Phosphonate Inhibitors Research Articles

Drugs Against Avian Influenza A Virus: Design of Novel Sulfonate Inhibitors of Neuraminidase N1

The outbreak of avian influenza A (H5N1) virus has raised a global concern for both the animal as well as human health. Besides vaccination, that may not achieve full protection in certain groups of patients, inhibiting neuraminidase or the transmembrane protein M2 represents the main measure of controlling the disease. Due to alarming emergence of influenza virus strains resistant to the currently available drugs, development of new neuraminidase N1 inhibitors is of utmost importance. The present paper provides an overview of the recent advances in the design of new antiviral drugs against avian influenza. It also reports findings in binding free energy calculations for nine neuraminidase N1 inhibitors (oseltamivir, zanamivir, and peramivir -carboxylate, -phosphonate, and -sulfonate) using the Linear Interaction Energy method. Molecular dynamics simulations of these inhibitors were performed in a free and two bound states - the so called open and closed conformations of neuraminidase N1. Obtained results successfully reproduce the experimental binding affinities of the already known neuraminidase N1 inhibitors, i.e. peramivir being a stronger binder than zanamivir that is in turn stronger binder than oseltamivir, or phosphonate inhibitors being stronger binders than their carboxylate analogues. In addition, the newly proposed sulfonate inhibitors are predicted to be the strongest binders - a fact to be confirmed by their chemical synthesis and a subsequent test of their biological activity. Finally, contributions of individual inhibitor moieties to the overall binding affinity are explicitly evaluated to assist further drug development towards inhibition of the H5N1 avian influenza A virus.

Metal-dependent amyloid β-degrading catalytic antibody construct

Catalytic antibodies (catabodies) that degrade target antigens rapidly are rare. We describe the metal-dependence of catabody construct 2E6, an engineered heterodimer of immunoglobulin light chain variable domains that hydrolyzes amyloid β peptides (Aβ) specifically. In addition to the electrophilic phosphonate inhibitor of serine proteases, the metal chelators ethylenediaminetetraacetic acid (EDTA) and 1,10-phenanthroline completely inhibited the hydrolysis of Aβ by catabody 2E6. Formation of catabody-electrophilic phosphonate inhibitor adducts was unaffected by EDTA, suggesting that the metal exerts a favorable effect on a catalytic step after the initial catabody nucleophilic attack on Aβ. The EDTA inactivated catabody failed to disaggregate fibrillar Aβ, indicating the functional importance of the Aβ hydrolytic activity. Treating the EDTA-inactivated catabody with Zn2+ or Co2+ restored the Aβ hydrolytic activity, and Zn2+-induced catabody conformational transitions were evident by fluorescence emission spectroscopy. The studies reveal the absolute catabody dependence on a metal cofactor.

Development and binding characteristics of phosphonate inhibitors of SplA protease from Staphylococcus aureus

Staphylococcus aureus is responsible for a variety of human infections, including life-threatening, systemic conditions. Secreted proteome, including a range of proteases, constitutes the major virulence factor of the bacterium. However, the functions of individual enzymes, in particular SplA protease, remain poorly characterized. Here, we report development of specific inhibitors of SplA protease. The design, synthesis, and activity of a series of α-aminoalkylphosphonate diaryl esters and their peptidyl derivatives are described. Potent inhibitors of SplA are reported, which may facilitate future investigation of physiological function of the protease. The binding modes of the high-affinity compounds Cbz-Phe(P) -(OC6 H4 -4-SO2 CH3 )2 and Suc-Val-Pro-Phe(P) -(OC6 H5 )2 are revealed by high-resolution crystal structures of complexes with the protease. Surprisingly, the binding mode of both compounds deviates from previously characterized canonical interaction of α-aminoalkylphosphonate peptidyl derivatives and family S1 serine proteases.

Glutathione-analogous peptidyl phosphorus esters as mechanism-based inhibitors of γ-glutamyl transpeptidase for probing cysteinyl-glycine binding site

γ-Glutamyl transpeptidase (GGT) catalyzing the cleavage of γ-glutamyl bond of glutathione and its S-conjugates is involved in a number of physiological and pathological processes through glutathione homeostasis. Defining its Cys-Gly binding site is extremely important not only in defining the physiological function of GGT, but also in designing specific and effective inhibitors for pharmaceutical purposes. Here we report the synthesis and evaluation of a series of glutathione-analogous peptidyl phosphorus esters as mechanism-based inhibitors of human and Escherichia coli GGTs to probe the structural and stereochemical preferences in the Cys-Gly binding site. Both enzymes were inhibited strongly and irreversibly by the peptidyl phosphorus esters with a good leaving group (phenoxide). Human GGT was highly selective for l-aliphatic amino acid such as l-2-aminobutyrate (l-Cys mimic) at the Cys binding site, whereas E. coli GGT significantly preferred l-Phe mimic at this site. The C-terminal Gly and a l-amino acid analogue at the Cys binding site were necessary for inhibition, suggesting that human GGT was highly selective for glutathione (γ-Glu-l-Cys-Gly), whereas E. coli GGT are not selective for glutathione, but still retained the dipeptide (l-AA-Gly) binding site. The diastereoisomers with respect to the chiral phosphorus were separated. Both GGTs were inactivated by only one of the stereoisomers with the same stereochemistry at phosphorus. The strict recognition of phosphorus stereochemistry gave insights into the stereochemical course of the catalyzed reaction. Ion-spray mass analysis of the inhibited E. coli GGT confirmed the formation of a 1:1 covalent adduct with the catalytic subunit (small subunit) with concomitant loss of phenoxide, leaving the peptidyl moiety that presumably occupies the Cys-Gly binding site. The peptidyl phosphonate inhibitors are highly useful as a ligand for X-ray structural analysis of GGT for defining hitherto unidentified Cys-Gly binding site to design specific inhibitors.

Novel method of stabilizing water pretreatment for reverse osmosis desalination

The preparation of functionalized filter media (FFM), its ability to release active antiscalant substances and its application for water treatment in water heating processes and as a pretreatment in reverse osmosis (RO) desalination were investigated in this study. FFM is a novel method of stabilizing water pretreatment designed to be free of the disadvantages of conventional methods of water scaling potential reduction which require expensive equipment, produce significant amounts of waste, have high reagent consumption or are strongly dependent on water composition. FFM was derived from anion exchange resin modified with phosphonate and polymer scale inhibitors. In order to choose the most suitable anion exchange resin for preparation of FFM, different types of resins were tested for sorption of phosphonate compounds. The effect of the anionic composition of water and the effect of the polymer inhibitor on the rate of phosphonate release were investigated. Finally, FFM was tested as a scaling reduction water treatment for a water heating process with heating coils and as a pretreatment before reaching the RO unit. In both cases the results obtained show great promise for the functionalized material in antiscaling water pretreatment.

Design, synthesis, functional and structural characterization of an inhibitor of N-acetylneuraminate-9-phosphate phosphatase: Observation of extensive dynamics in an enzyme/inhibitor complex

The design, synthesis and characterization of a phosphonate inhibitor of N-acetylneuraminate-9-phosphate phosphatase (HDHD4) is described. Compound 3, where the substrate C-9 oxygen was replaced with a nonlabile CH2 group, inhibits HDHD4 with a binding affinity (IC50 11μM) in the range of the native substrate Neu5Ac-9-P (compound 1, Km 47μM). Combined SAR, modeling and NMR studies are consistent with the phosphonate group in inhibitor 3 forming a stable complex with native Mg2+. In addition to this key interaction, the C-1 carboxylate of the sugar interacts with a cluster of basic residues, K141, R104 and R72. Comparative NMR studies of compounds 3 and 1 with Ca2+ and Mg2+ are indicative of a highly dynamic process in the active site for the HDHD4/Mg2+/3 complex. Possible explanations for this observation are discussed.

Partial deletion of β9 loop in pancreatic lipase-related protein 2 reduces enzyme activity with a larger effect on long acyl chain substrates

Structural studies on pancreatic lipase have revealed a complex architecture of surface loops surrounding the enzyme active site and potentially involved in interactions with lipids. Two of them, the lid and β9 loop, expose a large hydrophobic surface and are considered as acyl chain binding sites based on their interaction with an alkyl phosphonate inhibitor. While the role of the lid in substrate recognition and selectivity has been extensively studied, the implication of β9 loop in acyl chain stabilization remained hypothetical. The characterization of an enzyme with a natural deletion of the lid, guinea pig pancreatic lipase-related protein 2 (GPLRP2), suggests however an essential contribution of the β9 loop in the stabilization of the acyl enzyme intermediate formed during the lipolysis reaction. A GPLRP2 mutant with a seven-residue deletion of β9 loop (GPLRP2-Δβ9) was produced and its enzyme activity was measured using various substrates (triglycerides, monoglycerides, galactolipids, phospholipids, vinyl esters) with short, medium and long acyl chains. Whatever the substrate tested, GPLRP2-Δβ9 activity is drastically reduced compared to that of wild-type GPLRP2 and this effect is more pronounced as the length of substrate acyl chain increases. Changes in relative substrate selectivity and stereoselectivity remained however weak. The deletion within β9 loop has also a negative effect on the rate of enzyme inhibition by alkyl phosphonates. All these findings indicate that the reduced enzyme turnover observed with GPLRP2-Δβ9 results from a weaker stabilization of the acyl enzyme intermediate due to a loss of hydrophobic interactions.

A Novel and Comprehensive Study of Polymeric and Traditional Phosphonate Inhibitors for High-Temperature Scale Control

Summary A novel barite-inhibition assay based on the nucleation and inhibition model has been proposed and used to evaluate the thermal stability of phosphonates and polymeric scale inhibitors with regard to their potential application in high-temperature wells. Systematic experiments have been conducted to investigate the time (minutes to days) and temperature (up to 200°C) dependence of inhibitor thermal degradation, the impact of stainless steel and iron on the degradation of inhibitors at high temperatures, and the difference between aging tests with inhibitors in solution and with those inhibitors adsorbed on core materials. The results not only enable a more accurate understanding of the thermal degradation of scale inhibitors but also facilitate the selection and placement of scale inhibitors for high-temperature oil and gas production.

Crystal Structure of Proteus mirabilis Lipase, a Novel Lipase from the Proteus/Psychrophilic Subfamily of Lipase Family I.1

Bacterial lipases from family I.1 and I.2 catalyze the hydrolysis of triacylglycerol between 25–45°C and are used extensively as biocatalysts. The lipase from Proteus mirabilis belongs to the Proteus/psychrophilic subfamily of lipase family I.1 and is a promising catalyst for biodiesel production because it can tolerate high amounts of water in the reaction. Here we present the crystal structure of the Proteus mirabilis lipase, a member of the Proteus/psychrophilic subfamily of I.1lipases. The structure of the Proteus mirabilis lipase was solved in the absence and presence of a bound phosphonate inhibitor. Unexpectedly, both the apo and inhibitor bound forms of P. mirabilis lipase were found to be in a closed conformation. The structure reveals a unique oxyanion hole and a wide active site that is solvent accessible even in the closed conformation. A distinct mechanism for Ca2+ coordination may explain how these lipases can fold without specific chaperones.

Human Neutrophil Elastase Phosphonic Inhibitors with Improved Potency of Action

Herein, we present the synthesis and the measurement of the inhibitory activity of novel peptidyl derivatives of α-aminoalkylphosphonate diaryl esters as human neutrophil elastase inhibitors. Their selectivity against other serine proteases, including porcine pancreatic elastase, chymotrypsin, and trypsin, was also demonstrated. We also describe the preparation of single peptide diastereomers. The most active and selective compound developed possessed a k(inact)/K(I) of 2353000 M(-1) s(-1), which is the most potent irreversible peptidyl inhibitor of human neutrophil elastase reported to date. The peptidyl inhibitors were demonstrated to be stable in PBS buffer and human plasma, as were their complexes with HNE.

Red vs. Green Scale Inhibitors for Extending Squeeze life—A Case Study From the North Sea, Norwegian Sector—Part II

SummaryOver the years, environmental legislation has forced changes in the types of scale-inhibitor molecule that can be deployed in certain regions of the world. These regulations have resulted in changes from phosphonate scale inhibitor to polymer-based chemistry, particularly in the Norwegian and UK continental shelf where phosphonates have been either on the substitution list or phased out for many applications. Over the past 10 years, significant improvements in inhibitor properties of the so-called "green" scale inhibitors have been made. However, for one particular operator, the squeeze application of this green scale inhibitor resulted in poorer than expected treatment lifetimes and significant operating cost because of the frequency of retreatment. To overcome the increasing operating cost, an evaluation was made of the current treatment chemicals vs. the older, more-established phosphonate scale inhibitors. The results for the laboratory evaluation suggested that the older chemistry would extend treatment life and reduce operating cost. A case was made to the legislative authority, who approved the use of the phosphonate scale inhibitor, and field applications started. The squeeze lifetimes for the red phosphonate chemistry were shown to be significantly better than the existing yellow/green inhibitors. During the following months, other scale inhibitors with improved environmental characteristics were developed and evaluated. One such molecule was shown to have similar coreflood retention to that of the applied red phosphonate and presented no formation damage. This paper presents the laboratory evaluation of the new scale inhibitor, and illustrates the improvement observed with this new inhibitor through field squeeze-treatment results from a well treated with both the red and new yellow environmental profile inhibitor chemicals.This paper outlines the challenges with environmental legislation and how it has been possible to develop technical solutions (in terms of environmental vs. safety issues and with new inhibitor chemicals) to meet the challenges of offshore scale control.

Scale Prediction and Inhibition for Oil and Gas Production at High Temperature/High Pressure

Summary With the advance of new exploration and production technologies, oil and gas production has gone to deeper and tighter formations than ever before. These developments have also brought challenges in scale prediction and inhibition, such as the prevention of scale formation at high temperatures (150–200°C), pressures (1,000–1,500 bar), and total dissolved solids (TDS) (>300,000 mg/L) commonly experienced at these depths. This paper will discuss (1) the challenges of scale prediction at high temperatures, pressures, and TDS; (2) an efficient method to study the nucleation kinetics of scale formation and inhibition at these conditions; and (3) the kinetics of barite-crystal nucleation and precipitation in the presence of various scale inhibitors and the effectiveness of those inhibitors. In this study, nine scale inhibitors have been evaluated at 70–200°C to determine if they can successfully prevent barite precipitation. The results show that only a few inhibitors can effectively inhibit barite formation at 200°C. Although it is commonly believed that phosphonate scale inhibitors may not work for high-temperature inhibition applications, the results from this study suggest that barite-scale inhibition by phosphonate inhibitors was not impaired at 200°C under strictly anoxic condition in NaCl brine. However, phosphonate inhibitors can precipitate with Ca2+ at high temperatures and, hence, can reduce efficiency. In addition, the relationships of scale inhibition to types of inhibitors and temperature are explored in this study. This paper addresses the limits of the current predition of mineral solubility at high-temperature/high-pressure (HT/HP) conditions and sheds light on inhibitior selection for HT/HP application. The findings from this paper can be used as guidelines for applications in an HT/HP oilfield environment.

A New Squeeze Scale Inhibitor for a Sandstone Reservoir with a Stimulation Effect

Tannic acid, a nontoxic, easily biodegradable compound, is evaluated for scale inhibitor squeeze application in a sandstone reservoir. For this purpose, we designed a new inhibitor squeeze evaluation system, a combined core flood–tube blocking apparatus with appropriate pressure–temperature and data logging arrangement, that can simulate the near-wellbore reservoir as well as downhole locations. Berea sandstone core plugs represented the formation rock and the extended flow tube represented the wellbore. Backflow tests were conducted to measure scale precipitation within the core plug and wellbore flow restriction. A 10 pore volume (PV) tannic acid solution was squeezed through the core plug and after a 14-hr adsorption period, anion and cation brines were squeezed separately through the core plug followed by the tube. The tests were compared with a control and a commonly used phosphonate scale inhibitor. The results showed that a fast rate of permeability reduction due to scale deposition took place in the control experiment starting at the initial brine injection stage and, in the case of the phosphonate scale inhibitor squeeze, there was significant reduction in permeability due to the squeeze itself. There was also progressive reduction of permeability due to less control of scaling phenomena. However, the tannic acid–treated core showed a slight improvement in core permeability and no scale deposition took place up to 250 PV of brine injection. Scanning electron microscopy and inductively coupled plasma (ICP) studies explain the formation stimulating mechanism of this new scale inhibitor.

Phosphonate Analogs of 2-Oxoglutarate Perturb Metabolism and Gene Expression in Illuminated Arabidopsis Leaves

Although the role of the 2-oxoglutarate dehydrogenase complex (2-OGDHC) has previously been demonstrated in plant heterotrophic tissues its role in photosynthetically active tissues remains poorly understood. By using a combination of metabolite and transcript profiles we here investigated the function of 2-OGDHC in leaves of Arabidopsis thaliana via use of specific phosphonate inhibitors of the enzyme. Incubation of leaf disks with the inhibitors revealed that they produced the anticipated effects on the in situ enzyme activity. In vitro experiments revealed that succinyl phosphonate (SP) and a carboxy ethyl ester of SP are slow-binding inhibitors of the 2-OGDHC. Our results indicate that the reduced respiration rates are associated with changes in the regulation of metabolic and signaling pathways leading to an imbalance in carbon-nitrogen metabolism and cell homeostasis. The inducible alteration of primary metabolism was associated with altered expression of genes belonging to networks of amino acids, plant respiration, and sugar metabolism. In addition, by using isothermal titration calorimetry we excluded the possibility that the changes in gene expression resulted from an effect on 2-oxoglutarate (2OG) binding to the carbon/ATP sensing protein PII. We also demonstrated that the 2OG degradation by the 2-oxoglutarate dehydrogenase strongly influences the distribution of intermediates of the tricarboxylic acid (TCA) cycle and the GABA shunt. Our results indicate that the TCA cycle activity is clearly working in a non-cyclic manner upon 2-OGDHC inhibition during the light period.

S1 pocket fingerprints of human and bacterial methionine aminopeptidases determined using fluorogenic libraries of substrates and phosphorus based inhibitors

Methionyl aminopeptidases (MetAPs) are metallo-dependent proteases responsible for removing of N-terminal methionine residue of peptides and proteins during protein maturation and activation. In this report we use a comprehensive strategy to screen the substrate specificity of three methionyl aminopeptidases: Homo sapiens MetAP-1, Homo sapiens MetAP-2 and Escherichia coli MetAP-1. By utilizing a 65-membered fluorogenic substrate library consisting of natural and unnatural amino acids we established detailed substrate preferences of each enzyme in the S1 pocket. Our results show that this pocket is highly conserved for all investigated MetAPs, very stringent for methionine, and that several unnatural amino acids with methionine-like characteristics were also well hydrolyzed by MetAPs. The substrate-derived results were verified using several phosphonate and phosphinate-based inhibitors.

Silica-Templated Synthesis of Novel Zinc-DTPMP Nanomaterials: Their Transport in Carbonate and Sandstone Media During Scale Inhibition

Summary In this study, a silica-templated synthesis route was adopted for fabrication of zinc phosphonate nanomaterials to expand their use in the delivery of phosphonate inhibitors into crushed formation materials for scale control. Transition divalent metal Zn2+ was chosen because of its ability to significantly increase inhibitor retention and effectiveness. Zinc chloride was first adsorbed onto the surface of 22-nm silica particles, followed by gradual addition of diethylenetriaminepentakis (methylenephosphonic acid) (DTPMP) to form nanometer-sized particles in the presence of sodium dodecylbenzene sulfonate (SDBS) surfactant. The physical and chemical properties of the synthesized Si-Zn-DTPMP nanomaterial suspension (nanofluid) have been carefully evaluated. The nanofluid was stable at 70°C in 1% KCl at pH 6.7 for over 12 hours. The transport of the synthesized nanofluid through columns of crushed calcite and sandstone has been investigated using column breakthrough experiments and modeled with a 1D advection-dispersion equation. The nanofluid was transportable through these media, and near-total breakthrough could be obtained by preflushing the media with an anionic SDBS surfactant solution. The nanofluid was transformed from an amorphous phase into a crystalline phase during diafiltration. The crystalline-phase materials demonstrated a much longer inhibitor lifetime compared with the untreated ones. The long-term flowback performance of the crystalline nanofluid was examined with a laboratory squeeze-simulation test where the nanomaterials gradually returned phosphonate inhibitors in the flowback brine solution, and the normalized return volume was greater than the previously reported nanomaterials squeeze treatments.

Investigation into the effects of phosphonic inhibitors on the formation of calcium sulfate scales

The effects of several phosphonic additives on the formation of calcium sulfate scale on metal surface in a flow system were investigated thoroughly using multiple pipe flow system. Their chemical structure, concentration and run time are to be the main factors that influence the inhibitory capability of these anti-scalants. The relationships between these factors and the scale rate were established. The Langmuir adsorption isotherm is applied to predict the critical concentrations above which complete inhibition of scaling will occur. The inhibition efficiency order of the investigated organic additives is found as follows:EDTP > NTMP > NDPA > HEDP> NPDA, which corresponds to the number of phosphonate groups that the additives possess.

A Novel Approach to the Design of Phosphonate Inhibitors of Protein Tyrosine Phosphatase

Inhibition of some PTPases by mono-, bis-, and tetrakisphosphorylated calix[4]arenes have been investigated. Kinetics and computational docking studies provide a basis for understanding the mechanism of enzyme–inhibitor complexation, which may be useful for the development of potent inhibitors of the PTPases.

Covalent anchoring of a racemization catalyst to CALB-beads: towards dual immobilization of DKR catalysts

The preparation of a heterogeneous bifunctional catalytic system, combining the catalytic properties of an organometallic catalyst (racemization) with those of an enzyme (enantioselective acylation) is described. A novel ruthenium phosphonate inhibitor was synthesized and covalently anchored to a lipase immobilized on a solid support (CALB, Novozym® 435). The immobilized bifunctional catalytic system showed activity in both racemization of (S)-1-phenylethanol and selective acylation of 1-phenylethanol.

Simple phosphonic inhibitors of human neutrophil elastase

Herein, we describe the synthesis and resulting activity of a complex series of α-aminophosphonate diaryl esters as irreversible human neutrophil elastase inhibitors and their selectivity preference for human neutrophil elastase over several other serine proteases such as porcine pancreatic elastase, trypsin, and chymotrypsin. We synthesized and examined the inhibitory potency of several new simple Cbz-protected α-aminoalkylphosphonate diaryl esters that yielded several new HNE inhibitors, where one of the obtained compounds Cbz-Val(P)(OC(6)H(4)-4-COOMe)(2) displayed an apparent second-order inhibition value at 33,015 M(-1) s(-1).