The EU-OPENSCREEN (EU-OS) European Research Infrastructure Consortium (ERIC) is a multinational, not-for-profit initiative that integrates high-capacity screening platforms and chemistry groups across Europe to facilitate research in chemical biology and early drug discovery. Over the years, the EU-OS has assembled a high-throughput screening compound collection, the European Chemical Biology Library (ECBL), that contains approximately 100 000 commercially available small molecules and a growing number of thousands of academic compounds crowdsourced through our network of European and non-European chemists. As an extension of the ECBL, here we describe the computational design, quality control and use case screenings of the European Fragment Screening Library (EFSL) composed of 1056 mini and small chemical fragments selected from a substructure analysis of the ECBL. Access to the EFSL is open to researchers from both academia and industry. Using EFSL, eight fragment screening campaigns using different structural and biophysical methods have successfully identified fragment hits in the last two years. As one of the highlighted projects for antibiotics, we describe the screening by Bio-Layer Interferometry (BLI) of the EFSL, the identification of a 35 mu M fragment hit targeting the beta-ketoacyl-ACP synthase 2 (FabF), its binding confirmation to the protein by X-ray crystallography (PDB 8PJ0), its subsequent rapid exploration of its surrounding chemical space through hit-picking of ECBL compounds that contain the fragment hit as a core substructure, and the final binding confirmation of two follow-up hits by X-ray crystallography (PDB 8R0I and 8R1V). The process by which researchers from all over the world can apply for projects using the EFSL poised to the ECBL at the EU-OPENSCREEN screening facilities and optimize the hits identified through our network of chemistry partners.

1. Myo-inositol (MI) is an essential metabolite for cell function in animals and humans. The aim of this study was to characterise the transport mechanism of MI in the small intestine of laying hens as there is a lack of knowledge about the MI uptake mechanisms. The hypothesised secondary active, cation coupled transport of MI was assessed by electrophysiological measurements with Ussing chambers, and was compared to the electrophysiology of glucose transport.2. Twenty-six laying hens were used. The potential ion-dependent transport was tested in tissue of the small intestine. Barrier function of the tissue was shown by determining the transepithelial resistance. During the experiments, mucosal and serosal buffers were sampled to measure time-dependent changes in MI concentrations. Samples from eight hens were further used for Western blot analyses of the jejunal apical membranes.3. Active MI transport, indicated by changes in the short circuit current after MI addition, could not be demonstrated in the Ussing chambers experiments. MI was further not detectable in the serosal buffer, nor in the lysates of mucosal tissue cytoplasm nor lipids. Thus, there was no evidence for a MI transport or absorption. However, Western blot analyses of the jejunal apical membrane revealed signals indicated the expression of the MI transport proteins SMIT-1 and SMIT-2.4. In conclusion, the MI transport process in the chicken intestine is more complex than it was presumed and is probably influenced by still unknown regulations or metabolic processes.

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.

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.

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 InsP 8 –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 InsP 8 regulates plant Pi homeostasis by controlling the oligomeric state and hence the promoter binding capability of PHRs via their SPX receptors.

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).

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.

ABSTRACT In plants, phosphate (P i ) homeostasis is regulated by the interaction of P i starvation response transcription factors (PHRs) with stand-alone SPX proteins, which act as sensors for inositol pyrophosphates (PP-InsPs). Recently, ITPK1 was shown to generate the PP-InsP InsP 7 from InsP 6 in vitro , but the importance of this activity in P i signaling remained unknown. Here, we show that uncontrolled P i accumulation in ITPK1-deficient plants is accompanied by impaired P i -dependent InsP 7 and InsP 8 synthesis. Reciprocal grafting demonstrates that P i starvation responses are mainly controlled by ITPK1 activity in shoots. Nuclear magnetic resonance assays and PAGE analyses with recombinant protein reveal that besides InsP 6 phosphorylation, ITPK1 is also able to catalyze ATP synthesis using 5-InsP 7 but not any other InsP 7 isomer as a P-donor when ATP is low. Additionally, we show that the dynamic changes in InsP 7 and InsP 8 to cellular P i are conserved from land plant species to human cells, suggesting that P i -dependent PP-InsP synthesis is a common component of P i signaling across kingdoms. Together, our study demonstrates how P i -dependent changes in nutritional and energetic states modulate ITPK1 activities to fine-tune the synthesis of PP-InsPs.

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.

In recent years, labile phosphorylation sites on arginine, histidine, cysteine, and lysine as well as pyrophosphorylation of serine and threonine have gained more attention in phosphoproteomic studies. However, the analysis of these delicate posttranslational modifications via tandem mass spectrometry remains a challenge. Common fragmentation techniques such as collision-induced dissociation (CID) and higher energy collisional dissociation (HCD) are limited due to extensive phosphate-related neutral loss. Electron transfer dissociation (ETD) has shown to preserve labile modifications, but is restricted to higher charge states, missing the most prevalent doubly charged peptides. Here, we report the ability of electron transfer/higher energy collisional dissociation (EThcD) to fragment doubly charged phosphorylated peptides without losing the labile modifications. Using synthetic peptides that contain phosphorylated arginine, histidine, cysteine, and lysine as well as pyrophosphorylated serine residues, we evaluated the optimal fragmentation conditions, demonstrating that EThcD is the method of choice for unambiguous assignment of tryptic, labile phosphorylated peptides.

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-β32P, 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.

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.

The analysis of inositol poly- and pyrophosphates, an important group of eukaryotic messengers, is enabled by applying 13C-labeled inositol.

Non-enzymatic post-translational modifications of proteins can occur when a nucleophilic or redox-sensitive amino acid side chain encounters a reactive metabolite. In many cases, the biological function of these modifications is limited by their irreversibility, and consequently these non-enzymatic modifications are often considered as indicators of stress and disease. Certain non-enzymatic post-translational modifications, however, can be reversed, which provides an additional layer of regulation and renders these modifications suitable for controlling a diverse set of cellular processes ranging from signaling to metabolism. Here we summarize recent examples of irreversible and reversible non-enzymatic modifications, with an emphasis on the latter category. We use two examples, lysine glutarylation and pyrophosphorylation, to highlight principles of the regulation of reversible non-enzymatic post-translational modifications in more detail. Overall, a picture emerges that goes well beyond nonspecific chemical reactions and cellular damage, and instead portrays multifaceted functions of non-enzymatic post-translational modifications.

AbstractMany eukaryotic proteins regulating phosphate (Pi) homeostasis contain SPX domains. We have previously shown that these domains act as cellular receptors for inositol pyrophosphate (PP-InsP) signaling molecules, suggesting that PP-InsPs may regulate Pi homeostasis. Here we report that simultaneous deletion of two diphosphoinositol pentakisphosphate kinases VIH1 and 2 in Arabidopsis impairs plant growth and leads to constitutive Pi starvation responses. We demonstrate that VIH1 and VIH2 are bifunctional cytosolic enzymes able to generate and break-down PP-InsPs. Point-mutants targeting the kinase and phosphatase active sites have opposing effects on plant Pi content and Pi starvation responses, while VIH1 and VIH2 protein levels remain constant in different Pi growth conditions. Enzymatic assays reveal that ATP-Mg2+ substrate levels can shift the relative kinase and phosphatase activities of full-length diphosphoinositol pentakisphosphate kinases. Deletion of phosphate starvation response transcription factors rescues vih1 vih2 mutant phenotypes, placing diphosphoinositol pentakisphosphate kinases and PP-InsPs in plant phosphate signal transduction cascades. We propose that VIH1 and VIH2 relay changes in cellular ATP concentration to changes in PPInsP levels, allowing plants to maintain cellular Pi concentrations constant and to trigger Pi starvation responses.