Bioactivity and Structural Properties of Novel Synthetic Analogues of the Protozoan Toxin Climacostol

Abstract

Climacostol (5-[(2Z)-non-2-en-1-yl]benzene-1,3-diol) is a resorcinol produced by the protozoan Climacostomum virens for defence against predators. It exerts a potent antimicrobial activity against bacterial and fungal pathogens, inhibits the growth of several human and rodent tumour cells, and is now available by chemical synthesis. In this study, we chemically synthesized two novel analogues of climacostol, namely, 2-methyl-5 [(2Z)-non-2-en-1-yl]benzene-1,3-diol (AN1) and 5-[(2Z)-non-2-en-1-yl]benzene-1,2,3-triol (AN2), with the aim to increase the activity of the native toxin, evaluating their effects on prokaryotic and free-living protists and on mammalian tumour cells. The results demonstrated that the analogue bearing a methyl group (AN1) in the aromatic ring exhibited appreciably higher toxicity against pathogen microbes and protists than climacostol. On the other hand, the analogue bearing an additional hydroxyl group (AN2) in the aromatic ring revealed its ability to induce programmed cell death in protistan cells. Overall, the data collected demonstrate that the introduction of a methyl or a hydroxyl moiety to the aromatic ring of climacostol can effectively modulate its potency and its mechanism of action.

Natural Function and Structural Modification of Climacostol, a Ciliate Secondary Metabolite

“…In particular, experiments performed on human tumor squamous carcinoma A431 cells, and human promyelocytic leukemia HL60 cells demonstrated that climacostol exerts its action by inhibiting cell growth and triggering a mitochondrion-dependent apoptotic program. Subsequent extensive in vitro screenings and in vivo experiments confirmed the previous observations [4,21,[29][30][31].…”

“…The pro-apoptotic features of AN1 and AN2 on immortalized cell lines of both tumor (B16-F10, GL261, SK-N-BE, and CT26) and nontumor (C 2 C 12 ) origin were analyzed and compared with that of climacostol. The results indicated that cell viability was negatively affected by the two analogues with a comparable strength (Table 1), and both AN1 and AN2 displayed similar or even lower potencies when compared to climacostol [31]. Furthermore, as in the case of various tumor cells exposed to climacostol [4,29,30], immunostaining techniques revealed that B16-F10 melanoma cells expressed high levels of active caspase 3 after AN1 and AN2 treatment ( Figure 6), thus demonstrating the activation of an apoptotic pathway induced by both analogues.…”

“…Although climacostol cytotoxicity appears more selective against tumors than certain immortalized nontumor cells [21,29], recent data suggest that climacostol effects are not necessarily correlated to the cancerous origin of cells [31]. However, the possibility that climacostol preferentially affects cancerous vs. normal (nontransformed non-immortalized) cells requires further investigation.…”

“…Two analogues, methyl-5 [(2Z)-non-2-en-1-yl]benzene-1,3-diol (AN1) and 5-[(2Z)-non-2-en-1-yl]benzene-1,2,3-triol (AN2), respectively carrying an additional methyl group and a hydroxyl group in the aromatic ring, were analyzed for their biological activity [31] (Figure 5). The choice to introduce the aforementioned moieties into the aromatic ring of climacostol was supported by the observation that similar modification performed on some polyphenols and phenolic lipids resulted in a significant improvement of their cytotoxic and antimicrobial activity [37,38].…”

“…An exception was represented by the ciliate Euplotes aediculatus which displayed the activation of programmed cell death triggered by AN2. In fact, Terminal deoxyribonucleotidyl transferse (TdT)-mediated biotin-16-dUTP Nick-End Labelling (TUNEL) fluorescence assay and light microscopy observations on Euplotes specimens exposed to AN2 revealed the typical sequence of events associated with canonical apoptosis, such as the progressive fragmentation of the macronucleus during the early stages in the absence of major changes in cell morphology, and the lack of severe necrotic damage, such as cell swelling and rupture [31] (Figure 7). To summarize, whereas similar effects were substantially observed for climacostol and its analogues on mammalian cells, AN1 was the most active compound against bacterial and fungal pathogens, and protists.…”