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Screening systems for detecting inhibitors of cell wall transglycosylation in Enterococcus. Cell wall
N Mani1, P Sancheti, Z D Jiang
1Drug discovery department, Millennium Pharmaceuticals, Cambridge, MA 02139, USA.
Abstract:
We devised two screening systems to detect cell wall transglycosylation inhibitors. One screen utilizes a mutant of Enterococcus faecalis strain A256 that is dependent on vancomycin or moenomycin for growth. In the absence of transglycosylation inhibitors the strain fails to grow, while in the presence of inhibitors, cells are rescued. A second screening organism E. faecalis strain MDD212 utilizes a translational fusion of the lacZ gene to the vanH promoter in a derivative of E. faecalis that contains a vancomycin resistance determinant. Induction of beta-galactosidase occurs when cells are exposed to inhibitors of transglycosylation. Our natural products drug source of fungal fermentations was tested with these screens. Several cultures that produced the same family of compounds, called the thielavins, were detected. Thielavin B inhibited the formation of peptidoglycan in an in vitro assay, suggesting that these screening systems can detect compounds that interfere with cell wall transglycosylation.
Insights
New screening systems effectively identify cell wall transglycosylation inhibitors. These methods detected thielavins, compounds that inhibit peptidoglycan formation, validating their utility in drug discovery.
Area of Science:
- Microbiology
- Biochemistry
- Drug Discovery
Background:
- Cell wall biosynthesis is a critical target for antibacterial agents.
- Transglycosylation is a key enzymatic step in bacterial cell wall formation.
- Developing novel inhibitors of transglycosylation is essential for combating antibiotic resistance.
Purpose of the Study:
- To develop and validate novel screening systems for identifying inhibitors of bacterial cell wall transglycosylation.
- To discover new compounds targeting transglycosylation from natural product sources.
Main Methods:
- Utilized two distinct screening systems employing Enterococcus faecalis mutants.
- One system relied on growth dependency on specific antibiotics, while the other used a reporter gene assay (lacZ fusion to vanH promoter).
- Screened fungal fermentation extracts to identify potential inhibitors.
Main Results:
- Successfully detected compounds belonging to the thielavin family from fungal cultures.
- Thielavin B demonstrated inhibition of peptidoglycan formation in vitro.
- The developed screening systems proved effective in identifying transglycosylation inhibitors.
Conclusions:
- The devised screening systems are effective for detecting inhibitors of cell wall transglycosylation.
- Thielavins represent a promising class of compounds with potential antibacterial activity by interfering with cell wall synthesis.
- These methods facilitate the discovery of novel antibacterial agents targeting essential bacterial processes.