The cyanobacterial metabolite nocuolin a is a natural oxadiazine that triggers apoptosis in human cancer cells

Abstract

Oxadiazines are heterocyclic compounds containing N-N-O or N-N-C-O system within a six membered ring. These structures have been up to now exclusively prepared via organic synthesis. Here, we report the discovery of a natural oxadiazine nocuolin A (NoA) that has a unique structure based on 1,2,3-oxadiazine. We have identified this compound in three independent cyanobacterial strains of genera Nostoc, Nodularia, and Anabaena and recognized the putative gene clusters for NoA biosynthesis in their genomes. Its structure was characterized using a combination of NMR, HRMS and FTIR methods. The compound was first isolated as a positive hit during screening for apoptotic inducers in crude cyanobacterial extracts. We demonstrated that NoA-induced cell death has attributes of caspase-dependent apoptosis. Moreover, NoA exhibits a potent anti-proliferative activity (0.7-4.5 μM) against several human cancer lines, with p53-mutated cell lines being even more sensitive. Since cancers bearing p53 mutations are resistant to several conventional anti-cancer drugs, NoA may offer a new scaffold for the development of drugs that have the potential to target tumor cells independent of their p53 status. As no analogous type of compound was previously described in the nature, NoA establishes a novel class of bioactive secondary metabolites.

Fig 1. Chemical structure of nocuolin A.

Panel (A) demonstrates nocuolin A molecule with highlighted spin systems A, B, and C as obtained by NMR—COSY and ¹H-¹³C HSQC-TOSCY. Panel (B) displays the crucial NMR correlations. See also S1–S12 Figs.High resolution mass spectrometry (HRMS) measurement of NoA provided ions corresponding to a protonated molecule [M+H]⁺, sodium adduct [M+Na]⁺, potassium adduct [M+K]⁺, and sodium adduct of a dimer [2M+Na]⁺ (S2 Fig), which allowed us to determine the exact mass of the protonated molecule (299.2233) and to calculate the neutral formula to C₁₆H₃₀N₂O₃ with high accuracy (∆ 1.2 ppm in FTMS). In parallel we have found production of this compound in extracts of two other cyanobacterial strains, Anabaena sp. PCC 7108 (axenic strain) and Nodularia sp. HBU26, via HPLC-HRMS/MS measurements (S1B and S1C Fig). The analysis revealed the occurrence of a molecular ion perfectly matching that of NoA in molecular mass, fragmentation pattern and retention behaviour in extracts of both strains (S1 Fig). However, due to the extremely low level of NoA production in PCC 7108 and HBU26 we have performed the structure elucidation on the compound purified from strain Nostoc sp. CCAP 1453/38.

Fig 2. MS fragments of nocuolin A demonstrating the connection of the A/B and C spin systems obtained by NMR.

We suppose the formation of secondary linear resonance structure with interrupted N-O bond of the oxadiazine ring and formation of additional N-N double bond during the ionization process. For the full MS spectra see S2 Fig and for detail fragmentation see S10 Fig.

Fig 3

Predicted and experimental FTIR spectra of cyclic nocuolin A form (A) and hypothetical linearized structure (B). In the case of cyclic NoA form the theoretically predicted ab initio spectrum (red line) show exceptionally good overlap with the experimental (black line). Substantially different was the predicted spectrum obtained for hypothetical linear NoA form. The main vibration bands and corresponding structures are highlighted. For details see S11 and S12 Figs.

Fig 4. Nocuolin A triggers caspase-dependent apoptosis.

(A) NoA-induced cell death morphologically resembles apoptosis (blebbing) as illustrated by the time-lapse microscopy images obtained with HeLa cells exposed to 6.7 μM NoA over 24 hours. (B, C, D) The cell death is caspase-dependent. (B) The protease activity of caspase-3/7 for the DEVD sequence was measured as a luminescence signal at various time points. The relative luminescence units were normalised per cell (nRLU). NoA (a/b/c) at 6.7 μM stands for three independent experiments. (C) The enhanced activity of caspase-3/7 (nRLU) was reduced when the cells treated with NoA and positive controls, were pre-treated with caspase inhibitor Z-VAD-FMK (10 μM). (D) NoA treatment (6.7 μM) resulted in weak enhancement of activated caspase-3 fragments (19/17 kDa) and a consistent increase in PARP-1 cleavage. These apoptosis markers were completely absent when the caspase inhibitor Q-VD-OPh (10 μM) was added prior to NoA (lines 5,7,9,11). As a positive control, taxol (B) and staurosporine (STS) (C, D) were used both at concentration 1 μM and for indicated time.

Fig 5. Structure of the noc gene cluster in four cyanobacterial strains.

Gene arrangement, functional annotation of nocA–T genes, and domain structure of the PKS/NRPS genes. A–adenylation domain; AT–acyltransferase; C–condensation domain; DH–dehydratase domain; FAAL–fatty acyl-AMP ligase; KR–ketoreductase; KS–ketosynthetase; NRPS–non-ribosomal peptide synthetase; PKS–polyketide synthetase; T–thiolation domain (acyl or peptidyl carrier protein); TE–thioesterase. See also S1 Table.