Discovery of Potent and Selective Methylenephosphonic Acid CD73 Inhibitors.

Solid tumors are often associated with high levels of extracellular ATP. Ectonucleotidases catalyze the sequential hydrolysis of ATP to adenosine, which potently suppresses T-cell and NK-cell functions via the adenosine receptors (A2a and A2b). The ectonucleotidase CD73 catalyzes the conversion of AMP to adenosine. Thus, increased CD73 enzymatic activity in the tumor microenvironment is a potential mechanism for tumor immune evasion and has been associated with poor prognosis in the clinic. CD73 inhibition is anticipated to restore immune function by skirting this major mechanism of adenosine generation. We have developed a series of potent and selective methylenephosphonic acid CD73 inhibitors via a structure-based design. Key binding interactions of the known inhibitor adenosine-5'-(α,ß-methylene)diphosphate (AMPCP) with hCD73 provided the foundation for our early designs. The structure-activity relationship study guided by this structure-based design led to the discovery of 4a, which exhibits excellent potency against CD73, exquisite selectivity against related ectonucleotidases, and a favorable pharmacokinetic profile.

Determination of Ascorbic Acid, Total Ascorbic Acid, and Dehydroascorbic Acid in Bee Pollen Using Hydrophilic Interaction Liquid Chromatography-Ultraviolet Detection.

Ascorbic acid (AA) is one of the essential nutrients in bee pollen, however, it is unstable and likely to be oxidized. Generally, the oxidation form (dehydroascorbic acid (DHA)) is considered to have equivalent biological activity as the reduction form. Thus, determination of the total content of AA and DHA would be more accurate for the nutritional analysis of bee pollen. Here we present a simple, sensitive, and reliable method for the determination of AA, total ascorbic acids (TAA), and DHA in rape (Brassica campestris), lotus (Nelumbo nucifera), and camellia (Camellia japonica) bee pollen, which is based on ultrasonic extraction in metaphosphoric acid solution, and analysis using hydrophilic interaction liquid chromatography (HILIC)-ultraviolet detection. Analytical performance of the method was evaluated and validated, then the proposed method was successfully applied in twenty-one bee pollen samples. Results indicated that contents of AA were in the range of 17.54 to 94.01 µg/g, 66.01 to 111.66 µg/g, and 90.04 to 313.02 µg/g for rape, lotus, and camellia bee pollen, respectively. In addition, percentages of DHA in TAA showed good intra-species consistency, with values of 13.7%, 16.5%, and 7.6% in rape, lotus, and camellia bee pollen, respectively. This is the first report on the discriminative determination between AA and DHA in bee pollen matrices. The proposed method would be valuable for the nutritional analysis of bee pollen.

Excellency of pyrimidinyl moieties containing α-aminophosphonates over benzthiazolyl moieties for thermal and structural stability of stem bromelain.

An efficient approach has been made for the synthesis of a series of novel di α-aminophosphonates by the reaction of terephthalaldehyde with various pyrimidine/benzthiazole amines and diethyl phosphite using sulfonated graphitic carbon nitride - SA@g-C3N4 as catalyst under room temperature and solvent free conditions. Later, the different effects of these newly synthesized α-aminophosphonates as a function of concentration gradient has been scrutinized on the thermal and structural stability of stem bromelain (SBM) through combining the results of various spectroscopic techniques like UV-vis, steady state fluorescence and circular dichroism (CD). Lastly the competitive and distinctive behaviour of α-aminophosphonates towards the stability of SBM has been envisaged using molecular docking simulations which suggest that nature of α-aminophosphonates plays a crucial role for their interactions with SBM. Molecular docking results clearly show that α-aminophosphonates with pyrimidine ring are having more number of hydrogen bonding interaction with amino acid residues of SBM than α-aminophosphonates with benzthiazolyl ring. Sequentially for thermal and structure stability of SBM, concentration of α-aminophosphonates plays an inexorable role and through these results it must be concluded that most of the α-aminophosphonates are stabilizing the SBM upto the 0. 1 mM concentration.

Determination of cellular vitamin C dynamics by HPLC-DAD.

A redox-sensitive inter-conversion between ascorbic acid (ASC) and its oxidized form dehydroascorbic acid (DHA) in the intracellular environment has been of exceptional interest to recent metabolomics and pharmaceutical research. We developed a chromatographic protocol to instantly determine these vitamers with each identity from cellular extracts, without any labeling and pretreatments. Owing to its simplicity, one can readily continue the assay for hours, an otherwise difficult to cover timescale at which the intracellular DHA-ASC conversion comes into play. The method was validated for the analysis of pancreatic cancer cells, to our knowledge the first-ever study on a nucleated cell type, to trace in detail their kinetics of glucose transporter-dependent DHA uptake and, simultaneously, that for the intracellular ASC conversion. The simplest of all the relevant techniques and yet with the unique ability to provide each vitamer identity on a high-throughput basis, this method should offer the most practical option for VC-involved physiological and pharmaceutical studies including high-dose VC cancer therapy.

DNA-linked inhibitor antibody assay (DIANA) as a new method for screening influenza neuraminidase inhibitors.

Influenza neuraminidase is responsible for the escape of new viral particles from the infected cell surface. Several neuraminidase inhibitors are used clinically to treat patients or stockpiled for emergencies. However, the increasing development of viral resistance against approved inhibitors has underscored the need for the development of new antivirals effective against resistant influenza strains. A facile, sensitive, and inexpensive screening method would help achieve this goal. Recently, we described a multiwell plate-based DNA-linked inhibitor antibody assay (DIANA). This highly sensitive method can quantify femtomolar concentrations of enzymes. DIANA also has been applied to high-throughput enzyme inhibitor screening, allowing the evaluation of inhibition constants from a single inhibitor concentration. Here, we report the design, synthesis, and structural characterization of a tamiphosphor derivative linked to a reporter DNA oligonucleotide for the development of a DIANA-type assay to screen potential influenza neuraminidase inhibitors. The neuraminidase is first captured by an immobilized antibody, and the test compound competes for binding to the enzyme with the oligo-linked detection probe, which is then quantified by qPCR. We validated this novel assay by comparing it with the standard fluorometric assay and demonstrated its usefulness for sensitive neuraminidase detection as well as high-throughput screening of potential new neuraminidase inhibitors.

Substituted phosphonic analogues of phenylglycine as inhibitors of phenylalanine ammonia lyase from potatoes.

A series of phosphonic acid analogues of phenylglycine variously substituted in phenyl ring have been synthesized and evaluated for their inhibitory activity towards potato l-phenylalanine ammonia lyase. Most of the compounds appeared to act as moderate (micromolar) inhibitors of the enzyme. Analysis of their binding performed using molecular modeling have shown that they might be bound either in active site of the enzyme or in the non-physiologic site. The latter one is located in adjoining deep site nearby the to the entrance channel for substrate into active site.

Cultivar and Harvest Month Influence the Nutrient Content of Opuntia spp. Cactus Pear Cladode Mucilage Extracts.

Mucilage extracted from cactus pear cladodes is a hydrocolloid gum. It is a novel, natural, low-kilojoule, cost-effective texture-modifying ingredient in functional food products. Yet, the cultivar with the most optimal nutrient content and the preferred harvest times are as yet unknown. For this reason, mucilage from three Opuntia ficus-indica (Algerian, Morado and Gymno-Carpo) and one Opuntia robusta (Robusta) cultivar were investigated to determine their nutrient content over six months. Nutrients that contribute energy (10.2 kJ/g) were low. The mineral content was high (ash 17.7/100 g), particularly calcium (3.0 g/100 g) and phosphorous (109.5 mg/kg). Low insoluble acid-detergent fibre (1.4 g/kg) and neutral-detergent fibre (2.1 g/kg) values indicated that mucilage was mostly soluble fibre. Calcium oxalate crystals were not detected in dried mucilage. Opuntia robusta powders had higher protein, extractable fat and potassium content, while Opuntia ficus-indica mucilage powders had higher polyunsaturated (Linoleic and α-Linolenic acid) fat content. O. robusta Robusta mucilage, harvested after the fruit harvest (February) had the lowest energy content and the highest mineral and protein content. Mucilage powders were highly soluble, low-kilojoule and mineral-rich. This is a functional ingredient that is produced from an easily cultivated crop, as cactus pears grow in areas with poor soil, extremely high daytime temperatures and limited water supplies.

Discovery of potent and selective inhibitors of human aminopeptidases ERAP1 and ERAP2 by screening libraries of phosphorus-containing amino acid and dipeptide analogues.

A collection of fifty phosphonic and phosphinic acids was screened for inhibition of ERAP1 and ERAP2, the human endoplasmic reticulum aminopeptidases. The cooperative action of these enzymes is manifested by trimming a variety of antigenic precursors to be presented on the cell surface by major histocompatibility class I. The SAR studies revealed several potent compounds, particularly among the phosphinic dipeptide analogues, that were strong inhibitors of ERAP2 (Ki=100-350nM). A wide structural diversity of the applied organophosphorus compounds, predominantly non-proteinogenic analogues, allowed identification of representatives selective toward only one form of ERAP. For example, N'-substituted α,ß-diaminophosphonates and phosphinates exhibited potency only toward ERAP2, which is in agreement with the P1 basic substrate-oriented specificity. Such discriminating ligands are invaluable tools for elucidating the precise role of a particular aminopeptidase in the concerted function of antigen processing and in human diseases.

Mesoporous Silica Nanoparticles with pH-Sensitive Nanovalves for Delivery of Moxifloxacin Provide Improved Treatment of Lethal Pneumonic Tularemia.

We have optimized mesoporous silica nanoparticles (MSNs) functionalized with pH-sensitive nanovalves for the delivery of the broad spectrum fluoroquinolone moxifloxacin (MXF) and demonstrated its efficacy in treating Francisella tularensis infections both in vitro and in vivo. We compared two different nanovalve systems, positive and negative charge modifications of the mesopores, and different loading conditions-varying pH, cargo concentration, and duration of loading-and identified conditions that maximize both the uptake and release capacity of MXF by MSNs. We have demonstrated in macrophage cell culture that the MSN-MXF delivery platform is highly effective in killing F. tularensis in infected macrophages, and in a mouse model of lethal pneumonic tularemia, we have shown that the drug-loaded MSNs are much more effective in killing F. tularensis than an equivalent amount of free MXF.

Self-assembled monolayers of cyclohexyl-terminated phosphonic acids as a general dielectric surface for high-performance organic thin-film transistors.

A novel self-assembled monolayer (SAM) on AlOy /TiOx is terminated with cyclohexyl groups, an unprecedented terminal group for all kinds of SAMs. The SAM-modified AlOy /TiOx functions as a general dielectric, enabling organic thin-film transistors with a field-effect mobility higher than 5 cm(2) V(-1) s(-1) for both holes and electrons, good air stability with low operating voltage, and general applicability to solution-processed and vacuum-deposited n-type and p-type organic semiconductors.

Substrate profiling of Finegoldia magna SufA protease, inhibitor screening and application to prevent human fibrinogen degradation and bacteria growth in vitro.

SufA, which belongs to the subtilisin-like serine protease family, contains a non-canonical Asp-His-Ser catalytic triad. Under in vitro conditions, SufA is capable of human fibrinogen hydrolysis leading to inhibition of fibrin network formation, thus suggesting its important role in the development and progression of Finegoldia magna infections. In addition, it has been demonstrated that SufA can hydrolyze antibacterial peptides such as LL-37 and the chemokine MIG/CXCL 9, hence evading host defence mechanisms. Although the SufA protease from F. magna was discovered several years ago, its optimal substrate preference has not yet been identified. Considering the role of SufA, we have focused on the profiling of its substrate sequence preference spanning S1-S3 binding pockets using the FRET (fluorescence resonance energy transfer) approach. Next, based on the structure of the P1 residue of the developed substrate, we narrowed the inhibitor screening to the phosphonic analogues of amino acids containing an arginine-like side chain. Among all the compounds tested, only Cbz-6-AmNphth(P)(OPh)2 showed any inhibitory activity against SufA displaying k2/Ki value of 10,800 M(-1) s(-1). In addition, it prevented SufA-mediated human fibrinogen hydrolysis in vitro and exhibited potent antibacterial activity against F. magna, Staphylococcus aureus and Escherichia coli. Herein, we report on the substrate specificity, synthesis and kinetic evaluation of phosphonic inhibitors of SufA protease from F. magna which could help to establish its function in pathogenesis development and may lead to the elaboration of new antibacterial drugs.

Development of lysophosphatidic acid pathway modulators as therapies for fibrosis.

Lysophosphatidic acid (LPA) is a class of bioactive phospholipid that displays a wide range of cellular effects via LPA receptors, of which six have been identified (LPAR1-6). In serum and plasma, LPA production occurs mainly by the hydrolysis of lysophosphatidylcholine by the phospholipase D activity of autotaxin (ATX). The involvement of the LPA pathway in driving chronic wound-healing conditions, such as idiopathic pulmonary fibrosis, has suggested targets in this pathway could provide potential therapeutic approaches. Mice with LPAR1 knockout or tissue-specific ATX deletion have demonstrated reduced lung fibrosis following bleomycin challenge. Therefore, strategies aimed at antagonizing LPA receptors or inhibiting ATX have gained considerable attention. This Review will summarize the current status of identifying small-molecule modulators of the LPA pathway. The therapeutic utility of LPA modulators for the treatment of fibrotic diseases will soon be revealed as clinical trials are already in progress in this area.

Characterizing inorganic crystals grown on organic self-assembled bilayers with scanning probe and electron microscopies.

Combined microscopy techniques are used to establish the usability of phosphonic acid layers as promoters of hydroxyapatite (HAp) growth. Using spread coating, octadecylphosphonic acid (OPA) self-assembled bilayers are delivered to the thin natural oxide layer of a titanium film surface with no prior treatment. These bilayers aggregate two major advantages of phosphonic moieties to titanium surfaces: nucleation of hydroxyapatite crystals from ionic solution and affinity for both titanium oxide surface and HAp crystals. The functionalized substrates and bare titanium (control) samples are immersed in an aqueous solution containing calcium and phosphorus ions. Over a 4-week immersion time, OPA-functionalized substrates present numerous large agglomerates of inorganic crystals, in contrast to control samples, with no significant amount of deposits. Initial sample characterization was performed with atomic force microscopy (AFM). Compositional and structural characterization of these agglomerates (using TEM, EDS, and electron diffraction), revealed that they are indeed HAp, the main component of the inorganic bone matrix.

Discovery of an acyclic nucleoside phosphonate that inhibits Mycobacterium tuberculosis ThyX based on the binding mode of a 5-alkynyl substrate analogue.

The urgent need for new antibiotics poses a challenge to target un(der)exploited vital cellular processes. Thymidylate biosynthesis is one such process due to its crucial role in DNA replication and repair. Thymidylate synthases (TS) catalyze a crucial step in the biosynthesis of thymidine 5-triphosphate (TTP), an elementary building block required for DNA synthesis and repair. To date, TS inhibitors have only been successfully applied in anticancer therapy due to their lack of specificity for antimicrobial versus human enzymes. However, the discovery of a new family of TS enzymes (ThyX) in a range of pathogenic bacteria that is structurally and biochemically different from the "classic" TS (ThyA) has opened the possibility to develop selective ThyX inhibitors as potent antimicrobial drugs. Here, the interaction of the known inhibitor 5-(3-octanamidoprop-1yn-1yl)-2'-deoxyuridine-5'-monophosphate (1) with Mycobacterium tuberculosis ThyX enzyme is explored using molecular modeling starting from published crystal structures, with further confirmation through NMR experiments. While the deoxyuridylate (dUMP) moiety of compound 1 occupies the cavity of the natural substrate in ThyX, the rest of the ligand (the "5-alkynyl tail") extends to the outside of the enzyme between two of its four subunits. The hydrophobic pocket that accommodates the alkyl part of the tail is formed by displacement of Tyr 44.C, Tyr 108.A and Lys 165.A. Changes to the resonance of the Lys 165 NH3 group upon ligand binding were monitored in a titration experiment by 2D HISQC NMR. Guided by the results of the modeling and NMR studies, and inspired by the success of acyclic antiviral nucleosides, compounds where a 5-alkynyl uracyl moiety is coupled to an acyclic nucleoside phosphonate (ANP) were synthesized and evaluated. Of the compounds evaluated, sodium (6-(5-(3-octanamidoprop-1-yn-1-yl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)hexyl)phosphonate (3 e) exhibited 43 % of inhibitory effect on ThyX at 50 µM. While only modest activity was achieved, this is the first example of an ANP inhibiting ThyX, and these results can be used to further guide structural modifications to this class to develop more potent compounds with potential application as antibacterial agents acting through a novel mechanism of action.

Discovery of the antibiotic phosacetamycin via a new mass spectrometry-based method for phosphonic acid detection.

Naturally occurring phosphonates such as phosphinothricin (Glufosinate, a commercially used herbicide) and fosfomycin (Monurol, a clinically used antibiotic) have proved to be potent and useful biocides. Yet this class of natural products is still an under explored family of secondary metabolites. Discovery of the biosynthetic pathways responsible for the production of these compounds has been simplified by using gene based screening approaches, but detection and identification of the natural products the genes produce have been hampered by a lack of high-throughput methods for screening potential producers under various culture conditions. Here, we present an efficient mass-spectrometric method for the selective detection of natural products containing phosphonate and phosphinate functional groups. We have used this method to identify a new phosphonate metabolite, phosacetamycin, whose structure, biological activity, and biosynthetic gene cluster are reported.

Physico-chemical studies and emulsifying properties of N-propyl-N-methylene phosphonic chitosan.

Chitosan is a modified, natural carbohydrate polymer derived by deacetylation of chitin. Due to the presence of two functional groups can undergo many chemical modifications. In a previous work we described the synthetic strategy and characterization of a novel soluble derivative: N-propyl-N-methylene phosphonic chitosan (PNMPC). In the study of some physicochemical properties, results showed that this modified chitosan aggregates in several steps when the concentration is increased. By addition of NaOH the initially coiled molecules stretch exposing more phosphonic acid groups to neutralization and finally give a cooperative reaction with OH((). PNMPC has emulsifying properties and gives O/W emulsions with quasi-monodisperse small droplets. Emulsions with 0.18% PNMPC and 30:70 o:w ratio exhibited the best emulsifying properties within the test range. This emulsion ratio showed high stability to long time storage and several successive freeze/thaw and heating/cooling cycles.

Microrough cobalt-chromium alloy surfaces for paclitaxel delivery: preparation, characterization, and in vitro drug release studies.

Cobalt-chromium (Co-Cr) alloys have extensive biomedical applications including drug-eluting stents (DES). This study investigates the use of eight different microrough Co-Cr alloy surfaces for delivering paclitaxel (PAT) for potential use in DES. The eight different surfaces include four bare microrough and four self-assembled monolayer (SAM) coated microrough surfaces. The bare microrough surfaces were prepared by grit blasting Co-Cr with glass beads (50 and 100 µm in size) and Al(2)O(3) (50 and 110 µm). The SAM coated surfaces were prepared by depositing a -COOH terminated phosphonic acid monolayer on the different microrough surfaces. PAT was then deposited on all the bare and SAM coated microrough surfaces. The surfaces were characterized using scanning electron microscopy (SEM), 3D optical profilometry, and Fourier transform infrared spectroscopy (FTIR). SEM showed the different morphologies of microrough surfaces without and with PAT coating. An optical profiler showed the 3D topography of the different surfaces and the changes in surface roughness and surface area after SAM and PAT deposition. FTIR showed ordered SAMs were formed on glass bead grit blasted surfaces, while the molecules were disordered on Al(2)O(3) grit blasted surfaces. Also, FTIR showed the successful deposition of PAT on these surfaces. The PAT release was investigated for up to two weeks using high performance liquid chromatography. Al(2)O(3) grit blasted bare microrough surfaces showed sustained release profiles, while the glass bead grit blasted surfaces showed burst release profiles. All SAM coated surfaces showed biphasic drug release profiles, which is an initial burst release followed by a slow and sustained release. SAM coated Al(2)O(3) grit blasted surfaces prolonged the sustained release of PAT in a significant amount during the second week of drug elution studies, while this behavior was not observed for any other surfaces used in this study. Thus, this study demonstrates the use of different microrough Co-Cr alloy surfaces for delivering PAT for potential applications in DES and other medical devices.

Hydrogen bonding mediated by key orbital interactions determines hydration enthalpy differences of phosphate water clusters.

Electronic structure calculations have been carried out to provide a molecular interpretation for dihydrogen phosphate stability in water relative to that of metaphosphate. Specifically, hydration enthalpies of biologically important metaphosphate and dihydrogen phosphate with one to three waters have been computed with second-order Møller-Plesset perturbation and density functional theory (B3LYP) with up to the aug-cc-pvtz basis set and compared to experiment. The inclusion of basis set superposition error corrections and supplemental diffuse functions are necessary to predict hydration enthalpies within experimental uncertainty. Natural bond orbital analysis is used to rationalize underlying hydrogen bond configurations and key orbital interactions responsible for the experimentally reported difference in hydration enthalpies between metaphosphate and dihydrogen phosphate. In general, dihydrogen phosphate forms stronger hydrogen bonds compared to metaphosphate due to a greater charge transfer or enhanced orbital overlap between the phosphoryl oxygen lone pairs, n(O), and the antibonding O-H bond of water. Intramolecular distal lone pair repulsion with the donor n(O) orbital of dihydrogen phosphate distorts symmetric conformations, which improves n(O) and sigma*(O-H) overlap and ultimately the hydrogen bond strength. Unlike metaphosphate, water complexed to dihydrogen phosphate can serve as both a hydrogen bond donor and a hydrogen bond acceptor, which results in cooperative charge transfer and a reduction of the energy gap between n(O) and sigma*(O-H), leading to stronger hydrogen bonds. This study offers insight into how orbital interactions mediate hydrogen bond strengths with potential implications on the understanding of the kinetics and mechanism in enzymatic phosphoryl transfer reactions.

[Anticholinesterase activity of certain carboranyl-containing thio- and selenoesters of pentavalent phosphorus acids]. / Antikholinésteraznaia aktivnost' nekotorykh karboranilsoderzhashchikh tio- i selenoéfirov kislot piativalentnogo fosfora.

Anticholinesterase activity of carboranyl containing thio- and selenoesters of pentavalent phosphorus acids has been studied. Insertion of the carboranyl substituents in the thioester group of phosphororganic compounds was found to increase the anticholinesterase activity as compared with the thioalkyl analogues. The compounds with B-carboranyl group are less active inhibitors of cholinesterase than their isomers with C-carboranyl group.