Expedient synthesis and luminescence sensing of the inositol pyrophosphate cellular messenger 5-PP-InsP5

Inositol pyrophosphates are important biomolecules associated with apoptosis, cell growth and kinase regulation, yet their exact biological roles are still emerging and probes do not exist for their selective detection. We report the first molecular probe for the selective and sensitive detection of the most abundant cellular inositol pyrophosphate 5-PP-InsP5, as well as an efficient new synthesis. The probe is based on a macrocyclic Eu(III) complex bearing two quinoline arms providing a free coordination site at the Eu(III) metal centre. Bidentate binding of the pyrophosphate group of 5-PP-InsP5 to the Eu(III) ion is proposed, supported by DFT calculations, giving rise to a selective enhancement in Eu(III) emission intensity and lifetime. We demonstrate the use of time-resolved luminescence as a bioassay tool for monitoring enzymatic processes in which 5-PP-InsP5 is consumed. Our probe offers a potential screening methodology to identify drug-like compounds that modulate the activity of enzymes of inositol pyrophosphate metabolism

Multiple substrate recognition by yeast diadenosine and diphosphoinositol polyphosphate phosphohydrolase through phosphate clamping

The yeast diadenosine and diphosphoinositol polyphosphate phosphohydrolase DDP1 is a Nudix enzyme with pyrophosphatase activity on diphosphoinositides, dinucleotides, and polyphosphates. These substrates bind to diverse protein targets and participate in signaling and metabolism, being essential for energy and phosphate homeostasis, ATPase pump regulation, or protein phosphorylation. An exhaustive structural study of DDP1 in complex with multiple ligands related to its three diverse substrate classes is reported. This allowed full characterization of the DDP1 active site depicting the molecular basis for endowing multisubstrate abilities to a Nudix enzyme, driven by phosphate anchoring following a defined path. This study, combined with multiple enzyme variants, reveals the different substrate binding modes, preferences, and selection. Our findings expand current knowledge on this important structural superfamily with implications extending beyond inositide research. This work represents a valuable tool for inhibitor/substrate design for ScDDP1 and orthologs as potential targets to address fungal infections and other health concerns

Crystal structure and enzymology of solanum tuberosum inositol tris/tetrakisphosphate kinase 1 (StITPK1)

Inositol phosphates and their pyrophosphorylated derivatives are responsive to the phosphate supply and are agents of phosphate homeostasis and other aspects of physiology. It seems likely that the enzymes that interconvert these signals work against the prevailing milieu of mixed populations of competing substrates and products. The synthesis of inositol pyrophosphates is mediated in plants by two classes of ATP-grasp fold kinase: PPIP5 kinases, known as VIH, and members of the inositol tris/tetrakisphosphate kinase (ITPK) family, specifically ITPK1/2. A molecular explanation of the contribution of ITPK1/2 to inositol pyrophosphate synthesis and turnover in plants is incomplete: the absence of nucleotide in published crystal structures limits the explanation of phosphotransfer reactions, and little is known of the affinity of potential substrates and competitors for ITPK1. Herein, we describe a complex of ADP and StITPK1 at 2.26 Å resolution and use a simple fluorescence polarization approach to compare the affinity of binding of diverse inositol phosphates, inositol pyrophosphates, and analogues. By simple HPLC, we reveal the novel catalytic capability of ITPK1 for different inositol pyrophosphates and show Ins(3,4,5,6)P4 to be a potent inhibitor of the inositol pyrophosphate-synthesizing activity of ITPK1. We further describe the exquisite specificity of ITPK1 for the myo-isomer among naturally occurring inositol hexakisphosphates

Both D- and L-glucose polyphosphates mimic D-myo-inositol 1,4,5-trisphosphate: new synthetic agonists and partial agonists at the Ins(1,4,5)P3 receptor

Chiral sugar derivatives are potential cyclitol surrogates of the Ca2+-mobilizing intracellular messenger d-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]. Six novel polyphosphorylated analogues derived from both d- and l-glucose were synthesized. Binding to Ins(1,4,5)P3 receptors [Ins(1,4,5)P3R] and the ability to release Ca2+ from intracellular stores via type 1 Ins(1,4,5)P3Rs were investigated. β-d-Glucopyranosyl 1,3,4-tris-phosphate, with similar phosphate regiochemistry and stereochemistry to Ins(1,4,5)P3, and α-d-glucopyranosyl 1,3,4-tris-phosphate are full agonists, being equipotent and 23-fold less potent than Ins(1,4,5)P3, respectively, in Ca2+-release assays and similar to Ins(1,4,5)P3 and 15-fold weaker in binding assays. They can be viewed as truncated analogues of adenophostin A and refine understanding of structure-activity relationships for this Ins(1,4,5)P3R agonist. l-Glucose-derived ligands, methyl α-l-glucopyranoside 2,3,6-trisphosphate and methyl α-l-glucopyranoside 2,4,6-trisphosphate, are also active, while their corresponding d-enantiomers, methyl α-d-glucopyranoside 2,3,6-trisphosphate and methyl α-d-glucopyranoside 2,4,6-trisphosphate, are inactive. Interestingly, both l-glucose-derived ligands are partial agonists: they are among the least efficacious agonists of Ins(1,4,5)P3R yet identified, providing new leads for antagonist development

Substrate promiscuity of inositol 1,4,5-trisphosphate kinase driven by structurally-modified ligands and active site plasticity

D-myo-inositol 1,4,5-trisphosphate (InsP3) is a fundamental second messenger in cellular Ca2+ mobilization. InsP3 3-kinase, a highly specific enzyme binding InsP3 in just one mode, phosphorylates InsP3 specifically at its secondary 3-hydroxyl group to generate a tetrakisphosphate. Using a chemical biology approach with both synthetised and established ligands, combining synthesis, crystallography, computational docking, HPLC and fluorescence polarization binding assays using fluorescently-tagged InsP3, we have surveyed the limits of InsP3 3-kinase ligand specificity and uncovered surprisingly unforeseen biosynthetic capacity. Structurally-modified ligands exploit active site plasticity generating a helix-tilt. These facilitated uncovering of unexpected substrates phosphorylated at a surrogate extended primary hydroxyl at the inositol pseudo 3-position, applicable even to carbohydrate-based substrates. Crystallization experiments designed to allow reactions to proceed in situ facilitated unequivocal characterization of the atypical tetrakisphosphate products. In summary, we define features of InsP3 3-kinase plasticity and substrate tolerance that may be more widely exploitable

Fish Health Unit Report of Activities Undertaken in 2023

This report summarises the activities undertaken by the Fish Health Unit of the Marine Institute in 2023. Regulation (EU) 2016/429 lays down the rules for the prevention and control of animal diseases which are transmissible to animal or humans and the Marine Institute is the Competent Authority responsible for implementation of this regulation in Ireland. The purpose of this report is to provide all stakeholders with an improved understanding of the operations of the Marine Institute in fish health, and the findings encountered by the Fish Health Unit in 2023.Marine Institut

Depleting inositol pyrophosphate 5-InsP7 protected the heart against ischaemia–reperfusion injury by elevating plasma adiponectin

Aims Adiponectin is an adipocyte-derived circulating protein that exerts cardiovascular and metabolic protection. Due to the futile degradation of endogenous adiponectin and the challenges of exogenous administration, regulatory mechanisms of adiponectin biosynthesis are of significant pharmacological interest. Methods and results Here, we report that 5-diphosphoinositol 1,2,3,4,6-pentakisphosphate (5-InsP7) generated by inositol hexakisphosphate kinase 1 (IP6K1) governed circulating adiponectin levels via thiol-mediated protein quality control in the secretory pathway. IP6K1 bound to adiponectin and DsbA-L and generated 5-InsP7 to stabilize adiponectin/ERp44 and DsbA-L/Ero1-Lα interactions, driving adiponectin intracellular degradation. Depleting 5-InsP7 by either IP6K1 deletion or pharmacological inhibition blocked intracellular adiponectin degradation. Whole-body and adipocyte-specific deletion of IP6K1 boosted plasma adiponectin levels, especially its high molecular weight forms, and activated AMPK-mediated protection against myocardial ischaemia–reperfusion injury. Pharmacological inhibition of 5-InsP7 biosynthesis in wild-type but not adiponectin knockout mice attenuated myocardial ischaemia–reperfusion injury. Conclusion Our findings revealed that 5-InsP7 is a physiological regulator of adiponectin biosynthesis that is amenable to pharmacological intervention for cardioprotection

Crystal structure and enzymology of Solanum tuberosum inositol tris/tetrakisphosphate kinase 1 (StITPK1)

Inositol phosphates and their pyrophosphorylated derivatives are responsive to the phosphate supply and are agents of phosphate homeostasis and other aspects of physiology. It seems likely that the enzymes that interconvert these signals work against the prevailing milieu of mixed populations of competing substrates and products. The synthesis of inositol pyrophosphates is mediated in plants by two classes of ATP-grasp fold kinase: PPIP5 kinases, known as VIH, and members of the inositol tris/tetrakisphosphate kinase (ITPK) family, specifically ITPK1/2. A molecular explanation of the contribution of ITPK1/2 to inositol pyrophosphate synthesis and turnover in plants is incomplete: the absence of nucleotide in published crystal structures limits the explanation of phosphotransfer reactions, and little is known of the affinity of potential substrates and competitors for ITPK1. Herein, we describe a complex of ADP and StITPK1 at 2.26 Å resolution and use a simple fluorescence polarization approach to compare the affinity of binding of diverse inositol phosphates, inositol pyrophosphates, and analogues. By simple HPLC, we reveal the novel catalytic capability of ITPK1 for different inositol pyrophosphates and show Ins(3,4,5,6)P4 to be a potent inhibitor of the inositol pyrophosphate-synthesizing activity of ITPK1. We further describe the exquisite specificity of ITPK1 for the myo-isomer among naturally occurring inositol hexakisphosphates

Substrate promiscuity of inositol 1,4,5-trisphosphate kinase driven by structurally-modified ligands and active site plasticity

D-myo-inositol 1,4,5-trisphosphate (InsP3) is a fundamental second messenger in cellular Ca2+ mobilization. InsP3 3-kinase, a highly specific enzyme binding InsP3 in just one mode, phosphorylates InsP3 specifically at its secondary 3-hydroxyl group to generate a tetrakisphosphate. Using a chemical biology approach with both synthetised and established ligands, combining synthesis, crystallography, computational docking, HPLC and fluorescence polarization binding assays using fluorescently-tagged InsP3, we have surveyed the limits of InsP3 3-kinase ligand specificity and uncovered surprisingly unforeseen biosynthetic capacity. Structurally-modified ligands exploit active site plasticity generating a helix-tilt. These facilitated uncovering of unexpected substrates phosphorylated at a surrogate extended primary hydroxyl at the inositol pseudo 3-position, applicable even to carbohydrate-based substrates. Crystallization experiments designed to allow reactions to proceed in situ facilitated unequivocal characterization of the atypical tetrakisphosphate products. In summary, we define features of InsP3 3-kinase plasticity and substrate tolerance that may be more widely exploitable

Inositol polyphosphates and analogues: synthesis & chemical biology

In this work, myo-inositol phosphates and pyrophosphates were investigated to facilitate further understanding of their chemical biology and biological functions. The synthesis of novel carbohydrate-based polyphosphate analogues of lower inositol phosphates, including the second messenger ᴅ-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃], was undertaken, and novel routes for some naturally-occurring inositol pyrophosphates were designed. Synthetic routes for the meso myo-inositol pyrophosphates 5-PP-InsP₅ (5-IP₇) and 5- PP-InsP₄, as well as both the racemic and chiral versions of D-1,5-(PP)₂-InsP₄ (D-1,5-IP₈), were successfully executed. These approaches incorporated the hitherto little-used methylsulfonylethyl (MSE)-protecting group, allowing its versatility to be explored. Final products were then used with collaborators to investigate: substrate binding to the yeast diphosphoinositol polyphosphate phosphohydrolase through a structural biology approach; the potential scope of a novel pyrophosphate-monitoring, europium-based luminescence assay; and structure-activity (SAR) aspects of the inositol 1,4,5,6-tetrakisphosphate binding site in the histone deacetylase 3 - SMRT corepressor (HDAC3-SMRT) complex. The SAR investigation for HDAC3-SMRT featured seven glucose-based polyphosphate compounds (two known, five novel) that were designed, synthesised and biologically evaluated. The ʟ-glucose-based ligands were found to be the first carbohydrate-based ligands able to activate the complex, while their ᴅ-glucose-based enantiomers were inactive. A binding site region suspected to be able to accommodate steric bulk was explored, and it also was concluded that inositol pyrophosphates are unlikely to be physiologically relevant endogenous activators for the HDAC3-SMRT complex. A further six novel glucose-based polyphosphates were synthesised to investigate calcium release via the Ins(1,4,5)P₃ receptor (InsP₃R). Two ligands, α-ᴅ-glucopyranosyl 1,3,4-trisphosphate and β-ᴅ-glucopyranosyl 1,3,4-trisphosphate, were full agonists, the latter equipotent to Ins(1,4,5)P₃ itself. These two ligands were also substrates for Arabidopsis inositol tetrakisphosphate kinase-1 (ITPK1). Although unconfirmed, it is proposed that ITPK1 phosphorylates the 6-position primary hydroxyl. Both ligands were uniquely co-crystallised with Ins(1,4,5)P₃ 3-kinase. The crystallographicallysolved ternary complexes also revealed the primary hydroxyl group to be well-situated for potential phosphorylation. Two of the InsP₃R-study ligands, methyl α-ʟ-glucopyranoside 2,3,6-trisphosphate and methyl α-ʟ-glucopyranoside 2,4,6-trisphosphate, were low-affinity, low-efficacy partial agonists. From the presumed binding mode of the latter, structurally-inspired myo-inositol pyrophosphate-containing compounds were designed, targeting a potential higher-affinity partial agonist or antagonist. Using techniques earlier developed, three novel inositol pyrophosphates were synthesized, 4-PP-Ins(5)P, 5-PPIns(4)P and 4-PP-Ins(1,5)₂, which are the first examples of an Ins(1,4,5)P₃ pyrophosphate analogue modified at the 4,5-vicinal bisphosphate pharmacophore. Biological evaluation of these compounds is currently underway