Previous Article | Next Article 
Antimicrobial Agents and Chemotherapy, August 2005, p. 3428-3434, Vol. 49, No. 8
0066-4804/05/$08.00+0 doi:10.1128/AAC.49.8.3428-3434.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Development and Validation of a Whole-Cell Inhibition Assay for Bacterial Methionine Aminopeptidase by Surface-Enhanced Laser Desorption Ionization-Time of Flight Mass Spectrometry
Kenneth D. Greis,
*
Songtao Zhou,
Richard Siehnel,
Chuck Klanke,
Alan Curnow,
Jeremy Howard, and
Gerlinde Layh-Schmitt,
Procter & Gamble Pharmaceuticals, Mason, Ohio 45040
Received 9 February 2005/ Returned for modification 22 March 2005/ Accepted 30 May 2005
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.
* Corresponding author. Mailing address: Procter & Gamble Pharmaceuticals, Health Care Research Center, 8700 Mason Montgomery Road, Mason, OH 45040. Phone: (513) 622-2670. Fax: (513) 622-1196. E-mail: greis.kd{at}pg.com.
These authors contributed equally to the work.
Present address: Cincinnati Children's Hospital Medical Center, 3333 Burnet Ave., Cincinnati, OH 45229.
Antimicrobial Agents and Chemotherapy, August 2005, p. 3428-3434, Vol. 49, No. 8
0066-4804/05/$08.00+0 doi:10.1128/AAC.49.8.3428-3434.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
This article has been cited by other articles:
-
Granieri, L., Miller, O. J., Griffiths, A. D., Merten, C. A.
(2009). A competition-based assay for the screening of species-specific antibiotics. J Antimicrob Chemother
64: 62-68
[Abstract] [Full Text]
Copyright © 2009 by the American Society for Microbiology. For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»