The peptidoglycan layers surrounding bacterial membranes are essential for bacterial cell survival and provide an important target for antibiotics. Many antibiotics have mechanisms of action that involve binding to Lipid II, the prenyl chain-linked donor of the peptidoglycan building blocks. One of these antibiotics, the pore-forming peptide nisin uses Lipid II as a receptor molecule to increase its antimicrobial efficacy dramatically. Nisin is the first example of a targeted membranepermeabilizing peptide antibiotic. However, it was not known whether Lipid II functions only as a receptor to recruit nisin to bacterial membranes, thus increasing its specificity for bacterial cells, or whether it also plays a role in pore formation. We have developed a new method to produce large amounts of Lipid II and variants thereof so that we can address the role of the lipidlinked disaccharide in the activity of nisin. We show here that Lipid II is not only the receptor for nisin but an intrinsic component of the pore formed by nisin, and we present a new model for the pore complex that includes Lipid II.The cell wall is an essential structure of a bacterium, providing its shape and protecting it from bursting because of the high osmotic pressures of the cytoplasm. This wall is a threedimensional network built of identical subunits consisting of two amino sugars, N-acetylglucosamine (GlcNAc) and N-acetylmu-is attached to the carboxyl group of MurNAc. These subunits are assembled in the cytosol of bacteria using UDP-activated precursors on a special lipid carrier, undecaprenyl phosphate (for a review see Ref. 1). The integral membrane protein MraY and the peripherally membraneassociated MurG that synthesize the precursors Lipid I and II, respectively, are the key enzymes in the last two cytoplasmic steps in the formation of the subunits (Fig. 1). Subsequently, Lipid II is transported across the plasma membrane via an as of yet unknown mechanism. Thereafter, the subunits are polymerized and inserted into the pre-existing cell wall by means of the penicillin-binding proteins (for review see Ref.2). Numerous antibiotics target the cell wall synthesis, including a diverse group of antibiotics that bind to Lipid II. Perhaps the best known of these antibiotics is vancomycin, the antibiotic of last resort to treat MRSA infections (3). However, there are many others, including the polypeptide nisin, that kill cells by permeabilizing bacterial membranes. Efficient membrane permeabilization by nisin requires an interaction with Lipid II (4, 5). This designates nisin as the first example of a targeted poreforming peptide antibiotic.Two recent studies have shed light on the structural requirements within nisin for the interaction with Lipid II. A genetic approach indicated that the N terminus of nisin is involved in the interaction with Lipid II (6), and more recently we could map the binding interface toward specific residues in the N terminus using 15 N-labeled nisin (7). It is not clear yet what events lead to pore formation after the...

Commonly used methods for isolated enzyme inhibitor screening typically rely on fluorescent or chemiluminescent detection techniques that are often indirect and/or coupled assays. Mass spectrometry (MS) has been widely reported for measuring the conversion of substrates to products for enzyme assays and has more recently been demonstrated as an alternative readout system for inhibitor screening. In this report, a high-throughput mass spectrometry (HTMS) readout platform, based on the direct measurement of substrate conversion to product, is presented. The rapid ionization and desorption features of a new generation matrix-assisted laser desorption ionization-triple quadrupole (MALDI-QqQ) mass spectrometer are shown to improve the speed of analysis to greater than 1 sample per second while maintaining excellent Z′ values. Furthermore, the readout was validated by demonstrating the ability to measure IC 50 values for several known kinase inhibitors against cyclic AMP-dependent protein kinase (PKA). Finally, when the assay performance was compared with a common ADPaccumulation readout system, this HTMS approach produced better signal-to-background ratios, higher Z′ values, and a reagent cost of about $0.03 per well compared with about $0.60 per well for the fluorescence assay. Collectively, these data demonstrate that a MALDI-QqQ-MS-based readout platform offers significant advantages over the commonly used assays in terms of speed, sensitivity, reproducibility, and reagent cost. INTRODUCTION HIGH-THROUGHPUT SCREENING (HTS) plays a central role in the drug discovery process starting at the early "hit" discovery stage then continuing through to lead optimization. As such, the screening community has continuously developed new technologies to meet the broadening assay needs and to expedite the work flow of drug discovery. Within the pharmaceutical and biotechnology enzyme screening community, fluorescent-and chemiluminescent-based detection methods continue to be the routine assay platforms of choice for isolated enzyme assays. These assays are desirable because they have been developed to be simple homogeneous assays that often offer universal methods to evaluate a variety of targets using the same reagents. However, 1 of the primary challenges for these approaches continues to be how to maintain the high speed of analysis while minimizing false-positive or false-negative readouts. Inherent to this challenge is the nature of fluorescence and chemiluminescence readout, which can lend itself to false readouts because of the properties of various compound classes that enhance or quench the signals. Thus a comparable approach in speed, robust readout, and cost that further minimizes the potential for false readouts would be a benefit to the field.One approach that offers promise in this arena is mass spectrometry. A variety of mass spectrometry (MS) techniques have been used to measure enzyme activity, enzyme kinetics, inhibition, and more recently for HTS applications (reviewed in the studies of Greis 1 and De Boer et...

A novel synthesized watersoluble variant of lipid II (LII) was used to evaluate the noncovalent interactions between a number of glycopeptide antibiotics and their receptor by bioaffinity electrospray ionization mass spectrometry (ESI-MS). The water-soluble variant of lipid II is an improved design, compared to the traditionally used tripeptide, of the target molecule on the bacterial cell wall. A representative group of glycopeptide antibiotics was selected for this study to evaluate the validity of the novel cell-wall-mimicking target LII. Structure ± function relationships of various glycopeptide antibiotics were investigated by means of 1) bioaffinity mass spectrometry to evaluate solution-phase molecular interactions with both LII and KAA, 2) fluorescence leakage experiments to study the interactions with the membrane-embedded lipid II, and 3) minimum inhibitory concentrations against the indicator strain Micrococcus flavus. Our results with the novel LII molecule reveal that some antibiotics interact differently with KAA and LII. Additionally, our data cast doubt on the hypothesis that antibiotic selfdimerization assists in the invivo efficacy. Finally, the water-soluble lipid II proved to be a better model of the bacterial cell wall.

A sheath-flow capillary electrophoresis-mass spectrometry (CE-MS) system utilizing a fully integrated large-bore stainless-steel emitter electrode tapered at the end for micro-ionspray operation has been developed and evaluated. A separation capillary with an outer diameter of up to 360 microm was inserted into the electrode thus forming a void volume of less than 15 nL between the capillary end and the electrospray ionisation (ESI) tip. The sheath liquid, usually methanol-water (80:20) with 0.1% formic acid for positive ion mode or methanol for negative ion mode, was delivered at 0.5-1.0 microL/min. Unlike previously reported CE-MS interfaces, the CE-MS probe was incorporated directly onto an Applied Biosystems/MDS SCIEX orthogonal-spray Turbo "V" ion source for ease of use and automatic operation. This integration enables fast and facile coupling and replacement of the separation capillary without interrupting the ion source configuration, and the sheath liquid supply. The reusable electrospray electrode was precisely fabricated and aligned with the length of the nebulizing gas tube for improved reproducibility. Automation was achieved through software control of both CE and tandem MS (MS/MS) for unattended batch sample analysis. The system was evaluated for attomole- to low femtomole-level profiling of model peptides and protein mixtures, bisphosphates, as well as antiviral nucleosidic drugs in cellular extracts.

Many analytical approaches are available to evaluate (bio)molecular interactions, all of which have their particular advantages and disadvantages. In recent years, two relatively new techniques have emerged that may be used by the bioanalytical community to evaluate such interactions, namely affinity capillary electrophoresis (ACE) and bioaffinity electrospray ionization-mass spectrometry (ESI-MS). In this paper, we describe and evaluate the use of both these techniques for the investigation of the interactions of glycopeptide antibiotics with peptides that mimic the bacterial cell wall binding site. We focus particularly on the effect of the sugar moieties attached to the antibiotic peptide backbone and on the noncovalent dimerization of these glycopeptide antibiotics.