two phosphatase activities enhanced the observed growth phenotypes. Taken together, PPIP5K, PFA-DSP and NUDT inositol pyrophosphate phosphatases regulate growth and development by collectively shaping plant PP-InsP pools.

ABSTRACT Inositol pyrophosphate 1,5-IP8 regulates expression of a fission yeast phosphate homeostasis regulon, comprising phosphate acquisition genes pho1, pho84, and tgp1, via its action as an agonist of precocious termination of transcription of the upstream lncRNAs that repress PHO mRNA synthesis. 1,5-IP8 levels are dictated by a balance between the Asp1 N-terminal kinase domain that converts 5-IP7 to 1,5-IP8 and three inositol pyrophosphatases—the Asp1 C-terminal domain (a histidine acid phosphatase), Siw14 (a cysteinyl-phosphatase), and Aps1 (a Nudix enzyme). In this study, we report the biochemical and genetic characterization of Aps1 and an analysis of the effects of Asp1, Siw14, and Aps1 mutations on cellular inositol pyrophosphate levels. We find that Aps1’s substrate repertoire embraces inorganic polyphosphates, 5-IP7, 1-IP7, and 1,5-IP8. Aps1 displays a ~twofold preference for hydrolysis of 1-IP7 versus 5-IP7 and aps1∆ cells have twofold higher levels of 1-IP7 vis-à-vis wild-type cells. While neither Aps1 nor Siw14 is essential for growth, an aps1∆ siw14∆ double mutation is lethal on YES medium. This lethality is a manifestation of IP8 toxicosis, whereby excessive 1,5-IP8 drives derepression of tgp1, leading to Tgp1-mediated uptake of glycerophosphocholine. We were able to recover an aps1∆ siw14∆ mutant on ePMGT medium lacking glycerophosphocholine and to suppress the severe growth defect of aps1∆ siw14∆ on YES by deleting tgp1. However, the severe growth defect of an aps1∆ asp1-H397A strain could not be alleviated by deleting tgp1, suggesting that 1,5-IP8 levels in this double-pyrophosphatase mutant exceed a threshold beyond which overzealous termination affects other genes, which results in cytotoxicity.IMPORTANCERepression of the fission yeast PHO genes tgp1, pho1, and pho84 by lncRNA-mediated interference is sensitive to changes in the metabolism of 1,5-IP8, a signaling molecule that acts as an agonist of precocious lncRNA termination. 1,5-IP8 is formed by phosphorylation of 5-IP7 and catabolized by inositol pyrophosphatases from three distinct enzyme families: Asp1 (a histidine acid phosphatase), Siw14 (a cysteinyl phosphatase), and Aps1 (a Nudix hydrolase). This study entails a biochemical characterization of Aps1 and an analysis of how Asp1, Siw14, and Aps1 mutations impact growth and inositol pyrophosphate pools in vivo. Aps1 catalyzes hydrolysis of inorganic polyphosphates, 5-IP7, 1-IP7, and 1,5-IP8 in vitro, with a ~twofold preference for 1-IP7 over 5-IP7. aps1∆ cells have twofold higher levels of 1-IP7 than wild-type cells. An aps1∆ siw14∆ double mutation is lethal because excessive 1,5-IP8 triggers derepression of tgp1, leading to toxic uptake of glycerophosphocholine.

Summary: The universally conserved YchF/Ola1 ATPases regulate stress response pathways in prokaryotes and eukaryotes. Deletion of YchF/Ola1 leads to increased resistance against environmental stressors, such as reactive oxygen species, while their upregulation is associated with tumorigenesis in humans. The current study shows that in E. coli, the absence of YchF stimulates the synthesis of the alternative sigma factor RpoS by a transcription-independent mechanism. Elevated levels of RpoS then enhance the transcription of major stress-responsive genes. In addition, the deletion of ychF increases the levels of polyphosphate kinase, which in turn boosts the production of the evolutionary conserved and ancient chemical chaperone polyphosphate. This potentially provides a unifying concept for the increased stress resistance in bacteria and eukaryotes upon YchF/Ola1 deletion. Intriguingly, the simultaneous deletion of ychF and the polyphosphate-degrading enzyme exopolyphosphatase causes synthetic lethality in E. coli, demonstrating that polyphosphate production needs to be fine-tuned to prevent toxicity.

Summary: Phosphate (Pi) serves countless metabolic pathways and is involved in macromolecule synthesis, energy storage, cellular signaling, and bone maintenance. Herein, we describe the coordination of Pi uptake and efflux pathways to maintain mammalian cell Pi homeostasis. We discover that XPR1, the presumed Pi efflux transporter, separately supervises rates of Pi uptake. This direct, regulatory interplay arises from XPR1 being a binding partner for the Pi uptake transporter PiT1, involving a predicted transmembrane helix/extramembrane loop in XPR1, and its hitherto unknown localization in a subset of intracellular LAMP1-positive puncta (named “XLPVs”). A pharmacological mimic of Pi homeostatic challenge is sensed by the inositol pyrophosphate IP8, which functionalizes XPR1 to respond in a temporally hierarchal manner, initially adjusting the rate of Pi efflux, followed subsequently by independent modulation of PiT1 turnover to reset the rate of Pi uptake. These observations generate a unifying model of mammalian cellular Pi homeostasis, expanding opportunities for therapeutic intervention.

Fused deposition modeling (FDM) is well suited for microfluidic prototyping due to its low investment cost and a wide range of accessible materials. Nevertheless, most commercial FDM materials exhibit low chemical and thermal stability. This reduces the scope of applications and limits their use in research and development, especially for on-chip chemical synthesis. In this paper, we present FDM fabrication of microfluidic chips with polyvinylidene fluoride (PVDF) for applications that require high thermal or chemical resistance. Embedded microchannels with a minimum channel width and heights of ~200 µm × 200 µm were fabricated, and the resistance against common solvents was analyzed. A procedure was developed to increase the optical transmission to result in translucent components by printing on glass. Chips for fluid mixing were printed, as well as microreactors that were packed with a catalytically active phase and used for acetal deprotection with a conversion of more than 99%. By expanding the use of fluorinated polymers to FDM printing, previously challenging microfluidic applications will be conducted with ease at the lab scale.

Polyphosphate (polyP) is an evolutionary ancient inorganic molecule widespread in biology, exerting a broad range of biological activities. The intracellular polymer serves as an energy storage pool and phosphate/calcium ion reservoir with implications for basal cellular functions. Metabolisms of the polymer are well understood in procaryotes and unicellular eukaryotic cells. However, functions, regulation, and association with disease states of the polymer in higher eukaryotic species such as mammalians are just beginning to emerge. The review summarises our current understanding of polyP metabolism, the polymer’s functions, and methods for polyP analysis. In-depth knowledge of the pathways that control polyP turnover will open future perspectives for selective targeting of the polymer.

Abstract NADH:ubiquinone oxidoreductase, respiratory complex I, plays a major role in cellular energy metabolism by coupling electron transfer with proton translocation. Electron transfer is catalyzed by a flavin mononucleotide and a series of iron-sulfur (Fe/S) clusters. As a by-product of the reaction, the reduced flavin generates reactive oxygen species (ROS). It was suggested that the ROS generated by the respiratory chain in general could damage the Fe/S clusters of the complex. Here, we show that the binuclear Fe/S cluster N1b is specifically damaged by H2O2, however, only at high concentrations. But under the same conditions, the activity of the complex is hardly affected, since N1b can be easily bypassed during electron transfer.

ABSTRACT Inositol pyrophosphate 1,5-IP8 is a signaling molecule that regulates phosphate and polyphosphate homeostasis in the fission yeast Schizosaccharomyces pombe. 1,5-IP8 levels are dictated by a balance between the Asp1 kinase domain that converts 5-IP7 to 1,5-IP8 and two pyrophosphatases—the Asp1 pyrophosphatase domain (histidine acid phosphatase family) and the Aps1 pyrophosphatase enzyme (Nudix family)—that hydrolyze the β-phosphates of 1,5-IP8. Here, we characterize S. pombe Siw14 (SpSiw14), a cysteinyl-phosphatase family member and a homolog of Saccharomyces cerevisiae Siw14, as a third fission yeast pyrophosphatase implicated in inositol pyrophosphate catabolism. We find that SpSiw14’s substrate repertoire embraces inorganic pyrophosphate, inorganic polyphosphate, and the inositol pyrophosphates 5-IP7, 1-IP7, and 1,5-IP8, in addition to the generic substrate p-nitrophenylphosphate. Genetic analyses revealed that (i) elimination of the SpSiw14 protein or inactivation of the SpSiw14 pyrophosphatase by the C189S mutation had no effect on S. pombe growth but was lethal in the absence of Aps1 and (ii) the synthetic lethality of siw14∆ aps1∆ depended on the synthesis of 1,5-IP8 by the Asp1 kinase. We conclude that SpSiw14 and Aps1 pyrophosphatases have essential but redundant functions in fission yeast, and that their synthetic lethality is a consequence of the toxic effects of too much 1,5-IP8. Suppression of siw14∆ aps1∆ lethality by loss-of-function mutations of components of the fission yeast 3′-processing/termination machinery fortifies the case for overzealous transcription termination as the basis for 1,5-IP8 toxicosis. IMPORTANCE The inositol pyrophosphate signaling molecule 1,5-IP8 modulates fission yeast phosphate homeostasis via its action as an agonist of RNA 3′-processing and transcription termination. Cellular 1,5-IP8 levels are determined by a balance between the activities of the inositol polyphosphate kinase Asp1 and several inositol pyrophosphatase enzymes. Here, we characterize Schizosaccharomyces pombe Siw14 (SpSiw14) as a cysteinyl-phosphatase-family pyrophosphatase enzyme capable of hydrolyzing the phosphoanhydride substrates inorganic pyrophosphate, inorganic polyphosphate, and inositol pyrophosphates 5-IP7, 1-IP7, and 1,5-IP8. Genetic analyses implicate SpSiw14 in 1,5-IP8 catabolism in vivo, insofar as: loss of SpSiw14 activity is lethal in the absence of the Nudix-type inositol pyrophosphatase enzyme Aps1; and siw14∆ aps1∆ lethality depends on synthesis of 1,5-IP8 by the Asp1 kinase. Suppression of siw14∆ aps1∆ lethality by loss-of-function mutations of 3′-processing/termination factors points to precocious transcription termination as the cause of 1,5-IP8 toxicosis.

Eukaryotic cells control inorganic phosphate to balance its role as essential macronutrient with its negative bioenergetic impact on reactions liberating phosphate. Phosphate homeostasis depends on the conserved INPHORS signaling pathway that utilizes inositol pyrophosphates and SPX receptor domains. Since cells synthesize various inositol pyrophosphates and SPX domains bind them promiscuously, it is unclear whether a specific inositol pyrophosphate regulates SPX domains in vivo, or whether multiple inositol pyrophosphates act as a pool. In contrast to previous models, which postulated that phosphate starvation is signaled by increased production of the inositol pyrophosphate 1-IP7, we now show that the levels of all detectable inositol pyrophosphates of yeast, 1-IP7, 5-IP7, and 1,5-IP8, strongly decline upon phosphate starvation. Among these, specifically the decline of 1,5-IP8 triggers the transcriptional phosphate starvation response, the PHO pathway. 1,5-IP8 inactivates the cyclin-dependent kinase inhibitor Pho81 through its SPX domain. This stimulates the cyclin-dependent kinase Pho85-Pho80 to phosphorylate the transcription factor Pho4 and repress the PHO pathway. Combining our results with observations from other systems, we propose a unified model where 1,5-IP8 signals cytosolic phosphate abundance to SPX proteins in fungi, plants, and mammals. Its absence triggers starvation responses.

Polyphosphate kinases (PPKs) have become popular biocatalysts for nucleotide 5'-triphosphate (NTP) synthesis and regeneration. Two unrelated families are described: PPK1 and PPK2. They are structurally unrelated and use different catalytic mechanisms. PPK1 enzymes prefer the usage of adenosine 5'-triphosphate (ATP) for polyphosphate (polyP) synthesis while PPK2 enzymes favour the reverse reaction. With the emerging use of PPK enzymes in biosynthesis, a deeper understanding of the enzymes and their thermodynamic reaction course is of need, especially in comparison to other kinases. Here, we tested four PPKs from different organisms under the same conditions without any coupling reactions. In comparison to other kinases using phosphate donors with comparably higher phosphate transfer potentials that are characterised by reaction yields close to full conversion, the PPK-catalysed reaction reaches an equilibrium in which about 30% ADP is left. These results were obtained for PPK1 and PPK2 enzymes, and are supported by theoretical data on the basic reaction. At high concentrations of substrate, the different kinetic preferences of PPK1 and PPK2 can be observed. The implications of these results for the application of PPKs in chemical synthesis and as enzymes for ATP regeneration systems are discussed.

Inositol pyrophosphates (PP-IPs) regulate immunity and phosphate homeostasis. Here, the authors’ structural dissection of an Arabidopsis thaliana PP-IP phosphatase reveals that substrates drive their own hydrolysis and identifies a highly elusive metaphosphate-like reaction intermediate.

ABSTRACT Expression of the fission yeast Schizosaccharomyces pombe phosphate regulon is sensitive to the intracellular level of the inositol pyrophosphate signaling molecule 1,5-IP8. IP8 dynamics are determined by Asp1, a bifunctional enzyme consisting of an N-terminal kinase domain and a C-terminal pyrophosphatase domain that catalyze IP8 synthesis and catabolism, respectively. Here, we report structures of the Asp1 kinase domain, crystallized with two protomers in the asymmetric unit, one of which was complexed with ligands (ADPNP, ADP, or ATP; Mg2+ or Mn2+; IP6, 5-IP7, or 1,5-IP8) and the other which was ligand-free. The ligand-free enzyme adopts an “open” conformation that allows ingress of substrates and egress of products. ADPNP, ADP, and ATP and associated metal ions occupy a deep phospho-donor pocket in the active site. IP6 or 5-IP7 engagement above the nucleotide favors adoption of a “closed” conformation, in which surface protein segments undergo movement and a disordered-to-ordered transition to form an inositol polyphosphate-binding site. In a structure mimetic of the kinase Michaelis complex, the anionic 5-IP7 phosphates are encaged by an ensemble of nine cationic amino acids: Lys43, Arg223, Lys224, Lys260, Arg274, Arg285, Lys290, Arg293, and Lys341. Alanine mutagenesis of amino acids that contact the adenosine nucleoside of the ATP donor underscored the contributions of Asp258 interaction with the ribose 3′-OH and of Glu248 with adenine-N6. Changing Glu248 to Gln elicited a gain of function whereby the kinase became adept at using GTP as phosphate donor. Wild-type Asp1 kinase can utilize N6-benzyl-ATP as phosphate donor. IMPORTANCE The inositol pyrophosphate signaling molecule 1,5-IP8 modulates fission yeast phosphate homeostasis via its action as an agonist of RNA 3′-processing and transcription termination. Cellular IP8 levels are determined by Asp1, a bifunctional enzyme composed of an N-terminal kinase and a C-terminal pyrophosphatase domain. Here, we present a series of crystal structures of the Asp1 kinase domain, in a ligand-free state and in complexes with nucleotides ADPNP, ADP, and ATP, divalent cations magnesium and manganese, and inositol polyphosphates IP6, 5-IP7, and 1,5-IP8. Substrate binding elicits a switch from open to closed conformations, entailing a disordered-to-ordered transition and a rearrangement or movement of two peptide segments that form a binding site for the phospho-acceptor. Our structures, along with structure-guided mutagenesis, fortify understanding of the mechanism and substrate specificity of Asp1 kinase, and they extend and complement structural and functional studies of the orthologous human kinase PPIP5K2.

The “anti-branching rule”, introduced in 1950, excludes branched polyphosphates from biological relevance due to their supposedly rapid hydrolysis. Here, the authors synthesize monodisperse branched polyphosphates and demonstrate their unexpected stability in water, as well as provide evidence for their competence in phosphorylation.

Inositol phosphates constitute a family of highly charged messenger molecules that play diverse roles in cellular processes. The various phosphorylation patterns they exhibit give rise to a vast array of different compounds. To fully comprehend the biological interconnections, the precise molecular identification of each compound is crucial. Since the myo-inositol scaffold possesses an internal mirror plane, enantiomeric pairs can be formed. Most commonly employed methods for analyzing InsPs have been geared towards resolving regioisomers, but they have not been capable of resolving enantiomers. In this study, we present a general approach for enantiomer assignment using NMR measurements. To achieve this goal, we used 31P-NMR in the presence of L-arginine amide as a chiral solvating agent, which enables the differentiation of enantiomers. Using chemically synthesized standard compounds allows for an unambiguous assignment of the enantiomers. This method was applied to highly phosphorylated inositol pyrophosphates, as well as to lowly phosphorylated inositol phosphates and bisphosphonate analogs. Our method will facilitate the assignment of biologically relevant isomers when isolating naturally occurring compounds from biological specimens.

Inositol pyrophosphates (PP-InsPs), derivatives of inositol hexakisphosphate (phytic acid, InsP6) or lower inositol polyphosphates, are energy-rich signaling molecules that have critical regulatory functions in eukaryotes. In plants, the biosynthesis and the cellular targets of these messengers are not fully understood. This is because, in part, plants do not possess canonical InsP6 kinases and are able to synthesize PP-InsP isomers that appear to be absent in yeast or mammalian cells. This review will shed light on recent discoveries in the biosynthesis of these enigmatic messengers and on how they regulate important physiological processes in response to abiotic and biotic stresses in plants.

ABSTRACT Inositol pyrophosphates (IPPs) are signaling molecules that regulate cellular phosphate homeostasis in diverse eukaryal taxa. In fission yeast, mutations that increase 1,5-IP8 derepress the PHO regulon while mutations that ablate IP8 synthesis are PHO hyper-repressive. Fission yeast Asp1, the principal agent of 1,5-IP8 dynamics, is a bifunctional enzyme composed of an N-terminal IPP kinase domain and a C-terminal IPP pyrophosphatase domain. Here we conducted a biochemical characterization and mutational analysis of the autonomous Asp1 kinase domain (aa 1–385). Reaction of Asp1 kinase with IP6 and ATP resulted in both IP6 phosphorylation to 1-IP7 and hydrolysis of the ATP γ-phosphate, with near-equal partitioning between productive 1-IP7 synthesis and unproductive ATP hydrolysis under optimal kinase conditions. By contrast, reaction of Asp1 kinase with 5-IP7 is 22-fold faster than with IP6 and is strongly biased in favor of IP8 synthesis versus ATP hydrolysis. Alanine scanning identified essential constituents of the active site. We deployed the Ala mutants to show that derepression of pho1 expression correlated with Asp1’s kinase activity. In the case of full-length Asp1, the activity of the C-terminal pyrophosphatase domain stifled net phosphorylation of the 1-position during reaction of Asp1 with ATP and either IP6 or 5-IP7. We report that inorganic phosphate is a concentration-dependent enabler of net IP8 synthesis by full-length Asp1 in vitro, by virtue of its antagonism of IP8 turnover. IMPORTANCE Expression of the fission yeast phosphate regulon is sensitive to the intracellular level of the inositol pyrophosphate (IPP) signaling molecule 1,5-IP8. IP8 dynamics are determined by Asp1, a bifunctional enzyme comprising N-terminal IPP 1-kinase and C-terminal IPP 1-pyrophosphatase domains that catalyze IP8 synthesis and catabolism, respectively. Here, we interrogated the activities and specificities of the Asp1 kinase domain and full length Asp1. We find that reaction of Asp1 kinase with 5-IP7 is 22-fold faster than with IP6 and is strongly biased in favor of IP8 synthesis versus the significant unproductive ATP hydrolysis seen during its reaction with IP6. We report that full-length Asp1 catalyzes futile cycles of 1-phosphate phosphorylation by its kinase component and 1-pyrophosphate hydrolysis by its pyrophosphatase component that result in unproductive net consumption of the ATP substrate. Net synthesis of 1,5-IP8 is enabled by physiological concentrations of inorganic phosphate that selectively antagonize IP8 turnover.

Myo-Inositol phosphates (InsPs) and pyrophosphates (PP-InsPs) are important second messengers but their analysis remains challenging. Here, the authors develop a capillary electrophoresis-mass spectrometry method for the identification and quantitation of InsP and PP-InsP isomers in cells and tissues.

Summary: Polyphosphate (polyP) is a polymer of hundreds of phosphate residues present in all organisms. In mammals, polyP is involved in crucial physiological processes, including coagulation, inflammation, and stress response. However, after decades of research, the metabolic enzymes are still unknown. Here, we purify and identify Nudt3, a NUDIX family member, as the enzyme responsible for polyP phosphatase activity in mammalian cells. We show that Nudt3 shifts its substrate specificity depending on the cation; specifically, Nudt3 is active on polyP when Zn2+ is present. Nudt3 has in vivo polyP phosphatase activity in human cells, and importantly, we show that cells with altered polyP levels by modifying Nudt3 protein amount present reduced viability upon oxidative stress and increased DNA damage, suggesting that polyP and Nudt3 play a role in oxidative stress protection. Finally, we show that Nudt3 is involved in the early stages of embryo development in zebrafish.

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.

Photocages have been successfully applied in cellular signaling studies for the controlled release of metabolites with high spatio-temporal resolution. Commonly, coumarin photocages are activated by UV light and the quantum yields of uncaging are relatively low, which can limit their applications in vivo. Here, syntheses, the determination of the photophysical properties, and quantum chemical calculations of 7-diethylamino-4-hydroxymethyl-thiocoumarin (thio-DEACM) and caged adenine nucleotides are reported and compared to the widely used 7-diethylamino-4-hydroxymethyl-coumarin (DEACM) caging group. In this comparison, thio-DEACM stands out as a phosphate cage with improved photophysical properties, such as red-shifted absorption and significantly faster photolysis kinetics.

Photocaged inositol-pyrophosphates offer a tool to study cellular signalling, but their challenging synthesis has precluded any biological studies so far. Here, the authors report the synthesis and cellular delivery of a photocaged analogue, and show that it mediates protein translocation in cellulo.

The HCT116 cell line, which has a pseudo-diploid karotype, is a popular model in the fields of cancer cell biology, intestinal immunity, and inflammation. In the current study, we describe two batches of diverged HCT116 cells, which we designate as HCT116NIH and HCT116UCL. Using both gel electrophoresis and HPLC, we show that HCT116UCL cells contain 6-fold higher levels of InsP8 than HCT116NIH cells. This observation is significant because InsP8 is one of a group of molecules collectively known as 'inositol pyrophosphates' (PP-InsPs)-highly 'energetic' and conserved regulators of cellular and organismal metabolism. Variability in the cellular levels of InsP8 within divergent HCT116 cell lines could have impacted the phenotypic data obtained in previous studies. This difference in InsP8 levels is more remarkable for being specific; levels of other inositol phosphates, and notably InsP6 and 5-InsP7, are very similar in both HCT116NIH and HCT116UCL lines. We also developed a new HPLC procedure to record 1-InsP7 levels directly (for the first time in any mammalian cell line); 1-InsP7 comprised

Pharmacological tools-'chemical probes'-that intervene in cell signaling cascades are important for complementing genetically-based experimental approaches. Probe development frequently begins with a high-throughput screen (HTS) of a chemical library. Herein, we describe the design, validation, and implementation of the first HTS-compatible strategy against any inositol phosphate kinase. Our target enzyme, PPIP5K, synthesizes 'high-energy' inositol pyrophosphates (PP-InsPs), which regulate cell function at the interface between cellular energy metabolism and signal transduction. We optimized a time-resolved, fluorescence resonance energy transfer ADP-assay to record PPIP5K-catalyzed, ATP-driven phosphorylation of 5-InsP7 to 1,5-InsP8 in 384-well format (Z' = 0.82 ± 0.06). We screened a library of 4745 compounds, all anticipated to be membrane-permeant, which are known-or conjectured based on their structures-to target the nucleotide binding site of protein kinases. At a screening concentration of 13 μM, fifteen compounds inhibited PPIP5K >50%. The potency of nine of these hits was confirmed by dose-response analyses. Three of these molecules were selected from different structural clusters for analysis of binding to PPIP5K, using isothermal calorimetry. Acceptable thermograms were obtained for two compounds, UNC10112646 (Kd = 7.30 ± 0.03 μM) and UNC10225498 (Kd = 1.37 ± 0.03 μM). These Kd values lie within the 1-10 μM range generally recognized as suitable for further probe development. In silico docking data rationalizes the difference in affinities. HPLC analysis confirmed that UNC10225498 and UNC10112646 directly inhibit PPIP5K-catalyzed phosphorylation of 5-InsP7 to 1,5-InsP8; kinetic experiments showed inhibition to be competitive with ATP. No other biological activity has previously been ascribed to either UNC10225498 or UNC10112646; moreover, at 10 μM, neither compound inhibits IP6K2, a structurally-unrelated PP-InsP kinase. Our screening strategy may be generally applicable to inhibitor discovery campaigns for other inositol phosphate kinases.