Increased in vitro Anti-HIV Activity of Caffeinium-Functionalized Polyoxometalates

Abstract

Polyoxometalates (POMs), molecular metal oxide anions, are inorganic clusters with promising antiviral activity. Herein we report increased anti-HIV-1 activity of a POM when electrostatically combined with organic counter-cations. To this end, Keggin-type cerium tungstate POMs have been combined with organic methyl-caffeinium (Caf) cations, and their cytotoxicity, antiviral activity and mode of action have been studied. The novel compound, Caf₄ K[β₂ -CeSiW₁₁ O₃₉ ]×H₂ O, exhibits sub-nanomolar antiviral activity and inhibits HIV-1 infectivity by acting on an early step of the viral infection cycle. This work demonstrates that combination of POM anions and organic bioactive cations can be a powerful new strategy to increase antiviral activity of these inorganic compounds.

Keywords: Antiviral Activity; Cations; HIV; Polyoxometalates; Self-Assembly.

Figure 1

Effect of Caf‐POM and Ce‐POM on a) HIV‐1 gp120/41 and b) VSV‐G pseudotype infection. Virions were exposed to serial dilutions of the compounds and used to inoculate TZM‐bl cells. 2 days later, infection rates were determined by quantifying ß‐galactosidase activities in cellular lysates. Shown are mean values derived from three independent experiments each performed in triplicates (± standard deviation).

Figure 2

Effect of Caf‐ and Ce‐POMs on the metabolic activity of TZM‐bl cells. 10.000 TZM‐bl reporter cells were incubated for 48 hours with the indicated concentrations of POMs before cell viability was determined by measuring intracellular ATP levels. Values shown are mean values derived from one experiment performed in triplicates (± standard deviation).

Figure 3

Caf‐POM inhibits an early step in the HIV‐1 life cycle. Caf‐POM was either pre‐incubated with TZM‐bl reporter cells (blue lines) for one day prior to infection, or mixed with HIV‐1 pseudoparticle (black lines) and then added onto TZM‐bl cells. Infection rates were determined two day later by quantifying ß‐galactosidase activities in cellular lysates. Shown are mean values derived from three independent experiments each performed in triplicates (± standard deviation).

Figure 4

The antiviral activity of Caf‐ and Ce‐POMs increases over time. HIV‐1 particles were exposed for indicated times to serial dilutions of Caf‐POM (a) and Ce‐POM (b). Thereafter, mixtures were used to inoculated on TZM‐bl cells and infection rates were determined two days later. Shown are mean values±standard deviation of two independent experiments performed in triplicates. (c) IC₅₀ values of experiments shown in a and b were calculated using Graph Pad Prism. Statistical differences between IC₅₀ values was assessed with the unpaired t test. * p<0.05; ** p<0.01; n=2±SEM.

Figure 5

POMs disrupt membranes of virus‐like liposomes. Virus‐like liposome (VLL) with a membrane composition of DOPC/SM/Chol (45/25/30 mol%) were synthesized with 50 mM carboxyfluorescein cargo and purified by SEC. 2.25×10⁹ particles were added to 96 well plates and baseline fluorescence measured for 5 min while incubating at 37 deg C. (a) Indicated concentrations of POMs were then added (first dashed line) and plates incubated for 1 h. Finally, Triton X‐100 was added to all wells at a final concentration of 1 % (second dashed line) to induce total lysis; values were baseline‐subtracted and normalized to the signal obtained for total lysis. (b) DMSO equivalent to the amounts used in POM preparations did not induce leakage. (c) POM in PBS at the highest dose tested (100 μΜ) did not exhibit autofluorescence or an increase in fluorescence after 1 h incubation. Data shown are means±SEM form two experiments performed in triplicates.