New tool in antibiotics discovery
has led to new way of developing assays for antibacterial activity
and accelerate the search for novel antibiotic classes.
This welcome development comes at a time when drug companies have been reducing their investment in antibiotic development, even as bacteria resistant to the current generation of drugs are on the increase.
To date, all the antibiotics on the market have been identified in one of two ways; either they have been screened against bacteria in whole cell assays or against an assay designed to find inhibitors of a specific biochemical reaction or another predefined target.
Whole bacterial cell assays have fallen out of favour because it is hard to distinguish a specific antibacterial effect from a general toxic reaction. This means that target-based assays have become the workhorses of antibiotic discovery, but they are limited by one fundamental flaw - the target must be known and validated in advance and a suitable assay must be designed to fit.
Bayer and Genedata have focused on an alternative to the target-based approach known as a promoter-inducible reporter assay. These assays systems involve finding a bacterial 'promoter' gene that is central to a metabolic pathway that, when interrupted, inhibits bacterial growth.
The promoter gene should also be one whose expression is increased by exposure to an antibiotic. This means that compound libraries can be screened against the gene and, if expression increases, they are registered as potential antibacterial compounds.
The problem with these promoter-inducible reporter assays is that only a limited number of suitable genes have been identified. Faced with this obstacle, Bayer and Genedata set out to devise a way of tracking down additional antibiotic-responsive genes.
Researchers at the companies exposed the bacterium Bacillus subtilis to a range of antibiotics with different mechanisms of action, and selected those samples which showed an inhibitor in growth. The samples were then screened on microarrays carrying virtually all B subtilis's 4100 genes to see which of them were expressed after antibiotic exposure.
To narrow down the search, they focused on a particular metabolic pathway - involved in fatty acid synthesis (FAS) - that is considered a good prospect for new antibiotics because it is absolutely essential to the bacteria's survival.
Using computational methods developed by Genedata, the researchers pinpointed a number of genes in the FAS pathway that responded to antibiotic exposure. In turn, this allowed them to develop a genetically-modified B subtilis strain with the promoter gene linked to a marker for use as an assay in high-throughput screening. This assay not only correctly identified to drugs known to affect the FAS pathway, but also uncovered a number of small-molecule leads when tested against a compound library.
This strategy effectively removes a bottleneck that had been impeding the rational design of new antibiotics at a time when some important pathogens such as enterococci are becoming resistant even to last-line antibiotics.
