Squalamine as a broad-spectrum systemic antiviral agent with therapeutic potential

Abstract

Antiviral compounds that increase the resistance of host tissues represent an attractive class of therapeutic. Here, we show that squalamine, a compound previously isolated from the tissues of the dogfish shark (Squalus acanthias) and the sea lamprey (Petromyzon marinus), exhibits broad-spectrum antiviral activity against human pathogens, which were studied in vitro as well as in vivo. Both RNA- and DNA-enveloped viruses are shown to be susceptible. The proposed mechanism involves the capacity of squalamine, a cationic amphipathic sterol, to neutralize the negative electrostatic surface charge of intracellular membranes in a way that renders the cell less effective in supporting viral replication. Because squalamine can be readily synthesized and has a known safety profile in man, we believe its potential as a broad-spectrum human antiviral agent should be explored.

Fig. 1.

Squalamine strongly displaces Rac1 from model membranes because of charge density matching. (A) Squalamine chemical structure. (B) Diffraction patterns from lipid membrane vesicles, Rac1–membrane complexes, squalamine–membrane complexes, and membranes incubated with both Rac1 and squalamine (bottom to top). The diffraction pattern of the final case is nearly identical to the diffraction signature of squalamine–membrane complexes, indicating strong suppression of Rac1–membrane binding [lipid compositions 1,2-Dioleoyl-sn-Glycero-3-[Phospho-l-Serine] (sodium salt)/1,2-Dioleoyl-sn-Glycero-3-Phosphocholine (DOPC)/1,2-Dioleoyl-sn-Glycero-3-Phosphoethanolamine = 20/20/60, Rac1 to lipid ratio = 1:50, squalamine to lipid ratio = 1:15; all are molar ratios]. (C–E) Representative configurations from molecular dynamics simulations of solutions of Rac1 (C), squalamine (D), and both Rac1 and squalamine (E), respectively, in the presence of a coarse-grained membrane in which 20% of the lipids are charged. Rac1 and squalamine are present at concentrations that yield the same net charge on all molecules, each higher than needed to neutralize the membrane. Squalamine was found to exhibit nearly two times stronger electrostatic membrane binding than Rac1, and it displaced 56% of Rac1 from the membrane.

Fig. 2.

Treatment of YF in Syrian hamsters. Survival of Syrian hamsters treated before (A) or after (C) viral inoculation. Dosing regimens are indicated and described in Materials and Methods. Serum ALT was sampled at day 6 postinfection for the group receiving squalamine before (B) or after (D) viral inoculation [treatment groups (n = 10) and D5W (5% dextrose) placebo (n = 20)]. All animals that had survived 14 d remained alive until day 21 and were designated as cured. [We should note that, in the treatment experiment (C), a cohort that received 15 mg/kg 1 d before virus inoculation (a control prevention study) achieved a 40% survival rate and has been omitted from the graph for the sake of clarity.]

Fig. 3.

Treatment of EEEV in Syrian hamsters. Survival (A) and average body weight (B) of Syrian hamsters infected with EEEV and treated s.c. with either squalamine (10 mg/kg) or placebo (D5W) on days −1 to 6 after infection. (C) Viremia of Syrian hamsters on days 1–4 after infection with EEEV that were treated with either squalamine or placebo. Error bars indicate the SDs of the means. The limit of detection for the assay was 100 pfu/mL [treatment group (n = 10) and D5W placebo (n = 6)]. P = 0.003 by one-way ANOVA.

Fig. 4.

Treatment of MCMV in BALB/c mice. MCMV titers from organs harvested at 3, 7, or 14 d after viral inoculation (n = 6 animals for each data point). The limit of detection was 10 pfu/mL. Error bars indicate SDs of the means. *P < 0.05, **P < 0.01, and ***P < 0.001 by one-way ANOVA compared with D5W control.