In Vitro and In Vivo Activity of (Trifluoromethyl)pyridines as Anti- Chlamydia trachomatis Agents

Abstract

Chlamydia trachomatis is the leading pathogen in sexually transmitted bacterial infections across the globe. The development of a selective treatment against this pathogen could be an attractive therapeutic option that will reduce the overuse of broad-spectrum antibiotics. Previously, we reported some sulfonylpyridine-based compounds that showed selectivity against C. trachomatis. Here, we describe a set of related compounds that display enhanced anti-chlamydial potency when compared to our early leads. We found that the active molecules are bactericidal and have no impact on Staphylococcus aureus or Escherichia coli strains. Importantly, the molecules were not toxic to mammalian cells. Furthermore, a combination of molecule 20 (the most active molecule) and azithromycin at subinhibitory concentrations acted synergistically to inhibit chlamydial growth. Molecule 20 also eradicated Chlamydia in a 3D infection model and accelerated the recovery of Chlamydia-infected mice. This work presents compounds that could be further developed to be used alone or in combination with existing treatment regimens against chlamydial infections.

Keywords: 3D culture; Chlamydia trachomatis; sexually transmitted diseases.

Figure 1.

(A) Chemical structure of lead compounds 1–2, and the most active compound 3 in the second generation; (B) approaches to improving the activity of this scaffold of compounds.

Figure 2.

Field alignment of representative derivatives of the new generation with the three parent molecules; colored code and 2D structure are provided for each compound.

Figure 3.

Quantification of infectious progeny yield in the presence of selected derivatives (numbered), as compared to compound 1 and untreated (UTD) control at 24 h (A) and 48 h (B–D) post-infection. Timeframe of treatment is indicated in parentheses. The results are reported on a log₁₀ scale. Symbols indicate individual replicates. The dashed line indicates the average of the 24 h untreated sample, and the dotted line indicates the average of the 48 h untreated sample (same untreated samples for all drug treatments). Data represent three biological replicates. All values were log₁₀ transformed to achieve equal distribution prior to statistical analysis. For each graph, transformed values were analyzed by ordinary two-way analysis of variance (ANOVA) with Dunnet’s post hoc multiple comparisons test. Significance values for each sample compared to the untreated control are shown on the graph. (A) # = p < 0.0001, n.s. = not significant. (B–D) ** = p < 0.01, *** = p < 0.001, and # = p < 0.0001.

Figure 4.

Indirect IFA of 1, 17, and 20 at 50 and 25 μg/mL at 48 hpi. In the upper panels (48 h), cells were treated at 6 hpi, and drug-containing media was maintained throughout the experiment. For the tests for inhibiting developmental cycle progression, the cells were treated with the drugs at 24 hpi (+@24 h). The media was maintained throughout the remainder of the experiment. In the reactivation panels, the cells were treated from 6 to 24hpi, at which point the drug-containing medium was removed and replaced with a drug-free medium. Samples were stained for Ctr L2 using antibodies against the major outer membrane protein (MOMP; green), for mitochondria using MitoTracker (red) and for DNA using DAPI (blue). Scale bar = 10 μm.

Figure 5.

Dose–response curve for the effect of 17 and 20 in comparison with 1 in twofold dilutions starting from the common effective dose of the three derivatives. Data represent two biological replicates [significant difference (P < 0.05, ANOVA)].

Figure 6.

Effect of AZM and 20 combinations on Chlamydia growth showing the degree of growth in color codes; red (no growth); very faint green (<50% growth); light green (50% growth); and dark green (>50% growth) in comparison with an untreated sample; (A) representation of checkerboard assay; columns indicate 20 at different concentrations, rows show AZM at several dilutions, and the box indicates the combinations. For example, the upper left most well has a concentration of 20 at 200 μg/mL and AZM at 4 μg/mL. Data represent three biological replicates. (B) Titration assay diagram of AZM and 20 combinations. Stock solutions of AZM and 20 were mixed, in two directions. Then, 1 μL of each well was transferred into Chlamydia-infected HEp-2 cells (in triplicate) 6 h after infection to obtain the concentrations, as shown in the diagram (μg/mL). For example, well A1 has a concentration of 20 at 25 μg/mL and AZM at 0.5 μg/mL, whereas well B2 has a concentration of 20 at 6.25 μg/mL and AZM at 0.25 μg/mL. (C) Infection output in the case of untreated sample and well C4; samples were stained for MOMP (Ctr L2; green) and DNA (DAPI; blue). (D) Representation of quantified Ctr growth by IFU assay after second round of infection compared to untreated (UTD) and DMSO treatment as controls. Data represent three biological replicates.

Figure 7.

Immunofluorescence images of the fixed tissues; compound 20 was used in a concentration of 50 μg/mL in comparison with azithromycin (2 μg/mL), untreated (INF), and uninfected samples. The infection was detected by human sera staining; in green, chlamydial inclusions and in blue, HaCaT nuclei. The white bottom lines show the bottom of the 3D culture. Data represent three biological replicates.

Figure 8.

Groups of mice (n = 4–5) were pre-treated with Depo-Provera 5 days before intravaginal infection with 5 × 10⁴ IFU of C. muridarum. Mice were injected intraperitoneally with 100 mg/kg of 1, 17, or 20, in DMSO, or DMSO alone each day from days 0 to 5 after chlamydial inoculation. Chlamydial shedding at indicated time points was measured. Significant difference (p ≤ 0.001, ANOVA) in the AUC of chlamydial shedding is indicated as * between the indicated group and DMSO-alone treated animals and ¥ between 20 and 1.

Scheme 1.

Synthesis of Derivatives with Different Heterocyclics at the Right Part

Scheme 2.

Synthesis of Derivatives with the Longer Alkyl Chain