Activity of a novel sulfonamide compound 2-nitro-N-(pyridin-2-ylmethyl)benzenesulfonamide against Leishmania donovani

Figures & data

Figure 1 Molecular structure of 2-nitro-N-(pyridin-2-ylmethyl)benzenesulfonamide (2NB).

Figure 2 Activity of 2-nitro-N-(pyridin-2-ylmethyl)benzenesulfonamide (2NB) against Leishmania donovani promastigotes.

Notes: Promastigotes were treated with indicated concentrations of compound 2NB and the % of cell viability was checked at 24-hour interval for 3 consecutive days. Cell viability was reduced in a dose-dependent manner. In each test, there were three replicates, and the data are mean ± SD at each time point. *P<0.01 for 30 µg/mL and 40 µg/mL and **P<0.001 for 50 µg/mL and 60 µg/mL versus untreated control at day 3.
Abbreviation: SD, standard deviation.

Table 1 Reversion of resistant and sensitive phenotype by inhibition with the compound, 2-nitro-N-(pyridin-2-ylmethyl) benzenesulfonamide (2NB), in in vitro AmB sensitivity assay

Experimental sets	LD50 value (µg/mL)	Fold changea
Wild-type resistant + AmB	0.80±0.014	–
Wild-type sensitive + AmB	0.11±0.011	–
2NB + resistant + AmB	0.35*±0.042	~2.5 (decrease)
Sirtinol + resistant + AmB	0.22*±0.038	~3.6 (decrease)
2NB + sensitive + AmB	0.10±0.009	–

Notes: LD₅₀, lethal dose 50 means the concentration of drug at which 50% cells will be dead or 50% cells will be viable. Sirtinol (at a concentration of 15 µM), inhibitor of Sir2, was used as a positive control. After treatment with 2NB, the resistant property of the AmB-resistant parasites was reversed as the LD₅₀ value of AmB of the resistant parasites was decreased ~2.5-fold. Asterisk (*) denotes that the data are significantly different from wild-type resistant and sensitive strains, P<0.05.

a Fold increase or decrease from the LD₅₀ value of the wild-type sensitive and resistant strains. LD₅₀ values are represented as concentration of AmB ± standard deviation.

Abbreviations: AmB, amphotericin B; Sir2, silent information regulator 2.

Figure 3 Modulation of AmB-resistant property of AmB-resistant L. donovani by our compound of interest 2NB.

Notes: (A) Determination of LD₅₀ of AmB of the resistant parasites after inhibition with the compound 2NB. The concentration of 2NB (20 µg/mL) used for inhibition study was not toxic to the parasites. The LD₅₀ of AmB of the resistant parasites was decreased ~2.5-fold when pretreated with 2NB. (B) Measurement of the deacetylase activity of Sir2 of resistant parasites after inhibition with 2NB (20 µg/mL). NAD (0.6 mM) was used as the cofactor of the enzyme and was used to show the activity of the Sir2 in the parasites. Sirtinol (15 µM), known inhibitor of Sir2, was used as a positive control.
Abbreviations: AmB, amphotericin B; 2NB, 2-nitro-N-(pyridin-2-ylmethyl)benzenesulfonamide; LD₅₀, 50% lethal dose; Sir2, silent information regulator 2; min, minutes; R, AmB resistant; NAD, nicotinamide adenine dinucleotide.

Figure 4 Macrophage infection assay (ex vivo) to determine the effect of compound 2NB on intracellular amastigotes.

Notes: (A) Evaluation of macrophage toxicity of 2NB. Adhered murine peritoneal macrophages were incubated with indicated concentrations of 2NB. Cell viability was evaluated using alamar blue. (B) Determination of the numbers of macrophages infected with at least one amastigote, and then the percentage of infected macrophages was determined. (C) Peritoneal macrophages were infected with L. donovani promastigotes at a ratio of 1:10= acrophages:parasite. Infected macrophages were then treated with indicated concentrations of 2NB for 48 hours. In another set of experiments, AMT (15 mM) (as an NO synthase inhibitor) was given along with 2NB. Intracellular amastigotes number was determined by Giemsa staining. The number of intracellular amastigotes per 100 macrophages was determined microscopically. The bar diagrams show the number of parasites per 100 peritoneal macrophages. Miltefosine (2 µM) was used as a positive control. In each test, there are three replicates, and the data are mean ± SD at each time point. Asterisk (*, **) denotes (*P<0.01, **P<0.001) that the data are significantly different from the control.
Abbreviations: 2NB, 2-nitro-N-(pyridin-2-ylmethyl)benzenesulfonamide; AMT, 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine; SD, standard deviation.

Figure 5 2NB treatment induces ROS and NO generation as well as modulates different cytokines response.

Notes: (A) 2NB induced generation of ROS from infected and uninfected peritoneal macrophages. ROS generation was measured by H₂DCFDA probe. (B–E) Effect of 2NB on NO generation and cytokine response. Peritoneal macrophages from BALB/c mice were infected with L. donovani promastigotes (macrophage/parasite, 1:10). Noningested promastigotes were removed by washing, and macrophages were cultured for another 20 hours. Infected macrophages were then treated with 2NB (120 µg/mL) for 24 hours. NO production (B) was determined by measuring the accumulation of nitrite in the culture medium by Griess reagent. iNOS expressions at mRNA (C) levels were evaluated by semiquantitative RT-PCR. GAPDH PCR product was used as loading control. C (i) shows the gel image and C (ii) shows the densitometric analysis of the band intensity. Peritoneal macrophages were treated with 2NB (120 µg/mL) for 48 hours, and levels of IL-12 and TNF-α (D) and IL-10 and TGF-β (E) in culture supernatants were determined by ELISA. Error bars represent mean ± SD. The data shown are representative of three independent experiments. *P<0.01 and **P<0.001 versus corresponding infected or uninfected control. (F–H) Time- and dose-dependent increase in the level of nitric oxide, IL-12, and TNF-α after treatment with indicated concentrations of 2NB. Bars marked AG83 refer to the infected macrophages.
Abbreviations: 2NB, 2-nitro-N-(pyridin-2-ylmethyl)benzenesulfonamide; ROS, reactive oxygen species; iNOS, inducible nitric oxide; mRNA, messenger RNA; RT-PCR, reverse transcription–polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IL, interleukin; TNF-α, tumor necrosis factor α; TGF-β, transforming growth factor-β; ELISA, enzyme-linked immunosorbent assay; SD, standard deviation.