S10 Inhibitor binding data were plotted, and the set of 5 data points bracketing the midpoint of the saturation curve were analyzed for the of binding by fitting the data to Eqn. masses of 20-120 kDa and can be broadly divided into two groups, the high molecular mass (HMM) PBPs and the low molecular mass (LMM) PBPs . HMM PBPs are essential for bacterial survival and are the lethal targets for Clactam antibiotics, whereas LMM PBPs are non-essential for cell viability. An enigmatic feature of the PBPs is usually that, while LMM PBPs give readily detectible activity against peptide substrates, purified HMM PBPs give either low or undetectable activity against natural or synthetic cell wall-related peptide substrates (reviewed in [5, 6]). This has impeded the development of convenient assays for the HMM PBPs. Two approaches which have had some success for demonstrating the activity of the HMM PBPs are the use of thiolester-based substrates [7, 8], and assays based on the use of lipid II [9-12], which is a precursor to the nascent peptidoglycan substrate of the PBPs. However, neither of these assays appear well suited for microtiter plate based high throughput assays C the thiolesters because of their high background Beta-Lapachone rate of hydrolysis , and lipid II because of its difficult isolation  and synthesis [11, 12]. In an effort to circumvent the limitations of these and other HMM PBP assays, deSousa and coworkers have developed scintillation proximity assays to measure membrane associated peptidoglycan synthesis in membrane preparations [14-17], but these assays also appear difficult and cumbersome. We describe here a general assay for screening and characterizing HMM PBP inhibitors. This approach is based on the fact that this HMM PBPs, essentially by definition, bind Clactams. This assay uses a Clactam-biotin conjugate (BIO-AMP) previously described for the detection of PBPs in Western Blots [18-20]. In the present study purified PBPs were immobilized onto microtiter plate wells, and labeled with BIO-AMP. Treatment of the BIO-AMP labeled PBP with a strepavidin-horse radish peroxidase (HRP) conjugate followed by a fluorogenic HRP substrate (Amplex Red) allowed the efficient detection Beta-Lapachone of immobilized PBPs. Binding curves for BIO-AMP conversation with PBPs were then measured, and used to calculate apparent for binding vs various PBPs PBPs turnover Clactams (albeit usually very slowly). To assess the for BIO-AMP binding to a given PBP, the microtiter plate bound PBP was treated with serially diluted (actions of 5) concentrations of BIO-AMP, and the remaining steps of the assay performed as described above. Signals were plotted, and the set of 5 data points bracketing the midpoint Beta-Lapachone of the saturation curve were analyzed for the of binding by fitting the data to Eqn. S7 (Supplementary Material). RFU =?RFU0 +?(RFUmax?[I])?M?(+?[I]) Eqn. S7 Application to HMM PBP-inhibitor screening and characterization For inhibitor screening and characterization, BIO-AMP was used at a fixed concentration equal to the decided for a PBP. This is high enough Rabbit Polyclonal to LGR4 to give 1/2 of the maximum possible signal and low enough to still allow inhibition to be readily detected. To demonstrate this capability, NG PBP2 was characterized for inhibition by ampicillin. NG PBP2 was first attached to the wells of a microtiter plate as described above. Competitive ampicillin/BIO-AMP binding was performed by adding 100 L samples of serially (actions of 5) diluted solutions of ampicillin in the presence of 1.1 M BIO-AMP (the for BIO-AMP vs NG PBP2, Table 1). After 15 minutes the binding reactions were stopped and developed as described above. With [BIO-AMP] = +?2) Eqn. S10 Inhibitor binding data were plotted, and the set of 5 data points bracketing the midpoint of the saturation curve were analyzed for the of binding by fitting the data to Eqn. S10. Table 1 Microtiter plate decided values for BIO-AMP with several PBPs..