Microtiter Plate-Based Assay for Inhibitors of Penicillin-Binding Protein 2a from Methicillin-Resistant Staphylococcus aureus

Abstract

Penicillin-binding protein 2a (PBP2a), the molecular determinant for high-level ␤-lactam resistance in methicillin-resistant Staphylococcus aureus (MRSA), is intrinsically resistant to most ␤-lactam antibiotics. The development and characterization of new inhibitors targeting PBP2a would benefit from an effective and convenient assay for inhibitor binding. This study was directed toward the development of a fluorescently detected ␤-lactam binding assay for PBP2a from MRSA. Biotinylated ampicillin and biotinylated cephalexin were tested as tagging reagents for fluorescence detection by using a streptavidin-horseradish peroxidase conjugate. Both bound surprisingly well to PBP2a, with binding constants of 1.6 ؎ 0.4 M and 13.6 ؎ 0.8 M, respectively. Two forms of the assay were developed, a one-step direct competition form of the assay and a two-step indirect competition form of the assay, and both forms of the assay gave comparable results. This assay was then used to characterize PBP2a binding to ceftobiprole, which gave results consistent with previous studies of ceftobiprole-PBP2a binding. This assay was also demonstrated for screening for PBP2a inhibitors by screening a set of 13 randomly selected ␤-lactams for PBP2a inhibition at 750 M. Meropenem was observed to give substantial inhibition in this screen, and a follow-up titration experiment determined its apparent K i to be 480 ؎ 70 M. The availability of convenient and sensitive microtiter-plate based assays for the screening and characterization of PBP2a inhibitors is expected to facilitate the discovery and development of new PBP2a inhibitors for use in combating the serious public health problem posed by MRSA.Bacterial infections were the major cause of death and morbidity prior to the development of modern antibiotics, and the increasing resistance of pathogenic bacteria to commonly used antibacterial agents is of major public health concern. One organism of particular concern is methicillin-resistant Staphylococcus aureus (MRSA) (1, 7, 11). The high level of ␤-lactam resistance seen in MRSA, compared to methicillin-sensitive S. aureus (MSSA), is due to the presence of a novel acquired penicillin-binding protein (PBP): PBP2a (10; recently reviewed in references 13, 14, and 19). PBP2a is a high-molecular-mass (HMM) PBP that is intrinsically resistant to most ␤-lactam antibiotics. Assay methods for inhibitor binding to PBP2a have been described based on radiolabeled ␤-lactam binding (17) or on nitrocefin (9) (a chromogenic cephalosporin derivative) or BOCILIN-FL (a fluorescently tagged penicillin derivative) binding (8,20). However, these assays are incompatible with a microtiter plate format required for efficient high-throughput inhibitor screening and characterization. Given the high intrinsic resistance of PBP2a to ␤-lactams, it was uncertain whether a microtiter plate ␤-lactam binding assay of the type we have described recently for other HMM PBPs (16), e.g., based on biotinylated ␤-lactams, would work with PBP2a. In the study reported here, we investi...

Peptidoglycan Cross-Linking Preferences of Staphylococcus aureus Penicillin-Binding Proteins Have Implications for Treating MRSA Infections

“…Previous studies showed that PBP2a can be inactivated by the fifth-generation cephalosporins, ceftaroline and ceftobiprole, and is weakly inhibited by meropenem, but not other β-lactams. 2a,4b,11 We found that incubation of PBP2a with ceftaroline prior to adding PBP2-TP* and Gly 5 -Lipid II abolished formation of the dimer and hydrolyzed monomer peaks in the LC/MS assay (Fig. 2a).…”

“…7 Meropenem inhibited PBP2a, but was about twenty-fold less potent than ceftaroline, consistent with results obtained from competition binding assays for these β-lactams. 11–12 …”

Using the Predicted Structure of the Amot Coiled Coil Homology Domain to Understand Lipid Binding

“…The lysate was then collected by centrifugation at 15000 rpm at 4°C for 30 minutes using a JA-10 rotor. Protein was purified using batch purification of glutathione resin (Ali, Chachadi, Petrosyan, & Cheng, 2012; Bobba, Ponnaluri, Mukherji, & Gutheil, 2011; Petrosyan, Ali, Verma, Cheng, & Cheng, 2012). Overnight thrombin cleavage on the resin separated the GST tag from the protein of interest (Arnau, Lauritzen, Petersen, & Pedersen, 2006; Terpe, 2003).…”

Development of New Drugs for an Old Target — The Penicillin Binding Proteins

“…Gel electrophoresis has been replaced by filtration using 96-well filter plates in assays developed for PBP2a [ 48 ]. Another approach was the immobilization of PBPs on microtiter plates by using an ELISA-like protocol [ 49 ] or a GST-PBP2a fusion protein [ 50 ].…”