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.

Figure 1.

Purified Mtb DnaG. (A) A Coomassie-stained 12% sodium dodecyl sulphate–polyacrylamide gel electrophoresis of the purified Mtb DnaG showing its homogeneity. (B) The S-200 size-exclusion chromatogram of Mtb DnaG demonstrating its monomeric state.

Figure 2.

A schematic of the coupled colorimetric primase (DnaG)–pyrophosphatase (PPiase) assay.

Figure 3.

Synthesis of radiolabelled RNA primers by Mtb DnaG and its inhibition by 10 µM doxorubicin (D) and suramin (S). Phosphorimager analysis of a 12% of urea–polyacrylamide gel electrophoresis showing RNA products of the priming reaction. The priming reaction was performed with 1 mM of Mg²⁺, 2 mM of Mn²⁺ and both on a 38-mer ssDNA template 5′-CTGGTGGGCCCAAACTTGATGCTCTAATACCGACGCGT-3′ in the presence of a mixture of the four nucleotides containing α-³²P-GTP.

Figure 4.

Representative coupled primase–pyrophosphatase reactions (a 30-min time point) with wild-type (wt) and E268Q mutant of Mtb DnaG.

Figure 5.

Activity of Mtb DnaG in the presence of different divalent metals, at 2 mM. The grey bars indicate the absorbance at time 0 in the reaction.

Figure 6.

Optimization of conditions of the primase–pyrophosphatase assay. (A) A representative time course of the priming reaction, monitored by quantifying released PP_i. (B) Dependence of activity of Mtb DnaG on the concentration of MnCl₂. (C) Activity of Mtb DnaG as a function of the concentration of potassium glutamate. (D) Activity of Mtb DnaG in a variety of buffers (20 mM) at different pH in the range 6.0–9.0. The grey bars indicate the absorbance at time 0 in the reaction. (E) The steady-state rate of PP_i release during primer synthesis by Mtb DnaG as a function of DNA concentration. The reactions were carried out for 30 min at [Mtb DnaG] = 0.7 µM, [NTP] = 110 µM. (F) The steady-state rate of PP_i release by Mtb DnaG as a function of NTP concentration. The reactions were carried out for 30 min at [Mtb DnaG] = 0.7 µM, [DNA] = 1.25 µM.

Figure 7.

High-throughput coupled Mtb DnaG-Mtb PPiase assays. (A) A control assay used to calculate the Z′ score. The last two columns of wells contain 30 mM of EDTA, used as a positive inhibition control in the HTS. (B) The scatterplot of the pilot assay with 2560 diverse compounds. Red squares indicate full inhibition by 30 mM of EDTA (as in panel A). Compounds that do not exhibit inhibitory properties (serving as negative controls) are shown by the blue squares, and the rest of the compounds are shown by the green squares. The red line indicates a 3σ level of inhibition, where σ is the standard deviation of the negative controls.

Figure 8.

Dose–response assays with three hit compounds from the pilot HTS, suramin, doxorubicin and ellagic acid (the chemical structures are shown as insets). The assays were performed with 1.25 µM of DNA, 110 µM of NTP and 0.5 µM of Mtb DnaG.

Figure 9.

Inhibition of Mtb DnaG activity by suramin (S) and doxorubicin (D). The assay was performed under similar optimized reaction conditions and with the same DNA template as in the HTS and visualized and quantified as described in Figure 3.

Figure 10.

Measurements of the steady-state rate of PPi release on primer synthesis by Mtb DnaG in the presence of suramin (A and B) and doxorubicin (C and D). The assays were carried out at a fixed DNA concentration of 1.25 µM as a function of concentration of NTP (A and C) and at a fixed NTP concentration of 110 µM as a function of concentration of DNA (B and D). In all panels, the data collected without inhibitor are shown as open triangles, with 1.25 µM inhibitor as open squares, 2.5 µM inhibitor as open diamonds, 5 µM inhibitor as filled circles, 10 µM inhibitor as filled squares, 20 µM inhibitor as filled diamonds and 40 µM inhibitor as filled triangles. The insets display representative Lineweaver–Burk plots of the same data.