Antioxidant and anti-protease activities of diazepinomicin from the sponge-associated Micromonospora strain RV115

Abstract

Diazepinomicin is a dibenzodiazepine alkaloid with an unusual structure among the known microbial metabolites discovered so far. Diazepinomicin was isolated from the marine sponge-associated strain Micromonospora sp. RV115 and was identified by spectroscopic analysis and by comparison to literature data. In addition to its interesting preclinical broad-spectrum antitumor potential, we report here new antioxidant and anti-protease activities for this compound. Using the ferric reducing antioxidant power (FRAP) assay, a strong antioxidant potential of diazepinomicin was demonstrated. Moreover, diazepinomicin showed a significant antioxidant and protective capacity from genomic damage induced by the reactive oxygen species hydrogen peroxide in human kidney (HK-2) and human promyelocytic (HL-60) cell lines. Additionally, diazepinomicin inhibited the proteases rhodesain and cathepsin L at an IC₅₀ of 70-90 µM. It also showed antiparasitic activity against trypomastigote forms of Trypanosoma brucei with an IC₅₀ of 13.5 µM. These results showed unprecedented antioxidant and anti-protease activities of diazepinomicin, thus further highlighting its potential as a future drug candidate.

Keywords: Micromonospora; actinomycetes; anti-protease; antioxidant; diazepinomicin.

Figure 1

Structure of diazepinomicin (1).

Figure 2

Ferric reducing antioxidant power (FRAP) of cell-free solutions of diazepinomicin assessed by the photometric quantification (*= significant vs. control).

Figure 3

Flow cytometric analysis for the antioxidant capacity of diazepinomicin in HL-60 cells treated with 50 µM H₂O₂ and diazepinomicin for 30 min (Δ= non significant vs. control, * = significant vs. control and ≠ = significant vs. H₂O₂).

Figure 4

DNA damage (% DNA in tail) measured with the comet assay after treatment of HK-2 cells with 100 µM H₂O_2, and diazepinomicin (5&15 µM) and 50 µM tempol for 30 min. (Δ= non significant vs. control, * = significant vs. control and ≠ = significant vs. H₂O₂).

Figure 5

Progress curves of hydrolysis of the substrate Cbz-Phe-Arg-AMC (10 µM) in the absence or presence (from top to bottom) of diazepinomicin ([I] = 0.01 – 0.02 – 0.04 – 0.05 – 0.06 – 0.08 – 0.1 mg/mL).

Figure 6

Dependency of IC₅₀ values of the inhibition of rhodesain by diazepinomicin on substrate concentration. Increasing IC₅₀ values at higher substrate concentrations shows diazepinomicin to be a competitive inhibitor. The K_i value (extrapolated IC₅₀ at 0 substrate concentration) was determined to be 0.0456 mg/mL (98 µM).