SF53 Assay – A positive screen for novel antibiotics
DNA replication is crucial for bacteria cells to survive and represents a novel target for developing of new antibiotics. By targeting DNA replication it may be more difficult for bacteria to develop resistance. A compound detected as a positive hit in the SF53 assay (i.e. inhibiting DNA replication) represents a candidate drug with a novel mode of action to overcome multidrug resistance.

SF53 - the Technology 
The SF53 strain works well in high-throughput screening by providing an alternative initiation pathway for replication which does not depend on DnaA activity. The SF53 assay presents a considerable advantage that the screening is performed in vivo and only cells in which the function of DnaA has been inhibited can grow. Therefore, in addition to being a positive screen system, it allows detection of compounds that actually work inside a bacterial cell. This should lead to the identification of hits that can cross the outer membrane of Gram-negative bacteria, which is considered the major permeability barrier in these strains and the major reason for lack of efficacy of antibacterial agents that are active against Gram-positive pathogens . (Fossum S, De Pascale G, Weigel C, Messer W, Donadio S, Skarstad K. A robust screen for novel antibiotics: specific knockout of the initiator of bacterial DNA replication. FEMS Microbiol Lett 281 (2008) 210-214)

• SF53, a mutant strain of  Escherichia coli

• SF53 grows normally at 42°C, but dies at 30°C due to the activity of the protein DnaA

• Inhibition of DnaA results in growth at 30°C

The principle of finding hits in the SF53 assay. If a compound results in inhibition of DnaA at 30° C the cells will grow, i.e a hit (drug candidate)

To check that the assay is functioning we have constructed a strain, SF58 as a positive control: this is SF53 with a plasmid carrying an IPTG-inducible gene encoding a protein that will inhibit DnaA, i.e. this strain grows at 30° C  but only if induced by IPTG.

Candidate drug detected by the SF53 assay:
• Antibiotics for Gram-negative bacteria
• Antibiotics with new mode of action - overcome known resistances
• Candidate drugs expected to work in Gram-positive bacteria - target is conserved among species

The assay possesses several advantages compared to other assays:
• DNA replication is a prerequisite for cell growth and many of the replication proteins are highly conserved in bacteria.
• The screening assay utilizes a Gram-negative bacterium (E. coli) with wild type permeability; compounds discovered will most probably be able to penetrate and affect virtually all types of bacteria
• A positive hit is detected as growth
• The mechanism of action is known
• Can target the whole (multifunctional) DnaA protein, i.e. possibility of several hits
• The assay can be used for hits from other assay systems to check for DnaA inhibition