Design, quality and validation of the EU-OPENSCREEN fragment library poised to a high-throughput screening collection.

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.

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 (IP also known as Phytic acid) present in the amoeba allowed the discovery of the more polar inositol pyrophosphates, IP and IP, 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.

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

In plants, phosphate (P) 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 P and dependent on the inositol phosphate kinase ITPK1. We found that inositol pyrophosphates are more responsive to P than lower inositol phosphates, a response conserved across kingdoms.

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.

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.

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.

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 C-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-InsP, 1PP-InsP, and 1,5(PP)-InsP] is reported, relying on the highly active inositol hexakisphosphate kinase A from and the kinase domain of human diphosphoinositol pentakisphosphate kinase 2.

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.

Harnessing C-labeled -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 C-labeled compounds ([C]-inositol, [C]InsP, [C]InsP, and [C]5PP-InsP), in combination with commonly accessible NMR technology.

Electron Transfer/Higher Energy Collisional Dissociation of Doubly Charged Peptide Ions: Identification of Labile Protein Phosphorylations.

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.

Features and regulation of non-enzymatic post-translational modifications.

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.

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.

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.