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.
Table 1.
The 1H, 13C, and 15N NMR data are presented (600.23 MHz for 1H, 150.93 MHz for 13C, 60.82 ppm for 15N, CD3CN, 303.2 K). 15N NMR of 15N labelled sample (70.93 MHz, CD3CN, 293.2 K) δ 222, 302. Asterisk (*) denotes HSQC readouts.
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.
Table 2.
IC50 values for nocuolin A anti-proliferative activity against nine human cancer cell lines at 72 h.
Metabolic activity was assessed using colorimetric PrestoBlue assay and the cell membrane permeability was measured by fluorescence probes Hoechst 33342 and Ethidium homodimer (Methods). The most sensitive and least sensitive cell lines were the glioma cancer cell lines U251 and U87, respectively. IC50 values equal to or below 1 μM are highlighted in bold. The p53 status is indicated as WT (wild type) or M (mutated); number (codon position) is followed with the sequence of WT/mutated codon, ins3 (insertion of 3 bases). The p53 status was adopted from IACR database (http://p53.iarc.fr/CellLines.aspx) and p53 Web Site (http://p53.free.fr/index.html). Values in the last column refer to the cell doubling time (h).
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.