Arylphosphonium salts interact with DNA to modulate cytotoxicity

Abstract

Arylphosphonium salts (APS) are compounds that have both lipophilic and cationic character, allowing them facile transport through plasma membranes or cell walls to accumulate in the cytoplasm or mitochondria of cells. APS molecules preferentially accumulate in tumor cells and are therefore under investigation as tumor imaging agents and mitochondrial targeting molecules. We have generated a systematic set of APS to study their ability to associate with DNA. The chemical structure of the APS determines the extent of its interaction with DNA and therefore its ability to aggregate the DNA. Also, APS compounds blocked DNA amplification in vitro at concentrations below the aggregation threshold, corroborating the structure/interaction relationship. Furthermore, the extent of APS:DNA interaction strongly correlates with bacterial toxicity, implying that APS molecules may deter cellular metabolic DNA pathways. Finally, DNA repair deficient and DNA bypass polymerase deficient bacterial strains were screened for sensitivity to APS. Interestingly, no single pathway for the repair or tolerance of these compounds was solely responsible for APS mediated toxicity. Taken together, these findings suggest that APS compounds may be capable of targeting and regulating unchecked cell growth and therefore show potential applications as a chemotherapeutic agent.

Figure 1

Synthesis and structure of triarylphosphonium salts (APS). (A) Synthesis scheme: Reflux triphenylphosphine and the corresponding bromide (Benzyl bromide shown) in hot toluene until arylphosphonium salt precipitates. Wash with cold toluene, dry and characterize by IR, NMR and LC-MS. (B) Table of compounds, Y represents the starting bromide compound. Structures were geometrically optimized using molecular mechanics with Hyperchem software. Angstrom measurements from the phosphonium cation to the para carbon of the benzene ring are presented on the right. Abbreviations: TPP-Br, Tetraphenylphosphonium Bromide; Bz-TPP, Benzyl triphenylphosphonium Bromide; Et-TPP, 2-phenylethyl triphenylphosphonium Bromide; Pr-TPP, 3-phenylpropyl triphenylphosphonium Bromide; CTP-Cl, Cinnamyl triphenylphosphonium Chloride.

Figure 2

Evaluation of DNA aggregation capabilities of APS compounds. (A) Representative data of CTP-Cl mediated aggregation of DNA. (B) Plots of the ratio of free DNA versus APS compound concentration. Mean values with error bars indicated were calculated from triplicate experiments. Lanes representing the absence of APS were set at 1.0 ratio free DNA. A background intensity of 0.4 persists. (C) Plot showing APS concentration needed to maintain 0.8 ratio of free DNA. (D) Correlation between APS chemical structures as shown by the angstrom distance of the P⁺-Y arm and the DNA aggregating ability. Linear trend reported r² value of 0.9064.

Figure 3

DNA Amplification Block by APS compounds: A) PCR amplification of plasmid DNA in the presence of 176mM CTP-Cl (lane 1), 17.6mM CTP-Cl (lane 2), No APS compounds (lane 3), 17.6mM CTP-Cl pre-incubated with primers (lane 4), no template (lane 5), 107mM TPP-Br (lane 6), No APS (lane 7), 10.7mM TPP-Br pre-incubated with primer (lane 8), 107mM TPP-Br (lane 9). Data presented is representative of three independent trials. B) DNA amplification via qPCR in the presence of 1.1 mM CTP-Cl (circles), 1.1 mM TPP-Br (squares), No APS compound (triangles) and no template (inverted triangles). Independent trials were run in triplicate. Representative sample of three trials is shown. C) Analysis of qPCR amplification endpoints for equimolar doses presented in 3B. The mean values of three independent trials with standard deviations indicated is shown.

Figure 4

Cytotoxicity study. (A) Cytotoxicity at 12 mM APS. All compounds were normalized to DMSO. Methylmethanesulfonate (12mM), a known DNA alkylating agent, served as a toxicity baseline. Discrete colonies were counted from three independent trials. Results represent mean values of all trials. (B) Correlation between DNA interaction, shown in Figure 2C, and APS cytotoxicity. Logarithmic r² value equals 0.8416.

Figure 5

Bacterial Cytotoxicity of DNA Repair and Bypass Polymerase Deficient Strains. Gray bars report bacterial growth after exposure to 0.5 mM CTP-Cl while black bars report growth after exposure to 2.5 mM TPP-Br. Results are mean values of three independent experiments with standard error indicated. (A) AB1157 is parental strain for GM5560, AB2500 and BW528. GM5560 is deficient in recA, a critical Homologous Recombination repair pathway protein. AB2500 contains three mutations (uvrA, thyA and deoB) rendering these cells deficient in Nucleotide Excision Repair. BW528 has the two AP endonucleases, nth and xfo, deleted making this strain deficient in Base Excision Repair. (B) W3110 serves as parent strain for deletion of the replication fork stall stabilizing protein recQ (HL951). (C) BW25113 is parent strain for JW4128 that is deficient in mismatch repair (mutL). (D) Strains were obtained from Yale E. Coli database (Keio Collection) with single deletions as follows: BW25113, Parental strain; JW0221, dinB deficient; JW1172, umuC deficient; JW1173, umuD deficient and JW3835, polA deficient. Results are the mean values of three independent experiments with standard deviations indicated.