Novel Very Long-Chain α-Methoxylated Δ5,9 Fatty Acids from the Sponge Asteropus niger Are Effective Inhibitors of Topoisomerases IB

Abstract

The novel fatty acids (2R,5Z,9Z)-2-methoxy-25-methyl-5,9-hexacosadienoic acid (1a) and (2R,5Z,9Z)-2-methoxy-24-methyl-5,9-hexacosadienoic acid (1b) were isolated in 80 % purity from the Caribbean sponge Asteropus niger by chloroform/methanol extraction followed by solvent partitioning and silica gel column chromatography. The compounds were characterized by utilizing a combination of gas chromatography-mass spectrometry, nuclear magnetic resonance, and circular dichroism. Acids 1a and 1b were not detected in the phospholipids (PtdCho and PtdIns) of the sponge, but rather as free FA and possibly in glycosylceramides. The mixtures of 1a and 1b displayed cytotoxicity towards THP-1 and HepG2 cells with EC50's between 41 and 35 μg/mL. Apoptosis was not the preferred mode of cell death induced by 1a-1b in the THP-1 cells. This implies other types of cytotoxicity mechanisms, such as membrane disruption and/or the inhibition (EC50 = 1.8 μg/mL) of the human topoisomerase IB enzyme (hTopIB), with a mechanism of inhibition different from the one displayed by camptothecin (CPT). In a separate experiment, the mixture of 1a and 1b also displayed cytotoxicity towards ex vivo mouse splenocytes infected with Leishmania infantum amastigotes (IC(50) = 0.17 mg/mL) and free living promastigotes (IC(50) = 0.34 mg/mL). It was also found that the FA were inhibitory of the Leishmania topoisomerase IB (LTopIB) with an EC(50) = 5.1 μg/mL. Taken together, 1a and 1b represent a new class of FA with potential as TopIB inhibitors that preferentially inhibit hTopIB over LTopIB.

Keywords: Asteropus niger; Cancer; Leishmania infantum; Leishmaniasis; Methoxylated fatty acids; Sponges; Topoisomerase IB.

Fig. 1

Structures of (2R,5Z,9Z)-2-Methoxy-25-methyl-5,9-hexacosadienoic acid (1a) and (2R,5Z,9Z)-2-Methoxy-24-methyl-5,9-hexacosadienoic acid (1b)

Fig. 2

Comparison of the inhibition of the relaxation activity of LTopIB (left) and hTopIB (right) by 1a–1b. One unit of both enzymes was assayed in a plasmid DNA relaxation assay for 30 min at 37 °C in the presence of 0.15 – 10 mg/mL of 1a–1b. Reaction products were resolved in agarose gel and subsequently visualized by ethidium bromide staining. The relative position of the negatively supercoiled DNA substrate is indicated by Sc, R is the relaxed DNA, whereas the ladder of relaxed DNA topoisomer bands is shown in between. Reactions were stopped with 1% SDS. Lane (DNA) contains 0.5 μg of pSK plasmid DNA and lane (control) is the reaction mixture plus 10% DMSO.

Fig. 3

Inhibition of LTopIB (left) and hTopIB (right) by 1a–1b in a CPT-independent mechanism. Both enzymes were assayed in the presence of DMSO (control), 100 μM CPT, 15 μg/mL and a mixture of 1a-1b at the indicated concentrations. Samples were incubated for 4 min at 25 °C, stopped with 1% SDS and digested for one extra hour at 37 °C in the presence of 1 mg/mL proteinase K. DNA was extracted with one volume of phenol-chloroform and samples were run on a 1% agarose gel containing ethidium bromide to a final concentration of 40 μg/mL in order to separate supercoiled (Sc) relaxed DNA. The nicked band (N) corresponds to the stabilized cleavage complexes. The results are representative of three independent trials.

Fig. 4

Cytotoxicity of 1a and 1b towards THP-1 and HepG2 Cells

Fig. 5

Flow cytometry analysis of PtdSer externalization in undifferentiated human monocytes THP-1 exposed to 1a–1b. THP-1 cells were incubated at 37 °C in the absence (left panel) and in the presence of growing concentrations of 1a–1b (three middle panels) for 15 h or NaF (right panel) as a positive control. Q1 to Q4 represent necrosis, late apoptosis, early apoptosis and living cells. Dead cells were excluded by propidium iodide incorporation. Surface plots are representative of three independent assays.

Fig. 6

IC₅₀ calculation after a 72 h period of exposure of 1a–1b to L. infantum-iRFP.70 amastigote-infecting mouse splenocytes and L. infantum-iRFP.70 promastigotes. Drugs were added in a twofold dilution series, being the highest concentration 1.5 mg/mL. IC₅₀ values were calculated from dose-response curves performed in triplicate and repeated twice after nonlinear fitting with the Sigma Plot program. The complete experimental methodology regarding the use, in these assays, of infrared fluorescent L. infantum strains (iRFP) was reported in [18].