Pyrophosphatase Activity Research Articles

Advanced electrochemical detection of pyrophosphatase activity in LNCaP cells utilizing in-situ redox of copper

The necessity for precise delineation and quantitation of pyrophosphatase (PPase) activities is underscored, especially in aggressive prostate cancer, driving the demand for more refined analytical techniques due to challenges associated with inadequate sensitivity and poor signal amplification. In response, a novel electrochemical method utilizing in-situ copper(II) (Cu(II))-based Fast Scan Cyclic Voltammetry (FSCV) is introduced for the advanced detection of PPase in LNCaP prostate cancer cells. This technique is characterized by the exploitation of competitive binding between Cu(II) and L-Phenylalanine (L-Phe) versus pyrophosphate (PPi), with FSCV technology enhanced by ohmic drop compensation to improve detection sensitivity and signal reliability. Through the application of the FSCV technique transitioned to Current(I)-Voltage(V) analysis, the signal generation is facilitated by an L-Phe-Cu(II) complex, driven by the redox reaction of in-situ Cu(II), specifically its rapid transition from the Cu(0) state back to the Cu(II) state. When this complex is anchored onto a gold substrate modified with cucurbit[7]uril (CB[7]) through supramolecular host–guest interactions, a significant FSCV signal is elicited. A detection limit of 0.05 mU/mL (S/N = 3) is subsequently achieved when the method is applied to the quantification of PPase concentration. Leveraging the FSCV method, we have effectively analyzed PPase activity in LNCaP cells, offering a fresh perspective on tumor marker analysis and fostering advancements in drug development.

Effect of Near-Freezing Temperature Storage on the Quality and Organic Acid Metabolism of Apple Fruit

Organic acids play critical roles in fruit physiological metabolism and sensory quality. However, the conventional storage of apple fruit at 0 ± 0.1 °C cannot maintain fruit acidity efficiently. This study investigated near-freezing temperature (NFT) storage for ‘Golden Delicious’ apples, and the quality parameters, organic acid content, and malate metabolism were studied. The results indicate that NFT storage at −1.7 ± 0.1 °C effectively maintained the postharvest quality of apple fruit when compared to traditional storage at 0 ± 0.1 °C. Fruit that underwent NFT storage showed a better appearance and lower respiratory rate, ethylene production, weight loss, and malondialdehyde (MDA) content but higher firmness and soluble solids content. Further, fruit after NFT storage contained higher titratable acid (18.75%), malate (51.61%), citrate (36.59%), and succinate (2.12%) content when compared to the control after 250 days. This was achieved by maintaining higher cytosolic NAD-dependent malate dehydrogenase (cyNAD-MDH), phosphoenolpyruvate carboxylase (PEPC), vacuolar H+-ATPase (V-ATPase), and vacuolar inorganic pyrophosphatase (V-PPase) activities that promote malate biosynthesis and accumulation while inhibiting enzyme activity that is responsible for malate decomposition, including phosphoenolpyruvate carboxylase kinase (PEPCK) as well as the cytosolic NAD phosphate-dependent malic enzyme (cyNADP-ME). Further, storage at NFTs maintained a higher expression of malate biosynthesis-related genes (MdcyNAD-MDH and MdPEPC) and transport-related genes (MdVHA and MdVHP) while suppressing malate consumption-related genes (MdcyME and MdPEPCK). The results demonstrate that NFT storage could be an effective application for apple fruit, which maintains postharvest quality and alleviates organic acid degradation.

Structural insights into the bifunctional enzyme human FAD synthase

Human flavin adenine dinucleotide synthase (hFADS) is a bifunctional, multi-domain enzyme that exhibits both flavin mononucleotide adenylyltransferase and pyrophosphatase activities. Here we report the crystal structure of full-length hFADS2 and its C-terminal PAPS domain in complex with flavin adenine dinucleotide (FAD), and dissect the structural determinants underlying the contribution of each individual domain, within isoforms 1 and 2, to each of the two enzymatic activities. Structural and functional characterization performed on complete or truncated constructs confirmed that the C-terminal domain tightly binds FAD and catalyzes its synthesis, while the combination of the N-terminal molybdopterin-binding and KH domains is the minimal essential substructure required for the hydrolysis of FAD and other ADP-containing dinucleotides. hFADS2 associates in a stable C2-symmetric dimer, in which the packing of the KH domain of one protomer against the N-terminal domain of the other creates the adenosine-specific active site responsible for the hydrolytic activity.

Phosphorus sorption–desorption changes phosphorus fraction dynamic in a desert revegetation chronosequence

AbstractUnderstanding the characteristics of soil phosphorus (P) sorption and desorption is essential for comprehending P biogeochemical cycling and effectively managing ecosystems in a desert revegetation chronosequence. The present study utilized the Freundlich model and enzymatic activity to characterize the features of P sorption–desorption, and microbial activity, which aims to elucidate the effect of P kinetics and microbial activity on P fractions in the soils of a desert revegetation chronosequence, consisting of 11, 31, 40, 57, and 65 years old revegetated deserts. The findings revealed that the 31 years old soil showed the highest alkaline phosphomonoesterase and phosphodiesterase activities, and the 40 years old displayed the highest inorganic pyrophosphatase activity. In revegetated desert soils, microbial activity changed P sorption–desorption kinetics by decreased or increased the parameters including sorption/desorption energy site, P sorption/desorption ability, and the maximum buffering capacity of P sorption/desorption. And in microbial activity soils of 31–40 years old, P desorption significantly decreased T‐P and A‐P concentrations (p < 0.05). P sorption process and enzyme activity explained 35.10% and 22.20% of P fraction variation, respectively; and P desorption process and enzyme activity explained 48.3% and 22.3% of P fraction variation, respectively. These findings provide valuable insights into the contribution of P kinetics coupled with microbial activity in desert ecosystems, aiding in the effective management of these fragile ecosystems.

The neuroprotective effect of dl-3-n-butylphthalide on the brain with experimental intracerebral hemorrhage

Intracerebral hemorrhage (ICH) is the most devastating subtype of stroke, nevertheless specific treatments with conclusive clinical benefit in improving outcomes of ICH remain lacking. The present study applied dl-3-n-butylphthalide (NBP), a compound approved for the treatment of ischemic stroke and rarely studied in ICH, to an experimental animal model of ICH, aiming to evaluate the therapeutic effects of NBP on ICH and the potential mechanisms. The results showed that rats receiving NBP administration exhibited a structural and functional restoration of brain after ICH mainly manifested as alleviation of neuronal apoptosis, suppression of neuroinflammation and oxidative stress, neurovascular remodeling, and eventually improvement of neurological deficits. In addition, several protein targets of NBP were revealed, which mainly play molecular functions of ribonucleoside triphosphate phosphatase activity, pyrophosphatase activity, hydrolase activity and GTPase activity, and participate in the biological process of brain development by regulating the formation of cellular components such as spindles, polymeric cytoskeletal fibers, microtubules and synapses, through mediating pathways such as VEGF signaling pathway, Fc epsilon RI signaling pathway, ECM-receptor interaction, Fc gamma R-mediated phagocytosis, peroxisome and so on, guiding the mechanism exploration of NBP therapy to some extent. Taken together, the study added some new evidence to the application of NBP in ICH treatment.

Phosphatase activities and available nutrients in soil aggregates affected by straw returning to a calcareous soil under the maize–wheat cropping system

The objective of this study was to investigate the effects of different rates of straw returning on soil aggregate stability, phosphatase activities, and the available nitrogen (N) and phosphorus (P) within different soil aggregate sizes. The experiment included five treatments: 1) no straw returning and no chemical fertilizer, 2) chemical fertilizer only (150 kg N ha-1, 75 kg P ha-1, and 75 kg K ha-1), 3) 20% straw returning with chemical fertilizer, 4) 60% straw returning with chemical fertilizer, and 5) 100% straw returning with chemical fertilizer. Soil samples were collected 3.5 years after the start of the experiment and separated into four aggregate sizes (<0.25 mm, 0.25–1 mm, 1–2 mm, and 2–7 mm) using the dry sieving method. Soil acid phosphomonoesterase (AcP) and alkaline phosphomonoesterase (AlP); phosphodiesterase (PD); pyrophosphatase (PrA) activities; and soil NO3−−N, NH4+−N, and resin-P were determined within soil aggregates. The results showed that straw returning rates did not significantly impact soil aggregate distribution. However, straw returning increased soil AcP, AlP, and PD in <2 mm aggregates, and high rates of straw returning led to high enzyme activities. Soil phosphatase activities were also higher in 1–2 mm aggregates. All straw returning and chemical fertilization treatments increased soil NO3−−N and resin-P concentrations but had much less effect on soil NH4+−N concentrations. Additionally, the study revealed that soil pH, the concentrations of NH4+−N, NO3−−N, resin-P, and CaCO3 significantly influenced soil phosphatase activities, but their impact varied across different sizes of aggregates.

Acetylation of inorganic pyrophosphatase by S-RNase signaling induces pollen tube tip swelling by repressing pectin methylesterase.

Self-incompatibility (SI) is a widespread genetically determined system in flowering plants that prevents self-fertilization to promote gene flow and limit inbreeding. S-RNase-based SI is characterized by the arrest of pollen tube growth through the pistil. Arrested pollen tubes show disrupted polarized growth and swollen tips, but the underlying molecular mechanism is largely unknown. Here, we demonstrate that the swelling at the tips of incompatible pollen tubes in pear (Pyrus bretschneideri [Pbr]) is mediated by the SI-induced acetylation of the soluble inorganic pyrophosphatase (PPA) PbrPPA5. Acetylation at Lys-42 of PbrPPA5 by the acetyltransferase GCN5-related N-acetyltransferase 1 (GNAT1) drives accumulation of PbrPPA5 in the nucleus, where it binds to the transcription factor PbrbZIP77, forming a transcriptional repression complex that inhibits the expression of the pectin methylesterase (PME) gene PbrPME44. The function of PbrPPA5 as a transcriptional repressor does not require its PPA activity. Downregulating PbrPME44 resulted in increased levels of methyl-esterified pectins in growing pollen tubes, leading to swelling at their tips. These observations suggest a mechanism for PbrPPA5-driven swelling at the tips of pollen tubes during the SI response. The targets of PbrPPA5 include genes encoding cell wall-modifying enzymes, which are essential for building a continuous sustainable mechanical structure for pollen tube growth.

A novel orally bioavailable small molecular ENPP1 inhibitor (OC-1) for cancer immunotherapy.

e15136 Background: Ectonucleotide pyrophosphatase/phosphodiesterase I (ENPP1) is a type II transmembrane glycoprotein with nucleotide pyrophosphatase and phosphodiesterase enzymatic activities. Inhibiting ENPP1 activity has emerged as an attractive approach in cancer immunotherapy, based on two distinct mechanisms. One mechanism is the stabilization of cyclic GMP–AMP (2’3’-cGAMP), which binds to stimulator of IFN genes (STING) and functions as an endogenous second messenger to activate the innate immune system; the other is the lowering of adenosine concentrations in the tumor microenvironment. ENPP1 inhibitors have shown promising monotherapy activity and synergy with various targeted therapies in preclinical models. Methods: OC-1 was characterized, for activity and selectivity, in the following in vitro assays: a luminescence-based enzymatic assay; cell proliferation assays; selectivity assays against 15 phosphodiesterases (PDE) based on inhibition of enzymatic activity; and a cytokine release assay in human THP cells and mouse splenocytes looking at changes in IFN-β/CXCL10. Oral bioavailability of OC-1 in mice and rats was studied following oral gavage. Plasma exposures were quantified using standard LC/MS/MS methods. In vivo tumor growth inhibition (TGI) of OC-1 was evaluated in syngeneic mouse tumor models (CT26 and MC38), as monotherapy and in combination with an anti-PD1 antibody. OC-1 was dosed orally in all in-vivo models. Results: We have discovered a novel small molecule inhibitor of ENPP1, OC-1, with a Ki value less than 10 nM in an ENPP1 enzymatic assay. Increase in gene expression of IFN-β and CXCL10/IP10 was observed in OC-1-treated THP1 cells. No inhibition in cell proliferation in various cancer lines was seen up to the highest concentration tested (30 µM). OC-1 was also selective against 15 PDEs with IC50 > 30 µM. The mean oral bioavailability of OC-1 was 72% and 63% in mice and rats, respectively. OC-1 showed single agent activity (20 – 40% TGI) and combination activity of approximately 75% TGI when it was combined with an anti-PD-1 antibody in in vivo efficacy studies with no impact on body weight. Conclusions: In summary, OC-1 is an orally bioavailable ENPP1 inhibitor with promising anti-tumor activity in preclinical models. Its physicochemical properties make it a suitable candidate for clinical investigation.

Protein engineering of NADH pyrophosphatase for efficient biocatalytic production of reduced nicotinamide mononucleotide.

Introduction: NADH pyrophosphatase, a hydrolase catalyzing the phosphate bond of NADH to reduced nicotinamide mononucleotide, has potential applications in the food, cosmetic and pharmaceutical industry. Methods: Here, we investigated the effects of vector screening, promoter and RBS strategies on NADH pyrophosphatase expression and protein engineering on its enzymatic activity and thermal stability. Results: In this study, we describe a NADH pyrophosphatase derived from Escherichia coli (EcNudc). Strategies focusing on expression regulation including screening vectors, optimizing promoters and ribosome binding sites were utilized to enhance the productivity of EcNudc (1.8U/mL). Moreover, protein engineering was adopted to further improve the catalytic properties of EcNudc, achieving 3.3-fold higher activity and 3.6-fold greater thermostability at 50°C. Furthermore, fermentation for the combined mutant R148A-H149E (EcNudc-M) production in a 7L fermenter was implemented and the enzyme activity of EcNudc-M reached 33.0U/mL. Finally, the EcNudc-M was applied in the catalysis of NADH with the highest NMNH yield of 16.65g/L. Discussion: In conclusion, we constructed a commercially available genetically engineered strain with high activity and thermal stability of NADH pyrophosphatase, laying a broad foundation for the biocatalytic industrial production of NMNH and expand its application range.

Comparative genomics of five Valsa species gives insights on their pathogenicity evolution.

Valsa is a genus of ascomycetes within the Valsaceae family. This family includes many wood destructive pathogens such as the well known Valsa mali and Valsa pyri which cause canker diseases in fruit trees and threaten the global fruit production. Lack of genomic information of this family is impeding our understandings about their evolution and genetic basis of their pathogenicity divergence. Here, we report genome assemblies of Valsa malicola, Valsa persoonii, and Valsa sordida which represent close relatives of Valsa mali and Valsa pyri with different host preferences. Comparative genomics analysis revealed that segmental rearrangements, inversions, and translocations frequently occurred among Valsa spp. genomes. Gene families that exhibited gene copy expansions tended to be associated with secondary metabolism, transmembrane transport, and pyrophosphatase activities. Orthologous genes in regions lost synteny exhibited significantly higher rate of synonymous substitution (KS) than those in regions retained synteny. Moreover, among these genes, membrane transporter families associated with antidrug (MFS, DHA) activities and nutrient transportation (SP and APCs) activities were significantly over-represented. Lineage specific synonymous substitution (KS) and nonsynonymous substitution (KA) analysis based on the phylogeny constructed from 11 fungal species identified a set of genes with selection signatures in Valsa clade and these genes were significantly enriched in functions associated with fatty acid beta-oxidation, DNA helicase activity, and ATPase activity. Furthermore, unique genes that possessed or retained by each of the five Valsa species are more likely part of the secondary metabolic (SM) gene clusters. SM gene clusters conserved across five Valsa species showed various degrees of diversification in both identity and completeness. All 11 syntenically conserved SM clusters showed differential expression during the infection of apple branch with Valsa mali suggesting involvements of secondary metabolism in the pathogenicity of Valsa species.

Withdrawn.

Mycobacterial inorganic pyrophosphatase (Mt-PPa) play essential role in bacterial in vitro and in vivo survival. This family of proteins reacts on inorganic pyrophosphate to release inorganic phosphate and protect bacteria from pyrophosphate toxicity. Rv3628 (Mt-PPa) encodes inorganic pyrophosphatase protein which is a type I pyrophosphatase with metal ion dependent activity. Apart from the inorganic pyrophosphatase activity, Rv3628 also showed binding with nucleotide tri phosphate (GTP/ATP). The binding of GTP and Rv3628 was analyzed by isothermal calorimetry. However, the GTP binding activity was mild but important to consider, as GTP is the secondary messenger and involved in number of mycobacterial cellular metabolic pathways therefore this interaction might be an important part of signaling cascades. This article thus represents a trilateral approach that considers computational and mutational analysis which was further complemented by virtual screening of natural compounds to find potential drug candidates. Computational analysis showed that Mt-PPa showed extensive interaction with GTP and ATP family genes. Epitope prediction showed the occurrence of amino acid regions in PPa that can act as immunological epitopes. PPa is sensitive to phosphorylation by several kinases that phosphorylate PPa at different Ser/Thr/Tyr sites. Mutational analysis showed W102G, V150G, F44G, I119G, L93F, F3G, F122G, I108G, L32G, M82G, Y17G, L59G, V5G, V26G, I7G, W140D, W140G, W140A, F80G, W140S, L49G, L56G, I9G, V60G, V19G, V92G, L28G, L61G, Y126E and F123G were the top 30 mutational hits and Y126G, Y42G, R30G, E8G, K16G are top mutational hits in active site of Mt-PPa. Mt-PPa activity is temperature and pH sensitive as the mutation in the His 21 and His 86 residues shift the optimal pH. Virtual screening of 700 compounds from herbal ingredient targets (HITs) subset of zinc database showed ZINC000003979028, ZINC000003870413, ZINC000003870412, ZINC000150338758, ZINC0000070450948, ZINC000150338754, ZINC000095098891, ZINC000000119985 and ZINC000005085286 as the top target hits and Mac0182344 and NAV_2729 as the top GTPase inhibitor that would target and hinders Mt-PPa activity. This article is protected by copyright. All rights reserved.

A higher plant FAD synthetase is fused to an inactivated FAD pyrophosphatase

The riboflavin derivatives FMN and flavin adenine dinucleotide (FAD) are critical cofactors for wide-ranging biological processes across all kingdoms of life. Although it is well established that these flavins can be readily interconverted, in plants, the responsible catalysts and regulatory mechanisms remain poorly understood. Here, we report the cloning and biochemical characterization of an FAD synthetase encoded by the gene At5g03430, which we have designated AtFADS1 (A.thaliana FADS1). The catalytic properties of the FAD synthetase activity are similar to those reported for other FAD synthetases, except that we observed maximum activity with Zn2+ as the associated divalent metal cation. Like human FAD synthetase, AtFADS1 exists as an apparent fusion with an ancestral FAD pyrophosphatase, a feature that is conserved across plants. However, we detected no pyrophosphatase activity with AtFADS1, consistent with an observed loss of a key catalytic residue in higher plant evolutionary history. In contrast, we determined that algal FADS1 retains both FAD synthetase and pyrophosphatase activity. We discuss the implications, including the potential for yet-unstudied biologically relevant noncatalytic functions, and possible evolutionary pressures that have led to the loss of FAD pyrophosphatase activity, yet universal retention of an apparently nonfunctional domain in FADS of land plants.

Inositol Pyrophosphate-Controlled Kinetochore Architecture and Mitotic Entry in S. pombe.

Inositol pyrophosphates (IPPs) comprise a specific class of signaling molecules that regulate central biological processes in eukaryotes. The conserved Vip1/PPIP5K family controls intracellular IP8 levels, the highest phosphorylated form of IPPs present in yeasts, as it has both inositol kinase and pyrophosphatase activities. Previous studies have shown that the fission yeast S. pombe Vip1/PPIP5K family member Asp1 impacts chromosome transmission fidelity via the modulation of spindle function. We now demonstrate that an IP8 analogue is targeted by endogenous Asp1 and that cellular IP8 is subject to cell cycle control. Mitotic entry requires Asp1 kinase function and IP8 levels are increased at the G2/M transition. In addition, the kinetochore, the conductor of chromosome segregation that is assembled on chromosomes is modulated by IP8. Members of the yeast CCAN kinetochore-subcomplex such as Mal2/CENP-O localize to the kinetochore depending on the intracellular IP8-level: higher than wild-type IP8 levels reduce Mal2 kinetochore targeting, while a reduction in IP8 has the opposite effect. As our perturbations of the inositol polyphosphate and IPP pathways demonstrate that kinetochore architecture depends solely on IP8 and not on other IPPs, we conclude that chromosome transmission fidelity is controlled by IP8 via an interplay between entry into mitosis, kinetochore architecture, and spindle dynamics.

Assay of inorganic pyrophosphatase activity based on a fluorescence “turn–off” strategy using carbon quantum dots@Cu-MOF nanotubes

A highly sensitive and selective sensor for the quantitative assay of inorganic pyrophosphatase (PPase) activity was developed based on a fluorescence “turn–off” strategy. Carbon quantum dots@Cu(II)-based metal–organic framework nanotubes (CQDs@Cu-MOF) with length less than 300 nm and width less than 20 nm were synthesized. CQDs in the nanotubes exhibited weak fluorescence owing to static quenching. The coordination reaction between pyrophosphate ion (PPi) and Cu(II) decomposed CQDs@Cu-MOF and led to the release of CQDs, of which the fluorescence recovered. In the presence of PPase, the hydrolysis of PPi generated phosphate ion (Pi). CQDs@Cu-MOF remained their structural stability and the fluorescence turned off. The fluorescence intensity difference of the mixture of CQDs@Cu-MOF and PPi in the absence and presence of PPase (-ΔF) was proportional to the PPase concentration from 0.1 to 5 mU mL−1 and that from 5 to 50 mU mL−1, and a limit of detection at 0.03 mU mL−1 was obtained. PPase activity in human serum was analyzed using the proposed fluorescence sensor and the recovery values were found to vary from 95.0% to 104 %.

Phosphate solubilizing bacteria can significantly contribute to enhance P availability from polyphosphates and their use efficiency in wheat

Rhizosphere microbes significantly enhance phosphorus (P) availability from a variety of unavailable P pools in agricultural soils. However, little is known about the contribution of root-associated microorganisms, notably P solubilizing bacteria (PSB), to enhance the use of polyphosphate (PolyP) fertilizers as well as the key mechanisms involved. This study assesses the ability of four PSB (Bacillus siamensis, Rahnella aceris, Pantoea hericii, Bacillus paramycoides) and their consortium (Cs) to enhance the release rate of available P from two types of PolyP (“PolyB” and “PolyC”) with a focus on the key role of phosphatase enzyme activities and organic acids production. Wheat growth performance and P acquisition efficiency were evaluated in response to co-application of PSB and PolyP. Results showed that inoculation with PSB, notably Cs, significantly enhanced available P from PolyC, PolyB and tri-calcium P. Increased available P in response to inoculation with PSB significantly correlated with medium acidification, organic acids production (notably glycolic acid) and induced activities of acid phosphatase and pyrophosphatase. In planta, the co-application of PSB-PolyP improved wheat plant biomass, root growth and P acquisition, with best results obtained from Cs-PolyP co-application as compared to uninoculated and unfertilized plants. At seedling stage, the co-application of Cs-PolyP (PolyB and PolyC) enhanced root hairs length (125 % and 131 %), root length (26 % and 37 %) and root inorganic P (Pi) content (160 % and 182 %), respectively compared to uninoculated plants. Similarly, at tillering stage, plant biomass (35 % and 47 %), Pi content (43 % and 253 %), P translocation (215 % and 315 %) and soil phosphatases (213 % and 219 %) significantly improved under PolyB and PolyC application, respectively. Findings from this study demonstrate the key role of PSB to enhance the use of PolyP through production of organic acids and phosphatases, exhibiting differential traits patterns between the two PolyP. Improved wheat growth and root P acquisition in response to PSB-PolyP co-application can be attributed to induced rhizosphere processes leading to enhanced available P taken up by roots.

First-in-human experience using RBS2418, an oral ENPP1 inhibitor within an expanded access protocol in combination with pembrolizumab in a patient with metastatic adrenal cancer.

e14550 Background: ENPP1 is a type II transmembrane protein with nucleotide pyrophosphatase and phosphodiesterase enzymatic activities. ENPP1 expression is associated with poor prognosis across cancer types. ENPP1 hydrolyzes extracellular cGAMP, nucleoside triphosphates (ATP, GTP) and dinucleotide polyphosphates. ENPP1 inhibition protects cGAMP and ATP and reduces adenosine levels in the tumor microenvironment, activates antigen-presenting cells and increases T-cell infiltration promoting anticancer immunity. RBS2418 is a potent oral inhibitor of ENPP1 enzyme activity and under evaluation in a Phase 1a/b study in subjects with advanced unresectable, recurrent or metastatic tumors as monotherapy and in combination with pembrolizumab. Methods: Expanded access to RBS2418 was granted by the FDA under a single patient IND to a subject with high grade adreno-cortical carcinoma. The subject received pembrolizumab at 200mg IV every 3 weeks with escalating doses of RBS2418 weekly, starting at 100 mg p.o. BID, followed by 200 and 400 mg p.o BID. Blood samples were collected at each dose level to determine plasma RBS2418 concentration and the corresponding serum ENPP1 level inhibition. Pharmacokinetic data supported a fixed dose of 200mg p.o. bid. Safety and target engagement were closely monitored and protein expression profiling by IHC in the pre-treatment tumor sample was done. Results: All evaluated dose levels of RBS2418 in combination with pembrolizumab were found to be safe and well tolerated. No dose-limiting drug-related toxicities were observed. The plasma concentration of RBS2418 was 29 ng/ml at 12 hours after the second 100 mg dose (Ctrough). This concentration corresponded to approximately 17-fold times the 90% inhibition level of ENPP1 in human serum (17x serum EC90). After the first dose of 200 mg RBS2418 a maximal plasma concentration of 473 ng/ml was measured, while the Ctrough at 12 hours after the second 200 mg dose was 69 ng/ml (corresponding to ̃40x serum EC90). There was ENPP1, but no detectable cGAMP in the baseline serum sample. In serum samples collected during treatment with RBS2418 there was no detectable ENPP1 enzyme activity and cGAMP was fully stable. Conclusions: RBS2418 with pembrolizumab was safe and well tolerated and no drug-related safety concerns were reported. The pharmacokinetics of RBS2418 showed excellent oral bioavailability, with plasma levels of RBS2418 > 17-fold and > 40-fold above serum EC90 of ENPP1 at the 100 and 200 mg BID dose levels, respectively. Dose levels are further studied in the current Phase 1 trial. ENPP1 activity was measurable in the serum of this subject and RBS2418 led to complete enzyme inhibition throughout the study at all dose levels investigated. These results support the continued development of RBS2418, the first member of this novel class of immunotherapy agents.

A FRET ratiometric fluorescence biosensor for the selective determination of pyrophosphate ion and pyrophosphatase activity based on difunctional Cu-MOF nanozyme

A ratiometric fluorescent method based on the difunctional copper (II)-based metal-organic framework (Cu-MOF) for label-free monitoring pyrophosphatase (PPase) activity and pyrophosphate ion (PPi) was developed. Difunctional Cu-MOF possessed peroxidase mimetic activity and fluorescent emission property. The catalytic activity of Cu-MOF would be inhibited by the coordination reaction between Cu2+ and PPi because of the good affinity between Cu2+ and PPi. However, the PPi can be hydrolyzed by PPase catalyzation to Pi which can hardly affect the Cu-MOF nanozyme activity. Once the PPase participated, the catalytic activity of Cu-MOF was recovered and catalyzed the decomposition of hydrogen peroxide to oxidize o-phenylenediamine (OPD) to form a 2,3-diaminophenazine (DAP) solution and turns on the Förster resonance energy transfer (FRET) from Cu-MOF as a donor to DAP as an acceptor. The LODs of the fluorescence sensing platform were 0.30 mU mL−1 for PPase and 0.53 μmol L−1 for PPi, respectively. This method will broaden the applications of nanozyme and shows a great potential application in the diagnosis of PPi or PPase-related diseases.

Characterization of an archaeal inorganic pyrophosphatase from Sulfolobus islandicus using a [31P]-NMR-based assay

Inorganic pyrophosphatase catalyzes the conversion of pyrophosphate to phosphate and is often critical for driving reactions forward in cellular processes such as nucleic acid and protein synthesis. Commonly used methods for quantifying pyrophosphatase enzyme activity employ reacting liberated phosphate with a second molecule to produce absorbance changes or employing a second enzyme in coupled reactions to produce a product with a detectable absorbance. In this investigation, a novel [31P]-NMR spectroscopy-based assay was used to quantitatively measure the formation of phosphate and evaluate the activity of inorganic pyrophosphatase from the thermoacidophilic Crenarchaeota Sulfolobus islandicus. The enzymatic activity was directly measured via integration of the [31P] resonance associated with the phosphate product (δ = 2.1 ppm). Sulfolobus islandicus inorganic pyrophosphatase preferentially utilized Mg2+ as divalent cation and had pH and temperature optimums of 6.0 of 50 °C, respectively. The Vmax value was 850 μmol/min/mg and the Km for pyrophosphate was 1.02 mM. Sequence analysis indicates the enzyme is a Family I pyrophosphatase. Sulfolobus islandicus inorganic pyrophosphatase was shown to be inhibited by sodium fluoride with a IC50 of 2.26 mM, compared to a IC50 of 0.066 mM for yeast inorganic pyrophosphatase. These studies reveal that a [31P]-NMR spectroscopy-based assay is an effective method for analyzing catalysis by phosphate-producing enzymes.

PpSnRK1α overexpression alters the response to light and affects photosynthesis and carbon metabolism in tomato.

Sucrose nonfermentation 1 (SNF1) related kinase 1 (SnRK1) is a central energy sensor kinase in plants and a key switch regulating carbon and nitrogen metabolism. Fruit quality depends on leaf photosynthetic efficiency and carbohydrate accumulation, but the role of peach (Prunus persica) SnRK1 α subunit (PpSnRK1α) in regulating leaf carbon metabolism and the light signal response remains unclear. We studied the carbon metabolism of tomato leaves overexpressing PpSnRK1α and the responses of PpSnRK1α-overexpressing tomato leaves to light signals. Transcriptome, metabolome, and real-time quantitative polymerase chain reaction analyses revealed that uridine 5'-diphosphoglucose, glutamate, and glucose-6-phosphate accumulated in tomato leaves overexpressing PpSnRK1α. The expression of genes (e.g., GDH2, SuSy) encoding enzymes related to carbon metabolism (e.g., glutamate dehydrogenase (GDH2; EC: 1.4.1.3), sucrose synthase (SS; EC: 2.4.1.13)) and chlorophyllase (CLH) encoding chlorophyllase (EC: 3.1.1.14), which regulates photosynthetic pigments and photosynthesis, was significantly increased in PpSnRK1α-overexpressing plants. PpSnRK1α overexpression inhibited the growth of hypocotyls and primary roots in response to light. The chlorophyll content of the leaves was increased, the activity of SS and ADPG pyrophosphatase (AGPase; EC: 2.7.7.27) was increased, and photosynthesis was promoted in PpSnRK1α-overexpressing plants relative to wild-type plants. Under light stress, the net photosynthetic rate of plants was significantly higher in plants overexpressing PpSnRK1α than in wild-type plants. This indicates that PpSnRK1α promotes the accumulation of carbohydrates by regulating genes related to carbon metabolism, regulating genes related to chlorophyll synthesis, and then responding to light signals to increase the net photosynthetic rate of leaves.

In situ Cu2+-triggered wavelength-tunable fluorescent sensor for highly sensitive sensing inorganic pyrophosphatase activity and its logic gate application

As inorganic pyrophosphatase (PPase) gets involved in the regulation of various pathophysiological procedures, rapid and effective detection of its activity is of great value for monitoring and managing PPase-related diseases. Herein, an in situ Cu2+-triggered wavelength-tunable fluorescent sensor for high sensitivity monitoring of PPase activity is constructed. The proposed sensor is based on the principle of Cu2+-triggered polymerization of dopamine (DA) and m-phenylenediamine (MPD), the stronger coupling effect of pyrophosphate ion (PPi) and Cu2+, and the effective hydrolysis of PPase to PPi. With the change of substituents of MPD, the wavelength of fluorescence polymer nanoparticles (FPNPs) exhibits tunable capacity from 535 nm to 565 nm. Compared with the traditional detection method, as developed sensor shows high sensitivity to PPase with a low detection limit of 0.333 mU/mL in green channel and 0.033 mU/mL in orange channel. Additionally, the sensor realizes PPase sensing in the diluted human serum with excellent recovery in both green and orange models. Furthermore, this sensor is also applicable for inhibition assay of PPase (NaF: IC50 of 134.7 μM) and logic gates construction. Summarily, as prepared sensor provides one better way for monitoring PPase in diagnostic as well as drug screening.