Development and Validation of a Whole-Cell Inhibition Assay for Bacterial Methionine Aminopeptidase by Surface-Enhanced Laser Desorption Ionization-Time of Flight Mass Spectrometry

Abstract

Bacterial methionine aminopeptidase (MAP) is a protease that removes methionine from the N termini of newly synthesized bacterial proteins after the peptide deformylase enzyme cleaves the formyl group from the initiator formylmethionine. MAP is an essential bacterial gene product and thus represents a potential target for therapeutic intervention. A fundamental challenge in the antibacterial drug discovery field is demonstrating conclusively that compounds with in vitro enzyme inhibition activity produce the desired antibacterial effect by interfering with the same target in whole bacterial cells. One way to address the activity of inhibitor compounds is by profiling cellular biomarkers in whole bacterial cells using compounds that are known inhibitors of a particular target. However, in the case of MAP, no specific inhibitors were available for such studies. Instead, a genetically attenuated MAP strain was generated in which MAP expression was placed under the control of an inducible arabinose promoter. Thus, MAP inhibition in whole cells could be mimicked by growth in the absence of arabinose. This genetically attenuated strain was used as a benchmark for MAP inhibition by profiling whole-cell lysates for unprocessed proteins using surface-enhanced laser desorption ionization-time of flight mass spectrometry (MS). Eight proteins between 4 and 14 kDa were confirmed as being unprocessed and containing the initiator methionine by adding back purified MAP to the preparations prior to MS analysis. Upon establishing these unprocessed proteins as biomarkers for MAP inhibition, the assay was used to screen small-molecule chemical inhibitors of purified MAP for whole-cell activity. Fifteen compound classes yielded three classes of compound with whole-cell activity for further optimization by chemical expansion. This report presents the development, validation, and implementation of a whole-cell inhibition assay for MAP.

RETRACTED: Targeting Mycobacterial Enzymes with Natural Products

“…This effect can be estimated within investigated compounds by profiling cellular biomarkers in whole bacterial cells or in animals using compounds that are known inhibitors or activators of a particular target (Greis et al, 2005).…”

Synthesis, Antimicrobial and Molecular Modeling Studies of Some Benzophenone-based Thiazole and 4-Thiazolidinone Derivatives

“…This study measured the percentage of inhibition of microbial growth for each compound. Specifically, the in vitro experiments to elucidate the antimicrobial activity of compounds ( 2, 3, 4a-g, 5a-d & 6) were performed utilizing whole cell growth inhibition assays [22][23] . The inhibition of growth was measured against the following five bacteria: Escherichia coli (E. coli) ATCC 25922, Klebsiella pneumoniae (K. pneumoniae) MDR ATCC 700603, Acinetobacter baumannii (A. Baumannii) ATCC 19606, Pseudomonas aeruginosa (P. Aeruginosa) ATCC 27853 and Staphylococcus aureus (S. aureus) MRSA ATCC 43300, and two fungi: Candida albicans (C. albicans) ATCC 90028 and Cryptococcus neoformans (C. Neoformans) ATCC 208821 24 .…”

New Urea Derivatives as Potential Antimicrobial Agents: Synthesis, Biological Evaluation, and Molecular Docking Studies

“…Samples were prepared in DMSO and water to a final testing concentration of 32 µg/mL or 20 µM (unless otherwise indicated in the datasheet), in 384-well, non-binding surface plate (NBS) for each bacterial/fungal strain, in duplicates (n = 2); the final DMSO concentration was kept to a maximum of 1% DMSO [29][30][31][32][33]. All sample preparations for the antimicrobial studies were done using liquid handling robots.…”