A novel non-radioactive primase–pyrophosphatase activity assay and its application to the discovery of inhibitors of Mycobacterium tuberculosis primase DnaG

Abstract

Bacterial DNA primase DnaG synthesizes RNA primers required for chromosomal DNA replication. Biochemical assays measuring primase activity have been limited to monitoring formation of radioactively labelled primers because of the intrinsically low catalytic efficiency of DnaG. Furthermore, DnaG is prone to aggregation and proteolytic degradation. These factors have impeded discovery of DnaG inhibitors by high-throughput screening (HTS). In this study, we expressed and purified the previously uncharacterized primase DnaG from Mycobacterium tuberculosis (Mtb DnaG). By coupling the activity of Mtb DnaG to that of another essential enzyme, inorganic pyrophosphatase from M. tuberculosis (Mtb PPiase), we developed the first non-radioactive primase–pyrophosphatase assay. An extensive optimization of the assay enabled its efficient use in HTS (Z′ = 0.7 in the 384-well format). HTS of 2560 small molecules to search for inhibitory compounds yielded several hits, including suramin, doxorubicin and ellagic acid. We demonstrate that these three compounds inhibit Mtb DnaG. Both suramin and doxorubicin are potent (low-µM) DNA- and nucleotide triphosphate-competitive priming inhibitors that interact with more than one site on Mtb DnaG. This novel assay should be applicable to other primases and inefficient DNA/RNA polymerases, facilitating their characterization and inhibitor discovery.

ATPase activity measurement of DNA replicative helicase from Bacillus stearothermophilus by malachite green method

“…1A showed the absorption spectra of standard sodium dihydrogen phosphate, in complex with dye reagent mentioned above, from the wavelength of 550e750 nm. The spectra exhibited a characteristic absorption peak approximately at 650 nm, which deviated slightly from previously proposed 620 nm [18,19] and 630 nm [20]. Then the standard curve was determined in the 0e300 mM range of sodium dihydrogen phosphate (Fig.…”

Evaluation of DNA Primase DnaG as a Potential Target for Antibiotics

“…Unexpectedly, depletion of LigA did not significantly affect the growth of mycobacteria (34), largely precluding LigA as a target for new anti-TB drugs. Recently, Biswas et al developed a colorimetric primase-phosphatase assay as a tool for screening for efficient DnaG inhibitors (32). These researchers used this assay to screen 2,556 small molecules and identified suramin, doxorubicin, and ellagic acid as potential DnaG inhibitors.…”

“…The genomes of fast-and slow-growing mycobacteria each carry a single gene that is homologous to bacterial dnaG (Msmeg4482 and Rv2343c for fast and slow growers, respectively). The activity of M. tuberculosis DnaG was recently confirmed in an in vitro study (32). We used a pT7 Pol-based E. coli expression system to overproduce and purify DnaG from M. smegmatis and M. tuberculosis, and we subsequently used the purified DnaG of M. smegmatis for the vaccination of a rabbit to obtain polyclonal antibodies.…”

“…1). DnaG activity was suppressed in the presence of doxorubicin (100 M) and suramin (10 M), known inhibitors of DnaG (32), which inhibited the activities of M. smegmatis and M. tuberculosis DnaG in vitro by about 85% and 70%, respectively ( Fig. 2A).…”

“…The DnaG of M. tuberculosis displays 48% similarity and 32% identity with its E. coli counterpart and as much as 89% similarity and 82% identity with the DnaG of M. smegmatis (see http://www.ncbi.nlm.nih.gov/blast/Blast.cgi). In contrast, bacterial primases are distinct from their eukaryotic and archaeal counterparts, which makes them an attractive antibacterial drug target (31,32). The genomes of fast-and slow-growing mycobacteria each carry a single gene that is homologous to bacterial dnaG (Msmeg4482 and Rv2343c for fast and slow growers, respectively).…”