Biological Activity and Stability of Aeruginosamides from Cyanobacteria

Abstract

Aeruginosamides (AEGs) are classified as cyanobactins, ribosomally synthesized peptides with post-translational modifications. They have been identified in cyanobacteria of genera Microcystis, Oscillatoria, and Limnoraphis. In this work, the new data on the in vitro activities of three AEG variants, AEG A, AEG625 and AEG657, and their interactions with metabolic enzymes are reported. Two aeruginosamides, AEG625 and AEG657, decreased the viability of human breast cancer cell line T47D, but neither of the peptides was active against human liver cancer cell line Huh7. AEGs also did not change the expression of MIR92b-3p, but for AEG625, the induction of oxidative stress was observed. In the presence of a liver S9 fraction containing microsomal and cytosolic enzymes, AEG625 and AEG657 showed high stability. In the same assays, quick removal of AEG A was recorded. The peptides had mild activity against three cytochrome P450 enzymes, CYP2C9, CYP2D6 and CYP3A4, but only at the highest concentration used in the study (60 µM). The properties of AEGs, i.e., cytotoxic activity and in vitro interactions with important metabolic enzymes, form a good basis for further studies on their pharmacological potential.

Keywords: aeruginosamides; cyanopeptides; cytochrome P450 enzymes; cytotoxicity; metabolic stability; miRNA.

Figure 1

Structures of aeruginosamides: (A) AEG A, (B) AEG625, and (C) AEG657.

Figure 2

Luciferase assay response curves for Huh7 reporter cells exposed for 24 to increasing concentrations of the three AEGs. Relative luciferase activity was calculated using the ratio of firefly (F) and Renilla (R) luciferase activity (F/R) and was expressed in relative luminescence units (RLU). All assays were performed in triplicate in a 96-well format and were normalized to luciferase expression of cells treated only with the transfection reagent. Data are expressed as means with a standard deviation.

Figure 3

(A) Cell viability and (B) hydrogen peroxide levels in Huh7-donor cells exposed to increasing concentrations of AEGs. Luminescence values were normalized to untreated cells. All assays were conducted for 24 h in triplicate in a 96-well format. Data are expressed as means with a standard deviation.

Figure 4

Concentrations of 7-EC, AEG A, AEG625, and AEG657 after 60 min exposure to S9 fraction containing microsomal and cytosolic enzymes. Results are expressed in relation to initial concentration of the compounds (%C T₀).

Figure 5

Effects of AEG A, AEG625, and AEG657, applied at different concentrations, on activity of CYP2C9 human P450 enzyme (after 60 min incubation). Data are expressed as means with a standard deviation.