An unconventional gatekeeper mutation sensitizes inositol hexakisphosphate kinases to an allosteric inhibitor.

Inositol hexakisphosphate kinases (IP6Ks) are emerging as relevant pharmacological targets because a multitude of disease-related phenotypes has been associated with their function. While the development of potent IP6K inhibitors is gaining momentum, a pharmacological tool to distinguish the mammalian isozymes is still lacking. Here, we implemented an analog-sensitive approach for IP6Ks and performed a high-throughput screen to identify suitable lead compounds. The most promising hit, FMP-201300, exhibited high potency and selectivity toward the unique valine gatekeeper mutants of IP6K1 and IP6K2, compared to the respective wild-type (WT) kinases. Biochemical validation experiments revealed an allosteric mechanism of action that was corroborated by hydrogen deuterium exchange mass spectrometry measurements. The latter analysis suggested that displacement of the αC helix, caused by the gatekeeper mutation, facilitates the binding of FMP-201300 to an allosteric pocket adjacent to the ATP-binding site. FMP-201300 therefore serves as a valuable springboard for the further development of compounds that can selectively target the three mammalian IP6Ks; either as analog-sensitive kinase inhibitors or as an allosteric lead compound for the WT kinases.

Fluorination Influences the Bioisostery of Myo-Inositol Pyrophosphate Analogs.

Inositol pyrophosphates (PP-IPs) are densely phosphorylated messenger molecules involved in numerous biological processes. PP-IPs contain one or two pyrophosphate group(s) attached to a phosphorylated myo-inositol ring. 5PP-IP5 is the most abundant PP-IP in human cells. To investigate the function and regulation by PP-IPs in biological contexts, metabolically stable analogs have been developed. Here, we report the synthesis of a new fluorinated phosphoramidite reagent and its application for the synthesis of a difluoromethylene bisphosphonate analog of 5PP-IP5 . Subsequently, the properties of all currently reported analogs were benchmarked using a number of biophysical and biochemical methods, including co-crystallization, ITC, kinase activity assays and chromatography. Together, the results showcase how small structural alterations of the analogs can have notable effects on their properties in a biochemical setting and will guide in the choice of the most suitable analog(s) for future investigations.

A Series of Ni Complexes Based on a Versatile ATCUN-Like Tripeptide Scaffold to Decipher Key Parameters for Superoxide Dismutase Activity.

The cellular level of reactive oxygen species (ROS) has to be controlled to avoid some pathologies, especially those linked to oxidative stress. One strategy for designing antioxidants consists of modeling natural enzymes involved in ROS degradation. Among them, nickel superoxide dismutase (NiSOD) catalyzes the dismutation of the superoxide radical anion, O2•-, into O2 and H2O2. We report here Ni complexes with tripeptides derived from the amino-terminal CuII- and NiII-binding (ATCUN) motif that mimics some structural features found in the active site of the NiSOD. A series of six mononuclear NiII complexes were investigated in water at physiological pH with different first coordination spheres, from compounds with a N3S to N2S2 set, and also complexes that are in equilibrium between the N-coordination (N3S) and S-coordination (N2S2). They were fully characterized by a combination of spectroscopic techniques, including 1H NMR, UV-vis, circular dichroism, and X-ray absorption spectroscopy, together with theoretical calculations and their redox properties studied by cyclic voltammetry. They all display SOD-like activity, with a kcat ranging between 0.5 and 2.0 × 106 M-1 s-1. The complexes in which the two coordination modes are in equilibrium are the most efficient, suggesting a beneficial effect of a nearby proton relay.

Inositol pyrophosphates activate the vacuolar transport chaperone complex in yeast by disrupting a homotypic SPX domain interaction.

Many proteins involved in eukaryotic phosphate homeostasis are regulated by SPX domains. In yeast, the vacuolar transporter chaperone (VTC) complex contains two such domains, but mechanistic details of its regulation are not well understood. Here, we show at the atomic level how inositol pyrophosphates interact with SPX domains of subunits Vtc2 and Vtc3 to control the activity of the VTC complex. Vtc2 inhibits the catalytically active VTC subunit Vtc4 by homotypic SPX-SPX interactions via the conserved helix α1 and the previously undescribed helix α7. Binding of inositol pyrophosphates to Vtc2 abrogates this interaction, thus activating the VTC complex. Accordingly, VTC activation is also achieved by site-specific point mutations that disrupt the SPX-SPX interface. Structural data suggest that ligand binding induces reorientation of helix α1 and exposes the modifiable helix α7, which might facilitate its post-translational modification in vivo. The variable composition of these regions within the SPX domain family might contribute to the diversified SPX functions in eukaryotic phosphate homeostasis.

A Simple Fluorescence Affinity Assay to Decipher Uranyl-Binding to Native Proteins.

Determining the affinity of proteins for uranyl is key to understand the toxicity of this cation and to further develop decorporation strategies. However, usual techniques to achieve that goal often require specific equipment and expertise. Here, we propose a simple, efficient, fluorescence-based method to assess the affinity of proteins and peptides for uranyl, at equilibrium and in buffered solution. We first designed and characterized an original uranyl-binding fluorescent probe. We then built a reference scale for uranyl affinity in solution, relying on signal quenching of our fluorescent probe in presence of high-affinity uranyl-binding peptides. We finally validated our approach by re-evaluating the uranyl-binding affinity of four native proteins. We envision that this tool will facilitate the reliable and reproducible assessment of affinities of peptides and proteins for uranyl.

Dissecting the activation of insulin degrading enzyme by inositol pyrophosphates and their bisphosphonate analogs.

Inositol poly- and pyrophosphates (InsPs and PP-InsPs) are densely phosphorylated eukaryotic messengers, which are involved in numerous cellular processes. To elucidate their signaling functions at the molecular level, non-hydrolyzable bisphosphonate analogs of inositol pyrophosphates, PCP-InsPs, have been instrumental. Here, an efficient synthetic strategy to obtain these analogs in unprecedented quantities is described - relying on the use of combined phosphate ester-phosphoramidite reagents. The PCP-analogs, alongside their natural counterparts, were applied to investigate their regulatory effect on insulin-degrading enzyme (IDE), using a range of biochemical, biophysical and computational methods. A unique interplay between IDE, its substrates and the PP-InsPs was uncovered, in which the PP-InsPs differentially modulated the activity of the enzyme towards short peptide substrates. Aided by molecular docking and molecular dynamics simulations, a flexible binding mode for the InsPs/PP-InsPs was identified at the anion binding site of IDE. Targeting IDE for therapeutic purposes should thus take regulation by endogenous PP-InsP metabolites into account.

New structural insights reveal an expanded reaction cycle for inositol pyrophosphate hydrolysis by human DIPP1.

Nudix hydrolases attract considerable attention for their wide range of specialized activities in all domains of life. One particular group of Nudix phosphohydrolases (DIPPs), through their metabolism of diphosphoinositol polyphosphates (PP-InsPs), regulates the actions of these polyphosphates upon bioenergetic homeostasis. In the current study, we describe, at an atomic level, hitherto unknown properties of human DIPP1.We provide X-ray analysis of the catalytic core of DIPP1 in crystals complexed with either natural PP-InsPs, alternative PP-InsP stereoisomers, or non-hydrolysable methylene bisphosphonate analogs ("PCP-InsPs"). The conclusions that we draw from these data are interrogated by studying the impact upon catalytic activity upon mutagenesis of certain key residues. We present a picture of a V-shaped catalytic furrow with overhanging ridges constructed from flexible positively charged side chains; within this cavity, the labile phosphoanhydride bond is appropriately positioned at the catalytic site by an extensive series of interlocking polar contacts which we analogize as "suspension cables." We demonstrate functionality for a triglycine peptide within a ß-strand which represents a non-canonical addition to the standard Nudix catalytic core structure. We describe pre-reaction enzyme/substrate states which we posit to reflect a role for electrostatic steering in substrate capture. Finally, through time-resolved analysis, we uncover a chronological sequence of DIPP1/product post-reaction states, one of which may rationalize a role for InsP6 as an inhibitor of catalytic activity.

Triplexed Affinity Reagents to Sample the Mammalian Inositol Pyrophosphate Interactome.

The inositol pyrophosphates (PP-InsPs) are a ubiquitous group of highly phosphorylated eukaryotic messengers. They have been linked to a panoply of central cellular processes, but a detailed understanding of the discrete signaling events is lacking in most cases. To create a more mechanistic picture of PP-InsP signaling, we sought to annotate the mammalian interactome of the most abundant inositol pyrophosphate 5PP-InsP5. To do so, triplexed affinity reagents were developed, in which a metabolically stable PP-InsP analog was immobilized in three different ways. Application of these triplexed reagents to mammalian lysates identified between 300 and 400 putative interacting proteins. These interactomes revealed connections between 5PP-InsP5 and central cellular regulators, such as lipid phosphatases, protein kinases, and GTPases, and identified protein domains commonly targeted by 5PP-InsP5. Both the triplexed affinity reagents, and the proteomic datasets, constitute powerful resources for the community, to launch future investigations into the multiple signaling modalities of inositol pyrophosphates.

InsP7 is a small-molecule regulator of NUDT3-mediated mRNA decapping and processing-body dynamics.

Regulation of enzymatic 5' decapping of messenger RNA (mRNA), which normally commits transcripts to their destruction, has the capacity to dynamically reshape the transcriptome. For example, protection from 5' decapping promotes accumulation of mRNAs into processing (P) bodies-membraneless, biomolecular condensates. Such compartmentalization of mRNAs temporarily removes them from the translatable pool; these repressed transcripts are stabilized and stored until P-body dissolution permits transcript reentry into the cytosol. Here, we describe regulation of mRNA stability and P-body dynamics by the inositol pyrophosphate signaling molecule 5-InsP7 (5-diphosphoinositol pentakisphosphate). First, we demonstrate 5-InsP7 inhibits decapping by recombinant NUDT3 (Nudix [nucleoside diphosphate linked moiety X]-type hydrolase 3) in vitro. Next, in intact HEK293 and HCT116 cells, we monitored the stability of a cadre of NUDT3 mRNA substrates following CRISPR-Cas9 knockout of PPIP5Ks (diphosphoinositol pentakisphosphate 5-kinases type 1 and 2, i.e., PPIP5K KO), which elevates cellular 5-InsP7 levels by two- to threefold (i.e., within the physiological rheostatic range). The PPIP5K KO cells exhibited elevated levels of NUDT3 mRNA substrates and increased P-body abundance. Pharmacological and genetic attenuation of 5-InsP7 synthesis in the KO background reverted both NUDT3 mRNA substrate levels and P-body counts to those of wild-type cells. Furthermore, liposomal delivery of a metabolically resistant 5-InsP7 analog into wild-type cells elevated levels of NUDT3 mRNA substrates and raised P-body abundance. In the context that cellular 5-InsP7 levels normally fluctuate in response to changes in the bioenergetic environment, regulation of mRNA structure by this inositol pyrophosphate represents an epitranscriptomic control process. The associated impact on P-body dynamics has relevance to regulation of stem cell differentiation, stress responses, and, potentially, amelioration of neurodegenerative diseases and aging.

Control of XPR1-dependent cellular phosphate efflux by InsP8 is an exemplar for functionally-exclusive inositol pyrophosphate signaling.

Homeostasis of cellular fluxes of inorganic phosphate (Pi) supervises its structural roles in bones and teeth, its pervasive regulation of cellular metabolism, and its functionalization of numerous organic compounds. Cellular Pi efflux is heavily reliant on Xenotropic and Polytropic Retrovirus Receptor 1 (XPR1), regulation of which is largely unknown. We demonstrate specificity of XPR1 regulation by a comparatively uncharacterized member of the inositol pyrophosphate (PP-InsP) signaling family: 1,5-bis-diphosphoinositol 2,3,4,6-tetrakisphosphate (InsP8). XPR1-mediated Pi efflux was inhibited by reducing cellular InsP8 synthesis, either genetically (knockout [KO] of diphosphoinositol pentakisphosphate kinases [PPIP5Ks] that synthesize InsP8) or pharmacologically [cell treatment with 2.5 µM dietary flavonoid or 10 µM N2-(m-trifluorobenzyl), N6-(p-nitrobenzyl) purine], to inhibit inositol hexakisphosphate kinases upstream of PPIP5Ks. Attenuated Pi efflux from PPIP5K KO cells was quantitatively phenocopied by KO of XPR1 itself. Moreover, Pi efflux from PPIP5K KO cells was rescued by restoration of InsP8 levels through transfection of wild-type PPIP5K1; transfection of kinase-dead PPIP5K1 was ineffective. Pi efflux was also rescued in a dose-dependent manner by liposomal delivery of a metabolically resistant methylene bisphosphonate (PCP) analog of InsP8; PCP analogs of other PP-InsP signaling molecules were ineffective. High-affinity binding of InsP8 to the XPR1 N-terminus (Kd = 180 nM) was demonstrated by isothermal titration calorimetry. To derive a cellular biology perspective, we studied biomineralization in the Soas-2 osteosarcoma cell line. KO of PPIP5Ks or XPR1 strongly reduced Pi efflux and accelerated differentiation to the mineralization end point. We propose that catalytically compromising PPIP5K mutations might extend an epistatic repertoire for XPR1 dysregulation, with pathological consequences for bone maintenance and ectopic calcification.

Modeling the Oxygen Vacancy at a Molecular Vanadium(III) Silica-Supported Catalyst.

Here we report on the use of a silanol-decorated polyoxotungstate, [SbW9O33( tBuSiOH)3]3- (1), as a molecular support to describe the coordination of a vanadium atom at a single-site on silica surfaces. By reacting [V(Mes)3·thf] (Mes = 2,4,6-trimethylphenyl) with 1 in tetrahydrofuran, the vanadium(III) derivative [SbW9O33( tBuSiO)3V(thf)]3- (2) was obtained. Compound 2 displays the paramagnetic behavior expected for a d2-VIII high spin complex (SQUID measurements) with a triplet electronic ground state (ca. 30 kcal·mol-1 more stable than the singlet, from DFT calculations). Compound 2 proves to be a reliable model for reduced isolated-vanadium atom dispersed on silica surfaces [(≡Si-O)3VIII(OH2)], an intermediate that is often proposed in a Mars-van Krevelen type mechanism for partial oxidation of light alcohols. Oxidation of 2 under air produced the oxo-derivative [SbW9O33( tBuSiO)3VO]3- (3). In compound 2, the d2-electrons are localized in degenerated d(V) orbitals, whereas in the electronically analogous bireduced-[SbW9O33( tBuSiO)3VO]5-, 3·(2e), one electron is localized on d(V) orbital and the second one is delocalized on the polyoxotungstic framework, leading to a unique case of a bireduced heteropolyanion derivative with completely decoupled d1-V(IV) and d1-W(V). Our body of experimental results (EPR, magnetic measurements, spectroelectrochemical studies, Raman spectroscopy) and theoretical studies highlights (i) the role of the apical ligand coordination, i.e., thf (σ-donor) vs oxo (π-donor), in destabilizing or stabilizing the d(V) orbitals relative to the d(W) orbitals, and (ii) a geometrical distortion of the O3VO entity that causes a splitting of the degenerated orbitals and the stabilization of one d(V) orbital in the bireduced compound 3·(2e).

Graftable SCoMPIs enable the labeling and X-ray fluorescence imaging of proteins.

Bio-imaging techniques alternative to fluorescence microscopy are gaining increasing interest as complementary tools to visualize and analyze biological systems. Among them, X-ray fluorescence microspectroscopy provides information on the local content and distribution of heavy elements (Z ≥ 14) in cells or biological samples. In this context, similar tools to those developed for fluorescence microscopy are desired, including chemical probes or tags. In this work, we study rhenium complexes as a convenient and sensitive probe for X-ray fluorescence microspectroscopy. We demonstrate their ability to label and sense exogenously incubated or endogenous proteins inside cells.

Labeling of Hyaluronic Acids with a Rhenium-tricarbonyl Tag and Percutaneous Penetration Studied by Multimodal Imaging.

Hyaluronic acids were labeled with a rhenium-tricarbonyl used as single core multimodal probe for imaging and their penetration into human skin biopsies was studied using IR microscopy and fluorescence imaging (labeled SCoMPI). The penetration was shown to be dependent on the molecular weight of the molecule and limited to the upper layer of the skin.

A rhenium tris-carbonyl derivative as a model molecule for incorporation into phospholipid assemblies for skin applications.

A rhenium tris-carbonyl derivative (fac-[Re(CO)3Cl(2-(1-dodecyl-1H-1,2,3,triazol-4-yl)-pyridine)]) was incorporated into phospholipid assemblies, called bicosomes, and the penetration of this molecule into skin was monitored using Fourier-transform infrared microspectroscopy (FTIR). To evaluate the capacity of bicosomes to promote the penetration of this derivative, the skin penetration of the Re(CO)3 derivative dissolved in dimethyl sulfoxide (DMSO), a typical enhancer, was also studied. Dynamic light scattering results (DLS) showed an increase in the size of the bicosomes with the incorporation of the Re(CO)3 derivative, and the FTIR microspectroscopy showed that the Re(CO)3 derivative incorporated in bicosomes penetrated deeper into the skin than when dissolved in DMSO. When this molecule was applied on the skin using the bicosomes, 60% of the Re(CO)3 derivative was retained in the stratum corneum (SC) and 40% reached the epidermis (Epi). Otherwise, the application of this molecule via DMSO resulted in 95% of the Re(CO)3 derivative being in the SC and only 5% reaching the Epi. Using a Re(CO)3 derivative with a dodecyl-chain as a model molecule, it was possible to determine the distribution of molecules with similar physicochemical characteristics in the skin using bicosomes. This fact makes these nanostructures promising vehicles for the application of lipophilic molecules inside the skin.

Magnetic control of protein spatial patterning to direct microtubule self-assembly.

Living systems offer attractive strategies to generate nanoscale structures because of their innate functional properties such as the dynamic assembly of ordered nanometer fibers, the generation of mechanical forces, or the directional transport mediated by molecular motors. The design of hybrid systems, capable of interfacing artificial building blocks with biomolecules, may be a key step toward the rational design of nanoscale devices and materials. Here, we have designed a bottom-up approach to organize cytoskeletal elements in space using the self-assembly properties of magnetic nanoparticles conjugated to signaling proteins involved in microtubule nucleation. We show that magnetic nanoparticles conjugated to signaling proteins involved in microtubule nucleation can control the positioning of microtubule assembly. Under a magnetic field, a self-organized pattern of biofunctionalized nanoparticles is formed and leads to the nucleation of a periodical network of microtubules in Xenopus laevis egg extract. Our method shows how bioactive nanoparticles can generate a biochemically active pattern upon magnetic actuation, which triggers the spatial organization of nonequilibrium biological structures.

Spectroscopic and structural investigations reveal the signaling mechanism of a luminescent molybdate sensor.

A heteroditopic ligand H(2)-L consisting of a dihydroxybenzene (catechol)-unit linked via an amide bond to a pyridyl-unit and its methyl-protected precursor Me(2)-L were synthesized, characterized, and their photophysical properties investigated. The three accessible protonation states of the ligand, H(3)-L(+), H(2)-L, and H-L(-), showed distinct (1)H NMR, absorption and emission spectroscopic characteristics that allow pH-sensing. The spectroscopic signatures obtained act as a guide to understand the signaling mechanism of the luminescent pH and molybdate sensor [Re(bpy)(CO)(3)(H(2)-L)](+). It was found that upon deprotonation of the 2-hydroxy group of H(2)-L, a ligand-based absorption band emerges that overlaps with the Re(dπ)→bpy metal-to-ligand charge transfer (MLCT) band of the sensor, reducing the quantum yield for emission on excitation in the 370 nm region. In addition, deprotonation of the catechol-unit leads to quenching of the emission from the Re(dπ)→bpy (3)MLCT state, consistent with photoinduced electron transfer from the electron-rich, deprotonated catecholate to the Re-based luminophore. Finally, reaction of 2 equiv of [Re(bpy)(CO)(3)(H(2)-L)](+) with molybdate was shown to give the zwitterionic Mo(VI) complex [MoO(2){Re(CO)(3)(bpy)(L)}(2)], as confirmed by electrospray ionization (ESI) mass spectrometry and X-ray crystallography. The crystal structure determination revealed that two fully deprotonated sensor molecules are bound via their oxygen-donors to a cis-dioxo-MoO(2) center.