High-Throughput Screen for Inhibitors of Transglycosylase and/or Transpeptidase Activities of Escherichia coli Penicillin Binding Protein 1b

Abstract

Penicillin binding protein (PBP) 1b of Escherichia coli has both transglycosylase and transpeptidase activities, which are attractive targets for the discovery of new antibacterial agents. A high-throughput assay that detects inhibitors of the PBPs was described previously, but it cannot distinguish them from inhibitors of the MraY, MurG, and lipid pyrophosphorylase. We report on a method that distinguishes inhibitors of both activities of the PBPs from those of the other three enzymes. Radioactive peptidoglycan was synthesized by using E. coli membranes. Following termination of the reaction the products were analyzed in three ways. Wheat germ agglutinin (WGA)-coated scintillation proximity assay (SPA) beads were added to one set, and the same beads together with a detergent were added to a second set. Type A polyethylenimine-coated WGA-coated SPA beads were added to a third set. By comparison of the results of assays run in parallel under the first two conditions, inhibitors of the transpeptidase and transglycosylase could be distinguished from inhibitors of the other enzymes, as the inhibitors of the other enzymes showed similar inhibitory concentrations (IC 50 s) under both conditions but the inhibitors of the PBPs showed insignificant inhibition in the absence of detergent. Furthermore, comparison of the results of assays run under conditions two and three enabled the distinction of transpeptidase inhibitors. Penicillin and other ␤-lactams showed insignificant inhibition with type A beads compared with that shown with WGA-coated SPA beads plus detergent. However, inhibitors of the other four enzymes (tunicamycin, nisin, bacitracin, and moenomycin) showed similar IC 50 s under both conditions. We show that the main PBP being measured under these conditions is PBP 1b. This screen can be used to find novel transglycosylase or transpeptidase inhibitors.

Domain requirement of moenomycin binding to bifunctional transglycosylases and development of high-throughput discovery of antibiotics

“…However, the limited availability of lipid II has hampered the development of effective enzymatic assays for identification of inhibitors. As a result, the majority of the screening methods that are used to search for TG inhibitors, including the low-throughput methods that use surface plasmon resonance (SPR) (12) or radioactive assays (13)(14)(15)(16), rely mainly on moenomycin (11). Development of a TG activity assay that is amenable to high-throughput screening (HTS) is thus desirable for inhibitor identification.…”

Microplate Assay for Inhibitors of the Transpeptidase Activity of PBP1b of Escherichia coli

“…2A, squares). Under these conditions, we have shown that the beads capture total peptidoglycan and also lipid II, 25 but under these conditions, negligible lipid II was synthesized ( Table 1). Hence, the values reflect total peptidoglycan made under these conditions.…”

“…Subsequently, a source of TP (i.e., PBP1b) was added, and the conversion of un-crosslinked peptidoglycan to cross-linked peptidoglycan was assayed by specific capture of the latter using wheat germ agglutininscintillation proximity assay (WGA-SPA) beads in the presence of N-laurylsarcosine. 25 The assay can be used to screen for inhibitors of the TP and has much higher throughput than earlier published assays. …”

“…25 Here we report a novel, homogenous, microplate method to directly measure the enzymatic activity of the peptidoglycan TP. It takes advantage of the mechanism of inhibition of the β-lactam antibiotics to inactivate the endogenous PBPs; these antibiotics inhibit the TP activity while not affecting the TG activity.…”

“…24,25 E. coli membranes were pretreated with ampicillin and incubated under conditions conducive to peptidoglycan synthesis (step 1 of the TP assay; Fig. 1B).…”