A mass spectrometric protocol for identifying ligands with a wide range of affinities (3-101 mum) and quantitative spectral analysis for non-covalent interactions have been developed using Src SH2 as a target. Dissociation constants of five compounds, three with a phospho moiety, one with a sulphonic acid group and one with carboxylic acid groups only, were determined using one-ligand one-binding-site, two-ligands one-binding site and one-ligand two-binding-sites models. The K-d values determined by ESI-MS of the three compounds containing the phospho moiety (3.2-7.9 muM) were comparable to those obtained from a solution equilibrium fluorescence polarization assay. The compound with a sulphonate group is a much weaker binding ligand (K-d = 101 muM by ESI, much greater than300 muM by FP) towards the Src SH2 protein. Two complexes with different stoichiometric ratios 1:1 and 2:1 (ligand-protein) were observed by ESI-MS for the ligand GIXXX630X. Analysis of binding isotherms indicated the presence of two binding sites for the ligand with K-d values of 9.3 and 193 muM. These data confirmed that, for these polar compounds, non-covalent ESI-MS can measure affinity which very closely reflects the affinity measured under true solution equilibrium conditions. ESI-MS has several key advantages over many solution methods: it can identify the existence of and measure the affinity of complexes other than simple 1:1 ligand-enzyme complexes. Moreover, ESI-MS competition experiments can be readily performed to yield data on whether two ligands bind simultaneously or competitively at the same time as measuring the affinity of the ligand. Copyright (C) 2003 John Wiley Sons, Ltd.

A series of chiral, (S)-proline-alpha-methylpyrrolidine-5,5-trans-lactam serine protease inhibitors has been developed as antivirals of human cytomegalovirus (HCMV). The SAR of the functionality on the proline nitrogen has shown that derivatives of para-substituted phenyl ureas > para-substituted phenyl sulfonamides > para-substituted phenyl carboxamide for activity against HCMV deltaAla protease, producing para-substituted phenyl ureas with single figure nM potency (K(i)) against the viral enzyme. The SAR of the functionality on the lactam nitrogen has defined the steric and electronic requirements for high human plasma stability while retaining good activity against HCMV protease. The combination of high potency against HCMV deltaAla protease and high human plasma stability has produced compounds with significant in vitro antiviral activity against human cytomegalovirus with the 6-hydroxymethyl benzothiazole derivative 72 being equivalent in potency to ganciclovir. The parent benzothiazole 56 had good pharmacokinetics in dogs with 29% bioavailability and good brain and ocular penetration in guinea pigs.

Mechanism-based inhibitors of HCMV protease, which are stable to human plasma (> or = 20 h) and have single-figure potency in the microM range against HCMV protease, have been developed based on the dansylproline alpha-methyl pyrrolidine-5,5-trans-lactam nucleus.

The stereospecific synthesis of a series of alpha-methylpyrrolidine-5,5-trans-lactam inhibitors of human cytomegalovirus (HCMV) protease is described. Examination of the SAR in this series has defined the size and chirality of the alpha-substituent, optimized the acyl substituent on the lactam nitrogen, and defined the steric constraint of this functionality. The SAR of the functionality on the pyrrolidine nitrogen of the trans-lactam has been investigated, and this has led to the discovery of potent serine protease inhibitors that are highly selective for the viral enzyme over the mammalian enzymes elastase, thrombin, and acetylcholine esterase. The mechanism of action of our lead compounds has been established by mass spectrometry, and enzymatic degradation of HCMV deltaAla protease acylated with these inhibitors showed that Ser 132 is the active site nucleophile. The crystal structure of HCMV protease was obtained and used to model the conformationally restricted, chiral (S)-proline-alpha-methyl-5,5-trans-lactams into the active site groove of the enzyme, enabling us to direct and rationalize the SAR in this series. The activity against HCMV deltaAla protease is the greatest with inhibitors based on the dansyl-(S)-proline alpha-methyl-5,5-trans-lactam template, which have low nanomolar activity against the viral enzyme.

Mechanism-based inhibitors of human cytomegalovirus (HCMV) protease have been designed based on the pyrrolidine-5,5-trans-lactam ring system. New routes to the beta-methyl-, desmethyl-, and alpha-methyl-pyrrolidine-5,5-trans-lactam templates have been developed from 2,4-diaminobutyric acid. ESI/MS studies have shown that these inhibitors can bind covalently and reversibly to the viral enzyme in a time-dependent manner by a mechanism which is consistent with acylation of HCMV deltaAla protease at the active site nucleophile Ser 132. SAR in this series of pyrrolidine-5, 5-trans-lactams has defined the relative stereochemisty of the methyl substituent adjacent to the lactam carbonyl, the functionality on the lactam nitrogen, and the mechanism of action of this novel series of serine protease inhibitors against the HCMV deltaAla protease. Activity decreases on moving from the alpha-methyl to the desmethyl to the beta-methyl series. This selectivity is the opposite of that observed for these templates against the elastase and thrombin enzymes. The activity against HCMV deltaAla protease is the greatest with inhibitors based on the Cbz-protected alpha-methyl-5,5-trans-lactam template which have low micromolar activity against the viral enzyme.

Mechanism based inhibitors of HCMV protease have been designed based on the monocyclic beta-lactam nucleus, which have been shown to acylate the viral enzyme in a time dependent manner. SAR in a series of monocyclic beta-lactam N-ureas, has defined the size and relative stereochemistry of the C-3 substituent producing a low micromolar inhibitor 17b with good aqueous stability and selectivity over the mammalian serine proteases.

An investigation into the interaction between human cytomegalovirus (HCMV) protease and several beta-lactams, with characterization of the resulting acylenzymes using mass spectrometry, is reported. The time dependence of the inhibitors is highlighted by making comparisons of values obtained for inhibition and acylation, Analysis of inactivated HCMV protease revealed a beta-lactam:protease stoichiometry of 1, Subsequent enzymatic digestion with trypsin, peptide mapping using liquid chromatography coupled with electrospray ionization mass spectrometry and sequencing by nanoelectpospray tandem mass spectrometry (NanoES-MS/MS) allowed the identification of the site of covalent modification and confirmed Ser 132 as the active site hydroxyl nucleophile. Further, treatment of the protease with a peptide chloromethylketone and sequence analysis using NanoES-MS/MS of the alkylated enzyme confirmed His 63 as the active site imidazole nucleophile. (C) 1998 John Wiley & Sons, Ltd.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTKinetic Characterization of the Inactivation of UDP-GlcNAc Enolpyruvyl Transferase by (Z)-3-Fluorophosphoenolpyruvate: Evidence for Two Oxocarbenium Ion Intermediates in Enolpyruvyl Transfer CatalysisDennis H. Kim, Watson J. Lees, Terry M. Haley, and Christopher T. WalshCite this: J. Am. Chem. Soc. 1995, 117, 5, 1494–1502Publication Date (Print):February 1, 1995Publication History Published online1 May 2002Published inissue 1 February 1995https://doi.org/10.1021/ja00110a005RIGHTS & PERMISSIONSArticle Views176Altmetric-Citations19LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (972 KB) Get e-Alerts Get e-Alerts

UDP-GlcNAc enolpyruvyl transferase (MurZ) catalyzes the transfer of an enolpyruvyl moiety from phosphoenolpyruvate (PEP) to the 3-OH of UDP-GlcNAc in a reaction that constitutes the first committed step in bacterial peptidoglycan assembly. In a preliminary communication (Kim et al. J. Am. Chem. Soc. 1994, 116, 6478-6479) we reported the formation of two enzyme-associated fluoro analogs-a covalent phosphofluorolactyl-enzyme adduct, 3, and a phosphofluorolactyl-uDP-GlcNAc tetrahedral adduct, 4-of the corresponding normal reaction intermediates, 1 and 2, during inactivation of MurZ by both (E)- and (Z)-3-fluorophosphoenolpyruvate (FPEP). In this paper we present a detailed study of the kinetics of formation, interconversion and decomposition of 3 and 4. Kinetic analysis of the formation of 3 and 4 using enzymatically-synthesized [P-32](Z)-FPEP demonstrates that the formation of these adducts is consistent with the measured limiting rate constant for inactivation of MurZ by (Z)-FPEP (k(inact) = 1.8 min(-1)). The kinetics of formation suggest that 3 and 4 are formed by parallel pathways from the enzyme UDP-GlcNAc (Z)-FPEP ternary complex, with an initial 5-fold kinetic preference for the formation of 4 over 3. Analysis of the inactivation reaction over a long time period suggests that 3 and 4 are nearly isoenergetic, reaching an equilibrium ratio of 1.2 (3:4) through a slow equilibration at the active site of MurZ, with rate constants for direct interconversion of 0.1 min(-1). Evidence from experiments following the washout of P-32 from 3 and 4 as well as data on the rate of deuterium incorporation from D2O into 4 strongly suggest that the two analogs interconvert directly, without involving C-H bond cleavage and FPEP formation. The rate constant for the return of 3 and 4 to FPEP, a measure of the reversibility of (Z)-FPEP inhibition, was 0.004 min(-1), corresponding to a ratio of similar to 400:1 in favor of enzyme bound with 3 and 4 versus enzyme in ternary complex with (Z)-FPEP and UDP-GlcNAc. The relative retardation in rate of breakdown of 4 relative to the analogous normal reaction intermediate, 2, is estimated to be > 10(6), accounting for the observed enzyme inactivation. Comparison of the kinetic data with (Z)-FPEP with rapid-quench data for the normal reaction with PEP (Brown et al. Biochemistry 1994, 33, 10638-10645) supports a mechanism in which formation and decomposition of the reaction tetrahedral intermediate, 2 or 4 as well as interconversion between 2 or 4 and the corresponding enzyme adduct, 1 or 3, involves oxocarbenium ion intermediates. The (Z)-FPEP data are suggestive of a branching mechanism for normal MurZ catalysis, in which the primary pathway of catalysis is through direct formation and breakdown of the tetrahedral intermediate, 2.

Fosfomycin [(1R,2S)-1,2-epoxypropylphosphonic acid] has been shown to exert its antibiotic effect through the inhibition of UDP-GlcNAc enolpyruvoyl transferase [Kahan, F. M., et al. (1974) Ann. N.Y. Acad. Sci. 235, 364], the enzyme responsible for catalyzing the first committed step in bacterial cell wall biosynthesis. Time-dependent inactivation of MurZ by fosfomycin was found to be greatly accelerated by the presence of cosubstrate UDP-GlcNAc but could also be speeded appreciably by the unreactive substrate analog 3-deoxy-UDP-GlcNAc. These results argue against a reaction-based participation of the cosubstrate and suggest that UDP-GlcNAc has a role in influencing active site conformation critical to the inactivation event. A study of the influence of UDP-GlcNAc and fosfomycin on the kinetics of inactivation allowed the determination of dissociation constants for fosfomycin (KF = 8.6 microM) and UDP-GlcNAc (KS = 14 microM), in addition to a limiting inactivation rate constant (k(inact) = 7.4 min-1) at saturating UDP-GlcNAc and fosfomycin concentrations. Mass spectrometry of inactivated MurZ demonstrated an increase in molecular weight of 138, consistent with the covalent addition of a molar equivalent of fosfomycin (136 kDa). Titration of MurZ with fosfomycin revealed a stoichiometry of 1 molecule of inhibitor per active site when assessed using either enzyme activity or mass spectrometry as an index of modification. Peptide mapping of tryptic digests of fosfomycin-inactivated MurZ revealed modification of a unique 41-mer, the sequence of which revealed that Cys115 was the site of attachment of fosfomycin.

The use of size exclusion chromatography (SEC) for the determination of molar mass distribution and fractionation of coal tars has been investigated. The construction of a calibration graph based on typical components of the tars for use in the determination of molar mass distributions has revealed anomalously low retention volumes for phenols and amines when THF is used as the mobile phase. Consequently the molar mass distribution for a tar with a high phenolic content was not reliable when determined in THF but chloroform was shown to be a suitable solvent for this purpose. However the effect was used to fractionate the tar into two components, one of which contained a mixture of mainly phenols and long chain paraffins while the other was composed predominantly of polynuclear aromatics. A coke oven tar of low phenolic content was also fractionated into two components according to molecular weight and the fractions characterised in detail.