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The current paradigm for the treatment of chronic hepatitis C virus (HCV) infection involves combinations of agents that act directly on steps of the HCV life cycle. Here we report the preclinical characteristics of ITMN-8187, a nonmacrocyclic inhibitor of the NS3/4A HCV protease. X-ray crystallographic studies of ITMN-8187 and simeprevir binding to NS3/4A protease demonstrated good agreement between structures. Low nanomolar biochemical potency was maintained against NS3/4A derived from HCV genotypes 1, 2b, 4, 5, and 6. In cell-based potency assays, half-maximal reduction of genotype 1a and 1b HCV replicon RNA was afforded by 11 and 4 nM doses of ITMN-8187, respectively. Combinations of ITMN-8187 with other directly acting antiviral agents in vitro displayed additive antiviral efficacy. A 30-mg/kg of body weight dose of ITMN-8187 administered for 4 days yielded significant viral load reductions through day 5 in a chimeric mouse model of HCV. A 3-mg/kg oral dose administered to rats, dogs, or monkeys yielded concentrations in plasma 16 h after dosing that exceeded the half-maximal effective concentration of ITMN-8187. Human microdose pharmacokinetics showed low intersubject variability and prolonged oral absorption with first-order elimination kinetics compatible with once-daily dosing. These preclinical characteristics compare favorably with those of other NS3/4A inhibitors approved for the treatment of chronic HCV infection.

The discovery and optimization of a novel class of quinolone small-molecules that inhibit NS5B polymerase, a key enzyme of the HCV viral life-cycle, is described. Our research led to the replacement of a hydrolytically labile ester functionality with bio-isosteric heterocycles. An X-ray crystal structure of a key analog bound to NS5B facilitated the optimization of this series of compounds to afford increased activity against the target enzyme and in the cell-based replicon assay system.

(1,1-Dioxo-2H-[1,2,4]benzothiadiazin-3-yl) azolo[1,5-a]pyridine and azolo[1,5-a]pyrimidine derivatives have been investigated as potential anti-HCV drugs. Their synthesis, HCV NS5B polymerase inhibition, and replicon activity are discussed.

Future treatments for chronic hepatitis C virus (HCV) infection are likely to include agents that target viral components directly. Here, the preclinical characteristics of ITMN-191, a peptidomimetic inhibitor of the NS3/4A protease of HCV, are described. ITMN-191 inhibited a reference genotype 1 NS3/4A protein in a time-dependent fashion, a hallmark of an inhibitor with a two-step binding mechanism and a low dissociation rate. Under preequilibrium conditions, 290 pM ITMN-191 half-maximally inhibited the reference NS3/4A protease, but a 35,000-fold-higher concentration did not appreciably inhibit a panel of 79 proteases, ion channels, transporters, and cell surface receptors. Subnanomolar biochemical potency was maintained against NS3/4A derived from HCV genotypes 4, 5, and 6, while single-digit nanomolar potency was observed against NS3/4A from genotypes 2b and 3a. Dilution of a preformed enzyme inhibitor complex indicated ITMN-191 remained bound to and inhibited NS3/4A for more than 5 h after its initial association. In cell-based potency assays, half-maximal reduction of genotype 1b HCV replicon RNA was afforded by 1.8 nM; 45 nM eliminated the HCV replicon from cells. Peginterferon alfa-2a displayed a significant degree of antiviral synergy with ITMN-191 and reduced the concentration of ITMN-191 required for HCV replicon elimination. A 30-mg/kg of body weight oral dose administered to rats or monkeys yielded liver concentrations 12 h after dosing that exceeded the ITMN-191 concentration required to eliminate replicon RNA from cells. These preclinical characteristics compare favorably to those of other inhibitors of NS3/4A in clinical development and therefore support the clinical investigation of ITMN-191 for the treatment of chronic hepatitis C.

As bacteria continue to develop resistance toward current antibiotics, we find ourselves in a continual battle to identify new antibacterial agents and targets. We report herein a class of boron-containing compounds termed borinic esters that have broad spectrum antibacterial activity with minimum inhibitory concentrations (MIC) in the low microgram/mL range. These compounds were identified by screening for inhibitors against Caulobacter crescentus CcrM, an essential DNA methyltransferase from gram negative alpha-proteobacteria. In addition, we demonstrate that borinic esters inhibit menaquinone methyltransferase in gram positive bacteria using a new biochemical assay for MenH from Bacillus subtilis. Our data demonstrate the potential for further development of borinic esters as antibacterial agents as well as leads to explore more specific inhibitors against two essential bacterial enzymes.

We have developed a nonradioactive assay method for DNA methyltransferases based on the ability to protect substrate DNA from restriction. DNA immobilized to a microplate well was treated sequentially with methyltransferase and an appropriate endonuclease. The amount of methylated DNA product is reflected by a proportional decrease in endonuclease cleavage, which is in turn reflected by increased retention of the end-labeled affinity probe. A single universal substrate was designed to assay multiple methyltransferases including those that do not have a cognate endonuclease. The methodology developed is suited to screen a large number of compounds for inhibitors of various methyltransferases.

The interaction of dihydrofolate (H 2 F) and NADPH with a fluorescent derivative of H 2 F reductase (DHFR) was studied by using transient and single-molecule techniques. The fluorescent moiety Alexa 488 was attached to the structural loop that closes over the substrates after they are bound. Fluorescence quenching was found to accompany the binding of both substrates and the hydride transfer reaction. For the binding of H 2 F to DHFR, the simplest mechanism consistent with the data postulates that the enzyme exists as slowly interconverting conformers, with the substrate binding preferentially to one of the conformers. At pH 7.0, the binding reaction has a bimolecular rate constant of 1.8 × 10 7 M -1 ·s -1 , and the formation of the initial complex is followed by a conformational change. The binding of NADPH to DHFR is more complex and suggests multiple conformers of the enzyme exist. NADPH binds to a different conformer than H 2 F with a bimolecular rate constant of 2.6–5.7 × 10 6 M -1 ·s -1 , with the former value obtained from single-molecule kinetics and the latter from stopped-flow kinetics. Single-molecule studies of DHFR in equilibrium with substrates and products revealed a reaction with ensemble average rate constants of 170 and 470 s -1 at pH 8.5. The former rate constant has an isotope effect of >2 when NADPD is substituted for NADPH and probably is associated with hydride transfer. The results from stopped-flow and single-molecule methods are complementary and demonstrate that multiple conformations of both the enzyme and enzyme–substrate complexes exist.

We have created a federated database for genome studies of Magnaporthe grisea, the causal agent of rice blast disease, by integrating end sequence data from BAC clones, genetic marker data and BAC contig assembly data. A library of 9216 BAC clones providing >25-fold coverage of the entire genome was end sequenced and fingerprinted by HindIII digestion. The Image/FPC software package was then used to generate an assembly of 188 contigs covering >95% of the genome. The database contains the results of this assembly integrated with hybridization data of genetic markers to the BAC library. AceDB was used for the core database engine and a MySQL relational database, populated with numerical representations of BAC clones within FPC contigs, was used to create appropriately scaled images. The database is being used to facilitate sequencing efforts. The database also allows researchers mapping known genes or other sequences of interest, rapid and easy access to the fundamental organization of the M.grisea genome. This database, MagnaportheDB, can be accessed on the web at http://www.cals.ncsu.edu/fungal_genomics/mgdatabase/int.htm.

Recently, an alternative has been offered to the concept of transition state (TS) stabilization as an explanation for rate enhancements in enzyme-catalyzed reactions. Instead, most of the rate increase has been ascribed to preorganization of the enzyme active site to bind substrates in a geometry close to that of the TS, which then transit the activation barrier impelled by motions along the reaction coordinate. The question as to how an enzyme achieves such preorganization and concomitant TS stabilization as well as potential coupled motions along the reaction coordinate leads directly to the role of protein dynamic motion. Dihydrofolate reductase (DHFR) is a paradigm in which the role of dynamics in catalysis continues to be unraveled by a wealth of kinetic, structural, and computational studies. DHFR has flexible loop regions adjacent to the active site whose motions modulate passage through the kinetically preferred pathway. The participation of residues distant from the DHFR active site in enhancing the rate of hydride transfer, however, is unanticipated and may signify the importance of long range protein motions. The general significance of protein dynamics in understanding other biological processes is briefly discussed. © 2002 The Japan Chemical Journal Forum and John Wiley & Sons, Inc." Chem Rec 2: 24–36, 2002

Ribosomal protein synthesis in eubacteria and eukaryotic organelles initiates with an N -formylmethionyl-tRNA i , resulting in N-terminal formylation of all nascent polypeptides. Peptide deformylase (PDF) catalyzes the subsequent removal of the N-terminal formyl group from the majority of bacterial proteins. Until recently, PDF has been thought as an enzyme unique to the bacterial kingdom. Searches of the genomic DNA databases identified several genes that encode proteins of high sequence homology to bacterial PDF from eukaryotic organisms. The cDNA encoding Plasmodium falciparum PDF (PfPDF) has been cloned and overexpressed in Escherichia coli. The recombinant protein is catalytically active in deformylating N-formylated peptides, shares many of the properties of bacterial PDF, and is inhibited by specific PDF inhibitors. Western blot analysis indicated expression of mature PfPDF in trophozoite, schizont, and segmenter stages of intraerythrocytic development. These results provide strong evidence that a functional PDF is present in P. falciparum. In addition, PDF inhibitors inhibited the growth of P. falciparum in the intraerythrocytic culture.

Peptide deformylase is an essential Fe2+ metalloenzyme that catalyzes the removal of the N-terminal formyl group from nascent polypeptides in eubacteria. In vivo, the deformylase is capable of deformylating most of the polypeptides in a bacterial cell, which contain diverse N-terminal sequences. In this work, we have developed a combinatorial method to systematically examine the sequence specificity of peptide deformylase. A peptide library that contains all possible N-terminally formylated tetrapeptides was constructed on TentaGel resin, with a unique peptide sequence on each resin bead. Limited treatment with the Escherichia coli deformylase resulted in the deformylation of those peptides that are the most potent substrates of the enzyme. By using an enzyme-linked assay, the beads containing the deformylated peptides were identified and isolated. Peptide sequence analysis using matrix-assisted laser desorption ionization mass spectrometry revealed a consensus sequence, formyl-Met-X-Z-Tyr (X = any amino acid except for aspartate and glutamate; Z = lysine, arginine, tyrosine, or phenylalanine), for the E. coli enzyme. The deformylase is also capable of efficient deformylation of formyl-Phe-Tyr-(Phe/Tyr) peptides. These results demonstrate that, despite being a broad-specificity enzyme, the peptide deformylase deformylates different peptides at drastically different rates. In addition, the selectivity of peptide deformylase for the N-formyl over the N-acetyl group has been studied with N-alpha-fluoroacetyl peptides, and the results suggest that both electronic and steric factors are responsible for the observed specificity. The deformylase was also shown to exhibit esterase activity. These results will facilitate the design of specific deformylase inhibitors as potential antibacterial agents. This combinatorial method should be generally applicable to the study of the substrate specificity of other acylases and peptidases.

Peptide deformylase catalyzes the removal of the N-terminal formyl group from nascent polypeptides during prokaryotic protein maturation and is essential for bacterial survival. Its absence from eukaryotic organisms makes it an attractive target for designing novel antibacterial agents. Peptidyl H-phosphonates were synthesized and shown to be competitive inhibitors of the deformylase.

Glucose oxidase (GOX) or lactate oxidase (LOX) were immobilized in an osmium-based three-dimensional redox hydrogel that electrically connected the enzyme's redox centers to electrodes. The enzyme "wiring" hydrogel was formed by cross-linking poly(1-vinylimidazole) (PVI) complexed with Os-(4,4'-dimethylbpy)2Cl (termed PVI15-dmeOs) with poly(ethylene glycol) diglycidyl ether (peg). Glucose and lactate sensors exhibited typical limiting current densities of 250 and 500 microA/cm2, respectively. When the electrodes were poised at 200 mV (SCE), the currents resulting from electrooxidation of ascorbate, urate, acetaminophen, and L-cysteine were negligible. When a Nafion film was employed, the linear range was extended from 6 to 30 mM glucose and from 4 to 7 mM lactate. The redox potential of the gel-forming polymer was 95 mV (SCE). Glucose and lactate measurements performed in bovine calf serum correlated well with a substrate calibration in phosphate buffer.

Enzyme electrodes based on a redox hydrogel formed upon complexing water-soluble poly(1-vinylimidazole) ('PVI') with (Os(bpy)2Cl)(+)('Os') and cross-linked with water-soluble poly(ethylene glycol) diglycidyl ether (molecular weight 400, peg 400) are described. The properties of the electrodes depended on their polymers' osmium content, its extent of cross-linking, on the pH, and the ionic strength in which they were used. The redox hydrogels' electron diffusion coefficients (D (sub e) increased with osmium content of their polymers.

Hepatitis C virus (HCV) infection is treated with a combination of peginterferon alfa-2a/b and ribavirin. To address the limitations of this therapy, numerous small molecule agents are in development, which act by directly affecting key steps in the viral life-cycle. Herein we describe our discovery of quinolone derivatives, novel small-molecules that inhibit NS5b polymerase, a key enzyme of the viral life-cycle. A crystal structure of a quinoline analog bound to NS5B reveals that this class of compounds binds to allosteric site-II (non-nucleoside inhibitor-site 2, NNI-2) of this protein.

The hepatitis C virus (HCV) nonstructural protein 3 (NS3) is known to possess multiple enzymatic activities. In addition to its well-characterized protease activity, HCV NS3 also has ATP hydrolase (ATPase) and nucleic acid unwinding (helicase) activities. We systematically studied the effect of common reagents on all three enzymatic activities with a view to improving assay sensitivity for compound screening and profiling. Inclusion of the detergent lauryl dimethylamine oxide (LDAO) improves protease and helicase activities significantly, allowing robust assays at much lower NS3 concentrations. These conditions enable a particularly sensitive protease assay that uses picomolar concentrations of NS3.

2-(1,1-Dioxo-2H-[1,2,4]benzothiadiazin-3-yl)-1-hydroxynaphthalene derivatives as potential anti-HCV drugs targeting NS5B polymerase have been investigated. Their synthesis, HCV NS5B polymerase inhibition and replicon activity are discussed.

3-(1,1-Dioxo-2H-[1,2,4]benzothiadiazin-3-yl)-4-hydroxy-2H-quinolizin-2-one derivatives as potential anti-HCV drugs targeting NS5B polymerase have been investigated. Their synthesis, HCV NS5B polymerase inhibition, and replicon activity are discussed.

H-transfer was studied in the complex kinetic cascade of dihydrofolate reductase. Intrinsic kinetic isotope effects, their temperature dependence, and other temperature-dependent parameters indicated H-tunneling, but no 1 degrees to 2 degrees coupled motion. The data also suggested environmentally coupled tunneling and commitment to catalysis on pre-steady-state isotope effects.

Recently, the participation in catalysis of residues spatially removed from the enzyme's active site has received considerable attention. The influence of the distal Gly-121 residue on the chemical step of hydride transfer in dihydrofolate reductase (DHFR) catalysis had been demonstrated previously [Cameron, C. E., and Benkovic, S. J. (1997) Biochemistry 36, 15792-15800]. In our continuing effort to identify catalytically important residues that are distal from the active site, we used sequence conservation information, kinetic data on site-directed mutants, dynamic motion information from NMR methods, and correlated motions from MD simulations to identify a subset of residues. Among them, the region spanning positions 41-45 is distal to the active site and was chosen as the focus for the mutagenesis and kinetic studies reported here. Specifically, the highly conserved Met-42 was selected for site-directed mutagenesis. While the reaction kinetics for the M42F mutant enzyme did not deviate from wild-type behavior, a 41-fold reduction in the forward hydride transfer rate was found for the M42W mutant. Given the established role of Gly-121 in the hydride transfer process, double mutant enzymes involving positions 42 and 121 were constructed and characterized. These double mutant enzymes generally showed little changes in substrate and cofactor binding but synergistic decreases in forward hydride transfer rates, while the decreases in reverse rates were additive. Along with supporting information from mixed quantum/classical MD simulations [Agarwal, P. K., Billeter, S. R., Rajagopalan, P. T., Benkovic, S. J., and Hammes-Schiffer, S. (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 2794-2799], the data suggest that a coupled dynamic motion of these distal residues enhances crossing of the chemical reaction barrier and imply an expanded nonstatic role for the protein fold in catalysis.

The thermodynamics and kinetics of the interaction of dihydrofolate reductase (DHFR) with methotrexate have been studied by using fluorescence, stopped-flow, and single-molecule methods. DHFR was modified to permit the covalent addition of a fluorescent molecule, Alexa 488, and a biotin at the N terminus of the molecule. The fluorescent molecule was placed on a protein loop that closes over methotrexate when binding occurs, thus causing a quenching of the fluorescence. The biotin was used to attach the enzyme in an active form to a glass surface for single-molecule studies. The equilibrium dissociation constant for the binding of methotrexate to the enzyme is 9.5 nM. The stopped-flow studies revealed that methotrexate binds to two different conformations of the enzyme, and the association and dissociation rate constants were determined. The single-molecule investigation revealed a conformational change in the enzyme-methotrexate complex that was not observed in the stopped-flow studies. The ensemble averaged rate constants for this conformation change in both directions is about 2–4 s −1 and is attributed to the opening and closing of the enzyme loop over the bound methotrexate. Thus the mechanism of methotrexate binding to DHFR involves multiple steps and protein conformational changes.

A network of coupled promoting motions in the enzyme dihydrofolate reductase is identified and characterized. The present identification is based on genomic analysis for sequence conservation, kinetic measurements of multiple mutations, and mixed quantum/classical molecular dynamics simulations of hydride transfer. The motions in this network span time scales of femtoseconds to milliseconds and are found on the exterior of the enzyme as well as in the active site. This type of network has broad implications for an expanded role of the protein fold in catalysis as well as ancillaries such as the engineering of altered protein function and the action of drugs distal to the active site.

A direct UV–VIS spectrophotometric assay has been developed for peptide deformylase. This assay employs a novel class of peptide mimetics as deformylase substrates which, upon enzymatic removal of the N-terminal formyl group, rapidly release free thiols. The released thiols are quantitated using Ellman's reagent. A variety of peptide analogues that contain β-thiaphenylalanine or β-thiamethionine as the N-terminal residue were synthesized and found to be excellent substrates of the peptide deformylase from Escherichia coli ( k cat / K M = 6.9 × 10 5 M −1 s −1 for the most reactive substrate). The deformylase reaction is conveniently monitored on a UV–VIS spectrophotometer in a continuous fashion. The versatility of the assay has been demonstrated by its application to kinetic characterization of the deformylase, pH profile studies, and enzyme inhibition assays. The assay can also be performed in an end-point fashion. The results demonstrate that this assay is a simple, highly sensitive, and rapid method to study kinetic properties of deformylases without the use of any coupling enzymes.

Peptide deformylase catalyzes the removal of the N-formyl group from newly synthesized polypeptides in prokaryotes. Its essential character and unique presence in prokaryotes make it an attractive target for antibacterial chemotherapy. However, purification and characterization of the peptide deformylase have remained a major challenge because this enzyme is extraordinarily labile under a variety of conditions (t1/2 approximately 1 min at room temperature). In this work, we show that this unusual instability is because of oxidation of the catalytic Fe2+ ion of the deformylase into catalytically inactive Fe3+ ion by atmospheric oxygen. Oxidation of Fe2+ is accompanied by the conversion of O2 into a yet unidentified reactive species, which covalently modifies the deformylase protein, most likely by oxidizing cysteine-90, a ligand residue of the Fe2+ ion, into a cysteine sulfonic acid. Enzymatic exclusion of O2 from the deformylase assays renders the deformylase highly stable under otherwise identical conditions. An improved, readily reproducible purification procedure has been developed that produces approximately 10 mg of pure, fully active Fe2+ deformylase from a liter of cells. In addition, active peptide deformylase can be reconstituted in vitro from the denatured deformylase.

Peptide deformylase (EC 3.5.1.31) catalyzes the removal of a formyl group from the N-termini of nascent ribosome-synthesized polypeptides, an obligatory step during protein maturation in eubacteria. Since its discovery in crude Escherichia coli extracts 3 decades ago, the deformylase has resisted all attempts of purification or characterization due to its extraordinary lability. By placing the coding sequence (def gene) of Escherichia coli deformylase behind a bacteriophage T7 promoter, we have, however, been able to overexpress this deformylase in Escherichia coli. Overproduction has allowed the purification of50 mg of deformylase enzyme from each liter of cell culture. Purified deformylase is highly active toward N-formylated peptide substrates. A new, sensitive assay for the deformylase has been developed by measuring the amount of released formate using a formate dehydrogenase. This has allowed for the assessment of the catalytic properties of peptide deformylase using a series of synthetic N-formylated peptides as substrates. The deformylase exhibits strong preference for an L-methionine or the isosteric norleucine at the N-terminus of a substrate and has broad specificity for the rest of the residues. Small divalent metal chelators strongly inhibit the E. coli deformylase. In particular, certain 1,2- and 1,3-dithiol compounds act as potent, time-dependent inhibitors of the peptide deformylase.

Lactate electrodes based on electron-conducting hydrogels made by cross-linking lactate oxidase and the redox polymer formed upon complexing polyvinyl imidazole with [Os(dmo-bpy)2Cl]+/2+ (dmo-bpy = 4,4'-dimethoxy-2,2'-bipyridine) on vitreous carbon electrode surfaces were investigated. The redox potential of the hydrogels was -69 mV, versus the standard calomel electrode (SCE), and their lactate electro-oxidation current reached a plateau at +50 mV (SCE). Urate and acetaminophen were not electro-oxidized at this potential at rates that would interfere with the lactate assays, but ascorbate was catalytically oxidized by the gel. At 6 mM lactate concentration, switching of the atmosphere from argon to O2, reduced the current by 40 percent, showing that the rate of electron transfer from the reduced enzyme to the gel was slow.