Targeting the chromosome partitioning protein ParA in tuberculosis drug discovery

Abstract

Objective: To identify inhibitors of the essential chromosome partitioning protein ParA that are active against Mycobacterium tuberculosis.

Methods: Antisense expression of the parA orthologue MSMEG_6939 was induced on the Mycobacterium smegmatis background. Screening of synthetic chemical libraries was performed to identify compounds with higher anti-mycobacterial activity in the presence of parA antisense. Differentially active compounds were validated for specific inhibition of purified ParA protein from M. tuberculosis (Rv3918c). ParA inhibitors were then characterized for their activity towards M. tuberculosis in vitro.

Results: Under a number of culture conditions, parA antisense expression in M. smegmatis resulted in reduced growth. This effect on growth provided a basis for the detection of compounds that increased susceptibility to expression of parA antisense. Two compounds identified from library screening, phenoxybenzamine and octoclothepin, also inhibited the in vitro ATPase activity of ParA from M. tuberculosis. Structural in silico analyses predict that phenoxybenzamine and octoclothepin undergo interactions compatible with the active site of ParA. Octoclothepin exhibited significant bacteriostatic activity towards M. tuberculosis.

Conclusions: Our data support the use of whole-cell differential antisense screens for the discovery of inhibitors of specific anti-tubercular drug targets. Using this approach, we have identified an inhibitor of purified ParA and whole cells of M. tuberculosis.

Figure 1.

pMindGFP vector map showing the placement of the open reading frame of the target gene in the antisense orientation (pink) downstream of the tetR gene and tetO operator (green). Addition of tetracycline is used to induce expression. The GFP gene (purple) was cloned downstream of the antisense gene fragment to detect gene antisense expression. Vector map created using PlasMapper. This figure appears in colour in the online version of JAC and in black and white in the print version.

Figure 2.

(a) Growth of M. smegmatis on agar at 72 h following electroporation with (i) pMindGFP, (ii) pMind6938GFP, (iii) pMind6939GFP and (iv) pMind6938 + 6939GFP. Electroporations were performed in triplicate in two independent experiments, yielding similar results. The results of one of these independent experiments are shown. (b) Induction of tetO operator by addition of 0.02 mg/L tetracycline (TET) as measured by GFP fluorescence. RFU, relative fluorescence units. Results are mean values of triplicates ± standard error.

Figure 3.

Growth of M. smegmatis containing pMindGFP, pMind6938GFP and pMind6939GFP under (a) nutrient-rich, (c) carbon-limitation and (e) nitrogen-limitation conditions as measured by optical density at 590 nm (OD₅₉₀). Viability of M. smegmatis containing pMindGFP, pMind6938GFP and pMind6939GFP under (b) nutrient-rich, (d) carbon limitation and (f) nitrogen limitation conditions as determined by cfu counts. Each growth assay was performed in 96-well plates in a total volume of 100 µL with an initial optical density at 590 nm (OD₅₉₀) of 0.01 (equivalent to an OD₆₀₀ of approximately 0.05 in a 10 mm path length cuvette). Cultures were incubated at 37°C in 96-well plates and cfu measurements were performed on the cultures after entry into stationary phase: 72 h for carbon- and nitrogen-limited conditions and 144 h for nutrient-rich conditions. Two independent experiments were conducted in quintuplicate yielding similar results. The results of one of these independent experiments are shown. Mean values ± standard error are illustrated.

Figure 4.

(a) ATP hydrolysis in the presence of ParA, ParB and ParABS (n = 2). (b) Saturation curve showing the relationship between the concentration of ATP and the rate of reaction (n = 10). Each reaction was performed using 3 µg of ParA and the data conformed to the Michaelis–Menten equation. (c) ATPase activity of ParA in the presence of DMSO and sodium metavanadate (n = 3). While DMSO had a minimal effect, addition of 50 µM and 100 µM metavanadate reduced the ATPase activity by approximately 30% and 50%, respectively. (d) ATPase activity of ParA in the presence of ethambutol, streptomycin, phenoxybenzamine and octoclothepin (n = 2). Addition of the standard tuberculosis drugs ethambutol and streptomycin did not cause a significant reduction in the ATPase activity of ParA, while phenoxybenzamine and octoclothepin decreased the activity of ParA ATPase by approximately 50% and 20%, respectively. Methiothepin decreased the ATPase activity of ParA to a similar level as octoclothepin (data not shown). All data points are normalized against spontaneous ATP decay and results are mean values of duplicates ± standard error. (e) Structural formula and molecular weight (MW) of phenoxybenzamine [N-benzyl-N-(2-chloroethyl)-1-phenoxypropan-2-amine], octoclothepin [1-(3-chloro-5,6-dihydrobenzo[b][1]benzothiepin-5-yl)-4-methylpiperazine] and methiothepin [1-methyl-4-(3-methylsulfanyl-5,6-dihydrobenzo[b][1]benzothiepin-5-yl)piperazine].

Figure 5.

In silico studies of inhibitors on ParA of M. tuberculosis. (a) Binding site of the ParA homology model (green ribbon) with ADP (carbon, magenta; nitrogen, blue; oxygen, red); ADP from pdb2bej superimposed onto model. (b) Docking results showing superposition of (R) and (S) isomers of phenoxybenzamine on ParA of M. tuberculosis. (c) Octoclothepin (cyan carbons) docked into the ligand binding site. The in silico analysis showed that phenoxybenzamine and (±)-octoclothepin maleate bind in the same pocket as the ADP molecule. Surface representation of the ligand binding site shown as a surface. All representations are in the same orientation. All figures were generated using PyMOL. This figure appears in colour in the online version and in black and white in the print version of JAC.

Figure 6.

(a) M. smegmatis/pMindGFP and (b) M. smegmatis/pMind6969GFP cells under nitrogen-limited conditions in the presence of 0.02 mg/L tetracycline. M. smegmatis/pMindGFP cells show a couple of chromosomal foci per cell while M. smegmatis/pMind6969GFP cells contain multiple chromosomal loci along the length of the cells. M. smegmatis/pMind6969GFP cells under nitrogen limitation in the presence of 0.02 mg/L tetracycline and 20 µM (c) phenoxybenzamine and (d) octoclothepin. Both (c) and (d) show elongated cells with multiple foci along the length of the cells. The lengths of 100 cells were measured for each culture and from (a) to (d) were 5.08 ± 1.45, 6.8 ± 1.3, 11.36 ± 2.6 and 14.78 ± 3.18 µm, respectively. Images were taken with a ×100 oil-immersion lens. The scale bar is 10 µm and applies to all images. This figure appears in colour in the online version and in black and white in the print version of JAC.