Triphenilphosphonium Analogs of Chloramphenicol as Dual-Acting Antimicrobial and Antiproliferating Agents

Abstract

In the current work, in continuation of our recent research, we synthesized and studied new chimeric compounds, including the ribosome-targeting antibiotic chloramphenicol (CHL) and the membrane-penetrating cation triphenylphosphonium (TPP), which are linked by alkyl groups of different lengths. Using various biochemical assays, we showed that these CAM-Cn-TPP compounds bind to the bacterial ribosome, inhibit protein synthesis in vitro and in vivo in a way similar to that of the parent CHL, and significantly reduce membrane potential. Similar to CAM-C4-TPP, the mode of action of CAM-C10-TPP and CAM-C14-TPP in bacterial ribosomes differs from that of CHL. By simulating the dynamics of CAM-Cn-TPP complexes with bacterial ribosomes, we proposed a possible explanation for the specificity of the action of these analogs in the translation process. CAM-C10-TPP and CAM-C14-TPP more strongly inhibit the growth of the Gram-positive bacteria, as compared to CHL, and suppress some CHL-resistant bacterial strains. Thus, we have shown that TPP derivatives of CHL are dual-acting compounds targeting both the ribosomes and cellular membranes of bacteria. The TPP fragment of CAM-Cn-TPP compounds has an inhibitory effect on bacteria. Moreover, since the mitochondria of eukaryotic cells possess qualities similar to those of their prokaryotic ancestors, we demonstrate the possibility of targeting chemoresistant cancer cells with these compounds.

Keywords: alkyl(triphenyl)phosphonium; antibiotic activity; antiproliferative activity; bacterial ribosome; chloramphenicol; molecular dynamics simulations.

Figure 1

Scheme of the chemical synthesis of triphenylphosphonium (TPP) analogs of CHL: CAM-C10-TPP and CAM-C14-TPP. Step 1: 1M hydrochloric acid (HCl) at 100 °C for 2 h. Step 2: (1) Boc-GABA-OSu, dimethylformamide (DMF), and diisopropylethylamine (DIPEA) at 25 °C for 24 h; and (2) trifluoroacetic acid (TFA) at 25 °C for 30 min. Step 3: benzene at 85 °C for 12 h. Step 4: (1) 5, 1-hydroxysuccinimide (HOSu), N,N′-dicyclohexylcarbodiimide (DCC), and dichloromethane (CH₂Cl₂) at 0 °C for 2 h, then overnight at RT; (2) 2, DIPEA, DMF, and stirring at RT for 5 h, then overnight at 4 °C. Step 5: (1) 5, HOSu, DCC, and dichloromethane (CH₂Cl₂) at 0 °C for 2 h, then overnight at RT; (2) 6, DIPEA, DMF, and stirring at RT for 5 h.

Figure 2

Binding affinity to bacterial ribosomes and the inhibition of protein synthesis by CAM-C10-TPP and CAM-C14-TPP. (A) A competition-binding assay to test the displacement of fluorescently labeled analogs of the erythromycin, BODIPY-ERY, from E. coli 70S ribosomes in the presence of increasing concentrations of CHL (black circles), CAM-C10-TPP (red squares), CAM-C14-TPP (blue rhombus), decyl(triphenyl)phosphonium bromide (C10-TPP, green triangles), or tetradecyl(triphenyl)phosphonium bromide (C14-TPP, purple triangles), measured by fluorescence anisotropy. All reactions were repeated four times. Error bars represent the standard deviation. The resulting values for the apparent dissociation constants (K_Dapp) are shown on the plot. (B) Testing of the CAM-C10-TPP and CAM-C14-TPP activity using E. coli BW25113 ΔtolC pDualrep2 reporter strain. The induction of the red fluorescent protein expression (green halo around the inhibition zone, pseudocolor) is triggered by DNA-damage, while the induction of Katushka2S protein (red halo) occurs in response to ribosome stalling. Levofloxacin (LEV), erythromycin (ERY), chloramphenicol (CHL), N-acetyl-chloramphenicol amine (CAM-Ac), C10-TPP, and C14-TPP are used as the controls. (C) The inhibition of protein synthesis by 30 µM of CHL, CAM-C10-TPP, or CAM-C14-TPP in vitro in the cell-free bacterial (black columns) and eukaryotic (transparent columns) transcription–translation coupled system. The relative enzymatic activity of in vitro synthesized firefly luciferase is shown. The error-bars represent the standard deviations of the mean of three independent measurements. (D) Ribosome stalling by CAM-Cn-TPP on trpL mRNA in comparison with CHL, as detected by a reverse-transcription primer-extension inhibition (toeprinting) assay in a cell-free translation system. The nucleotide sequences of trpL mRNA and their corresponding amino acid sequences are shown on the left. The black arrowhead marks the translation arrest at the start codon, while the colored arrowheads point to drug-induced arrest sites within the coding sequences of the mRNAs used. Note that due to the large size of the ribosome, the reverse transcriptase used in the toeprinting assay stops 16 nucleotides downstream of the codon located in the P-site.

Figure 3

Interactions between CAM-C10-TPP (A), CAM-C14-TPP (B), and E. coli A,A/P,P–ribosome, obtained using MD simulations. Hydrogen bonds are shown by black dashes. CAM-Cn-TPP nitrophenyl fragment is immersed in the “hydrophobic cavity” formed by the Ψ2504 and U2506 bases, forming stacking interactions with them. The arrangement of hydrogen bonds for the CAM-C10-TPP complex (A) corresponds to the non-canonically linked CHL [59]. For CAM-C14-TPP (B), a stable hydrogen bond between the O1-hydroxyl group of the CAM residue and O6 of G2061 is observed. The TPP fragment of CAM-C10-TPP interacts with the “hydrophobic cavity” of the macrolide binding site, forming developed hydrophobic contacts with the A2058, A2059, and C2610 bases. The long C14-linker in CAM-C14-TPP appears in the “hydrophobic cavity” between the A2058 and A2059 bases, and the TPP fragment is located deeper in the NPET adjacent to the residue, A2015. (C) The energy of the noncovalent interactions between CAM-C10-TPP (red columns) or CAM-C14-TPP (green columns) and the neighboring 23S rRNA residues of the E. coli ribosome, which are in the canonical A,A/P,P–state. E_noncov is shown with a negative sign for improved readability.

Figure 4

Dose-dependent effect of CAM-Cn-TPP on the kinetics of the membrane potential of B. subtilis cells, as assessed by DiS-C3-(5) (10 µM) fluorescence in a PBS buffer. To reach the desired concentrations, appropriate amounts of CAM-Cn-TPP were added at different moments, which are marked by the arrows. The Gramicidin A concentration was 0.5 ng/mL.

Figure 5

Effect of CAM-TPP derivatives on the cell viability of TNBC cells. MDA-MB-231 (A–D) and BT-549 (E–H) TNBC-sensitive (S) and chemoresistant (R) cells were grown at a 60% confluence for 3 days with the indicated concentrations of CAM-TPP derivatives. Representative images (A,E) were obtained at 40× magnification. The scale bar is 10 µm. Cells were subjected to viability assays. The results represent the mean of 3 independent experiments. The data indicate the mean ± SEM. The p-values, all relative to controls, were statistically significant (p < 0.05).

Figure 6

Effect of CAM-TPP derivatives on the cell viability of CSC-like TNBC cells. MDA-MB-231 (A–D) and BT-549 (E–H) TNBC-sensitive (S) and chemoresistant (R) cells were grown under non-adherent conditions for 5 days in the presence of CAM-TPP derivatives to form spheroids. Representative images (A,E) of CAM-C10-TPP-treated cells show a decrease in spheroid size. CSC-like cells were then tested for viability as before. The results are the mean of 3 independent experiments. The data indicate the mean ± SEM. The p-values, all relative to controls, were statistically significant (p < 0.05).