Phenylthiazole Antibacterial Agents Targeting Cell Wall Synthesis Exhibit Potent Activity in Vitro and in Vivo against Vancomycin-Resistant Enterococci

Abstract

The emergence of antibiotic-resistant bacterial species, such as vancomycin-resistant enterococci (VRE), necessitates the development of new antimicrobials. Here, we investigate the spectrum of antibacterial activity of three phenylthiazole-substituted aminoguanidines. These compounds possess potent activity against VRE, inhibiting growth of clinical isolates at concentrations as low as 0.5 μg/mL. The compounds exerted a rapid bactericidal effect, targeting cell wall synthesis. Transposon mutagenesis suggested three possible targets: YubA, YubB (undecaprenyl diphosphate phosphatase (UPPP)), and YubD. Both UPPP as well as undecaprenyl diphosphate synthase were inhibited by compound 1. YubA and YubD are annotated as transporters and may also be targets because 1 collapsed the proton motive force in membrane vesicles. Using Caenorhabditis elegans, we demonstrate that two compounds (1, 3, at 20 μg/mL) retain potent activity in vivo, significantly reducing the burden of VRE in infected worms. Taken altogether, the results indicate that compounds 1 and 3 warrant further investigation as novel antibacterial agents against drug-resistant enterococci.

Figure 1

Chemical structures of phenylthiazole compounds investigated.

Figure 2

Time-kill analysis of phenylthiazole compounds 1, 2, 3, and linezolid (all tested at 4 × MIC) over a 24 hour incubation period at 37 °C against A) vancomycin-resistant Enterococcus faecium ATCC 700221 and B) vancomycin-resistant Enterococcus faecalis HM-201. DMSO served as a negative control. The error bars represent standard deviation values obtained from triplicate samples used for each compound/antibiotic studied.

Figure 3

Percent viable mammalian cells (measured as average absorbance ratio (test agent relative to untreated cells)) for cytotoxicity analysis of phenylthiazole compounds 1, 2, and 3 at 5, 10, 20, and 40 μg/mL against human colorectal (HRT-18) cells using the MTS 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay. Untreated cells served as a negative control to determine a baseline measurement for the cytotoxic impact of each compound. Test agents were incubated with cells for either A) two hours or B) 24 hours. The absorbance values represent an average of a minimum of three samples analyzed for each compound. Error bars represent standard deviation values for the absorbance values. A one-way ANOVA (with post-hoc Dunnett’s multiple comparisons test), P ≤ 0.05, demonstrated statistical difference between the values obtained for compounds 1, 2, and 3 relative to untreated cells at 40 μg/mL (denoted by an asterisk, *).

Figure 4

Multi-step resistance selection of compounds 1, 2, 3, and linezolid against A) vancomycin-resistant Enterococcus faecium HM-968 and B) vancomycin-resistant Enterococcus faecalis ATCC 51299. Bacteria were serially passaged over a fourteen-day period and the broth microdilution assay was used to determine the minimum inhibitory concentration of each compound against VRE after each successive passage. A four-fold shift in the MIC is defined as resistance to the test agent.

Figure 5

Effects of compound 1 on cell wall biosynthesis in E. coli ΔtolC. (A, B, F, G) Untreated cells. (C, D, H, I) Cells treated with compound 1 for either 30 minutes or two hours at 5 × MIC (25 μg/mL). (E, J) Cells treated with ᴅ-cycloserine at 5 × MIC (125 μg/mL) for 30 minutes. Cells (F–J) were treated in the presence of 0.5 M sucrose to facilitate visualization of cell shape defects. Cells were stained with FM 4–64 (red), DAPI (blue), and SYTOX Green (green). Scale bar is 1 μm.

Figure 6

Comparison of cell shape defects in Bacillus subtilis treated with compound 1 or vancomycin. All cells were grown in LB in the presence of MSM at 37°C and are shown at two hours. Both compound 1 and vancomycin lead to slight bending of the cells and bulges, as indicated by the arrows.

Figure 7

Schematic illustration of some key steps in cell wall biosynthesis in many Gram-positive bacteria, and sites of action of known drugs and inhibitors discussed in the text.

Figure 8

Dose response curves for enzyme inhibition by 1, and its effects on the PMF. A, UPPP (YubB) inhibition; bacitracin control. B, UPPS inhibition, bisamidine NSC-50460 control. C, PMF collapse in E. coli IMVs, ATP driven PMF, CCCP control. D, As C but succinate/O₂-driven PMF generation.

Figure 9

Structures of proposed targets of compound 1. The UPPS structure is the X-ray structure of EcUPPS, PDB ID code 1X06. The membrane protein structures are models for UPPP (YubB), YubA and YubD. Compound 1 inhibits UPPS and UPPP at low μM levels and collapses the PMF (in EcIMVs), suggested to affect the activity of YubA or YubD, putative transporters identified as targets in the transposon mutagenesis experiment (together with UPPP/YubB).

Figure 10

In vivo examination of toxicity and antibacterial activity of phenylthiazole compounds 1 and 3 (tested at 20 μg/mL) in C. elegans AU37 infected with vancomycin-resistant Enterococcus faecalis HM-201. Linezolid served as a positive control. A) Worms (in L4 stage of growth) were treated with 20 μg/mL of compounds 1, 3, or linezolid and percent viable worms was determined after 24 hours of exposure. B) Worms (in L4 stage of growth) infected with vancomycin-resistant E. faecalis HM-201 for two hours before transferring 20–25 worms to wells of a 96-well plate. Test agents were added and incubated with worms for 18 hours. Worms were sacrificed and the number of viable colony-forming units of E. faecalis HM-201 in infected worms was determined for each treatment regimen. The figure shows the percent reduction of E. faecalis HM-201 (relative to untreated worms). Asterisks (*) denote statistical significance (P < 0.05) in bacterial reduction relative to the positive control (linezolid) using a two-tailed Student’s t-test (with post-hoc Holm-Sidak test for multiple comparisons).