A Microtiter Plate-Based Assay for Inhibitors of Penicillin-Binding

PBP2a, the molecular determinant for high level β-lactam resistance in 19 Methicillin-resistant Staphylococcus aureus (MRSA), is intrinsically resistant to most β20 lactam antibiotics. The development and characterization of new inhibitors targeting 21 PBP2a would benefit from an effective and convenient assay for inhibitor binding. This 22 study was directed towards the development of a fluorescently detected β-lactam binding 23 assay for PBP2a from MRSA. Biotinylated ampicillin and biotinylated cephalexin were 24 tested as tagging reagents for fluorescence detection using a strepavidin-HRP conjugate. 25 Both bound surprisingly well to PBP2a, with binding constants of 1.6 ± 0.4 μM and 13.6 ± 26 0.8 μM respectively. Two forms of the assay were developed, a one step direct 27 competition form of the assay and a two step indirect competition form of the assay, and 28 both forms of the assay gave comparable results. This assay was then used to 29 characterize PBP2a binding to ceftobiprole which gave results consistent with previous 30 studies of ceftobiprole-PBP2a binding. This assay was also demonstrated for screening 31 for PBP2a inhibitors by screening a set of thirteen randomly selected β-lactams for 32 PBP2a inhibition at 750 μM. Meropenem was observed to give substantial inhibition in 33 this screen, and a follow-up titration experiment determined its KI app to be 480 ± 70 μM. 34 The availability of convenient and sensitive microtiter-plate based assays for the 35 screening and characterization of PBP2a inhibitors is expected to facilitate the discovery 36 and development of new PBP2a inhibitors for use in combating the serious public health 37 problem posed by MRSA. 38 39 on D ecem er 4, 2017 by gest httpaac.asm .rg/ D ow nladed fom