A new G-quadruplex-specific photosensitizer inducing genome instability in cancer cells by triggering oxidative DNA damage and impeding replication fork progression

Abstract Photodynamic therapy (PDT) ideally relies on the administration, selective accumulation and photoactivation of a photosensitizer (PS) into diseased tissues. In this context, we report a new heavy-atom-free fluorescent G-quadruplex (G4) DNA-binding PS, named DBI. We reveal by fluorescence microscopy that DBI preferentially localizes in intraluminal vesicles (ILVs), precursors of exosomes, which are key components of cancer cell proliferation. Moreover, purified exosomal DNA was recognized by a G4-specific antibody, thus highlighting the presence of such G4-forming sequences in the vesicles. Despite the absence of fluorescence signal from DBI in nuclei, light-irradiated DBI-treated cells generated reactive oxygen species (ROS), triggering a 3-fold increase of nuclear G4 foci, slowing fork progression and elevated levels of both DNA base damage, 8-oxoguanine, and double-stranded DNA breaks. Consequently, DBI was found to exert significant phototoxic effects (at nanomolar scale) toward cancer cell lines and tumor organoids. Furthermore, in vivo testing reveals that photoactivation of DBI induces not only G4 formation and DNA damage but also apoptosis in zebrafish, specifically in the area where DBI had accumulated. Collectively, this approach shows significant promise for image-guided PDT.


General methods
Reagents and chemicals from commercial sources were used without further purification. Solvents were dried and purified using standard techniques. Silica gel chromatographies were performed on Aldrich (Saint-Louis, USA) silica gel (technical grade, pore size 60 Å, 230-400 mesh particle size) packed with analytical-grade solvents. Flexible plates ALUGRAM® Xtra SIL G UV254 from MACHEREY-NAGEL (Düren, Germany) were used for TLC. Compounds were detected by UV irradiation (Thermo Fisher Scientific, Waltham, MA, USA). NMR spectra were recorded with an AVANCE III 300 (1H, 300 MHz and 13C, 75MHz) from Bruker (Bruker, Billerica, MA, USA). Chemical shifts are given in ppm relative to TMS and coupling constants J in Hz. UV-vis spectra were recorded on a Shimadzu UV-1800 spectrometer (Shimadzu, Kyoto, Japan). High-resolution mass spectrometry (HRMS) was performed with a JEOL JMS-700 B/E (JEOL, Peabody, USA). Cyclic voltammetry was performed using a Biologic SP-150 potentiostat with positive feedback compensation in 0.10 M Bu4NPF6/CH2Cl2 (HPLC grade). Experiments were carried out in a one-compartment cell equipped with a platinum working electrode (2 mm of diameter) and a platinum wire counter electrode. A silver wire immersed in 0.10 M Bu4NPF6/CH2Cl2 was used as pseudo-reference electrode and checked against the ferrocene/ferrocenium couple (Fc/Fc+) before and after each experiment. The potentials were then expressed vs Fc/Fc+.
The organic phase was then extracted with CH2Cl2, dried over MgSO4 and concentrated under reduced pressure. The resulting 1-aminonaphthalene-2-thiol was directly engaged in the next step without further purification. The latter was blended with 4-bromo-1,8-naphthalic anhydride (11.0 g, 39.7 mmol) and potassium carbonate (5.49 g, 39.7 mmol). DMF (270 mL) under air was then added and the reaction mixture was stirred for 16 h at room temperature. Isopentyl nitrite (15.7 mL, 119.1 mmol) was then added. An orange precipitate appeared upon stirring at 60 °C. After 24 h, the latter was filtrated, successively washed with water, methanol and finally dried. The resulting powder was directly blended added to a flask containing 3-aminopentane (5.33 mL, 45.7 mmol) and imidazole (77 g). This mixture was stirred for 16 h at 100 °C before being cooled down to room temperature. Then, a 1 M aqueous solution of HCl was gently added and the organic phase was subsequently extracted with CH2Cl2, dried over MgSO4 and concentrated under reduced pressure. The crude was purified by column chromatography on silica gel (eluent: CH2Cl2) affording an orange solid (2.20 g, 10%

Spin-Orbit Coupling calculations
Ground and excited states geometry optimizations were carried out with the Gaussian16 code.(1) The global hybrid functional PBE0 was used both for ground state and excited state geometry optimisations.(2) This functional was chosen because of its accuracy to reproduce spectroscopic properties of benzothioxanthene imide derivatives.(3) Structural optimisations and subsequent frequency calculations for both the ground and excited states were performed using an all electron Pople triple zeta basis set with one polarisation function on all atoms and one diffuse function of heavier atoms, known as 6-311+G(d,p), for H, C, N, O and S atoms.(4) Bulk solvent effects were included using the Polarizable Continuum Model (PCM) of Tomasi and co-workers.(5) Default radii (from the UFF, scaled by 1.1) were used. Excited state geometry was obtained by minimizing the forces of the S1 state computed at the TD-DFT level by considering the 3 first excited states. The Dalton(6) program was used to compute the SOC between the three first triplet states (namely T1, T2 and T3) and the S1 state at the S1 optimized geometry using the quadric-response TD-DFT at the CAM-B3LYP/PCM level with the cc-pVDZ basis set adapted for the Douglas-Kroll calculations.(7) The Spin-Orbit Coupling was computed using the Douglas-Kroll Hamiltonian along with the spin-orbit mean field approach. (8) Absorption, emission and phosphorescence spectral signatures of DBI Absorption spectra were recorded on a JASCO V-650 spectrophotometer in diluted solution (ca. 10 -5 or 10 -6 M), using spectrophotometric grade solvents. Emission spectra were measured using Horiba-Jobin-Yvon Fluorolog-3 fluorimeter. The steady-state luminescence was excited by unpolarised light from a 450 W xenon continuous wave (CW) lamp and detected at an angle of 90° for measurements of dilute solutions (10 mm quartz cuvette) by using a Hamamatsu R928. Spectra were corrected for both excitation source light-intensity variation and emission spectral responses. 77K time-gated phosphorescence was measured with a 50 μs delay.
A summary of the main spectroscopic and photophysical data for the DBI is included in Supplementary

Fluorescence quantum yield
Luminescence quantum yields (ΦF) were measured in diluted solutions with an absorbance lower than 0.1, by using the following Equation 1: where A is the absorbance (or optical density) at the excitation wavelength, n the refractive index of the solvent and D the integrated luminescence intensity. "r" and "x" stand for reference and sample, respectively. Here, the reference is coumarin-153 in methanol (ΦF = 0.45). Excitations of reference and sample compounds were performed at the same wavelength. The reported results are the average of 4-5 independent measurements at various absorbances (comprised between 0.01-0.1) for both sample and reference. The plot of the integrated luminescence intensity vs. absorbance gives straight line with excellent correlation coefficients and the slope S can be determined for both sample (x) and reference (r). Equation 1 becomes Equation 2.

Determination of singlet oxygen quantum yield
Singlet oxygen quantum yield determination ΦΔ was calculated based on the following equation: The reported results are the average of 4-5 independent measurements at various absorbances (comprised between 0.01-0.1) for both sample and reference. The plot of the integrated singlet oxygen luminescence intensity vs. absorbance gives straight line with excellent correlation coefficients. The reference used is phenalenone (ΦΔ = 0.98 in dichloromethane). No Light + DNase + Light + DNase

In vitro G4 binding studies
Materials: Solvents, reagents, chemicals and biological templates were purchased from commercial suppliers (Sigma-Aldrich and Eurofins Genomics) and used without further modifications unless otherwise stated. Oligonucleotides were diluted with ultrapure water (DNase and RNase free) and stored at 5 °C. RNA oligos were diluted in DEPC-treated water. The exact oligonucleotide concentration was determined by UV/Vis spectroscopy using the molar extinction coefficients (ε260) provided in the Supplementary Table 3 and calculated by using an oligo analyzer on the IDT website. An aqueous stock solution (1 M) of TRIS buffer was prepared by dissolving tris(hydroxymethyl)aminomethane in water and the pH was then adjusted to 7.2.
General procedures: Steady-state emission spectra were recorded with a Jasco FP-6500 spectrofluorometer equipped with a JascoPeltier-type temperature controller (ETC2736, at Umeå University). 1.0 cm path length quartz cells were used throughout these measurements. Fluorescence lifetime decays were collected via Time-Correlated Single Photon Counting (TCSPC) setup by using a Fluorolog TCSPC (Horiba Jobin Yvon). The excitation source was a NanoLED with excitation peak at 490 nm and a long pass filter (>500 nm) was inserted into the excitation path. CD spectra were recorded with a Jasco J-1700 CD spectrometer or with a Jasco J-720 spectropolarimeter equipped with a JascoPeltier-type temperature controller (PTC-423L). Fluorescence polarization was recorded with a Biotek Synergy H4.
Oligonucleotide folding: Oligonucleotides were heated at 95 °C for 5 min in the presence of 100 mM KCl and then slowly allowed to reach room temperature overnight. The list of the oligonucleotides used in this study is provided in the Supplementary Table 3.  Figure 4a, of DBI complexed with various oligonucleotides was performed by dividing the emission of the complex (F) over the emission of DBI alone (F0) at 561 nm. Data representation was performed by using OriginPro 2020 software. Lifetimes were obtained by using bi-exponential (DBI-HIF-1α) or tri-exponential (DBI, DBI-mut HIF-1α and DBI-C-rich HIF-1α) decay functions. Then the averaged fluorescence lifetimes (τave) were calculated according to the following equation:

G4-binding screening:
where Ai is the i-th relative amplitude and τi is the i-th component of fluorescence lifetime, respectively.
Fluorescence polarization binding assay: DBI (1.0 μM, DMSO 1 % v/v) was titrated with incremental additions of oligonucleotides ranging from 0 to 10 equivalents. The binary mixtures (DBIoligonucleotide) were left to equilibrate for 1 hour before recording the signal. The variation in the fluorescence polarization signal of DBI was monitored upon the addition of incremental oligonucleotide concentrations and in certain systems provides typical saturation binding curves. Binding constants were obtained with Bindfit(9,10) by using multiple global fitting methods (Nelder-Mead method). Data representation was performed by using OriginPro 2020 software.

H NMR G4 studies. HIF-1α and mut
HIF1α were dissolved at a concentration of 110 μM in KCl 100 mM and 10 mM Tris buffer (pH = 7.2). 10% D2O was added to the solutions. All spectra were recorded at 298 K on a Bruker 850 MHz Avance III HD spectrometer equipped with a 5 mm TCI cryoprobe. Excitation sculpting was used in the 1H NMR titration experiments, and 256 scans were recorded. Processing was performed in Topspin 3.6 (Bruker Biospin, Germany).
CD-based thermal melting assays. CD melting data were acquired by increasing the temperature and monitoring the characteristic CD peaks of each sequence used. The oligonucleotide concentration was 2 μM and DBI concentration was 10 μM. Experiments were performed in TRIS buffer (10 mM, pH = 7.2) and KCl (5 mM). Melting values were estimated by fitting the normalized melting curves with a dose response function using OriginPro 2020 software. a Conventional 5ˊ to 3ˊ direction. b Molar extinction coefficient calculated by using oligo analyzer on the IDT web site. c ds-DNA sequences are formed by mixing an equimolar concentration of the two strands.

H NMR spectra in the imino-proton region for HIF-1α and mut HIF-1α
Supplementary Figure 12. 1 H NMR spectra in the imino-proton region for HIF-1α and mut HIF-1α (G4 template = 100 μM, TRIS buffer 10 mM, pH = 7.2 and KCl 100 mM). The absence of imino-proton signals in the mut HIF-1α spectrum indicates the absence of a G4 structure.

Molecular Dynamics simulations
Molecular dynamics simulations of G4s in the presence of two DBI molecules were performed using Amber20 (see Supplementary Table 5). The initial position of the DBI ligands mimics the binding pose of BMVC molecules in 6O2L structure.(11) Two stacked positions are modeled for each ligand, providing different environment around the rings and the sulfur atom. Parmbsc1 (12) and Gaff (13) parameters were used for G4 nucleobases and DBI respectively. In addition, DBI atomic charges were determined using RESP (14) approach at B3LYP/6-31+G(d,p)/IEFPCM(15) level of theory using Gaussian09.
Each system is solvated in a rectangular-shaped TIP3P water box to ensure a minimum distance of 12 Å between the solute and the edge of the periodic box. K + and Clions are added to neutralize the boxes and to reach an ionic concentration of about 0.1 M. After minimization of the systems, all the simulations were run using a timestep of 2 fs in combination with the SHAKE algorithm constraining the hydrogen covalent bonds, a 10 Å cutoff for intermolecular forces and the Particle Mesh Ewald method for electrostatic long-range interactions. We start with a heating procedure of 60 ps to increase the temperature from 0 to 300 K in the NVT ensemble, followed by a 1 ns equilibration step in the NPT ensemble with a Langevin thermostat (with a collision frequency of 1 ps -1 ) and a Berendsen barostat at 300 K and 1 bar. Finally, 5 replicas of 200 ns were produced per starting conformation in the same conditions. They were analyzed using CPPTRAJ and the MMPBSA.py module from Amber20. Binding free energy calculations have been performed on the last 20 ns of each replica.

Competitive binding assay between DBI and PhenDC3
Supplementary Figure 19. a) Schematic representation of the competitive binding assay. DBI is weakly fluorescent in its unbound state. Upon binding to parallel G4s its emissive properties are enhanced. Competition for the same G4-binding site using the well-known G4-end stacker PhenDC3 causes the replacement of DBI from the G4 template and the resulting fluorescence quenching. b) Experimental evidence for the competitive binding between DBI and PhenDC3 (DBI = 1μM, HIF-1α = 1 μM, PhenDC3 = 0 to 0.45 μM, TRIS buffer = 50 mM, pH = 7.2 and KCl = 100 mM. c) Color changes detected upon UV-irradiation (312 nm) for DBI, DBI-HIF-1α complex and PhenDC3-HIF-1α-DBI mixture.

EPR determination of radicals formed upon photoirradiation
2,2,6,6-tetramethyl-4-piperidine (TEMP) (Sigma-Aldrich) and DMPO (TCI chemicals) samples (5.10 -3 M) were prepared in air atmosphere in capillary tubes, in chloroform and DMSO, respectively. In each The concentration of DBI was 10 -4 M in all experiments. The irradiation performed using a Thorlab LED 530 nm, was directed into the EPR cavity while the spectrum was recorded. EPR assays were all carried out at room temperature using a Bruker E500 spectrometer operating at X-band (9.35GHz), sensitive cavity, with 100 KHz modulation frequency. The instrument settings were as follows: microwave power: 2-69 mW; modulation amplitude: 1 G; Hyperfine coupling constants a and g values were obtained with simulation of experimental spectra using easyspin (Matlab toolbox).