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

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

Itraconazole inhibits endothelial cell migration by disrupting inositol 2 pyrophosphate-dependent focal adhesion dynamics and cytoskeletal remodeling

The antifungal drug itraconazole has been repurposed to anti-angiogenic agent, but the mechanisms of action have been elusive. Here we report that itraconazole disrupts focal adhesion dynamics and cytoskeletal remodeling, which requires 5-diphosphoinositol 1,2,3,4,6-pentakisphosphate (5-InsP7). We find that inositol hexakisphosphate kinase 1 (IP6K1) binds Arp2 and generates 5-InsP7 to recruit coronin, a negative regulator of the Arp2/3 complex. IP6K1 also produces focal adhesion-enriched 5-InsP7, which binds focal adhesion kinase (FAK) at the FERM domain to promote its dimerization and phosphorylation. Itraconazole treatment elicits displacement of IP6K1/5-InsP7, thus augments 5-InsP7-mediated inhibition of Arp2/3 complex and reduces 5-InsP7-mediated FAK dimerization. Itraconazole-treated cells display reduced focal adhesion dynamics and actin cytoskeleton remodeling. Accordingly, itraconazole severely disrupts cell motility, an essential component of angiogenesis. These results demonstrate critical roles of IP6K1-generated 5-InsP7 in regulating focal adhesion dynamics and actin cytoskeleton remodeling and reveal functional mechanisms by which itraconazole inhibits cell motility

Evaluation of minimum unit pricing of alcohol: A mixed method natural experiment in Scotland

In May 2018, Scotland became the first country in the world to implement minimum unit pricing (MUP) for all alcoholic drinks sold in licensed premises in Scotland. The use of a Sunset Clause in the MUP legislation was a factor in successfully resisting legal challenges by indicating that the final decision on a novel policy would depend on its impact. An overarching evaluation has been designed and the results will provide important evidence to inform the parliamentary vote on the future of MUP in Scotland. The evaluation uses a mixed methods portfolio of in-house, commissioned, and separately funded studies to assess the impact of MUP across multiple intended and unintended outcomes related to compliance, the alcoholic drinks industry, consumption, and health and social harms. Quantitative studies to measure impact use a suitable control where feasible. Qualitative studies assess impact and provide an understanding of the lived experience and mechanism of change for key sub-groups. As well as providing important evidence to inform the parliamentary vote, adding to the international evidence on impact and experience of alcohol pricing policy across a broad range of outcomes, this approach to evaluating novel policy interventions may provide guidance for future policy innovations

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.</p

Itraconazole inhibits endothelial cell migration by disrupting inositol pyrophosphate-dependent focal adhesion dynamics and cytoskeletal remodeling

The antifungal drug itraconazole has been repurposed to anti-angiogenic agent, but the mechanisms of action have been elusive. Here we report that itraconazole disrupts focal adhesion dynamics and cytoskeletal remodeling, which requires 5-diphosphoinositol 1,2,3,4,6-pentakisphosphate (5-InsP7). We find that inositol hexakisphosphate kinase 1 (IP6K1) binds Arp2 and generates 5-InsP7 to recruit coronin, a negative regulator of the Arp2/3 complex. IP6K1 also produces focal adhesion-enriched 5-InsP7, which binds focal adhesion kinase (FAK) at the FERM domain to promote its dimerization and phosphorylation. Itraconazole treatment elicits displacement of IP6K1/5-InsP7, thus augments 5-InsP7-mediated inhibition of Arp2/3 complex and reduces 5-InsP7-mediated FAK dimerization. Itraconazole-treated cells display reduced focal adhesion dynamics and actin cytoskeleton remodeling. Accordingly, itraconazole severely disrupts cell motility, an essential component of angiogenesis. These results demonstrate critical roles of IP6K1-generated 5-InsP7 in regulating focal adhesion dynamics and actin cytoskeleton remodeling and reveal functional mechanisms by which itraconazole inhibits cell motility