Esther E. Biswas

Discovery, Characterization and Comparison of Inhibitors of Bacillus anthracis and Staphylococcus aureus Replicative DNA Helicases

Antibacterial compounds with new mechanisms of action are needed for effective therapy against drug-resistant pathogens in the clinic and in biodefense. Screens for inhibitors of the essential replicative helicases of Bacillus anthracis and Staphylococcus aureus yielded 18 confirmed hits (IC(50)25 microM). Several (5 of 18) of the inhibitors were also shown to inhibit DNA replication in permeabilized polA-deficient B. anthracis cells. One of the most potent inhibitors also displayed antibacterial activity (MIC approximately 5 microg/ml against a range of Gram-positive species including bacilli and staphylococci) together with good selectivity for bacterial versus mammalian cells (CC(50)/MIC>16) suitable for further optimization. This compound shares the bicyclic ring of the clinically proven aminocoumarin scaffold, but is not a gyrase inhibitor. It exhibits a mixed mode of helicase inhibition including a component of competitive inhibition with the DNA substrate (K(i)=8 microM) and is rapidly bactericidal at 4 x MIC.

Mechanisms of DNA binding and regulation of Bacillus anthracis DNA primase.

DNA primases are pivotal enzymes in chromosomal DNA replication in all organisms. In this article, we report unique mechanistic characteristics of recombinant DNA primase from Bacillus anthracis. The mechanism of action of B. anthracis DNA primase (DnaG(BA)) may be described in several distinct steps as follows. Its mechanism of action is initiated when it binds to single-stranded DNA (ssDNA) in the form of a trimer. Although DnaG(BA) binds to different DNA sequences with moderate affinity (as expected of a mobile DNA binding protein), we found that DnaG(BA) bound to the origin of bacteriophage G4 (G4ori) with approximately 8-fold higher affinity. DnaG(BA) was strongly stimulated (>or=75-fold) by its cognate helicase, DnaB(BA), during RNA primer synthesis. With the G4ori ssDNA template, DnaG(BA) formed short (<or=20 nucleotides) primers in the absence of DnaB(BA). The presence of DnaB(BA) increased the rate of primer synthesis. The observed stimulation of primer synthesis by cognate DnaB(BA) is thus indicative of a positive effector role for DnaB(BA). By contrast, Escherichia coli DnaB helicase (DnaB(EC)) did not stimulate DnaG(BA) and inhibited primer synthesis to near completion. This observed effect of E. coli DnaB(EC) is indicative of a strong negative effector role for heterologous DnaB(EC). We conclude that DnaG(BA) is capable of interacting with DnaB proteins from both B. anthracis and E. coli; however, between DnaB proteins derived from these two organisms, only the homologous DNA helicase (DnaB(BA)) acted as a positive effector of primer synthesis.