Candidats médicaments et sondes pour les structures secondaires des acides nucléiques

Sondes ciblant les acides nucléiques G-quadruplexes

 

Guanines exhibit a high propensity to self-assemble into quartets. These units stack readily in presence of potassium cations leading to highly stable tetrahelical structures called G-quadruplexes(G4). Consequently G-rich single-stranded nucleic domains are prone to fold into quadruplexes.

Guanine quartet (blue) and schematic representation of G-rich single strand folding into a G-quadruplex structure ( blue square = guanine, orange ribbon = phosphate sugar backbone).
Figure 1: Guanine quartet (blue) and schematic representation of G-rich single strand folding into a G-quadruplex structure ( blue square = guanine, orange ribbon = phosphate sugar backbone).

 

G4 structures have become the object of intense study with the aim of defining their potential as regulatory elements and/or therapeutic targets and, as a result, are increasingly considered as druggable targets. These structures can be stabilized by small synthetic molecules (G4-ligands) and currently many families of G4 ligands show strong preferential binding for G-quadruplex DNA as compared to duplex DNA.  Over the past two decades our team has contributed to develop several original chemical series for studying quadruplex DNA structures and their involvement in various biological processes ( Fig 2).

Main chemical classes of G4 binding compounds developed
Figure 2: Main chemical classes of G4 binding compounds developed

1 — The Bisquinolinium Dicarboxamide compound series: Benchmark probes for quadruplexes
The Phenanthroline derivative (PhenDC3) ranks amongst the most selective quadruplex binder reported so far (De Cian et al. J. Am. Chem . Soc. 2007). Its exceptional performances result from the U-shape and size of the scaffold which afford perfect p-p-overlap with a G-quartet surface as confirmed by the NMR structure of the PhenDC3-Cmyc quadruplex  (Fig 3) ( collaboration with Prof. A.T. Phan ,NTU-Singapore).  PhenDC3 combines high binding affinity (nanomolar Kd) for most G4  structures and exquisite selectivity over duplex DNA ( >100), both being prerequisite to unbiaised cellular investigations.  Therefore PhenDC3 is one of the most popular benchmark G4-probe and has been used successfully in various cellular models to support G4 formation at the level of DNA and RNA (Nucleic Acid Res. 2011, Cancer Res. 2011, ChemBiochem 2011, Nat.Struc. Mol. Biol. 2011, Nat. Comm 2017).  In particular, in collaboration with the team of A. Nicolas (UMR3244 , Institut Curie) PhenDC3 has been shown to inhibit G4-unwinding by Pif1 helicase thereby promoting quadruplex structures in human minisatellites sequences inserted in a yeast model (Nucleic Acid. Res. 2010, EMBO.J ; 2011, 2015).

Figure 1

Figure 3: A,B) NMR structure of PhenDC3 bound to the c-myc quadruplex, B) Top view showing the perfect p-overlap of the four guanines forming the top G-quartet.

Imaging G4 ligand distribution on metaphasic chromosomes: A current challenge in the field is to localize G4-drugs in cells to correlate/rationalize in vitro target specificity and in cellulo phenotype. In turn G4-drug localization approaches are expected to provide knowledge/evidence on G4 presence in various cellular contexts and conditions.  Recently we employed secondary ion mass spectroscopy imaging (nanoSIMS) for mapping the distribution of a bromo derivative of PhenDC3 (Br-PhenDC3), within metaphase human chromosomes. To increase the accuracy and the spatial resolution of the nanoSIMS imaging a statistical approach was implemented as a plug in for the image analysis software ImageJ. Strikingly, the results demonstrate the presence of Br-PhenDC3 both at terminal and interstitial regions of chromosomes in consistency with studies using G4 antibody staining and tritium-labelled G4 ligand (360A) but with an unprecedented resolution (Fig.4). More largely, our study constitutes a demonstration of the effectiveness of nanoSIMS imaging as an alternative method for accurate genome-wide mapping of DNA interactive drugs. D. Verga et al. J. Mol. Biol. & Mol. Imaging, 2017, 4(1), 1029-1035.

left) Br-PhenDC3 derivative, center) nanoSIMS analysis. Overlay of significant (p=0.9999) Br (orange) and P (green) nanoSIMS images depicting the localization of Br in discrete regions in chromosomes center, c) and nucleus (top, n). Left) Numerical zoom of single chromosome indicated by white arrow. Left P signal; Middle, overlay of Br (orange) and P (green) signals; right Br significant (p=0.9999) signal.
Figure 4: left) Br-PhenDC3 derivative, center) nanoSIMS analysis. Overlay of significant (p=0.9999) Br (orange) and P (green) nanoSIMS images depicting the localization of Br in discrete regions in chromosomes center, c) and nucleus (top, n). Left) Numerical zoom of single chromosome indicated by white arrow. Left P signal; Middle, overlay of Br (orange) and P (green) signals; right Br significant (p=0.9999) signal.

Using the parent compound PDC (aka 360A , Mailliet, Riou , Mergny et al. 2004) harboring a pyridine core easily derivatizable in para position, we developed a number of functional probes selective for quadruplex structures.  In particular fluorescent probes (E. Largy et al. Topics in Current Chem. 2013, 330, 111–177; see also A.Granzhan team), photocrosslinking derivatives that alkylate selectively quadruplex structures (Fig.5) D. Verga et al. Angew. Chem. Int. Ed. 2014, 53, 994-998 and biotin derivatives.

Structure of PDC derivatives developed for crosslinking, trapping and labelling G-quadruplexes
Figure 5: Structure of PDC derivatives developed for crosslinking, trapping and labelling G-quadruplexes

2 — Metallic complexes for targeting G4 in human telomeres.  We have been the first to show that tridentate complexes of transition metals (Pt2+, Pd2+) are able to establish a dual interaction based both on p-stacking and metallic coordination to loop bases surrounding external quartets of quadruplex forming sequences (Org.Biomol.Chem. 2009, Chem. Eur. J. 2011).  This design stems on the choice to develop monocoordinating complexes much less studied than chelating complexes (cisPt type). The very efficient coordination observed with the Pt-ttpy compound (Fig. 4) is attributed to a confinement of the compound inside the quadruplex matrix and to the loop sequence specificity bearing adenine residues (Fig. 5).  The importance of the large aromatic surface provided by the tolylterpyridine aromatic moiety has been clearly shown and confirmed by a recent NMR study (M. Trajkovski et al. Chem.Eur.J. 2015, Fig. 6) ( Collab.  Y.Plavec, Lubjlana, Slovenia)

Structure of the tolyterpyridine Pt(II) derivative Pt-ttPy and representation of the platination of adenine residues surrounding the top quartet in the telomeric (22AG) and the modified oncogene c-myc (myc22) quadruplex forming sequences ( bound Pt-ttpy in pink).
Figure 6: Structure of the tolyterpyridine Pt(II) derivative Pt-ttPy and representation of the platination of adenine residues surrounding the top quartet in the telomeric (22AG) and the modified oncogene c-myc (myc22) quadruplex forming sequences ( bound Pt-ttpy in pink).

Interestingly, the preferential action of this compound series (Pt-ttpy and Cu-ttpy) at telomeres has been confirmed by a recent study based on a telomere specific drug screening assay in cells (H.S. Lee et al. Cancer Res. 2018, 78, 6282-6296  (collab. Y.Pommier, V. Larionov NCI, Bethesda, MA, USA).

We are also contributing to two translational projects in collaboration with clinicians of the Institut Curie i) Optimized photosensitizers for Photodynamic treatment of retinoblastoma and ii) New radiosensitizers for treating radioresistant cancers (Glioblastoma) ( see anticancer drugs and Photosensitizer topics).