Affiliation:
1. Departments of Antimicrobial Research
2. Leads Discovery
3. Chemistry, Bristol-Myers Squibb Company, Wilmington, Delaware 19880
4. J-Star Research, Inc., South Plainfield, New Jersey 07080
Abstract
ABSTRACT
Escherichia coli
under-expressing
lepB
was utilized to test cellular inhibition of signal peptidase I (SPase). For the construction of a
lepB
regulatable strain, the
E. coli lepB
gene was cloned into pBAD, with expression dependent on
l
-arabinose. The chromosomal copy of
lepB
was replaced with a kanamycin resistance gene, which was subsequently removed. SPase production by the
lepB
regulatable strain in the presence of various concentrations of
l
-arabinose was monitored by Western blot analysis. At lower arabinose concentrations growth proceeded more slowly, possibly due to a decrease of SPase levels in the cells. A penem SPase inhibitor with little antimicrobial activity against
E. coli
when tested at 100 μM was utilized to validate the cell-based system. Under-expression of
lepB
sensitized the cells to penem, with complete growth inhibition observed at 10 to 30 μM. Growth was rescued by increasing the SPase levels. The cell-based assay was used to test cellular inhibition of SPase by compounds that inhibit the enzyme in vitro. MD1, MD2, and MD3 are SPase inhibitors with antimicrobial activity against
Staphylococcus aureus
, although they do not inhibit growth of
E. coli
. MD1 presented the best spectrum of antimicrobial activity. Both MD1 and MD2 prevented growth of
E. coli
under-expressing
lepB
in the presence of polymyxin B nonapeptide, with growth rescue observed when wild-type levels of SPase were produced. MD3 and MD4, a reactive analog of MD3, inhibited growth of
E. coli
under-expressing
lepB
. However, growth rescue in the presence of these compounds following increased
lepB
expression was observed only after prolonged incubation.
Publisher
American Society for Microbiology
Subject
Infectious Diseases,Pharmacology (medical),Pharmacology
Reference26 articles.
1. Allsop A. E. M. J. Ashby G. Brooks G. Bruton S. Coulton P. D. Edwards S. A. Elsmere I. K. Hatton A. C. Kaura S. D. McLean M. J. Pearson N. D. Pearson C. R. Perry T. C. Smale and R. Southgate. 1997. Inhibition of protein export in bacteria: the signaling of a new role for β-lactams p. 61-72. In P. H. Bentley and P. J. O'Hanlon (ed.) Anti-infectives. Recent advances in chemistry and structure-activity relationships. The Royal Society of Chemistry Cambridge United Kingdom.
2. Allsop, A. E., G. Brooks, P. D. Edwards, A. C. Kaura, and R. Southgate. 1996. Inhibitors of bacterial signal peptidase: a series of 6-(substituted oxyethyl) penems. J. Antibiot.49:921-928.
3. Allsop, A. E., G. Brooks, G. Bruton, S. Coulton, P. D. Edwards, I. K. Hatton, A. C. Kaura, S. D. McLean, N. D. Pearson, T. C. Smale, and R. Southgate. 1995. Penem inhibitors of bacterial signal peptidase. Bioorg. Med. Chem. Lett.5:443-448.
4. Amsterdan D. 1996. Susceptibility testing of antimicrobials in liquid medium p. 52-111. In V. Lorian (ed.) Antibiotics in laboratory medicine 4th ed. Williams & Wilkins Baltimore Md.
5. Evidence that the catalytic activity of prokaryote leader peptidase depends upon the operation of a serine-lysine catalytic dyad
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